WO2021263123A1

WO2021263123A1 - Rapid mass spectrometric methods for identifying microbes and antibiotic resistance proteins

Info

Publication number: WO2021263123A1
Application number: PCT/US2021/039126
Authority: WO
Inventors: William MC GEE; Scott KRONEWITTER; Stevan Horning; Leena Valmu; Tuomo Von Lerber; Roger GRIST; Suvi RAVELA; Johan Finell; Pirjo Wacklin; Anssi RANTAKARI; James Stephenson; Jason NEIL
Original assignee: Thermo Fisher Scientific Oy
Priority date: 2020-06-26
Filing date: 2021-06-25
Publication date: 2021-12-30

Abstract

The direct identification of antibiotic resistance proteins from clinical specimens remains a challenge. Disclosed herein are unique compositions and methods for overcoming the challenges presented by confounding proteins in positive blood cultures. The disclosed compositions and methods are especially applicable to mass spectrometric based analyses for the identification of microorganisms in a blood culture.

Description

RAPID MASS SPECTROMETRIC METHODS FOR IDENTIFYING MICROBES AND ANTIBIOTIC RESISTANCE PROTEINS

RELATED APPLICATIONS

[0001] This International application claims the benefit of U.S. Provisional Application No. 63/044,738 filed June 26, 2020, which disclosures are incorporated herein by reference.

BACKGROUND

[0002] The discovery, commercialization and routine administration of antimicrobial compounds to treat infections revolutionized modem medicine and changed the therapeutic paradigm. Antibiotics ushered in the development of complex medical approaches such as cutting-edge surgical procedures, solid organ transplantation and management of patients with cancer. After the introduction of antibiotics, bacterial infections were no longer among the top ten causes of death.

[0003] Unfortunately, the marked increase in antimicrobial resistance among common bacterial pathogens is now threatening this therapeutic accomplishment, jeopardizing the successful outcomes of critically ill patients. The World Health Organization has named antibiotic resistance as one of the three most important public health threats of the 21^st century.

[0004] Antimicrobial resistance is an expected result of the interaction of many organisms with their environment. When susceptible microbes are exposed to antimicrobial compounds, a subset of the microbes may develop mutations in genes that affect the activity of the antimicrobial compound on the microbe, resulting in cell survival or growth in the presence of the antimicrobial compound. Once a resistant mutant emerges, continued exposure of the microbial population to the antimicrobial compound eliminates the susceptible microbes and the resistant microbes then predominate. Resistance to the effects of antimicrobial compounds can be transferred to susceptible microbes through horizontal gene transfer.

[0005] In the clinic, it is critical to know whether an infecting microbe is resistant or susceptible to the action of an antimicrobial compound. This is accomplished by antibiotic susceptibility testing. Presently, antibiotic susceptibility testing relies largely on culture-based methods. In culture-based methods, microbial pathogens are initially grown from a patient specimen, a pure culture is isolated and ultimately challenged with different antibiotics. This process is time consuming, taking days to culture pathogens and then test for susceptibility. During this time clinicians will have likely already initiated sub-optimal antibiotic treatment.

[0006] The instant disclosure provides unique methodologies, based on mass spectrometric analyses, for identifying microbes and assessing whether a microbe possesses antibiotic resistance. These methodologies detect the presence or absence of antibiotic resistance proteins. The disclosed methodologies are rapid, significantly reducing time to answer by eliminating time consuming culture steps.

BRIEF SUMMARY

[0007] Rapid and reliable microbe identification, especially for pathogens, is crucial in medicine and clinical diagnosis for prescribing the most efficient treatment. Microbial identification is largely based on isolation, analysis of morphological characteristics, biochemical assays and molecular analysis, such as sequencing. In the clinical microbiology lab, after the microbe is identified, the microbe’s resistance to antimicrobial agents is often assessed. This is accomplished by growing microbes in the presence of antimicrobials. These assays can require a week or more to complete. Taken in whole, all of this represents a time consuming, laborious process in a situation requiring expediency.

[0008] The introduction of matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy to the clinical microbiology laboratory has improved microbial identification time, but still fails to detect microbial resistance proteins. Clinical MALDI-TOF mass spectroscopy relies on indirect methods for resistance detection. For instance, the detection of antimicrobial break-down products or changes in microbial growth rates in the presence of an antimicrobial.

[0009] Disclosed herein is an automated diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, and methods, for rapidly detecting and identifying microbes and antimicrobial resistance proteins by electrospray ionization (ESI) mass spectrometry, wherein the identification of the microbe is accomplished by MSI spectrum protein analysis, without MS2 protein spectrum analysis. In some instances, the antimicrobial resistance protein that is detected imparts resistance to b-lactam antibiotics. In some instances, the antimicrobial resistance protein that is detected and identified is penicillin binding protein 2a (PBP-2a). In other instances, the antimicrobial resistance protein that is detected and identified is a b-lactamase. In still other instances, the antimicrobial protein that is detected and identified is a carbapenemase.

DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 depicts a schematic diagram of a workflow of the automated rapid diagnostic system disclosed herein, the Acrion™ Analyzer. Extracted proteins from a sample are first characterized using MSI, producing an MSI spectrum. If the MSI spectrum possesses sufficient data, the MSI spectrum is further analyzed using an identification algorithm which compares the MSI spectrum resulting from the sample against a preformed database of MSI spectra. From this, an identification of the microbe present in the sample can be made. The microbe identification is made solely using MSI spectrum; not requiring an analysis of MS2 spectra. Identification of the presence of certain microbes triggers a reflex method for the detection and identification of antimicrobial resistance proteins. In the reflex method, the sample is once again subjected to MSI, followed by MS2 analysis, producing a MS2 spectrum. The MS2 spectrum is then used to identify the antimicrobial resistance protein present.

[0011] Figure 2 depicts the integrated rapid mass spectrometric instrument and work-flow for identifying microbes and underlying antimicrobial resistance protein identification. A sample is introduced into a vial, which in turn is inserted into the Acrion™ Analyzer. The Acrion™ Analyzer combines sample preparation, liquid chromatography and a Thermo Scientific™ Orbitrap™ high resolution mass spectrometer into a single seamless integrated process. Appearing on the far left of the figure is a cartoon of the Acrion™ Analyzer, just right of the analyzer in the figure is a sample vial in which liquid is being added to the sample vial. Moving to the right from the sample vials, the liquid chromatography column, the ProTrap™ column and liquid chromatography module are shown. Right of that, an internal representation of the Thermo Scientific™ Orbitrap™ high resolution mass spectrometer used by the analyzer, and on the farthest right, identification of the microbe by application of identification program which compares the sample MSI spectrum with that of a preformed MSI database of MSI spectra. A report is then produced with the microbe and antimicrobial resistance protein identification.

Mass spectrometric microbe identification is carried out in < 2 minutes run time per sample, while the reflex method for antimicrobial protein detection and identification occurs in < 6 minutes. [0012] Figure 3 shows a graph representing characteristics of the reflex method for identifying carbapenem resistance proteins. After identifying bacteria present in the sample, the Acrion™ Analyzer is programed to run a reflex analysis method to detect and identify antibacterial resistance proteins present. In the reflex method an aliquot from the same extracted protein sample used in the identification process is passed through a liquid chromatography column. The Protrap™ column bound proteins are subsequently eluted by running increasing concentrations of acetonitrile through the column over time and into the mass spectrometer; x- axis, time in minutes, left hand y-axis, percentage of acetonitrile, right-hand y-axis, MS resolution range. The elution time, acetonitrile concentration, and mass spectrometer resolution range of various antibacterial resistance proteins are depicted.

[0013] Figure 4 depicts a representative MSI spectra used for microbe identification. In this instance, the MSI spectra is that from the Gram-negative bacteria Proteus mirabilis grown on an agar plate.

[0014] Figure 5 shows an alternative visualization of the data shown in Figure 4. Here, the x-axis is time, while the y-axis provides monoisotopic masses. Intensity from Figure 4 is provided in grey scale, with light grey representing less intensity progressing to black, the highest intensity.

[0015] Figure 6 depicts a representative MSI spectra used for microbe identification. In this instance, the MSI spectra is that from the Gram-negative bacteria Acinetobacter pittii grown on an agar plate.

[0016] Figure 7 shows an alternative visualization of the data shown in Figure 6. Here, the x-axis is time, while the y-axis provides monoisotopic masses. Intensity from Figure 6 is provided in grey scale, with light grey representing less intensity progressing to black, the highest intensity.

[0017] Figure 8 depicts a representative MSI spectra used for microbe identification. In this instance, the MSI spectra is that from the Gram-negative bacteria Clostridioides difficile grown on an agar plate.

[0018] Figure 9 shows an alternative visualization of the data shown in Figure 8. Here, the x-axis is time, while the y-axis provides monoisotopic masses. Intensity from Figure 8 is provided in grey scale, with light grey representing less intensity progressing to black, the highest intensity.

[0019] Figure 10 depicts a representative MSI spectra used for microbe identification. In this instance, the MSI spectra is that from the Gram-positive bacteria Gordonia sputi grown on an agar plate.

[0020] Figure 11 shows an alternative visualization of the data shown in Figure 10. Here, the x-axis is time, while the y-axis provides monoisotopic masses. Intensity from Figure 10 is provided in grey scale, with light grey representing less intensity progressing to black, the highest intensity.

[0021] Figure 12 depicts a representative MSI spectra used for microbe identification. In this instance, the MSI spectra is that from the Gram-positive bacteria Streptococcus pyogenes grown on an agar plate.

[0022] Figure 13 shows an alternative visualization of the data shown in Figure 12. Here, the x-axis is time, while the y-axis provides monoisotopic masses. Intensity from Figure 12 is provided in grey scale, with light grey representing less intensity progressing to black, the highest intensity.

[0023] Figure 14 depicts a representative MSI spectra used for microbe identification. In this instance, the MSI spectra is that from the Gram-positive bacteria Bacteroides fragilis grown on an agar plate.

[0024] Figure 15 shows an alternative visualization of the data shown in Figure 14. Here, the x-axis is time, while the y-axis provides monoisotopic masses. Intensity from Figure 14 is provided in grey scale, with light grey representing less intensity progressing to black, the highest intensity.

[0025] Figure 16 depicts a representative MSI spectra used for microbe identification. In this instance, the MSI spectra is that from the Gram-negative bacteria Achromobacter xylosxidans grown in a blood culture.

[0026] Figure 17 shows an alternative visualization of the data shown in Figure 16. Here, the x-axis is time, while the y-axis provides monoisotopic masses. Intensity from Figure 16 is provided in grey scale, with light grey representing less intensity progressing to black, the highest intensity.

[0027] Figure 18 depicts a representative MSI spectra used for microbe identification. In this instance, the MSI spectra is that from the Gram-negative bacteria Bacteroides fragilis grown in a blood culture.

[0028] Figure 19 shows an alternative visualization of the data shown in Figure 18. Here, the x-axis is time, while the y-axis provides monoisotopic masses. Intensity from Figure 18 is provided in grey scale, with light grey representing less intensity progressing to black, the highest intensity.

[0029] Figure 20 depicts a representative MSI spectra used for microbe identification. In this instance, the MSI spectra is that from the Gram-negative bacteria Citrobacter freundii grown in a blood culture.

[0030] Figure 21 shows an alternative visualization of the data shown in Figure 20. Here, the x-axis is time, while the y-axis provides monoisotopic masses. Intensity from Figure 20 is provided in grey scale, with light grey representing less intensity progressing to black, the highest intensity.

[0031] Figure 22 depicts a representative MSI spectra used for microbe identification. In this instance, the MSI spectra is that from the Gram-positive bacteria Enterococcus avium grown in a blood culture.

[0032] Figure 23 shows an alternative visualization of the data shown in Figure 22. Here, the x-axis is time, while the y-axis provides monoisotopic masses. Intensity from Figure 22 is provided in grey scale, with light grey representing less intensity progressing to black, the highest intensity.

[0033] Figure 24 depicts a representative MSI spectra used for microbe identification. In this instance, the MSI spectra is that from the Gram-positive bacteria Gamella haenolysans grown in a blood culture.

[0034] Figure 25 shows an alternative visualization of the data shown in Figure 24. Here, the x-axis is time, while the y-axis provides monoisotopic masses. Intensity from Figure 24 is provided in grey scale, with light grey representing less intensity progressing to black, the highest intensity.

[0035] Figure 26 depicts a representative MSI spectra used for microbe identification. In this instance, the MSI spectra is that from the Gram-positive bacteria Streptococcus gallolyticus grown in a blood culture.

[0036] Figure 27 shows an alternative visualization of the data shown in Figure 26. Here, the x-axis is time, while the y-axis provides monoisotopic masses. Intensity from Figure 26 is provided in grey scale, with light grey representing less intensity progressing to black, the highest intensity.

[0037] Figure 28 depicts a representative MSI spectra used for microbe identification. In this instance, the MSI spectra is that from the yeast Aspergillus niger grown on a nutrient plate.

[0038] Figure 29 shows an alternative visualization of the data shown in Figure 28. Here, the x-axis is time, while the y-axis provides monoisotopic masses. Intensity from Figure 28 is provided in grey scale, with light grey representing less intensity progressing to black, the highest intensity.

[0039] Figure 30 depicts a representative MSI spectra used for microbe identification. In this instance, the MSI spectra is that from the yeast Aspergillus terms grown on a nutrient plate.

[0040] Figure 31 shows an alternative visualization of the data shown in Figure 30. Here, the x-axis is time, while the y-axis provides monoisotopic masses. Intensity from Figure 30 is provided in grey scale, with light grey representing less intensity progressing to black, the highest intensity.

[0041] Figure 32 depicts a representative MSI spectra used for microbe identification. In this instance, the MSI spectra is that from the yeast Candida albicans grown on a nutrient plate.

[0042] Figure 33 shows an alternative visualization of the data shown in Figure 32. Here, the x-axis is time, while the y-axis provides monoisotopic masses. Intensity from Figure 32 is provided in grey scale, with light grey representing less intensity progressing to black, the highest intensity.

[0043] Figure 34 depicts a representative MSI spectra used for microbe identification. In this instance, the MSI spectra is that from the yeast Candida auris grown on a nutrient plate. [0044] Figure 35 shows an alternative visualization of the data shown in Figure 34. Here, the x-axis is time, while the y-axis provides monoisotopic masses. Intensity from Figure 34 is provided in grey scale, with light grey representing less intensity progressing to black, the highest intensity.

[0045] Figure 36 depicts a representative MSI spectra used for microbe identification. In this instance, the MSI spectra is that from the yeast Candida neoformans grown on a nutrient plate.

