WO2015013177A2 - Rapid detection of carbapenemase and beta-lactamase producing bacteria - Google Patents

Rapid detection of carbapenemase and beta-lactamase producing bacteria Download PDF

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Publication number
WO2015013177A2
WO2015013177A2 PCT/US2014/047394 US2014047394W WO2015013177A2 WO 2015013177 A2 WO2015013177 A2 WO 2015013177A2 US 2014047394 W US2014047394 W US 2014047394W WO 2015013177 A2 WO2015013177 A2 WO 2015013177A2
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beta
composition
test
drug
tetrazolium
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PCT/US2014/047394
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French (fr)
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WO2015013177A3 (en
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Raymond Kwong
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Siemens Healthcare Diagnostics Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material

Definitions

  • the present invention relates generally to methods and systems for detecting drug- resistant bacteria, particularly carbapenemase and beta-lactamase producing bacteria.
  • Carbapenem and beta-lactam antibiotics are penicillin-related drugs used to combat bacterial infections.
  • Carbapanems are often thought of as a subset of beta- lactams, but for our purposes, beta-lactams refers to the cephems (cephalosporins or cephamycins), since the remaining beta-lactams are substantially not viable alternatives in terms of drug resistance.
  • Recent increases in drug resistant bacteria strains have limited the suitability of this class of drugs in use against bacterial infections. Specifically, the carbapenemase and beta-lactamases produced by resistant bacteria degrade the drugs, resulting in nullification of bacteriacidal activity and drug ineffectiveness.
  • CRE Carbapenem-Resistant Enterobacteriaceae
  • Some embodiments provide a method for determining a drug resistance of a bacteria sample, comprising providing a bacteria cell suspension using the bacteria sample; providing a drug test mixture including carbapenem or beta-lactam drug;
  • the monitoring step includes reading a color change of the reaction mixture following incubation of the reaction mixture at 35°C.
  • the step of preparing a drug test mixture includes adding a detection dye to the drug test mixture, and the color change results from a change in the detection dye.
  • the detection dye is resazurin.
  • the reaction vessel is at least one well of a 96-well panel.
  • the bacterial sample is not previously lysated. That is to say that a separate lysating step and separation are not required.
  • test plate comprising at least one test well, defined by at least one wall; wherein the at least one well is coated with one or more of an antibiotic composition, a metal cation or salt, and a cation membrane disrupting surfactant.
  • the at least one test well is coated with two or more of a beta-lactam antibiotic composition, a metal cation or salt, and a cation membrane disrupting surfactant.
  • the beta-lactam antibiotic composition comprises a carbapanem or a cephem.
  • the antibiotic composition comprises an antibiotic selected from a carbapenem and a beta-lactam.
  • the antibiotic composition is a carbapenem selected from imipenem, meropenem, ertapenem, doripenem, panipenem, and biapenem.
  • the antibiotic composition is a beta-lactam selected from ceftazidime, cefotaxime, ceftriaxone, cefepime, cefazolin, ceftaroline, or aztreonam.
  • compositions for use in detecting beta-lactamase activity comprising a buffer; a metal cation; a protein solubilizing surfactant; a cation membrane disrupting surfactant; a dye; and a beta-lactam antibiotic drug.
  • the composition further comprises a beta-lactamase inhibitor.
  • the beta-lactamase inhibitor is phenyl boronic acid.
  • the beta-lactam antibiotic drug is a carbapanem.
  • the beta-lactam antibiotic drug is a cephem selected from ceftazidime, cefotaxime, ceftriaxone, cefepime, cefazolin, ceftaroline, or aztreonam.
  • the buffer is present at about 0.06 to about 0.4 mg/mL of the test solution
  • the metal cation is present at about 0.01 mg/mL to about O.lmg/mL
  • the protein solubilizing surfactant is present at about 0.5 ⁇ g/mL to about 5 ⁇ g/mL of the test solution
  • the cation membrane disrupting surfactant is present at about 1 ⁇ g/mL to about 10 ⁇ g/mL of test solution
  • the dye is present at about 1.3 to about 8.33 ⁇ g/mL of test solution.