[0046] Figure 37 shows an alternative visualization of the data shown in Figure 36. Here, the x-axis is time, while the y-axis provides monoisotopic masses. Intensity from Figure 36 is provided in grey scale, with light grey representing less intensity progressing to black, the highest intensity.

[0047] Figure 38 shows a graph representing the mass spectrum for the antibacterial resistance protein PBP2a during the reflex analysis method. The inset represents MS2 data derived in this analysis.

[0048] Figure 39 shows a graph representing the characteristics of the antibacterial resistance protein KPC-2 during the reflex analysis method. The inset graph shows the tandem mass spectrum obtained. The light gray line represents the base peak chromatogram for the run while the thick black line represents the base peak MS2 chromatogram for KPC-2.

[0049] Figure 40 shows a graph representing the characteristics of the antibacterial resistance protein KPC-3 during the reflex analysis method. The inset graph shows the tandem mass spectrum obtained. The light gray line represents the base peak chromatogram for the run while the thick black line represents the base peak MS2 chromatogram for KPC-3.

[0050] Figure 41 depicts a comparison of the spectra of KPC-2 and KPC-3 shown in Figures 39 and 40. This demonstrates the ability of the Acrion™ Analyzer to detect and distinguish closely related variants. This ability to rapidly distinguish closely related antibacterial resistance proteins, in automated fashion, is unique to the Acrion™ Analyzer and associated methods.

[0051] Figure 42 shows a graph representing the antibacterial resistance protein OXA-232 during the reflex analysis method. The inset graph shows the tandem mass spectrum obtained. The light gray line represents the base peak chromatogram for the run while the thick black line represents the base peak MS2 chromatogram for OXA-232. The inset graphs show the transition ions from the precursor or isolation step generated by MS2.

[0052] Figure 43 depicts the full mass spectrum of a reflex analysis of OXA-232. The inset shows the diagnostic region.

[0053] Figure 44 shows a graph representing the antibacterial resistance protein VIM-1 during the reflex analysis method. The jagged light gray line is again the base peak chromatogram for the run. The thick dark line is a summation of the ion intensities of the key distinguishing product ions from VIM-1. All of these ions are shown in the seven insets in figure 10. The thicker gray line happens to be the base peak chromatogram derived from the precursor MS/MS signal. Please note that this optimum does not line up directly with the maximum of the thick black line. This is because there is another protein interfering in the isolation window with the target. In this instance, the interfering protein happens to be more intense, however, VIM-1 can diagnostic ions can still be detected.

[0054] Figure 45 depicts a graph representing the mass spectrum for the antibacterial resistance protein VIM-1 during the reflex analysis method. The insets show the lower intensity fragment ions. The peak of highest intensity corresponds to 974 m/z. This is because of the co isolation of proteins in this range. The interfering protein does not interfere with the detection and identification of VIM-1.

[0055] Figure 46 shows a graph representing the antibacterial resistance protein VIM-2 during the reflex analysis method. The jagged light gray line is again the base peak chromatogram for the run. The thick dark line is a summation of the ion intensities of the key distinguishing product ions from VIM-2. All of these ions are shown in the seven insets.

[0056] Figure 47 shows a graph representing the mass spectrum for the antibacterial resistance protein VIM-1 during the reflex analysis method.

DETAILED DESCRIPTION

[0057] Rapid and correct pathogen identification is crucial in medicine, veterinary science and food analysis. Mass spectrometry is very attractive for microbiology work because of its sensitivity, specificity and speed. Microbial identification can be accomplished in minutes after microbial biomarkers enter the mass spectrometer. Mass spectrometry can also determine whether microbes possess antimicrobial resistance. This combination of microbe identification and antimicrobial resistance determination is critically important to clinical microbiologists, infectious disease specialists and physicians.

[0058] MALDI (matrix-assisted laser desorption ionization) mass spectrometry based systems have been commercially introduced for identifying microbes and detecting antimicrobial resistance byproducts. Two main commercially available MALDI-based systems are the BioTyper (Bruker Daltonics, Bremen, Germany) and the VITEX MS (BioMerieux, Marcy l'Etoile, France). In these systems, a cultured microbe is mixed with a matrix, plated on a target and ionized. This produces a mass spectrum which is compared to a pre-formed mass spectral database for microbe identification. Antimicrobial resistance is detected indirectly by these systems. Detecting the metabolites produced but not the antimicrobial resistance biomolecules themselves.

[0059] While MALDI has found use in the microbiology laboratory, it suffers from several drawbacks. MALDI is relatively difficult to couple on-line, in an automated fashion, with separation methods. MALDI sample preparation requires a series of manual steps which are difficult to automate. Plating microbial colonies on a MALDI target plate is tedious. Some describe MALDI plating as an art form, suggesting that the manual preparatory steps can introduce errors and inconsistencies among samples. These factors can limit speed and through put of samples, which is especially important to clinical microbiology laboratories.

[0060] The instant disclosure solves the need for a rapid mass spectrometry based diagnostic modality for detecting the presence or absence of a microbe in a sample and, when present, the identity of the microbe and whether the microbe possesses an antimicrobial resistance protein, and, when present, the identity of antimicrobial resistance protein.

[0061] The rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station and an electrospray ionization (ESI) mass spectrometer, wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of an antimicrobial resistance protein, and when present, the identification of a resistance protein in the sample. In some instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified, but not more than 5 - 10 are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified, but not more than 5 - 10. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only one antimicrobial resistance protein is identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only two antimicrobial resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only three antimicrobial resistance proteins are identified. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum alone. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum alone. In still other instances, when more than one antimicrobial resistance protein is detected, one or more antimicrobial resistance proteins are identified by analysis of MSI and one or more antimicrobial resistance proteins are identified by analysis of MS2.

[0062] "MS 1 " refers to the detection and analysis of first ions entering the mass spectrometer, the ions having not been subjected to prior mass filtering or prior mass analysis. ESI generates charged biomolecule ions by dissolving the biomolecule in a solvent and then pumping this combination through a thin capillary or needle with an electrical potential. As a result of the electrical field the biomolecule containing solution exits the capillary or needle as a charged droplet. These charged droplets travel down a pressure and potential gradient through an orifice in the mass spectrometer. As the droplets traverse this path they become desolvated. As the solvent evaporates the analyte becomes protonated. This ultimately results in a same analyte population with different levels of protonation/charge. The mass spectrometer separates multiply charged ions based on a combination of mass and charge. [0063] Data generated from the mass spectrometer can be represented as a two dimensional plot referred to as a mass spectrum. One axis of the mass spectrum is the mass-to-charge ratio (m/z). The other axis of the mass spectrum is the intensity. "MSI spectrum" refers to the mass spectrum resulting from MS 1.

[0064] "MS2", also called MS/MS or tandem mass spectrometry, refers to the analysis of ions that have been selected by a mass filter or mass analysis step. The selected ions are then altered in some way, for instance, the fragmentation of a population of polypeptide ions into its constituent polypeptide ions or by the charge-altered ion following a proton transfer reaction. "MS2 spectrum" refers to the mass spectrum resulting from MS2.

[0065] "Rapid" refers to the determination of the presence or absence of a microbe in a sample, and if present, the identity of the microbe and whether the identified microbe possesses an antimicrobial resistance protein in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station and a mass spectrometer, wherein identification of the microbe, if present, is accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence and identification of a antimicrobial resistance protein in the sample. In some instances, rapid refers to the determination of the presence or absence of a microbe in a sample, and if present, the identity of the microbe and whether the identified microbe possesses an antimicrobial resistance protein in 5 - 45 minutes, in 5 - 40 minutes, in 5 - 30 minutes or in 5 - 20 minutes from placing a sample into a vessel and placing the vessel into the instrument encompassing a robotic sample preparation station and a mass spectrometer, wherein identification of the microbe, if present, is accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence and identification of a antimicrobial resistance protein in the sample. In some instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified, but not more than 5 - 10 are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified, but not more than 5 - 10. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only one antimicrobial resistance protein is identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only two antimicrobial resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only three antimicrobial resistance proteins are identified. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum alone. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum alone. In still other instances, when more than one antimicrobial resistance protein is detected, one or more antimicrobial resistance proteins are identified by analysis of MSI and one or more antimicrobial resistance proteins are identified by analysis of MS2.

[0066] "Automated," as it is applied to analysis of a sample using the disclosed instrument encompassing a robotic sample preparation station and a mass spectrometer, wherein the presence or absence of a microbe in the sample and, if present, the identification of the microbe is accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence and identification of a antimicrobial resistance protein in the sample, refers to a process or step(s) occurring independently of manual intervention. Such a process or step, or processes or steps, can be controlled, or are controlled by, operation of computer software. Initiation of the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station and a mass spectrometer, wherein the presence or absence of a microbe in the sample and, if present, the identification of the microbe is accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence and identification of an antimicrobial resistance protein in the sample. In some instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified, but not more than 5 10 are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified, but not more than 5 10 In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only one antimicrobial resistance protein is identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only two antimicrobial resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only three antimicrobial resistance proteins are identified. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum alone. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum alone. In still other instances, when more than one antimicrobial resistance protein is detected, one or more antimicrobial resistance proteins are identified by analysis of MSI and one or more antimicrobial resistance proteins are identified by analysis of MS2.

[0067] "Robotic" or "robotic system" or "robot" refers to a stand-alone system that performs both physical and computational activities. The physical activities may be performed using a wide variety of movable parts, such as a robotic arm, a liquid sample transfer device, such as a pipette and a container capper/decapper. The computational activities may be performed utilizing a suitable processor and memory stores, for example, a data memory storage device or a computer.

[0068] In some instances, the robotic sample preparation station encompasses a robotic arm. "Robotic arm" refers to an electromechanical device that translates a payload, for instance a pipettor or capper/decapper, in two spatial dimensions, X and Y, but not in a third spatial dimension Z. In some instances, the robotic arm translates a payload in three spatial dimensions, X, Y, and Z. In some instances, the robotic arm can translate a payload in radial dimensions, degrees, Q and f, but not in a third spatial dimension z. In some instances, the robotic arm can translate a payload in spherical coordinates, degrees, q, f, and z. [0069] Accordingly, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a robotic arm, and an ESI mass spectrometer, wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample. In some instances, the robotic arm translates the payload in only two spatial dimensions. In other instances, the robotic arm translates the payload in three spatial dimensions. In some instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified, but not more than 5 10 are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified, but not more than 5 10 In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only one antimicrobial resistance protein is identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only two antimicrobial resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only three antimicrobial resistance proteins are identified. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum alone. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum alone. In still other instances, when more than one antimicrobial resistance protein is detected, one or more antimicrobial resistance proteins are identified by analysis of MSI and one or more antimicrobial resistance proteins are identified by analysis of MS2.

[0070] "Sample preparation station" refers to the combination of necessary materials and methods to treat a sample in such a way as to extract protein analytes. The output of the sample preparation station is an "extracted sample." [0071] In some instance, the sample preparation station encompasses lysis reagents. "Lysis" refers to the rupturing of a cell membrane or cell wall, releasing the cell’s cytoplasm. In some instances, the sample preparation station encompasses reagents for the lysis of non-microbial cells. Non-microbial cell lysis can be brought about by building osmotic pressure differences between the external medium and the non-microbial cell’s cytoplasm. Water and ammonium chloride are examples of reagents that can cause osmotic imbalance and non-microbial cell lysis.

[0072] Reagents for the lysis of non-microbial cells often include detergents. Detergents can be non-ionic or ionic. Examples of detergents used for non-microbial cell lysis include CHAPS, Triton-X or saponin.

[0073] Thus, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non- microbial cells lysis reagent. In some instances, the lysis reagent encompasses water or ammonium chloride. In other instances, the lysis reagent encompasses a detergent. In still other instance, the detergent is selected from CHAPS, Triton-X or saponin. In some instances, the robotic arm translates the payload in only two spatial dimensions. In other instances, the robotic arm translates the payload in three spatial dimensions. In some instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified, but not more than 5 10 are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified, but not more than 5 10 In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only one antimicrobial resistance protein is identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only two antimicrobial resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only three antimicrobial resistance proteins are identified. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum alone. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum alone. In still other instances, when more than one antimicrobial resistance protein is detected, one or more antimicrobial resistance proteins are identified by analysis of MSI and one or more antimicrobial resistance proteins are identified by analysis of MS2.

[0074] In some instance, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a robotic arm, and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cells lysis reagent. In some instances, the lysis reagent encompasses water or ammonium chloride. In other instances, the lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the robotic arm translates the payload in only two spatial dimensions. In other instances, the robotic arm translates the payload in three spatial dimensions. In some instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified, but not more than 5 - 10 are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified, but not more than 5 - 10. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only one antimicrobial resistance protein is identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only two antimicrobial resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only three antimicrobial resistance proteins are identified. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum alone. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum alone. In still other instances, when more than one antimicrobial resistance protein is detected, one or more antimicrobial resistance proteins are identified by analysis of MSI and one or more antimicrobial resistance proteins are identified by analysis of MS2.

[0075] Accordingly, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cells lysis reagent, the non-microbial cells lysis reagent encompasses saponin. In some instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified, but not more than 5 10 are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified, but not more than 5 10 In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only one antimicrobial resistance protein is identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only two antimicrobial resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only three antimicrobial resistance proteins are identified. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum alone. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum alone. In still other instances, when more than one antimicrobial resistance protein is detected, one or more antimicrobial resistance proteins are identified by analysis of MSI and one or more antimicrobial resistance proteins are identified by analysis of MS2.

[0076] The rapid diagnostic modality disclosed herein, in some instances, encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a robotic arm, and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cells lysis reagent, the non- microbial cells lysis reagent encompasses saponin. In some instances, the robotic arm translates the payload in only two spatial dimensions. In other instances, the robotic arm translates the payload in three spatial dimensions. In some instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins. In still other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins, but not more than 5 - 10. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins, but not more than 5 - 10. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum.

[0077] In other instances, the sample preparation station encompasses reagents for the lysis of microbial cells. Reagents for the lysis of microbial cells includes organic solvents, for instance, acetonitrile, acids, for instance, formic acid, and bases, for instance, sodium hydroxide. In some instances, microbial cells are lysed by a combination of organic solvents and acids, such as the combination of acetonitrile and formic acid.