  • the buffer is tris(hydroxymethyl)aminomethane (TRIS), 4- (2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES), or 3-(N- morpholino)propanesulfonic acid (MOPS).
  • TMS tris(hydroxymethyl)aminomethane
  • HPES 4- (2-hydroxyethyl)-l-piperazineethanesulfonic acid
  • MOPS 3-(N- morpholino)propanesulfonic acid
  • the metal cation is provided as a metal salt selected from MgCl 2 , CaCl 2 , or ZnCl 2 .
  • the protein solubilizing surfactant is Triton X-100, NP-40, or Tween 20.
  • the cation membrane disrupting surfactant is benzalkonium chloride, cetyl trimethylammonium bromide, or dioctadecyldimethylammonium bromide.
  • the dye is Resazurin, New Methylene Blue N, Indigo Carmine- b, Laurths Violet (thionin acetate), Trypan Blue, 2,3,5-Triphenyl-2H- Tetrazolium Chloride (TCC or tetrazolium red), Tetrazolium Blue Chloride, Tetrazolium Violet, 2,3-Diphenyl-5-Thienyl-(2) Tetrazolium Chloride (STC), Janus Green B, Orange Tetrazolium, Nile Blue A (Basic Blue B), or 2,6-Dichloroindophenol (DCIP).
  • TCC 2,3,5-Triphenyl-2H- Tetrazolium Chloride
  • STC Tetrazolium Violet
  • STC 2,3-Diphenyl-5-Thienyl-(2) Tetrazolium Chloride
  • STC 2,3-Diphenyl-5-Thienyl-(2) Tetrazolium Chloride
  • Janus Green B Orange Tetrazolium
  • Nile Blue A
  • the antibiotic drug is a carbapenem selected from imipenem, meropenem, ertapenem, doripenem, panipenem, and biapenem or a beta- lactam selected from ceftazidime, cefotaxime, ceftriaxone, cefepime, cefazolin, ceftaroline, or aztreonam.
  • FIG. 1 is a graph depicting the evaluation of drug resistence in three samples.
  • Carbapenemase and beta-lactamase activity can be detected in bacteria using the Modified Hodge Test (an on-plate bacteria growth test) or via molecular markers (gene detection) using PCR or gene expression using RTPCR.
  • Other growth tests include kits where different types of drug disks are added to bacteria growth media and bacteria exclusion zones are read for enzyme activity. Additionally there are bacteria lysis and enzymatic reaction tests developed as kits for such detection. This includes the detection outlined by Nordmann et. al. 2012 which uses lysis of bacteria followed by treatment of the lysate with Imipenem and a pH indicator to detect the degraded enzyme.
  • the methods disclosed herein include a rapid detection of the CRE
  • carbapenemase or beta-lactamase activity using a microbiology system, such as a product from Siemens' MicroScan product line.
  • a microbiology system such as a product from Siemens' MicroScan product line.
  • the methods described herein use carbapenem and/or beta-lactam drug degradation by resistant bacteria and the subsequent drug degradation product detection via change in color of vital dye or pH indicator dye, to detect carbapenemase or beta-lactamase activity.
  • the proposed test uses bacteria cell suspensions made by customers or technicians with a bacteria test sample.
  • the bacteria suspension is either dispensed into a well of well plate, such as a 96-well panel, containing the carbapenemase or beta-lactamase test mixture, or both the bacteria suspension and enzyme testing mixture are dispensed into the appropriate testing container, and the enzymatic degradation of the drug is read via color change following incubation at 35°C.
  • the reaction rate depends on enzyme activity which in turn depends on the density of the bacteria suspension added.
  • the density of the bacteria suspension can be increased by first growing the bacterial sample in or on a growth medium.
  • Detection can be as fast as 15 minutes but can be extended up to 4 hours depending on the detection dye used, enzyme activity, amount of enzyme produced by the bacteria (strain dependent) or concentration of bacteria used.
  • This test is unique in that no additional steps are required. No bacterial lysis and extraction of lysate is needed (as per Nordmann et. al. 2012), nor any specialized equipment such as a PCR or PCR based machine.
  • the test is adaptable to various existing microbiology systems, such as the current MicroScan Panel, MicroScan AS-4 and MicroScan Walk-Away systems. For example, MicroScan customers can easily add this test to their current process as a presumptive early detection test (offline using the new invention, the carbapenemase test solution).
  • the assay can either use a Carabapenem or cephem the assay can be used to detect CREs as well as other beta-lactamase enzymes.
  • the system can be used to distinguish different classes of beta-lactimases, such as Metallo-beta-lactamases and Extended Spectrum Beta-lactimases.
  • a biological sample is provided from a subject suspected of having a bacterial infection, and possibly a drug-resistant bacterial infection.
  • the biological sample may be incubated and grown in or on a growth medium, in others, it may be used directly.
  • the biological sample is, without any prior lysis step, then contacted with a test solution which comprises a carbapanem or beta-lactam drug, a dye, and one or more of a buffer, a metal cation, a protein solubilizing surfactant, and a cation membrane disrupting surfactant to form a reaction mixture.
  • the reaction mixture is then allowed to react, in some instances incubating for a period of time.
  • the color of the reaction mixture is monitored.
  • a color change indicates the presence of carbapanemase or beta-lactamase activity, thus indicating drug-resistance, to whichever drug was present in the test mixture.
  • the color change is detected by visual inspection. In other embodiments, the color change may be detected by instrumental analysis.
  • the biological sample may be any biological sample obtained from a subject.
  • biological samples include fluids, tissues, cell samples, etc.
  • biological samples are whole blood, serum, plasma or urine.
  • the biological sample may be worked up so as to provide a sufficient amount of sample for analysis, by incubating the sample in or on a growth medium as will be known in the art. In some cases, the biological sample may be tested directly without any workup.
  • the biological sample is mixed with a test solution.
  • the test solution comprises several component parts: the drug (carbapanem or beta-lactam), a dye, and one or more of a buffer, a metal cation, a protein solubilizing surfactant, and a cation membrane disrupting surfactant.
  • Any suitable buffer may be used.
  • Exemplary buffers include
  • the buffer may be present at about O.Olmg/mL to about lmg/mL of the test solution. In some embodiments, the buffer is present at about 0.06 to about 0.4 mg/mL of the test solution. In some embodiments, the buffer is present at about 0.06mg/mL, about 0.08mg/mL, about O. lOmg/mL, about 0.2mg/mL, about 0.3mg/mL, about 0.4mg/mL or any value of range of values between any two of the recited values, including endpoints.
  • Metal cations are well known for use in enzymatic processes. Exemplary metal cations include, but are not limited to Magnesium, Calcium, and Zinc.
  • the metal cation may be provided by a metal salt, for example MgCl 2 , CaCl 2 , or ZnCl 2 .
  • the metal cation, in the form of a metal salt may be present at about 0.01 mg/mL to about 0. lmg/mL. In some embodiments, the metal salt is present at about 0.013 to 0.085 mg/mL of the test solution. In some embodiments, the metal salt is present at about O.Olmg/mL,
  • the protein solubilizing surfactant is a surfactant useful for solubilizing protein, such as Triton X-100, NP-40, or Tween 20.
  • the protein solubilizing surfactant is present at about 0.5 ⁇ g/mL to about 5 ⁇ g/mL of the test solution.
  • the protein solubilizing surfactant may be present at about 0.64 to about 4.17 ⁇ g/mL.