[0078] Thus, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a microbial cells lysis reagent. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid. In some instances, the robotic arm translates the payload in only two spatial dimensions. In other instances, the robotic arm translates the payload in three spatial dimensions. In some instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified, but not more than 5 10 are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified, but not more than 5 10 In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only one antimicrobial resistance protein is identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only two antimicrobial resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only three antimicrobial resistance proteins are identified. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum alone. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum alone. In still other instances, when more than one antimicrobial resistance protein is detected, one or more antimicrobial resistance proteins are identified by analysis of MSI and one or more antimicrobial resistance proteins are identified by analysis of MS2.

[0079] In some instance, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a robotic arm, and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a microbial cells lysis reagent. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid. In some instances, the robotic arm translates the payload in only two spatial dimensions. In other instances, the robotic arm translates the payload in three spatial dimensions. In some instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified, but not more than 5 - 10 are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified, but not more than 5 - 10. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only one antimicrobial resistance protein is identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only two antimicrobial resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only three antimicrobial resistance proteins are identified. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum alone. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum alone. In still other instances, when more than one antimicrobial resistance protein is detected, one or more antimicrobial resistance proteins are identified by analysis of MSI and one or more antimicrobial resistance proteins are identified by analysis of MS2.

[0080] Accordingly, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cells lysis reagent, the microbial cells lysis reagent encompasses acetonitrile and formic acid. In some instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified, but not more than 5 - 10 are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified, but not more than 5 - 10. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only one antimicrobial resistance protein is identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only two antimicrobial resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only three antimicrobial resistance proteins are identified. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum alone. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum alone. In still other instances, when more than one antimicrobial resistance protein is detected, one or more antimicrobial resistance proteins are identified by analysis of MSI and one or more antimicrobial resistance proteins are identified by analysis of MS2.

[0081] The rapid diagnostic modality disclosed herein, in some instances, encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a robotic arm, and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a microbial cells lysis reagent, the microbial cells lysis reagent encompasses acetonitrile and formic acid. In some instances, the robotic arm translates the payload in only two spatial dimensions. In other instances, the robotic arm translates the payload in three spatial dimensions. In some instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins. In still other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins, but not more than 5 - 10. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins, but not more than 5 - 10. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum.

[0082] The rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cell lysis reagent and microbial cell lysis reagent. In some instances, the lysis reagent encompasses water or ammonium chloride. In other instances, the lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid. In some instances, the robotic arm translates the payload in only two spatial dimensions. In other instances, the robotic arm translates the payload in three spatial dimensions. In some instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified, but not more than 5 - 10 are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified, but not more than 5 - 10. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only one antimicrobial resistance protein is identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only two antimicrobial resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only three antimicrobial resistance proteins are identified. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum alone. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum alone. In still other instances, when more than one antimicrobial resistance protein is detected, one or more antimicrobial resistance proteins are identified by analysis of MSI and one or more antimicrobial resistance proteins are identified by analysis of MS2.

[0083] In some instance, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a robotic arm, and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cell lysis reagent and microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid. In some instances, the robotic arm translates the payload in only two spatial dimensions. In other instances, the robotic arm translates the payload in three spatial dimensions. In some instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified, but not more than 5 - 10 are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified, but not more than 5 - 10. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only one antimicrobial resistance protein is identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only two antimicrobial resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only three antimicrobial resistance proteins are identified. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum alone. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum alone. In still other instances, when more than one antimicrobial resistance protein is detected, one or more antimicrobial resistance proteins are identified by analysis of MSI and one or more antimicrobial resistance proteins are identified by analysis of MS2.

[0084] Accordingly, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cell lysis reagent and microbial cell lysis reagent. In some instances, the non- microbial cell lysis reagent encompasses saponin. In some instances, non-microbial cells lysis encompasses acetonitrile and formic acid. In some instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least two or more resistance proteins are identified, but not more than 5 - 10 are identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, at least three or more resistance proteins are identified, but not more than 5 - 10. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only one antimicrobial resistance protein is identified. In other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only two antimicrobial resistance proteins are identified. In still other instances, when a microbe is present, and the presence of an antimicrobial resistance protein is detected, only three antimicrobial resistance proteins are identified. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum alone. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum alone. In still other instances, when more than one antimicrobial resistance protein is detected, one or more antimicrobial resistance proteins are identified by analysis of MSI and one or more antimicrobial resistance proteins are identified by analysis of MS2.

[0085] The rapid diagnostic modality disclosed herein, in some instances, encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a robotic arm, and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cell lysis reagent and microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses saponin and the microbial cell lysis encompasses acetonitrile and formic acid. In some instances, the robotic arm translates the payload in only two spatial dimensions. In other instances, the robotic arm translates the payload in three spatial dimensions. In some instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins. In still other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins, but not more than 5 - 10. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins, but not more than 5 - 10. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum.

[0086] In some embodiments, the sample preparation station encompasses a centrifuge.

[0087] Accordingly, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a centrifuge, and an ESI mass spectrometer, wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample. In some instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins. In still other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins, but not more than 5 - 10. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins, but not more than 5 - 10. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum. In some instances, the robotic sample preparation station encompasses a non- microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[0088] In other embodiments, the sample preparation station encompasses a centrifuge. Accordingly, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a robotic arm and a centrifuge, and an ESI mass spectrometer, wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample. In some instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins. In still other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins, but not more than 5 - 10. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins, but not more than 5 - 10. In some instances, the robotic arm translates the payload in only two spatial dimensions.

In other instances, the robotic arm translates the payload in three spatial dimensions. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non- microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[0089] Thus, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a centrifuge, and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cells lysis reagent. In some instances, the lysis reagent encompasses water or ammonium chloride. In other instances, the lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins. In still other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins, but not more than 5 - 10. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins, but not more than 5 - 10. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[0090] In some instance, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a robotic arm and a centrifuge, and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non- microbial cells lysis reagent. In some instances, the lysis reagent encompasses water or ammonium chloride. In other instances, the lysis reagent encompasses a detergent. In still other instance, the detergent is CHAPS, Triton-X or saponin. In some instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins. In still other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins, but not more than 5 - 10. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins, but not more than 5 - 10. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non- microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid. [0091] Accordingly, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a centrifuge, and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cells lysis reagent, the non- microbial cells lysis reagent encompasses saponin. In some instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins. In still other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins, but not more than 5 - 10. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins, but not more than 5 - 10. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non- microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid. [0092] The rapid diagnostic modality disclosed herein, in some instances, encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a robotic arm and a centrifuge, and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non- microbial cells lysis reagent, the non-microbial cells lysis reagent encompasses saponin. In some instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins. In still other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins, but not more than 5 - 10. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins, but not more than 5 - 10. In some instances, the robotic arm translates the payload in only two spatial dimensions.

[0093] In other embodiments, the sample preparation station encompasses a sonicator. "Sonicator" refers to a device effective to provide ultrasonic energy, for instance, by providing acoustic energy, typically in the form of vibrations, at ultrasonic frequencies. A sonicator can include a driving element which provides high-frequency vibrations. Many sonicators utilize piezoelectric materials to produce high-frequency vibrations. Piezoelectric ultrasound generators typically require high voltages about 200 volts (V) to about 400 V) to provide the needed alternating current drive to operate such generators.

[0094] Sonicators often have sonicator horns configured to direct ultrasonic energy to a tip portion. A tip portion is configured to effectuate energy transfer to a vessel, a wall of a vessel, or to a fluid contained within a vessel.

[0095] Accordingly, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a sonicator, and an ESI mass spectrometer, wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample. In some instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins. In still other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins, but not more than 5 - 10. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins, but not more than 5 - 10. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum. In some instances, the robotic sample preparation station encompasses a non- microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[0096] In other embodiments, the sample preparation station encompasses a centrifuge. Accordingly, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a robotic arm and a sonicator, and an ESI mass spectrometer, wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample. In some instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins. In still other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins, but not more than 5 10 In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins, but not more than 5 10 In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum. In some instances, the robotic sample preparation station encompasses a non- microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[0097] The rapid diagnostic modality disclosed herein, in some instances encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a sonicator and a centrifuge, and an ESI mass spectrometer, wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample. In some instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins. In still other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins, but not more than 5 - 10. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins, but not more than 5 - 10. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[0098] In other embodiments, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a robotic arm, a centrifuge and a sonicator, and an ESI mass spectrometer, wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample. In some instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins. In still other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins, but not more than 5 - 10. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins, but not more than 5 - 10. In some instances, the robotic arm translates the payload in only two spatial dimensions. In other instances, the robotic arm translates the payload in three spatial dimensions. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[0099] The rapid diagnostic modality disclosed herein, in other instances encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a sonicator, and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cells lysis reagent. In some instances, the lysis reagent encompasses water or ammonium chloride. In other instances, the lysis reagent encompasses a detergent. In still other instance, the detergent is CHAPS, Triton-X or saponin. In some instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins. In still other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins, but not more than 5 - 10. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins, but not more than 5 - 10. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum.

[00100] In some instance, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a sonicator, and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cells lysis reagent. In some instances, the lysis reagent encompasses water or ammonium chloride. In other instances, the lysis reagent encompasses a detergent. In still other instance, the detergent is CHAPS, Triton-X or saponin. In some instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins. In still other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins, but not more than 5 - 10. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins, but not more than 5 - 10. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum.

[00101] Accordingly, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a sonicator and a robotic arm, and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non- microbial cells lysis reagent, the non-microbial cells lysis reagent encompasses saponin. In some instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins. In still other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins, but not more than 5 - 10. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins, but not more than 5 - 10. In some instances, the robotic arm translates the payload in only two spatial dimensions.

In other instances, the robotic arm translates the payload in three spatial dimensions. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum.

[00102] The rapid diagnostic modality disclosed herein, in some instances, encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a robotic arm and a centrifuge and a sonicator, and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cells lysis reagent, the non-microbial cells lysis reagent encompasses saponin. In some instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins. In still other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins, but not more than 5 - 10. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins, but not more than 5 - 10. In some instances, the robotic arm translates the payload in only two spatial dimensions.

[00103] The rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a robotic arm, wherein the robotic arm translates the payload in only two spatial dimensions and a centrifuge and a sonicator, and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cell lysis reagent, the non- microbial cell lysis reagent encompasses saponin. In some instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins. In still other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least two or more resistance proteins, but not more than 5 - 10. In other instances, when a microbe is present, the absence or presence of an antimicrobial resistance protein is detected, and when present, the identification of at least three or more resistance proteins, but not more than 5 - 10. In some instances, the antimicrobial resistance protein is identified by analysis of an MSI spectrum. In other instances, the antimicrobial resistance protein is identified by analysis of an MS2 spectrum.

[00104] MS analysis of microorganisms present in complex biological samples obtained from food, water, and clinical specimens must often be preceded by purification and concentration. The complexity of microbial biomarkers might be reduced with various chromatography -based methods. Efficient separation approaches should be considered to achieve a fast and high-throughput analysis.

Liquid chromatography - Mass spectrometry (LC-MS)

[00105] Liquid chromatography is a technique used to separate an analyte from a mixture. This separation occurs based on the interactions of the compound with the mobile and stationary phases. Liquid-solid column chromatography involves a liquid mobile phase which passes through a solid stationary phase. As the mobile passes through the solid stationary phase in the column, the compounds are separated. As each compound is eluted from the column, each can be collected and analyzed.

[00106] Solid phase extraction (SPE) is another term used to describe liquid chromatography using a solid stationary phase. SPE can be divided into three general types: normal phase, reverse phase and ion exchange. Normal phase SPE typically involves a polar analyte, a mid- to nonpolar matrix and a polar stationary phase. Retention of an analyte under normal phase conditions is primarily due to interactions between polar functional groups of the analyte and the polar groups of the solid phase. Reverse phase separations involve a polar or moderately polar mobile phase and a nonpolar stationary phase. The analyte of interest is typically mid- to nonpolar. Ion exchange SPE is used for compounds that are charged when in solution.

[00107] A powerful analytical system is created by the combination of liquid chromatography with mass spectrometry. In such a combined system, the physical separation capabilities of liquid chromatography are combined with the mass analysis capabilities of a mass spectrometer. This LC-MS system is particularly suited to resolving complex mixtures, as can be encountered when detecting microorganisms in a sample, such as a patient, a veterinary or a food sample.

[00108] Accordingly, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, a liquid chromatography station and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample. [00109] The liquid chromatography station encompasses an inlet for communicating the extracted sample from the robotic sample preparation station to a chromatography column and an outlet for communicating the eluate to the electrospray ionization source. The chromatography column is a monolithic column. Differentiation in elution times through the stationary phase of various molecules is based, at least in part, on the molecules size or affinity for the column. By manipulating the properties of the fluid passing through the stationary phase, by pH or relative concentration of aqueous or organic solvents, the passage of a molecule, such as a protein, through the column is controlled.

[00110] The liquid chromatography station also encompasses one or more solvent reservoirs, one or more valves and one or more pumps.

[00111] The rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, a liquid chromatography station and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the liquid chromatography station encompasses an organic solvent, the organic solvent encompassing acetonitrile.

[00112] In some instances, wherein the organic solvent of the sample preparation station encompasses acetonitrile, the acetonitrile represents 1-50%, 1-45%, 1-40%, 1-35%, 1-30%, 1- 25%, 1-20%, 1-15%, 1-10%, l-5% vol/vol of the mobile phase passing through chromatography column. In some instances, wherein the organic solvent of the sample preparation station encompasses acetonitrile, the acetonitrile represents 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the mobile phase passing through chromatography column.

[00113] The rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a robotic arm, a liquid chromatography station and an ESI mass spectrometer, wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample.

[00114] In some instances, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, a liquid chromatography station and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cells lysis reagent. In some instances, the lysis reagent encompasses water or ammonium chloride. In other instances, the lysis reagent encompasses a detergent. In still other instance, the detergent is CHAPS, Triton-X and saponin.

[00115] In other instances, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a robotic arm, a liquid chromatography station and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cells lysis reagent. In some instances, the lysis reagent encompasses water or ammonium chloride. In other instances, the lysis reagent encompasses a detergent. In still other instance, the detergent is CHAPS, Triton-X or saponin.

[00116] In still other instances, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cells lysis reagent, the non-microbial cells lysis reagent encompasses saponin.

[00117] The rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a centrifuge, a liquid chromatography station and an ESI mass spectrometer, wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample.

[00118] In some instances, the sample preparation station encompasses a centrifuge. Accordingly, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a robotic arm and a centrifuge, a liquid chromatography station and an ESI mass spectrometer, wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample.

[00119] In still other instances, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a centrifuge, a liquid chromatography station and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cells lysis reagent. In some instances, the lysis reagent encompasses water or ammonium chloride. In other instances, the lysis reagent encompasses a detergent. In still other instance, the detergent is CHAPS, Triton-X and saponin. [00120] In some instance, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a robotic arm and a centrifuge, a liquid chromatography station and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cells lysis reagent. In some instances, the lysis reagent encompasses water or ammonium chloride. In other instances, the lysis reagent encompasses a detergent. In still other instance, the detergent is CHAPS, Triton-X and saponin.

[00121] In other instances, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a centrifuge, a liquid chromatography station and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cells lysis reagent, the non-microbial cells lysis reagent encompasses saponin.

[00122] In some instances, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a sonicator, a liquid chromatography station and an ESI mass spectrometer, wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample. [00123] In other instances, the sample preparation station encompasses a centrifuge. Accordingly, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a robotic arm and a sonicator, a liquid chromatography station and an ESI mass spectrometer, wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample.

[00124] In some instances, the rapid diagnostic modality disclosed herein, in some instances encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a sonicator and a centrifuge, a liquid chromatography station and an ESI mass spectrometer, wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample.

[00125] In other instances, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a robotic arm, a centrifuge and a sonicator, a liquid chromatography station and an ESI mass spectrometer, wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample.

[00126] The rapid diagnostic modality disclosed herein, in other instances encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a sonicator, a liquid chromatography station and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cells lysis reagent. In some instances, the lysis reagent encompasses water or ammonium chloride. In other instances, the lysis reagent encompasses a detergent. In still other instance, the detergent is CHAPS, Triton-X or saponin.

[00127] The rapid diagnostic modality disclosed herein, in some instances encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a sonicator and a robotic arm, a liquid chromatography station and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cells lysis reagent, the non-microbial cells lysis reagent encompasses saponin.

[00128] The rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a sonicator, a robotic arm, and a centrifuge, a liquid chromatography station and an ESI mass spectrometer wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence of antimicrobial resistance protein, and when present, identification of a resistance protein in the sample, wherein the robotic sample preparation station encompasses a non-microbial cells lysis reagent, the non-microbial cells lysis reagent encompasses saponin, wherein the liquid chromatography station encompasses an organic solvent, the organic solvent encompassing acetonitrile.

[00129] In some instances, wherein the organic solvent of the sample preparation station encompasses acetonitrile, the acetonitrile represents 1-50%, 1-45%, 1-40%, 1-35%, 1-30%, 1- 25%, 1-20%, 1-15%, 1-10%, l-5% vol/vol of the mobile phase passing through chromatography column. In some instances, wherein the organic solvent of the sample preparation station encompasses acetonitrile, the acetonitrile represents 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the mobile phase passing through chromatography column.

Samples

[00130] "Sample" refers to a liquid, a semi-solid or a solid, and may or may not include one or more cells. In some instances, the rapid diagnostic modality, disclosed herein, encompasses the automated analysis of a liquid using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence and identification of a resistance protein in the sample. In other instances, the rapid diagnostic modality, disclosed herein, encompasses the automated analysis of a semi-solid using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence and identification of a resistance protein. In still other instances, the rapid diagnostic modality, disclosed herein, encompasses the automated analysis of a solid using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence and identification of a resistance protein in the sample. These analyses are rapid, in that the determination of the presence or absence of a microbe in a sample, and if present, the identity of the microbe and whether the identified microbe is resistant in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a vessel containing the sample into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein identification of the microbe, if present, is accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence and identification of a resistance protein in the sample. In some instances, rapid refers to the determination of the presence or absence of a microbe in a sample, and if present, the identity of the microbe and whether the identified microbe is resistant in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein identification of the microbe, if present, is accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, the robotic sample preparation station encompasses a non- microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[00131] In some instances, the rapid diagnostic modality, disclosed herein, encompasses the automated analysis of a patient sample using a diagnostic instrument encompassing a robotic patient sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a microbe in the patient sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence and identification of a resistance protein.

[00132] A "patient sample" refers to a biological fluid or a biological tissue taken from a human donor. Examples of biological fluids include amniotic fluid, blood, cerebral spinal fluid, mucus, plasma, serum, saliva, semen, stool, sputum, tears and urine. Biological tissues are aggregates of cells, usually of a particular kind. Examples of biological tissues include organs and tumors. These analyses are rapid, in that the determination of the presence or absence of a microbe in a patient sample, and if present, the identity of the microbe and whether the identified microbe is resistant in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a patient sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a microbe in the patient sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence and identification of a resistance protein. In some instances, rapid refers to the determination of the presence or absence of a microbe in a patient sample, and if present, the identity of the microbe and whether the identified microbe is resistant in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a patient sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a microbe in the patient sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence and identification of a resistance protein. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[00133] In some instances, the rapid diagnostic modality, disclosed herein, encompasses the automated analysis of a veterinary sample using a diagnostic instrument encompassing a robotic patient sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a microbe in the patient sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence and identification of a resistance protein.

[00134] A "veterinary sample" refers to a biological fluid or a biological tissue taken from a non-human animal donor. Examples of biological fluids include amniotic fluid, blood, cerebral spinal fluid, mucus, plasma, serum, saliva, semen, stool, sputum, tears and urine. Biological tissues are aggregates of cells, usually of a particular kind. Examples of biological tissues include organs and tumors. These analyses are rapid, in that the determination of the presence or absence of a microbe in a veterinary sample, and if present, the identity of the microbe and whether the identified microbe is resistant in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a veterinary sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a microbe in the patient sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence and identification of a resistance protein. In some instances, rapid refers to the determination of the presence or absence of a microbe in a veterinary sample, and if present, the identity of the microbe and whether the identified microbe is resistant in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a veterinary sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a microbe in the veterinary sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence and identification of a resistance protein. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non- microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[00135] "Food sample" refers to any nutritional item that provides nourishment to an animal. The food sample can be a solid or a liquid. Food sample can also encompass a substrate, such as a paper or swab, placed in contact with food.

[00136] The patient sample, veterinary sample or a food sample can be introduced to a culture medium. The culture medium can be a liquid, semi-solid, or solid. The combination of culture medium and patient sample or veterinary sample can be incubated for minutes, hours, days or weeks, to promote or allow for microbial growth and thereby forming a "cultured sample." Examples of a cultured sample include microbial colonies picked from the surface or sub-surface of solid medium or semisolid medium or an aliquot from a liquid blood culture, a blood culture being when blood is introduced into a growth medium. Examples of commercially available brands of liquid culture medium used in a liquid blood culture sample are Thermo Scientific™ VersaTREK™, BD™ Bactec™, and bioMerieux BACT/ALERT™.

[00137] In some instances, the rapid diagnostic modality, disclosed herein, encompasses the automated analysis of a cultured sample using a diagnostic instrument encompassing a robotic cultured sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a microbe in the cultured sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence and identification of a resistance protein. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture.

[00138] These analyses are rapid, in that the determination of the presence or absence of a microbe in a cultured sample, and if present, the identity of the microbe and whether the identified microbe is resistant in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a cultured sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a microbe in the cultured sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence and identification of resistance. In some instances, rapid refers to the determination of the presence or absence of a microbe in a cultured sample, and if present, the identity of the microbe and whether the identified microbe is resistant in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a cultured sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a microbe in the cultured sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a microbe is present, the absence or presence and identification of a resistance protein. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

Resistance

[00139] Antimicrobial resistance has emerged as one of the most pressing public health problems of the 21^st century, threatening the effective prevention and treatment of an increasing range of infections. In the past, resistant infections were associated predominantly with hospitals and care settings, but over the last 20 years resistant infections have been seen in the wider community as well. With microbial resistance on the rise, we stand to lose the immense ground gained in health care over last century.

[00140] Resistance to antimicrobials is a natural process. In the presence of an antimicrobial, microbes are either killed or, if they carry resistance genes capable of expression, survive. These survivors will replicate, and their progeny will quickly become the dominant type throughout a given microbial population. Antimicrobial resistance is becoming an increasing problem now because overuse of antimicrobials has increased the rate at which resistance is developing and spreads.

[00141] Our ability to cure infections that were once considered benign is now damaged.

The rapid development of drug-resistant microbes combined with the fact that there are limited options for rapid and direct diagnostic tests to guide treatment, means that often “last line” drugs are the only option for successful therapy. Such empiric treatment regimens results in the selection of microbes resistant to even these last line drugs. After the last line drug fails, there are no more treatment options on the shelf.

[00142] The instant disclosure solves the need for a rapid diagnostic modality for detecting the presence or absence of a microbe from a sample and identifying, if present, the microbe, and determining whether the microbe is resistant. "Resistant" or "resistance" refers to the characteristics of certain microbes to remain viable or grow or proliferate in the presence of a defined concentration of an antimicrobial compound. An antimicrobial resistant microbe is one that remains viable or can grow or proliferate when the minimum inhibitory concentration that is 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-fold higher or more than that observed for microbe without resistance. An "antimicrobial resistance protein" is a polypeptide the presence of which within a microbe, or secreted into the immediate extra-cellular milieu, that renders antimicrobial resistance.

Bacteria

[00143] The problem of antimicrobial resistance is especially urgent regarding antibiotic resistance in bacteria. "Bacteria" (singular "bacterium") refers to a prokaryotic organism that reproduces by fission and exists as a single cell or in a cluster or aggregate of single cells. A cluster or aggregate of bacteria is often referred to as a "colony" or "colony forming unit."

[00144] Bacterial infections are routinely treated by the administration of antibiotics. "Antibiotic" refers to compounds capable of destroying or inhibiting the growth or reproduction of bacteria. Antibiotics that cause bacterial cell death are bactericides. Classes of bactericides include b-lactams, aminoglycosides, glycopeptides, ansamycins, quinolones, stretogramins and lipopeptides. Antibiotics that restrict bacterial growth and reproduction are bacteriostatic.

Classes of bacteriostatic compounds sulfonamides, chloramphenicol, tetracyclines, macrolides and oxazolidinomes.

[00145] "Antibiotic resistance" or "Antibiotic resistant" refers to the characteristics of certain bacteria to remain viable or grow or proliferate in the presence of a certain concentration of an antibiotic. An antibiotic resistant bacterium is one that remains viable or can grow or proliferate when the minimum inhibitory concentration that is 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9- 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-fold higher or more than that observed for bacteria without resistance. An "antibiotic resistance protein" is a polypeptide the presence of which within, or secreted into the immediate extra-cellular milieu, renders antibiotic resistance.

[00146] In some instances, the rapid diagnostic modality, disclosed herein, encompasses the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a bacterium is present, the absence or presence and identification of a resistance protein. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture.

[00147] These analyses are rapid, in that the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a bacterium is present, the absence or presence and identification of a resistance protein. In some instances, rapid refers to the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a bacterium is present, the absence or presence and identification of a resistance protein. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non- microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

Categorizing Bacteria: The Gram Stain

[00148] Bacteria can be divided into two groups by applying the Gram method, often called Gram staining. In a generalized version of the Gram method, bacteria in the first steps are contacted with crystal violet and iodide. Iodide is thought to trap crystal violet in the peptidoglycan cell wall of certain bacteria. This is followed usually with ethanol or acetone to remove crystal violet that is not trapped. The bacteria are then counterstained with safranin or less commonly carbol fuchsin. "Gram-positive" bacteria are those that retain the color of crystal violet and appear purplish or bluish under a light microscope. Examples of Gram-positive bacteria include Staphylococci and Streptococci. "Gram-negative" bacteria are those bacteria that appear red or pink under a light microscope after the Gram method. Examples of Gram negative bacteria include Klebsiella , Acintebactobacter , Pseudomonas and Escherichia. Gram negative bacteria have relatively thin peptidoglycan cell walls as compared to Gram-positive bacteria and Gram-negative bacteria are surrounded by an outer membrane containing lipopolysaccharide, providing a periplasmic space between the relatively thin peptidoglycan cell wall and outer lipopolysaccharide membrane.

[00149] In some instances, the rapid diagnostic modality, disclosed herein, encompasses the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a bacterium is present, the absence or presence and identification of a resistance protein. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non- microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[00150] These analyses are rapid, in that the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a bacterium is present, the absence or presence and identification of a resistance protein. In some instances, rapid refers to the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a bacterium is present, the absence or presence and identification of a resistance protein. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non- microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

Gram-positive

[00151] Examples of Gram-positive bacteria include bacteria of the following genera: Enterococcus , Streptococcus , Staphylococcus , Bacillus , Paenihacillus , Lactobacillus , Listeria , Peptostreptococcus , Propionibacterium , Clostridium , Bacteroides, Gardnerella , Kocuria , Lactococcus , Leuconostoc , Micrococcus, and Corynebacteria.

[00152] In some instances, the rapid diagnostic modality, disclosed herein, encompasses the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-positive bacterium in the sample is determined, the identification of the Gram-positive bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-positive bacterium is present, the absence or presence and identification of a resistance protein. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. [00153] These analyses are rapid, in that the determination of the presence or absence of a Gram-positive bacterium in a sample, and if present, the identity of the Gram-positive bacterium and whether the identified bacterium is resistant in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-positive bacterium in the sample is determined, the identification of the Gram-positive bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-positive bacterium is present, the absence or presence and identification of a resistance protein. In some instances, rapid refers to the determination of the presence or absence of a Gram-positive bacterium in a sample, and if present, the identity of the Gram-positive bacterium and whether the identified bacterium is resistant in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-positive bacterium in the sample is determined, the identification of the Gram-positive bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-positive bacterium is present, the absence or presence and identification of a resistance protein. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

Gram-negative

[00154] Examples of Gram-negative bacteria include bacteria of the following genera: Pseudomonas , Escherichia , Salmonella , Shigella , Enterohacter , Klebsiella , Serratia, Proteus , Campylobacter , Haemophilus , Morganella , Vibrio , Yersinia , Acinetobacter , Stenotrophomonas, Brevundimonas, Ralstonia , Achromobacter , Fusobacterium , Prevotella , Branhamella , Neisseria , Burkholderia , Citrobacter, Hafiiia , Edwardsiella , Aeromonas, Moraxella , Brucella , Pasteurella , Providencia , and Legionella.

[00155] In some instances, the rapid diagnostic modality, disclosed herein, encompasses the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-negative bacterium is present, the absence or presence and identification of a resistance protein. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture.