  • the protein solubilizing surfactant may be present at about 0.5 ⁇ g/mL, about 0.6 ⁇ g/mL, about 0.64 ⁇ g/mL, about 0.7 ⁇ g/mL, about 0.8 ⁇ g/mL, about 0.9 ⁇ g/mL, about 1.0 ⁇ g/mL, about 2.0 ⁇ g/mL, about 3.0 ⁇ g/mL, about 4.0 ⁇ g/mL, about 5.0 ⁇ g/mL, or any value of range of values between any two of the recited values, including endpoints.
  • the cation membrane disrupting surfactant can be benzalkonium chloride, cetyl trimethylammonium bromide, or dioctadecyldimethylammonium bromide.
  • the cation membrane disrupting surfactant may be present at about 1 ⁇ g/mL to about 10 ⁇ g/mL.
  • the cation membrane disrupting surfactant may be present at about 0.26 to about 1.67 ⁇ g/mL
  • the dye may be any suitable dye, such as a pH indicator, a vital dye, a metabolic indicator, a redox indicator, or the like.
  • exemplary dyes include, but are not limited to Resazurin, New Methylene Blue N, Indigo Carmine- b, Laurths Violet (thionin acetate), Trypan Blue, 2,3,5-Triphenyl-2H-Tetrazolium Chloride (TCC or tetrazolium red), Tetrazolium Blue Chloride, Tetrazolium Violet, 2,3-Diphenyl-5-Thienyl-(2) Tetrazolium Chloride (STC), Janus Green B, Orange Tetrazolium, Nile Blue A (Basic Blue B), 2,6- Dichloroindophenol (DCIP).
  • the dye may be present at about 1.3 to about 8.33 ⁇ g/mL of test solution.
  • Resazurin is a metabolic indicator and a redox indicator that is particularly well-suited for the methods and systems described herein.
  • the drug may be any carbapanem or beta-lactam.
  • the carbapanem is selected from imipenem, meropenem, ertapenem, doripenem, panipenem, and biapenem.
  • imipenem is used.
  • the beta-lactam may be a cephalosporin or a monobactam.
  • the beta-lactam is selected from ceftazidime, cefotaxime, ceftriaxone, cefepime, cefazolin, ceftaroline, or aztreonam.
  • the beta-lactam is selected from ceftazidime, cefotaxime, and ceftriaxone.
  • additional tests may be desirable for specific class of carbapenemases or beta-lactamases. This can be achieved by running a parallel sample with the addition of a carbapenemase or beta-lactamase inhibitor.
  • a carbapenemase or beta-lactamase inhibitor for detection of Metallo B-lactamase class carbapenemases, inhibition of the reaction with addition of ImM EDTA will give a negative carbapenemase test result (positive result for presence of Metallo-B-lactamase).
  • phenyl boronic acid or another beta-lactamase inhibitor such as Clavulanic Acid, Tazobactam,
  • Avibactam may be used.
  • a first sample is treated with the basic test solution described herein with either the carbapenem drug or the beta-lactam drug.
  • a second aliquot of the same biological sample is treated in parallel with the basic test solution plus an addition of the inhibitor.
  • the test reveals inhibitor sensitivity.
  • the inhibitor is ethylenediaminetetraacetic acid (EDTA).
  • a color change in the inhibitor free test accompanied by lack of color change in the inhibitor containing test indicates the presence of metallo-beta-lactamase. Such indication can provide valuable information to a trating physician as to which drugs he should or should not use to treat the patient.
  • the inhibitor to be added is phenyl-boronic acid or a beta-lactamase inhibitor such as Clavulanic Acid, Tazobactam, Avibactam, or the like.
  • a beta-lactamase inhibitor such as Clavulanic Acid, Tazobactam, Avibactam, or the like.
  • the test solution may be a single solution or multiple component parts which come together to complete the test solution.
  • the test solution is complete and awaits addition of the biological sample to begin the test reaction.
  • one or more component parts may be added in preparing the biological sample and then combined with the remaining component parts to initiate the test reaction.