[00156] These analyses are rapid, in that the determination of the presence or absence of a Gram-negative bacterium in a sample, and if present, the identity of the Gram-negative bacterium and whether the identified Gram-negative bacterium is resistant in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instalment encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-negative bacterium is present, the absence or presence and identification of a resistance protein. In some instances, rapid refers to the determination of the presence or absence of a Gram-negative bacterium in a sample, and if present, the identity of the Gram-negative bacterium and whether the identified Gram-negative bacterium is resistant in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-negative bacterium is present, the absence or presence and identification of a resistance protein. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In some instances, the robotic sample preparation station encompasses a non- microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid. Identifying Resistance to b-lactam Antibiotics

[00157] Until 2003, more than half of all commercially available antibiotics in use, by sales, were b-lactam antibiotics. The effectiveness of this important class of antibiotics has been tempered by the increasing prevalence of bacterial resistance to b-lactam antibiotics.

[00158] b-Lactam antibiotics, named after the active chemical component of the drug (the 4- membered b-lactam ring), include the 6-membered ring-structured penicillins, monobactams, and carbapenems; and the 7-membered ring-structured cephalosporins and cephamycins.

[00159] b-Lactam antibiotics impair the development of bacterial cell walls by interfering with transpeptidase enzymes responsible for the formation of the crossdinks between peptidoglycan strands. These enzymes are associated with a group of proteins; the penicillin binding proteins (PBPs). At least nine different PBPs comprise the cell wall. Different b-lactam antibiotics may target different PBPs, accounting for differences in spectrum and resistance.

[00160] During bacterial cell growth, while the peptidoglycan structure is being formed, autolysins continually cleave cell wall lattices, in anticipation of providing acceptor sites for new strands of bacterial cell synthesis. Normal bacterial growth depends on a balance between cell wall autolysis and synthesis. The b-lactam drug mimics the PBP substrate, thus inhibiting the PBP and thus cell wall synthesis. In the face of continued autolysin activity, the cell wall becomes deformed. The cell, which is generally hypertonic compared with its environment, is no longer impermeable to the flow of small molecules and is susceptible to osmotic lysis.

[00161] The effect of the b-lactams when present in sufficient concentrations is generally bactericidal toward most bacteria (an exception is in listeriosis for which penicillins are bacteriostatic and cephalosporins are ineffective). However, at sub-inhibitory concentrations, b- lactam antibiotics do exert residual effects on bacterial structure and function that, in turn, promote host-mediated cell death.

[00162] The rapid assessment of a patient’s bacterial infection as resistant or sensitive to b- lactam antibiotics is of critical importance. Every hour of delay in beginning effective antibiotic treatment increases the chances of death by an additional 10%.

[00163] To minimize delay in beginning appropriate antimicrobial therapy, disclosed herein are methods for the rapid detection and identification of b-lactam resistance proteins, the method encompassing the automated analysis of a sample using a diagnostic instalment encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a bacterium is present, the absence or presence and identification of a b-lactam resistance protein. In some instances, the sample is a solid or semi solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture.

[00164] These analyses are rapid, in that the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant, the resistance being antibiotic resistance, the antibiotic being a b-lactam in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a bacterium is present, the absence or presence and identification of a b-lactam resistance protein. In some instances, rapid refers to the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant, the resistance being antibiotic resistance, the antibiotic being a b-lactam in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a bacterium is present, the absence or presence and identification of a b-lactam resistance protein. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[00165] In some instances, the rapid diagnostic modality, disclosed herein, encompasses the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-positive bacterium in the sample is determined, the identification of the Gram-positive bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-positive bacterium is present, the absence or presence and identification of a b-lactam resistance protein. In some instances, the sample is a solid or semi solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture.

[00166] These analyses are rapid, in that the determination of the presence or absence of a Gram-positive bacterium in a sample, and if present, the identity of the Gram-positive bacterium and whether the identified bacterium is resistant, the resistance being antibiotic resistance, the antibiotic being a b-lactam in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-positive bacterium in the sample is determined, the identification of the Gram-positive bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-positive bacterium is present, the absence or presence and identification of a b-lactam resistance protein. In some instances, rapid refers to the determination of the presence or absence of a Gram-positive bacterium in a sample, and if present, the identity of the Gram-positive bacterium and whether the identified bacterium is resistant in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-positive bacterium in the sample is determined, the identification of the Gram-positive bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-positive bacterium is present, the absence or presence and identification of a b-lactam resistance protein. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[00167] In some instances, the rapid diagnostic modality, disclosed herein, encompasses the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-negative bacterium is present, the absence or presence and identification of a b-lactam resistance protein. In some instances, the sample is a solid or semi solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture.

[00168] These analyses are rapid, in that the determination of the presence or absence of a Gram-negative bacterium in a sample, and if present, the identity of the Gram-negative bacterium and whether the identified Gram-negative bacterium is resistant, the resistance being antibiotic resistance, the antibiotic being a b-lactam in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-negative bacterium is present, the absence or presence and identification of a b-lactam resistance protein. In some instances, rapid refers to the determination of the presence or absence of a Gram negative bacterium in a sample, and if present, the identity of the Gram-negative bacterium and whether the identified Gram-negative bacterium is resistant, the resistance being antibiotic resistance, the antibiotic being a b-lactam in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-negative bacterium is present, the absence or presence and identification of a b-lactam resistance protein. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi solid medium. In other instances, the cultured sample is a blood culture. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[00169] Resistance to b-lactams can be mediated by alterations in PBP expression or activity or by enzymatic inactivation of the b-lactam ring.

Alterations to PBP: Penicillin Binding Protein 2a (PBP2A)

[00170] PBP2a, also known as PBP2', is a variant PBP resistant to b-lactam antibiotics. Resistance is mediated by reduced affinity for b-lactam. The relatively low binding affinity of PBP2a to b-lactams enables peptidoglycan cell wall synthesis despite the presence of b-lactams concentrations that inhibit other PBPs. [00171] The ability of PBP2a producing bacteria to evade b-lactams is made readily apparent by methicillin-resistant Staphylococcus aureus (MRSA). Methicillin is a b-lactam, being a derivative of penicillin. It was first produced in the late 1950s and used to treat Staphylococcus infections. By the 1960s, methicillin was rendered useless for clinical purposes by the appearance of methicillin-resistant bacteria. Methicillin resistance is conferred by the expression of PBP2a. MRSA now kills more U.S. citizens that HIV/AIDS, Parkinson’s disease, emphysema and homicide combined.

[00172] To minimize delay in beginning appropriate antibacterial therapy, disclosed herein are methods for the rapid detection and identification of b-lactam resistance proteins, the method encompassing the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a bacterium is present, the absence or presence of PBP2a. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi solid medium. In other instances, the cultured sample is a blood culture.

[00173] These analyses are rapid, in that the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant, the resistance being antibiotic resistance, the antibiotic being a b-lactam in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a bacterium is present, the absence or presence of PBP2a. In some instances, rapid refers to the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant, the resistance being antibiotic resistance, the antibiotic being a b-lactam in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a bacterium is present, the absence or presence of PBP2a. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[00174] In some instances, the rapid diagnostic modality, disclosed herein, encompasses the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-positive bacterium in the sample is determined, the identification of the Gram-positive bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-positive bacterium is present, the absence or presence of PBP2a. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture.

[00175] These analyses are rapid, in that the determination of the presence or absence of a Gram-positive bacterium in a sample, and if present, the identity of the Gram-positive bacterium and whether the identified bacterium is resistant, the resistance being antibiotic resistance, the antibiotic being a b-lactam in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-positive bacterium in the sample is determined, the identification of the Gram-positive bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-positive bacterium is present, the absence or presence of PBP2a. In some instances, rapid refers to the determination of the presence or absence of a Gram-positive bacterium in a sample, and if present, the identity of the Gram-positive bacterium and whether the identified bacterium is resistant in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram positive bacterium in the sample is determined, the identification of the Gram-positive bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-positive bacterium is present, the absence or presence of PBP2a. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[00176] Disclosed herein are methods for the rapid detection and identification of b-lactam resistance proteins, the method encompassing the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a bacterium, the bacterium being a Staphylococcus is present, the absence or presence of PBP2a is detected.

In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi solid medium. In other instances, the cultured sample is a blood culture. In some instances, the bacterium is a Staphylococcus aureus.

[00177] These analyses are rapid, in that the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant, the resistance being antibiotic resistance, the antibiotic being a b-lactam in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a bacterium being a Staphylococcus is present, the absence or presence of PBP2a is detected. In some instances, rapid refers to the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant, the resistance being antibiotic resistance, the antibiotic being a b -lactam in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a bacterium being a Staphylococcus is present, the absence or presence of PBP2a. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In some instances, the bacterium is a Staphylococcus aureus. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non- microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[00178] In some instances, the rapid diagnostic modality, disclosed herein, encompasses the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-positive bacterium in the sample is determined, the identification of the Gram-positive bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-positive bacterium being a Staphylococcus is present, the absence or presence of PBP2a. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In some instances, the bacterium is a Staphylococcus aureus.

[00179] These analyses are rapid, in that the determination of the presence or absence of a Gram-positive bacterium in a sample, and if present, the identity of the Gram-positive bacterium and whether the identified bacterium is resistant, the resistance being antibiotic resistance, the antibiotic being a b-lactam in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-positive bacterium in the sample is determined, the identification of the Gram-positive bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-positive bacterium being a Staphylococcus is present, the absence or presence of PBP2a. In some instances, rapid refers to the determination of the presence or absence of a Gram-positive bacterium in a sample, and if present, the identity of the Gram-positive bacterium and whether the identified bacterium is resistant in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-positive bacterium in the sample is determined, the identification of the Gram-positive bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-positive bacterium being a Staphylococcus is present, the absence or presence of PBP2a. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In some instances, the bacterium is a Staphylococcus aureus. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non- microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid. b-lactamases

[00180] A mechanism of bacterial resistance to b-lactam antibiotics is enzymatic inactivation by cleavage of the 4-member b-lactam ring by b-lactamases. Cleavage results in the inability of the b-lactam antibiotics to bind to target PBPs. There currently are >1000 different b-lactamases, representing six major classes, with the enzyme varying with the organism and drugs targeted varying with the enzyme. The ever increasing number of b-lactamases reflects, in part, pressure brought with the increasingly widespread use of b-lactams and the continued manipulation of the drugs in an attempt to circumvent bacterial b-lactamase production.

[00181] Two classification schemes for b-lactamases are currently used. The Ambler scheme relies on amino acid sequence identity, dividing b-lactamases into 4 classes: A, B, C, and D. Enzymes in classes A, C, and D utilize an active-site serine to hydrolyze the b-lactam ring, serine^-lactamases. Class B enzymes require zinc at the active site for substrate hydrolysis. These metal requiring enzymes are called metallo^-lactamases.

[00182] An alternative classification scheme is the Jacoby-Bush scheme. This scheme classifies b-lactamases based on functional characteristics, substrate specificity and inhibitor profiles. Jacoby-Bush groupings generally correlate with primary structure: Group 1 (Ambler class C) are cephalosporinases; Group 2 (Ambler classes A and D) are broad-spectrum, inhibitor- resistance, extended-spectrum b-lactamases and serine carbapenemases; and Group 3 metallo-b- lactamases.

[00183] It is important to note that both classifications have caveats and they do not fully overlap. For instance, Ambler classes A and D enzymes are all considered within group 2 of the Jacoby-Bush scheme. In addition, while the Ambler classification seems to be easier to follow, the lack of correlation with functional characteristics of the enzymes may cause confusion. As an example, the Ambler class A group has enzymes with a wide range of biochemical activities, from narrow spectrum b-lactamases to enzymes capable of destroying almost all available b- lactams, including carbapenems.

[00184] Accordingly, disclosed herein are methods for the rapid detection and identification of a bacterium and a b-lactam resistance protein, the method encompassing the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined, and if present, the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a bacterium is present, detecting the absence or presence of a b-lactam resistance protein, the b-lactam resistance protein being a b-lactamase. In some instances, the b- lactam resistance protein is a serine b-lactamase. In still other instances, the b-lactam resistance protein is a metallo^-lactamase. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture.

In other instances, the cultured sample is a blood culture.

[00185] These analyses are rapid, in that the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant to a b-lactam by detecting and identifying a b-lactam resistance protein, the resistance protein being a b-lactamase occurs in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, the b -lactam resistance protein is a serine b-lactamase. In still other instances, the b- lactam resistance protein is a metallo^-lactamase. In some instances, the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant to a b-lactam by detecting and identifying a b-lactam resistance protein, the resistance protein being a b-lactamase occurs in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, the b-lactam resistance protein is a serine b-lactamase. In still other instances, the b-lactam resistance protein is a metallo^-lactamase. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In other instances, the cultured sample is a blood culture. In some instances, the robotic sample preparation station encompasses a non- microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[00186] In some instances, the rapid diagnostic modality disclosed herein, encompasses the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-negative bacterium is present detecting the absence or presence and identification of a b-lactam antibiotic resistance protein, the b-lactam antibiotic resistance protein being a b-lactamase. In some instances, the b-lactam resistance protein is a serine b-lactamase.

In still other instances, the b-lactam resistance protein is a metallo^-lactamase. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In some instances, the Gram-negative bacterium is of the order Enterobacterales. In other instances, the Gram-negative bacterium is of the genus Pseudomonas. In still other instances, the Gram-negative bacterium is of the genus Acinetobacter .

[00187] These analyses are rapid, in that the determination of the presence or absence of a Gram-negative bacterium in a sample, and if present, the identity of the Gram-negative bacterium and whether the identified Gram-negative bacterium is resistant by detecting and identifying a b-lactam antibiotic resistance protein, the b-lactam antibiotic resistance protein being a b-lactamase in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, the b-lactam resistance protein is a serine b-lactamase. In still other instances, the b-lactam resistance protein is a metallo^-lactamase. In some instances, rapid refers to the determination of the presence or absence of a Gram-negative bacterium in a sample, and if present, the identity of the Gram negative bacterium and whether the identified Gram-negative bacterium is resistant by detecting and identifying a b-lactam antibiotic resistance protein, the b-lactam antibiotic resistance protein being b-lactamase in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, the b-lactam resistance protein is a serine b-lactamase. In still other instances, the b-lactam resistance protein is a metallo^-lactamase. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In other instances, the cultured sample is a blood culture. In some instances, the Gram-negative bacterium is of the order Enterobacterales. In other instances, the Gram-negative bacterium is of the genus Pseudomonas. In still other instances, the Gram-negative bacterium is of the genus Acinetobacter . In some instances, the robotic sample preparation station encompasses a non- microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[00188] New b-lactamases have recently evolved that hydrolyze the carbapenem class of antimicrobials.

Carbapenemases

[00189] Carbapenems are used for treating life-threatening infections not responding to standard antibiotic therapy. Carbapenems are b-lactam antibiotics possessing the broadest spectrum of activity of all the b-lactam antibiotics. Carbapenems are active against many Gram negative and Gram-positive bacteria being used in treating infections caused by Streptococci , Enterococci , Staphylococci , Listeria , Enterbacteriaceae, and many Pseudomonas , Bacteroides, and Acinetobacter species.

[00190] Carbapenems are naturally occurring, being originally isolated from a species of Streptomyces. Current examples of carbapenems include imipenem, meropenem, doripenem, and ertapenem. Carbapenems are resistant to most b-lactamases.

[00191] b-lactamases that hydrolyze the carbapenem class of antimicrobials have been identified and are a global health threat. Common bacteria such as Klebsiella pneumoniae and Escherichia coli can acquire carbapenem resistant. The family of bacteria which are difficult to treat because they are resistant to carbapenems is often referred to as Carbapenem resistant Enterobacteriaceae (CRE).

[00192] Carbapenemases are a diverse group of b-lactamases that include enzymes belonging to Ambler class A, B and D. Class A carbapenemases include the Klebsiella pneumoniae carbapenemase (KPC) family. Class B carbapenemases, the metallo^-lactamases, include New Delhi metallo^-lactamase (NDM), the Verona Integron mediated metallo^-lactamase (VIM) family, and the active on imipenem (IMP) family, also called an imipenemase. Class D carbapenemases, oxicillinase (OXA), include OXA- 23, OXA-24, OXA-40, OXA-48 and OXA- 58 as well as others. Both KPC and OXA carbapenemases are serine -b-lactamases. [00193] In the Jacoby-Bush classification scheme, OXA and KPC members occupy Group 2; 2d, 2de, 2df and 2f. NDM, IMP and VIM members occupy Group 3; 3a.

[00194] Carbapenem resistance is an issue expected to occupy the medical community for many years to come, since few antibiotic alternatives to carbapenems are readily available. Therefore, prompt and reliable detection of carbapenem resistant bacteria is indispensable for therapeutic, epidemiological, and infection control purposes.

[00195] Accordingly, disclosed herein are methods for the rapid detection and identification of a bacteria and a b-lactam resistance protein, the method encompassing the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined, and if present, the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a bacterium is present, detecting the absence or presence of a b-lactam resistance protein, the b-lactam resistance protein being a carbapenemase. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample.

In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In other instances, the cultured sample is a blood culture. In some instances, the Gram-negative bacterium is of the order Enterobacterales. In other instances, the Gram-negative bacterium is of the genus Pseudomonas. In still other instances, the Gram-negative bacterium is of the genus Acinetobacter .

[00196] These analyses are rapid, in that the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant to a b-lactam by detecting and identifying a b-lactam resistance protein, the resistance protein being a carbapenemase occurs in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant to a b- lactam by detecting and identifying a b-lactam resistance protein, the resistance protein being a carbapenemase occurs in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In other instances, the cultured sample is a blood culture. In some instances, the Gram-negative bacterium is of the order Enterobacterales. In other instances, the Gram-negative bacterium is of the genus Pseudomonas. In still other instances, the Gram negative bacterium is of the genus Acinetobacter . In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid. [00197] In some instances, the rapid diagnostic modality disclosed herein, encompasses the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-negative bacterium is present detecting the absence or presence and identification of a b-lactam antibiotic resistance protein, the b-lactam antibiotic resistance protein being a carbapenemase. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture.

In some instances, the Gram-negative bacterium is of the order Enterobacterales. In other instances, the Gram-negative bacterium is of the genus Pseudomonas. In still other instances, the Gram-negative bacterium is of the genus Acinetobacter .

[00198] These analyses are rapid, in that the determination of the presence or absence of a Gram-negative bacterium in a sample, and if present, the identity of the Gram-negative bacterium and whether the identified Gram-negative bacterium is resistant by detecting and identifying a b-lactam antibiotic resistance protein, the b-lactam antibiotic resistance protein being a carbapenemase in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, rapid refers to the determination of the presence or absence of a Gram-negative bacterium in a sample, and if present, the identity of the Gram-negative bacterium and whether the identified Gram-negative bacterium is resistant by detecting and identifying a b-lactam antibiotic resistance protein, the b- lactam antibiotic resistance protein being carbapenemase in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In other instances, the cultured sample is a blood culture. In some instances, the Gram-negative bacterium is of the order Enterobacterales. In other instances, the Gram-negative bacterium is of the genus Pseudomonas. In still other instances, the Gram-negative bacterium is of the genus Acinetobacter . In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

Serine Carbapenemases

[00199] As provided above, some b-lactamases possess an active site serine that mediates the hydrolysis of a b-lactam ring. Other b-lactamases possess zinc at the active site that mediates the hydrolysis of a b-lactam ring. This dichotomy extends to carbapenemases.

[00200] Disclosed herein are methods for the rapid detection and identification of a bacteria and a b-lactam resistance protein, the method encompassing the automated analysis of a sample using a diagnostic instalment encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined, and if present, the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a bacterium is present, detecting the absence or presence of a b-lactam resistance protein, the b- lactam resistance protein being a serine^-carbapenemase. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In other instances, the cultured sample is a blood culture. In some instances, the Gram-negative bacterium is of the order Enterobacterales. In other instances, the Gram-negative bacterium is of the genus Pseudomonas. In still other instances, the Gram negative bacterium is of the genus Acinetobacter .

[00201] These analyses are rapid, in that the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant to a b-lactam by detecting and identifying a b-lactam resistance protein, the resistance protein being a serine^-carbapenemase occurs in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant to a b-lactam by detecting and identifying a b-lactam resistance protein, the resistance protein being a serine^-carbapenemase occurs in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In other instances, the cultured sample is a blood culture. In some instances, the Gram-negative bacterium is of the order Enterobacterales. In other instances, the Gram-negative bacterium is of the genus Pseudomonas. In still other instances, the Gram-negative bacterium is of the genus Acinetobacter . In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[00202] In some instances, the rapid diagnostic modality disclosed herein, encompasses the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-negative bacterium is present detecting the absence or presence and identification of a b-lactam antibiotic resistance protein, the b-lactam antibiotic resistance protein being a serine^-carbapenemase. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture.

[00203] These analyses are rapid, in that the determination of the presence or absence of a Gram-negative bacterium in a sample, and if present, the identity of the Gram-negative bacterium and whether the identified Gram-negative bacterium is resistant by detecting and identifying a b-lactam antibiotic resistance protein, the b-lactam antibiotic resistance protein being a serine^-carbapenemase in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, rapid refers to the determination of the presence or absence of a Gram-negative bacterium in a sample, and if present, the identity of the Gram-negative bacterium and whether the identified Gram-negative bacterium is resistant by detecting and identifying a b-lactam antibiotic resistance protein, the b- lactam antibiotic resistance protein being serine^-carbapenemase in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram -negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In other instances, the cultured sample is a blood culture. In some instances, the Gram-negative bacterium is of the order Enterobacterales. In other instances, the Gram-negative bacterium is of the genus Pseudomonas. In still other instances, the Gram-negative bacterium is of the genus Acinetobacter . In some instances, the robotic sample preparation station encompasses a non- microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

Klebsiella pneumoniae carbapenemase (KPC)

[00204] The Klebsiella pneumoniae carbapenemase (KPC) carbapenemase was first described in 1996 in North Carolina, but since then has disseminated widely.

[00205] Accordingly, disclosed herein are methods for the rapid detection and identification of a bacteria and a b-lactam resistance protein, the method encompassing the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined, and if present, the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a bacterium is present, detecting the absence or presence of a b-lactam resistance protein, the b-lactam resistance protein being a carbapenemase, the carbapenemase being Klebsiella pneumoniae carbapenemase (KPC). In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In other instances, the cultured sample is a blood culture. In some instances, the Gram-negative bacterium is of the order Enterobacterales. In other instances, the Gram-negative bacterium is of the genus Pseudomonas. In still other instances, the Gram negative bacterium is of the genus Acinetobacter .

[00206] These analyses are rapid, in that the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant to a b-lactam by detecting and identifying a b-lactam resistance protein, the b-lactam resistance protein being a carbapenemase, the carbapenemase being Klebsiella pneumoniae carbapenemase (KPC) occurs in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant to a b- lactam by detecting and identifying a b-lactam resistance protein, the b-lactam resistance protein being a carbapenemase, the carbapenemase being Klebsiella pneumoniae carbapenemase (KPC) occurs in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture.

In other instances, the cultured sample is a blood culture. In some instances, the Gram-negative bacterium is of the order Enterobacterales. In other instances, the Gram-negative bacterium is of the genus Pseudomonas. In still other instances, the Gram-negative bacterium is of the genus Acinetobacter . In some instances, the robotic sample preparation station encompasses a non- microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[00207] In some instances, the rapid diagnostic modality disclosed herein, encompasses the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-negative bacterium is present detecting the absence or presence and identification of a b-lactam antibiotic resistance protein, the b-lactam antibiotic resistance protein being a carbapenemase, the carbapenemase being Klebsiella pneumoniae carbapenemase (KPC). In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi solid medium. In other instances, the cultured sample is a blood culture. In some instances, the Gram-negative bacterium is of the order Enterobacterales. In other instances, the Gram-negative bacterium is of the genus Pseudomonas. In still other instances, the Gram-negative bacterium is of the genus Acinetobacter .

[00208] These analyses are rapid, in that the determination of the presence or absence of a Gram-negative bacterium in a sample, and if present, the identity of the Gram-negative bacterium and whether the identified Gram-negative bacterium is resistant by detecting and identifying a b-lactam antibiotic resistance protein, the b-lactam antibiotic antibiotic resistance protein being a carbapenemase, the carbapenemase being Klebsiella pneumoniae carbapenemase (KPC) in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, rapid refers to the determination of the presence or absence of a Gram-negative bacterium in a sample, and if present, the identity of the Gram-negative bacterium and whether the identified Gram-negative bacterium is resistant by detecting and identifying a b-lactam antibiotic resistance protein, the b- lactam antibiotic resistance protein being carbapenemase, the carbapenemase being Klebsiella pneumoniae carbapenemase (KPC) in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

Metallo-P-Lactamases

[00209] Metal Io-b-lactamases (MBLs) represent a unique subfamily of antibiotic resistance enzymes. MBLs rely on zinc ions in the catalytic site for b-lactam hydrolysis. The MBL family includes New Delhi metallo^-lactamase (NDM), the Verona Integron mediated metallo-b- lactamase (VIM) family, and the active on Imipenem, as called Imipenemase, (IMP) family.

[00210] New Delhi metallo^-lactamase (NDM) is a member of the MBLs and has rapidly emerged as the leading threat to the treatment of infections. Originally identified in a Klebsiella pneumonia isolate in 2008, it has now been found in Enerobacterales order, Pseudomonaceae and Moraxacellaceae families. Because few antibiotics can be used to treat infections caused by bacteria harboring NDM, NDM has become a major public health problem worldwide.

[00211] Verona Integron mediated metallo^-lactamase (VIM) enzymes are among the most widely distributed MBLs, with more than 40 variants reported. VIM-1 was identified in 1997, in an Italian clinical isolate of Pseudomonas aeuroginosa. Soon after, the variant VIM-2 was reported, arising from an earlier clinical isolate from France in 1996. More than 20 different VIM allotypes are known. VIM show even broader substrate specificities than IMP (discussed below), being able to hydrolyze 6-a-methoxy -penicillins. VIM-type enzymes have relatively high affinity for carbapenems as compared to other MBLs.

[00212] Imipenemase (IMP) was first identified in Japan in the 1990s. The majority of IMP enzymes were see in Acinetobacter and Pseudomonas species as well as the Enterobacteriaceae family. IMP -type enzymes have broad substrate specificity with relatively high affinity for carbapenems and cephalosporins, but relatively little affinity (or activity) against temocillin.

[00213] MBLs are particularly potent b-lactam resistance enzymes, being capable of hydrolyzing all known classes of b-lactam antibiotics except for monobactams. Rapid diagnostic techniques, with the isolation of infected patients and carriers, are the main factors used in attempting to contain the spread of bacteria harboring MBLs, which threaten the efficacy of modem medicine.

[00214] Accordingly, disclosed herein are methods for the rapid detection and identification of a bacteria and a b-lactam resistance protein, the method encompassing the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined, and if present, the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a bacterium is present, detecting the absence or presence of a b-lactam resistance protein, the b-lactam resistance protein being a metallo^-carbapenemase. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In other instances, the cultured sample is a blood culture. In some instances, the Gram-negative bacterium is of the order Enterobacterales. In other instances, the Gram-negative bacterium is of the genus Pseudomonas. In still other instances, the Gram-negative bacterium is of the genus Acinetobacter .

[00215] These analyses are rapid, in that the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant to a b-lactam by detecting and identifying a b-lactam resistance protein, the resistance protein being a metallo^-carbapenemase occurs in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant to a b-lactam by detecting and identifying a b-lactam resistance protein, the resistance protein being a metallo^-carbapenemase occurs in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In other instances, the cultured sample is a blood culture. In some instances, the Gram-negative bacterium is of the order Enterobacterales. In other instances, the Gram-negative bacterium is of the genus Pseudomonas. In still other instances, the Gram-negative bacterium is of the genus Acinetobacter . In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[00216] In some instances, the rapid diagnostic modality disclosed herein, encompasses the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-negative bacterium is present detecting the absence or presence and identification of a b-lactam antibiotic resistance protein, the b-lactam antibiotic resistance protein being a metallo^-carbapenemase. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In some instances, the Gram-negative bacterium is of the order Enterobacterales. In other instances, the Gram-negative bacterium is of the genus Pseudomonas. In still other instances, the Gram-negative bacterium is of the genus Acinetobacter .