  • one or more component of the test solution may be dried in a sample vessel, such as a microtitre well, and reconstituted by adding the remaining components of the test solution.
  • the treating physician can be alerted to the fact that the particular bacteria involved is resistant to a particular drug. The physician is then free to tailor his treatment, avoiding the resistance. In most instances, it is therefore desirable to test for both carbapenemase and beta-lactamase activity. Thus, in some instances, side by side tests will be performed, in separate vessels or wells analyzing both carbapenemase and beta-lactamase activity. Where one or the other activity is detected, the physician can be alerted to tailor the treatment towards the appropriate drug.
  • test solution is substantially the same whether testing for carbapenemase or beta-lactamase activity.
  • the test solution is prepared as separate components in a kit, where the drug is separate from one or more of the other components. These can be separate solutions, or the drug may be present, dried or wet, in the sample vessel or well.
  • a previously prepared well plate may contain at least one well containing carbapenem and at least one well containing a beta-lactam. Each well may be in in certain location designated for evaluating drug resistance.
  • a standard 96-well plate could have two wells assigned for evaluation of carbapenemase and beta-lactamase activity. The remaining cells could be assigned for other common tests or analyses of the same biological sample, or could contain the same tests for other biological samples.
  • a microtiter well plate may contain at least two wells.
  • a first of the two well is designated for carbapenemase evaluation and a second of the two wells is designated for beta-lactamase evaluation.
  • the first well is provided with a quantity of a carbapenem drug, such as imipenem.
  • the second well is provided with a quantity of a beta-lactam drug, such as cefotaxime or ceftazidime.
  • the drug may be dried for ease of storage until use.
  • one or more of the other test solution components may also be present along with the drug.
  • the cation, in the form of a metal salt is particularly well suited for drying in a well plate.
  • each of the first and second wells may contain the drug to be evaluated, the metal cation (in the form of a salt), and/or the cation membrane disrupting surfactant.
  • a test solution containing the buffer, dye, and protein solubilizing surfactant is provided separately. The test solution is added to the two wells, along with the biological sample. Once the test solution and biological samples are added, the wells are incubated at 35 °C and color change is monitored. Color change indicates the presence of a drug resistant bacteria.
  • FIG. 1 illustrates some exemplary results.
  • Three biological samples were tested with imipenem.
  • An E. coli sample and two K. pneumonia CREs were evaluated to show how susceptible strains (the E. coli) and resistant strains change over time.
  • the Y-axis shows values that come off the instrument. In this instance, negative 4,000 is baseline at time point of zero. In this case the instrument acts like a spectrophotometer, the 560 wave length in 30 minute intervals.
  • the substantially horizontal line represents a E. coli strain known not to be resistant to carbapenems.
  • the other two lines represent K. pneumoniae strains known to resistant to carbapenems. It is expected, that the K.
  • pneumoniae would degrade imipenem and as shown between 60 and 90 minutes there is a steep and continuous decline representative of a change from the dye going from blue to purple to pink, indicating the breakdown of the imipnem by the carbapenemase containing bacteria.
  • a treating physician could be notified within about 60 to 90 minutes that the bacteria in question is carbapenem resistant, and the patient's treatment tailored accordingly. In most instances, the resistant nature can be readily identified with about 4 hours. Compared to current techniques which take overnight, this is a significant improvement.
  • carbapenemase activity will yield steeper and faster drop offs. By accumulating additional data, it will be possible to detect and predict the activity of a particular bacteria, to better advise treating physicians on which drugs to use and perhaps in what concentrations.
  • An exemplary test solution includes 20mM Tris, 0.5mM magnesium chloride, 1% Triton X-100, 0.4mg/L benzalkonium chloride (Zephiran) and a dye. Dyes include resazurin at 0.02g/L or phenol red at 0.04g/L (these are minimal dye concentrations). Test Solution has final pH of 7.8. In some embodiments, the drug is either 2.5mg/mL
  • Imipenem or cefotaxime or ceftazidime are particularly well suited for the reaction.