[00217] These analyses are rapid, in that the determination of the presence or absence of a Gram-negative bacterium in a sample, and if present, the identity of the Gram-negative bacterium and whether the identified Gram-negative bacterium is resistant by detecting and identifying a b-lactam antibiotic resistance protein, the b-lactam antibiotic resistance protein being a metallo^-carbapenemase in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, rapid refers to the determination of the presence or absence of a Gram-negative bacterium in a sample, and if present, the identity of the Gram-negative bacterium and whether the identified Gram-negative bacterium is resistant by detecting and identifying a b-lactam antibiotic resistance protein, the b- lactam antibiotic resistance protein being metallo^-carbapenemase in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram -negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In other instances, the cultured sample is a blood culture. In some instances, the Gram-negative bacterium is of the order Enterobacterales. In other instances, the Gram-negative bacterium is of the genus Pseudomonas. In still other instances, the Gram-negative bacterium is of the genus Acinetobacter . In some instances, the robotic sample preparation station encompasses a non- microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[00218] Disclosed herein are methods for the rapid detection and identification of a bacteria and a b-lactam resistance protein, the method encompassing the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined, and if present, the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a bacterium is present, detecting the absence or presence of a b-lactam resistance protein, the b- lactam resistance protein being a carbapenemase, the carbapenemase being a New Delhi metallo- b-lactamase (NDM) protein. In other instances, the carbapenemase is a Verona Integron mediated metallo^-lactamase (VIM) family protein. In still other instances, the carbapenemase is an Imipenemase (IMP) family protein. In some instances, the sample is a solid or semi-solid.

In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In other instances, the cultured sample is a blood culture. In some instances, the Gram-negative bacterium is of the order Enterobacterales. In other instances, the Gram-negative bacterium is of the genus Pseudomonas. In still other instances, the Gram-negative bacterium is of the genus Acinetobacter .

[00219] These analyses are rapid, in that the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant to a b-lactam by detecting and identifying a b-lactam resistance protein, the b-lactam resistance protein being a carbapenemase, the carbapenemase being a New Delhi metallo^-lactamase (NDM) protein occurs in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In other instances, the carbapenemase is a Verona Integron mediated metallo^-lactamase (VIM) family protein. In still other instances, the carbapenemase is an Imipenemase (IMP) family protein. In some instances, the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant to a b- lactam by detecting and identifying a b-lactam resistance protein, the b-lactam resistance protein being a carbapenemase, the carbapenemase being a New Delhi metallo^-lactamase (NDM) protein occurs in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In other instances, the carbapenemase is a Verona Integron mediated metallo^-lactamase (VIM) family protein. In still other instances, the carbapenemase is an Imipenemase (IMP) family protein. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In other instances, the cultured sample is a blood culture. In some instances, the Gram-negative bacterium is of the order Enterobacterales. In other instances, the Gram-negative bacterium is of the genus Pseudomonas. In still other instances, the Gram negative bacterium is of the genus Acinetobacter . In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[00220] In some instances, the rapid diagnostic modality disclosed herein, encompasses the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-negative bacterium is present detecting the absence or presence and identification of a b-lactam antibiotic resistance protein, the b-lactam antibiotic resistance protein being a carbapenemase, the carbapenemase being a New Delhi metallo^-lactamase (NDM) protein. In other instances, the carbapenemase is a Verona Integron mediated metallo-b- lactamase (VIM) family protein. In still other instances, the carbapenemase is an Imipenemase (IMP) family protein. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In some instances, the Gram-negative bacterium is of the order Enterobacterales. In other instances, the Gram-negative bacterium is of the genus Pseudomonas. In still other instances, the Gram-negative bacterium is of the genus Acinetobacter .

[00221] These analyses are rapid, in that the determination of the presence or absence of a Gram-negative bacterium in a sample, and if present, the identity of the Gram-negative bacterium and whether the identified Gram-negative bacterium is resistant by detecting and identifying a b-lactam antibiotic resistance protein, the b-lactam antibiotic antibiotic resistance protein being a carbapenemase, the carbapenemase being a New Delhi metallo^-lactamase (NDM) protein in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In other instances, the carbapenemase is a Verona Integron mediated metallo^-lactamase (VIM) family protein. In still other instances, the carbapenemase is an Imipenemase (IMP) family protein. In some instances, rapid refers to the determination of the presence or absence of a Gram-negative bacterium in a sample, and if present, the identity of the Gram-negative bacterium and whether the identified Gram-negative bacterium is resistant by detecting and identifying a b-lactam antibiotic resistance protein, the b- lactam antibiotic resistance protein being carbapenemasem, the carbapenemase being a New Delhi metallo^-lactamase (NDM) protein in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In other instances, the carbapenemase is a Verona Integron mediated metallo^-lactamase (VIM) family protein. In still other instances, the carbapenemase is an Imipenemase (IMP) family protein. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample.

In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

Oxacillin b-lactamase - oxacillinases

[00222] The OXA b-lactamases were among the earliest b-lactamases detected; however, these molecular class D b-lactamases were originally relatively rare. They had a substrate profile limited to the penicillin, but some became able to confer resistance to cephalosporins. From the 1980s onwards, isolates of Acinetobacter baumannii that were resistant to the carbapenems emerged, manifested by the b-lactamases (OXA-23, OXA-40, and OXA-58) categorized as OXA enzymes because of their sequence similarity to earlier OXA b-lactamases. It was soon found that every A. baumannii strain possessed a chromosomally encoded OXA b-lactamase (OXA-51- like), some of which could confer resistance to carbapenems when the genetic environment around the gene promoted its expression. Similarly, Acinetobacter species closely related to A. baumannii also possessed their own chromosomally encoded OXA b-lactamases; some could be transferred to A. baumannii , and they formed the basis of transferable carbapenem resistance in this species.

[00223] The OXA-48 carbapenemase was first reported in a Klebsiella pneumoniae isolate in 2001. Since the discovery of OXA-48, several variants have been identified which differ from OXA-48 by one to five amino acid substitutions and/or by a four amino acid deletions, which results in a modified b-lactam hydrolysis spectrum. These OXA-48-like variants include OXA- 162, OXA- 163, OXA-181, OXA-199, OXA-204, OXA-244, OXA-245, OXA-370 and OXA- 405.

[00224] OXA-48 and OXA-48-like oxacillinases have migrated into the Enterobacterales and are becoming a significant cause of carbapenem resistance. Accordingly, disclosed herein are methods for the rapid detection and identification of a bacteria and a b-lactam resistance protein, the method encompassing the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined, and if present, the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a bacterium is present, detecting the absence or presence of a b-lactam resistance protein, the b-lactam resistance protein being an OCA-b-lactamase. In some instances, the sample is a solid or semi solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In other instances, the cultured sample is a blood culture. In some instances, the Gram-negative bacterium is of the order Enterobacterales. In other instances, the Gram-negative bacterium is of the genus Pseudomonas. In still other instances, the Gram-negative bacterium is of the genus Acinetobacter .

[00225] These analyses are rapid, in that the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant to a b-lactam by detecting and identifying a b-lactam resistance protein, the resistance protein being an OCA-b-lactamase occurs in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, the determination of the presence or absence of a bacterium in a sample, and if present, the identity of the bacterium and whether the identified bacterium is resistant to a b- lactam by detecting and identifying a b-lactam resistance protein, the resistance protein being an OCA-b-lactamase occurs in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a bacterium in the sample is determined and if present the bacterium is identified, the identification of the bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In other instances, the cultured sample is a blood culture. In some instances, the Gram-negative bacterium is of the order Enterobacterales. In other instances, the Gram-negative bacterium is of the genus Pseudomonas. In still other instances, the Gram negative bacterium is of the genus Acinetobacter. In some instances, the robotic sample preparation station encompasses a non-microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

[00226] In some instances, the rapid diagnostic modality disclosed herein, encompasses the automated analysis of a sample using a diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, and when a Gram-negative bacterium is present detecting the absence or presence and identification of a b-lactam antibiotic resistance protein, the b-lactam antibiotic resistance protein being an OCA-b-lactamase. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture.

[00227] These analyses are rapid, in that the determination of the presence or absence of a Gram-negative bacterium in a sample, and if present, the identity of the Gram-negative bacterium and whether the identified Gram-negative bacterium is resistant by detecting and identifying a b-lactam antibiotic resistance protein, the b-lactam antibiotic resistance protein being an OCA-b-lactamase in 120 minutes or less, 110 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less, 70 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram-negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, rapid refers to the determination of the presence or absence of a Gram-negative bacterium in a sample, and if present, the identity of the Gram-negative bacterium and whether the identified Gram-negative bacterium is resistant by detecting and identifying a b-lactam antibiotic resistance protein, the b- lactam antibiotic resistance protein being an OCA-b-lactamase in 5 - 25 minutes, in 5 - 20 minutes, 5 - 15 minutes or from 5 - 10 minutes from placing a sample into a vessel and placing the vessel into the diagnostic instrument encompassing a robotic sample preparation station, a centrifuge and a mass spectrometer, wherein the presence or absence of a Gram -negative bacterium in the sample is determined, the identification of the Gram-negative bacterium being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum. In some instances, the sample is a solid or semi-solid. In other instances, the sample is a liquid. In still other instances, the sample is a patient sample. In some instances, the sample is a veterinary sample. In other instances, the sample is a cultured sample. In some instances, the cultured sample is a colony picked from the surface or sub-surface of a solid medium or semi-solid medium. In other instances, the cultured sample is a blood culture. In other instances, the cultured sample is a blood culture. In some instances, the Gram-negative bacterium is of the order Enterobacterales. In other instances, the Gram-negative bacterium is of the genus Pseudomonas. In still other instances, the Gram-negative bacterium is of the genus Acinetobacter . In some instances, the robotic sample preparation station encompasses a non- microbial cell lysis reagent. In other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent. In still other instances, the robotic sample preparation station encompasses a microbial cell lysis reagent and a non-microbial cell lysis reagent. In some instances, the non-microbial cell lysis reagent encompasses water or ammonium chloride. In other instances, the non-microbial cell lysis reagent encompasses a detergent. In still other instances, the detergent is CHAPS, Triton-X or saponin. In some instances, the microbial lysis reagent encompasses an organic solvent, base or acid. In other instances, the microbial lysis reagent encompasses an organic solvent. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid. In still other instances, the microbial lysis reagent encompasses an organic solvent and an acid, wherein the organic solvent is acetonitrile and the acid is formic acid.

Yeast

[00228] Yeasts are rising in clinical importance, especially as the numbers of immunosuppressed, cancer and HIV patients increase. In these patients, Ascomycota fungi comprise the largest group of pathogenic fungi responsible for life threatening infections. Most of these pathogens are members of the anamorphic genus Candida and to a lesser extent, yeasts classified within the genera Saccharomyces , Pichia (including species formerly assigned to Hansenula ), and Magnusiomyces (formerly Dipodascus , Blastoschizomyces ). The precision with which such taxa are identified markedly contributes to treatment success. This is true particularly for emerging species that have undertaken host jumps, such as Candida blankiior , Candida rugose , and also Candida auris.

[00229] Accordingly, the rapid diagnostic modality disclosed herein encompasses the automated analysis of a sample using an instrument encompassing a robotic sample preparation station, the robotic sample preparation station encompassing a robotic arm, and an ESI mass spectrometer, wherein the presence or absence of a microbe in the sample is determined, the identification of the microbe being accomplished by analysis of an MSI spectrum alone, without using an MS2 spectrum, wherein the microbe is a yeast. In some instances, the robotic arm translates the payload in only two spatial dimensions. In other instances, the robotic arm translates the payload in three spatial dimensions.

EXAMPLES

[00230] Generalized Methodology for Isolation and Analysis of Microbes from Blood Culture

[00231] 100 - 200 mΐ. aliquot of a blood culture sample is introduced into a blood culture sample vial containing saponin and trypsin. The saponin and trypsin being dried on the bottom of the sample vial. After introduction of the blood culture to the blood culture sample vial, the vial is subjected to sonication for 10 seconds, after which the vial is centrifuged for 2 minutes. The resulting supernatant is removed and 200 mΐ. of blood culture wash buffer is introduced into the blood culture sample vial. This is subjected to 12 seconds of sonication, followed by centrifugation. The resulting supernatant is removed and 100 mΐ. of Acrion Reagent 5 is introduced. This is then sonicated for 30 seconds. Then 100 mΐ. of Acrion Reagent 4 is introduced. This is then sonicated for 5 seconds, followed by centrifugation for 5 minutes. This sample is then used for introduction into the liquid chromatography module for continued analysis.

Accurate Identification of Species within the Enterobacter cloacae Complex Using the Acrion System

[00232] A total of 20 strains of Enterobacter asburiae (x2), E. cancerogenus (x3), E. cloacae (x4), E. hormaechei (x6), E. kobei (x2), E. roggenkampii (xl), Lelliottia nimipressuralis (xl) and Enterobacter ludwigii (xl) were included in the study, sourced from culture collections and clinical laboratories. Strains were cultured on Columbia Blood Agar with Sheep Blood (Oxoid, PB0123A) for 24 hrs at 35°C in C0_2.

[00233] A loopfull of a bacterial mass was picked from an agar plate and transferred to an Acrion Plate Culture Vial. Prepared samples were inserted to the automated Acrion Analyzer for sample processing. The Acrion Analyzer combines sample preparation, liquid chromatography and Thermo Scientific™ Orbitrap™ high resolution mass spectrometry into a single seamless process. On-board the Acrion Analyzer the bacterial cells were disrupted and the proteins extracted. The prepared protein extracts were each loaded on to an Acrion ProTrap. The Acrion ProTrap combines protein extract purification and liquid chromatographic separation. The liquid chromatographic separation and mass spectrometric detection was carried out with <2 minutes run time per sample (in steady state).

[00234] 16 Strains were sequenced for reference identification with Illumina technology

(2xl50bp read length, 180-300x coverage) by Eurofms Genomics (Konstanz, Germany) and whole genome sequencing data was assembled with CLC Genomics (QIAGEN) software. Identification to species level was conducted with a Neighbor-Joining concatenated cladogram of seven MLSTgenes dnaA, fusA, gyrB, leuS, pyrG, rplB, rpoB using the Jukes-Cantor nucleotide distance model. The cladogram was computed with 1000 bootstrap support replicates with CLC Genomics software. Reference genomes of type strains available on NCBI were downloaded and included in the phylogeny. Strains of E. cancerogenus and strains of E. hormaecheiwere identified by gyrase B (gyrB) gene BLAST against NCBI nucleotide public database following Sanger sequencing carried out by Eurofms Genomics (Ebersberg, Germany).

[00235] The DB contains mass spectra from more than 600 microbial species with multiple strains and isolate entries for each. It contains at least five strains each per claimed species. The MSI database comprises a minimum of 8 replicates analyzed per each strain.

[00236] The samples were identified by the identification algorithm matching MSI spectrum of an unknown sample against diagnostic spectral features in a taxon database. Classification algorithm sorts the species by distance of the matched features of the measured spectrum and delivers species level classification.

[00237] The Action System correctly identified 99.3% of the tested samples from plate cultures and 100% of the tested samples from positive blood culture (identification results of the Action System. Only one L. nimipressuralis replicate from a colony sample yielded a partial identification result. No replicates were wrongly identified from either of the sample types. The species level identification of all isolates also matched with the sequencing results.

[00238] The high phenotypic similarity between members of the Enterobacter cloacae complex has posed a challenge for their accurate identification, and members of the ECC, particularly E. hormaechei and E. cloacae have often been misidentified in clinics across the globe. Furthermore, the prevalence of Enterobacter species belonging to the ECC in hospitals with the concurrent spread of carbapenemase-producing strains has led to an increased interest in accurate and rapid diagnostic technologies by the scientific community.

[00239] The Action System offers a fully automated analysis in a mass-spectrometry based platform which accurately identifies the most clinically relevant members of the ECC to species level including E. cloacae and E. hormaechei from both plate cultures and positive blood cultures. The combination of species level identification accuracy as well as rapid sample throughout brought by the new Action System provides an effective solution to the diagnosis of this challenging group of bacteria.

Identification of Microorganisms from Clinical Blood Culture using the Acrion System [00240] A l-2mL aliquot of 295 signal positive blood culture samples were collected at Barts Health NHS Trust, London UK, from the routine workflow. The samples were grown in a bioMerieux Bactec system. After signaling positive, the blood culture bottles were removed from the system and 1-2 mL aliquots were frozen into -70°C within five hours. The samples were shipped frozen to Thermo Fisher Scientific (Vantaa, Finland) with dry ice, and maintained frozen until use, days or months later.

[00241] After thawing the frozen aliquot, 150 mΐ of each positive blood culture sample was placed into an Acrion blood culture vial and inserted to the Acrion Analyzer. The microbial identification was performed automatically with the analyzer which includes sample preparation, liquid-chromatography followed by Thermo Scientific™Orbitrap™ high resolution mass spectrometry analysis, and results compared against a taxon database.

[00242] The Acrion System identification results of clinical samples were compared to identification results of the clinical laboratory. Hospital results were based mainly on MALDI- TOF, supplemented with DNA sequencing and other testing to provide species level identification.

[00243] In conjunction with Acrion identification analysis, all samples were subcultured onto solid media and incubated under appropriate conditions to evaluate growth. The CFU/ml was determined for each positive blood culture sample. Samples where there was no recovery of viable organisms or the CFU/ml was below the determined Limit of Detection (LoD) value (ranging from 5.7x 10⁶ to 1 4x 10⁹ CFU/mL) were excluded from the analysis.

[00244] In total, 206 positive blood culture samples representing 28 different bacterial species were included in the study. The Acrion System identified the correct microorganisms with 95.1 % accuracy (196/206) directly from positive blood cultures, with 151 Gram positive and 55 Gram negative isolates tested. None of the clinical blood culture samples were wrongly identified. All samples collected for the study were from the normal blood culture workflow in the clinical laboratory without preselecting the samples. Using high resolution mass spectrometry, the Acrion System enables rapid identification of microorganisms with high accuracy directly from positive blood culture. The system offers a diagnostic solution with fully automated analysis providing rapid and reliable results. Detection and Identification of Intact Carbapenemases Protein using Liquid Chromatography-Mass Spectrometry from Microbial Lysates

[00245] Isolates sourced from culture collections and clinical laboratories were revived from frozen stock cultures on Columbia blood agar (PB0123A, Oxoid) and incubated 18-24 h at 35°C in 5% CO2. Subsequent subculture was inoculated and incubated similarly. Initial research explored the dependency of antibiotic resistance marker expression on the presence of antibiotics, and found that for carbapenemases, typically high levels of expression are observed and no additional induction is necessary.

[00246] A loopfull of a bacterial mass was picked from an agar plate and transferred to a vial. Prepared samples were inserted to the automated analyzer for sample processing. The automated analyzer combines sample preparation, liquid chromatography and Thermo Scientific™ Orbitrap™ high resolution mass spectrometer into a single seamless process. On board the automated analyzer the bacterial cells were disrupted and the proteins extracted. The prepared protein extracts were each loaded on to an Acrion™ ProTrap. The Acrion ProTrap combines protein extract purification and liquid chromatographic separation. The liquid chromatographic separation and mass spectrometric detection was carried out with <6 minute run time per sample (steady state). Tandem mass spectrometry (MS/MS) is used to determine the presence of carbapenemases by dissociating the intact protein(s) in a data-independent manner followed by evaluation of fragments produced. Reproducible fragmentation patterns are trackable from specific intact proteins, provided the dissociation conditions are held constant. This allows specific diagnostic fragments to be used to determine the presence or absence of resistance markers.

[00247] The samples were identified by the identification algorithm matching MSI spectrum of an unknown sample against diagnostic spectral features in a preformed taxon database. The classification algorithm sorts the species by distance of the matched features of the measured spectrum and delivers species level classification. When a relevant species is detected, the sample is automatically re-analyzed for carbapenamases. Diagnostic fragments produced directly from unique individual carbapenemases, each possessing specific characteristics as mass-to- charge (m/z) and charge state (z), are used to determine the presence of specific carbapenemases. A series of acceptance criteria filters must be met for any one resistance marker to be claimed as positive. All resistance markers determined to be positive for a single sample will be reported.

[00248] The automated analyzer, the Acrion™ System offers the ability to directly detect intact carbapenemases from bacterial cell lysates. To date, Acrion System can be used to determine the presence or absence of KPC, VIM, IMP, NDM and OXA-48-like in Enterobacteriales; KPC, VIM and IMP in Pseudomonas, and VIM in Acinetobacter, based on isolate availability. Multiple variants from each of these resistance markers are detected. The Acrion System allows the automated detection of the big five carbapenemases, with the duration of the CARBA assay lasting under 6 minutes (steady state). This detection method presented here takes advantage both of the ability of liquid chromatography to simplify complex protein mixtures and the high-resolution, targeted capabilities of mass spectrometry for confident direct detection of carbapenemases.

Direct Detection of PBP2a Protein by High Resolution Orbitrap™ Mass Spectrometry and Rapid Discrimination of Antibiotic Resistance in Staphylococcus aureus using the Acrion System

[00249] Development of the PBP2a detection assay was performed through an initial enrichment, long liquid chromagraphy (LC) separation, and detection using a Thermo Scientific Orbitrap™ MS system and a variety of fragmentation approaches. The identification of intact PBP2a protein ions yielded unique mass and sequence information as confirmed through protein sequence fragment ions. Validation of these fragments was also confirmed with synthetic peptides. For samples processed with the Acrion System, onboard protein enrichment and LC separation was accomplished using the Acrion ProTrap. Separate trap columns and LC separations were used for each sample and MRSA identification analyses. Reflex identification of the PBP2a protein in samples processed by the Acrion System relies on isolation and fragmentation of a subset of these unique fragments following a two-step fragmentation and isolation process.

[00250] During assay development, processing of intact and fragmented protein data was performed manually, with in-house developed software, or commercially available software such as Xtract™ (Thermo Scientific). As stated above, some of this information was confirmed with synthetic analogs. For MRSA samples processed by the Acrion System, the initial MS-based identification was accomplished by matching spectra data to those in a taxon database and a subsequent S. aureus identification. Reflex analysis of the same S. aureus samples using the targeted MRSA method for PBP2a detection was processed using a MRSA specific protein fragment database. If the PBP2a fragment criteria are satisfied, then the sample is classified as MRSA.

[00251] While previous MALDI MS detection approaches for identifying MRSA have depended on uncharacterized protein signatures or more recently on the Phenol-Soluble Modulin (PSM)-mec marker, the MS detection strategy presented here relies on the direct detection of the expressed PBP2a protein. Since PBP2a is directly linked to the antibiotic resistance phenotype, its direct detection is a much more powerful diagnostic readout. Polymerase chain reaction (PCR) and immunochromatographic detection approaches also provide a direct readouts of PBP2a expression. However, they are slower than the Action Plate Culture MRSA method described here, as they are uncoupled with the species identification of the sample, or reliant on reagent design and stability. The MS detection method described here also proved successful across different SCCmec types and MRSA strains with different genetic backgrounds.

[00252] When tested in a clinical lab the Action Plate Culture MRSA method was very reliable, exhibiting > 95% accuracy in the detection of the PBP2a protein and MRSA antibiotic resistance phenotype. The ability of the Action System to automate bacterial species identification and antibiotic resistance detection has the potential to streamline and improve the efficiency of clinical microbiology workflows.

High Accuracy Identification of Common Blood Culture Isolates and Detection of Methicillin-Resistance Staphylococcus aureus using the Acrion System

[00253] Bacterial isolates were sourced from clinical laboratories and reference collections, and their identity had previously been confirmed by DNA sequencing. Bacteria were revived from frozen stocks by culturing on suitable agar plates. Generally, aerobes and facultative anaerobes were cultured on CBA (PB0123A, Oxoid Ltd, UK) and incubated 24-72 h at 35°C in 5% CO2. Anaerobic organisms were cultured on FAA (PB0124A, Oxoid Ltd, UK) for 48-72 h at 35°C under anaerobic conditions.

[00254] Cells from agar plates were resuspended in PBS (10010-015, Gibco) and diluted in series to produce a seeding suspension with low cell count. BACT/ALERT FA PLUS or FN PLUS (410851/410852, bioMerieux) and BACTECPlus Aerobic (442192, BD) bottles were inoculated with ~10² CFU and 8 ml of EDTA-treated human blood. Cultures were incubated until signaling positive in BACT/ALERT3D 60 (bioMerieux) and BACTEC9050 (BD) systems, respectively.

[00255] Positive blood cultures were sampled for analysis in Acrion System. An aliquot (150 mΐ) was transferred to Acrion Blood Culture Sample vial and inserted into the Acrion System for analysis. Organisms representing common blood culture related species were analyzed in 20-26 replicates, and MRSA strains in triplicates. Identification was performed against a blood culture taxon database.

[00256] For the fifteen most common blood culture bacteria, Acrion System demonstrates high accuracy (97.4 %) when used directly on positive blood cultures, comparable to DNA sequencing methods. By contrast, identification accuracy of MALDI-TOF mass spectrometry- detection methods on blood cultures have been reported to be within 80-94 % success rate. In addition to species identification, the Acrion System enables detection of MRSA directly from blood cultures with 95 % accuracy. MRSA test can be set to run automatically as a reflex test, following S. aureus identification. The Acrion System provides a lean workflow to clinical laboratories, especially the automated sample preparation ensures a significant reduction of workload minimizing human intervention during sample handling.

Identification of Clinically Relevant Yeast using the Acrion System

[00257] A total of 39 yeast species, with a minimum of 2 strains per species were included in this performance study. Organisms were sourced from the Westerdijk Fungal Biodiversity Centre and from clinical laboratories in the European Union, Asia, South America, Africa and North America. Strains were cultured on Sabouraud Dextrose Agar (CM0041Oxoid Ltd, UK) for 48 h at 25°C.

[00258] A loopfull of a yeast cells were picked from an agar plate and transferred to an Acrion Plate Culture Vial. Prepared samples were inserted to the automated Acrion Analyzer for sample processing. The Acrion Analyzer combines sample preparation, liquid chromatography and Thermo Scientific Orbitrap high resolution mass spectrometry into a single seamless process. On-board the Acrion Analyzer the yeast cells were disrupted and the proteins extracted. The prepared protein extracts were each loaded on to an Acrion ProTrap. The Acrion ProTrap combines protein extract purification and liquid chromatographic separation. The liquid chromatographic separation and mass spectrometric detection was carried out with <2 minutes run time per sample.

[00259] Data analysis was performed with a variety of Thermo Scientific proprietary algorithms, which includes signal deconvolution, charge state assignment and mass recognition of proteins, down to clustering and classification of organisms given the obtained data. A total of >1800 LC-MS measurements had been taken to create the database, and these independent test strains (39 species, including species outside the claimed species list) were analyzed to evaluate its performance accuracy.

[00260] Identification success is >95% for all yeast species tested. This technology demonstrates robust and taxonomically accurate yeast identification.

Claims

CLAIMS What we claim:

1. A rapid diagnostic method, the method comprising an automated instrument and a sample, the automated instrument comprising a robotic sample preparation station, a centrifuge and a mass spectrometer, the automated instrument removing a first portion of the sample for analysis, a first analysis portion, ionizing the first analysis portion, thereby forming a first ionized portion, transporting the first ionized portion into the mass spectrometer, detecting ionized proteins in the first ionized portion, an MSI spectrum, comparing the MSI spectrum to a first reference mass spectrum, and thereby identifying a microbe in the sample without comparing a MS2 spectrum to a reference mass spectrum; the automated instrument removing a second portion of the sample, a second analysis portion, ionizing the second analysis portion, thereby forming a second ionized portion, transporting the second ionized portion into the mass spectrometer, detecting ions present in the second ionized portion, a MS2 spectrum, comparing the MS2 spectrum to a second reference mass spectrum, and thereby identifying a antimicrobial resistance protein in the sample.

2. The method of claim 1, wherein the ionization of the first analysis portion and the ionization of the second analysis portion occur within 20 minutes of each other.

3. The method of claim 1, wherein the ionization of the first analysis portion and the ionization of the second analysis portion occur within 10 minutes of each other.

4. The method of claim 1, wherein the ionization of the first analysis portion and the ionization of the second analysis portion occur within 6 minutes of each other.

5. The method of claim 1, wherein the sample is selected from a patient sample, a veterinary sample, a food sample or a cultured sample.

6. The method of claim 5, wherein the cultured sample is a blood culture.

7. The method of claim 1, wherein the microbe is a bacteria.

8. The method of claim 1, wherein the antimicrobial resistance protein provides resistance to an antibiotic.

9. The method of claim 8, wherein the antimicrobial resistance protein is penicillin-binding protein 2A.

10. The method of claim 1, wherein the antimicrobial resistance protein is a b-lactamase.

11. The method of claim 10, wherein the b-lactamase is a serine-P-lactamase or a metallo- b- lactamase.

12. The method of claim 11, wherein the b-lactamase is a carbapenemase.

13. The method of claim 12, wherein the carbapenemase is selected from: imipenemase, Klebsiella pneumoniae carbapenemase, oxacillinase, New Delhi metallo^-lactamase, and Verona integrin-encoded metallo^-lactamase.

14. A rapid diagnostic instrument for identifying a microbe and a resistant protein in a sample, the instrument comprising a robotic sample preparation station, a centrifuge, a mass spectrometer, an on-board instrument control software, a microbe identification software, and a resistance protein identification software, wherein the identification of a microbe in the sample is solely accomplished by comparing an MSI mass spectrum to a reference MSI mass spectrum, without comparison of a MS2 mass spectrum to a reference MS2 mass spectrum, with the preparation of the sample and the identification being completed in less than 10 minutes.