  • cefotaxime or ceftazidime drugs are particularly well suited for the reaction.
  • carbapenemase Imipenem drug is particularly well suited for the reaction.
  • This invention fills a gap in the current offerings and adds to the testing options offered by current microbiology systems.
  • the system can be used in the microtiter and/or 96-well microtiter formats.
  • Examples of Rapid Carbapenemase testing. [0052] Example 1 : Use of panel as vessel only.
  • Test solution containing the following: [0054] 0.4mg/mL Tris, [0055] 0.085mg/mL MgCl 2 , [0056] 4. ⁇ g/mL Triton X-100, [0057] 1.67 ⁇ g/mL Benzalkonium Chloride, [0058] 8.33 ⁇ g/mL resazurin.
  • Solution was pH 7.8, and a 2X concentrate created.
  • Imipenem is added to a final concentration of 0.75mg/mL
  • reaction solution was blue in color and opaque when transferred to a 35C incubator.
  • Example 3 [0068] A modification of the above reaction (Test 1), where EDTA
  • a modification to the above reaction (Test 1), where phenyl-boronic acid or a beta-lactamase inhibitor (Clavulanic Acid, Tazobactam, Avibactam, etc..) will be added can be used to distinguish the classes of carbapenemases or beta-lactamases that are sensitive to beta-lactamase inhibitor drugs. At least two wells will be required. One containing the standard setup as in Test 1 , another well with Test 1 + phenyl-boronic acid or a beta-lactamase inhibitor. Where the blue to pink color change happens in Test 1 but not in Test 1 + phenyl-boronic acid or a beta-lactamase inhibitor, it will indicate a beta- lactamase inhibitor sensitive carbapenemase or beta-lactamase.
  • Example 5 In panel testing.
  • Test solution containing the following:
  • Solution was pH 7.8, and a 25X concentrate created.
  • IX concentrate is added to a 0.5 McFarland standard inoculum of bacteria.
  • the test solution was added to series of wells on a custom Siemens MicroScan panel with the following drug dilutions:
  • Ertapenem, Imipenem and Meropenem are Carbapanems; Ceftriaxone, Ceftazidime and Cefotaxime are Cephalosporins; and Aztreonam is a monobactam beta- lactam drug)
  • Panels are then incubated and read via a Siemens MicroScan Walk-Away instrument at 30 minute increments starting at time zero up to 6 hours.
  • phenyl-boronic acid or a beta-lactamase inhibitor (Clavulanic Acid, Tazobactam, Avibactam, etc..) will allow for detection of those carbapenemases and beta- lactamases that are sensitive to the beta-lactamase inhibitors. At least two wells will be required. One containing the standard setup as in Test 5, another well with Test 5 + phenyl-boronic acid or a beta-lactamase inhibitor. Where the blue to pink color change happens in Test 1 but not in Test 5 + phenyl-boronic acid or a beta-lactamase inhibitor, it will indicate a beta-lactamase inhibitor sensitive carbapenemase or beta-lactamase.

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Abstract

Disclosed are methods, reagents, kits, and systems for detecting drug-resistant bacteria, particularly carbapenemase and beta-lactamase producing bacteria. The methods involve contacting unlysated bacterial samples with a test composition including an antibiotic drug composition, a dye and monitoring a color change produced as a result of degradation of the antibiotic drug.

Description

RAPID DETECTION OF CARBAPENEMASE AND BETA-LACTAMASE
PRODUCING BACTERIA
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional application Serial No.
61/858,217, filed July 25, 2013, which is incorporated herein by reference in its entirety.
TECHNOLOGY FIELD
[0002] The present invention relates generally to methods and systems for detecting drug- resistant bacteria, particularly carbapenemase and beta-lactamase producing bacteria.
BACKGROUND
[0003] Carbapenem and beta-lactam antibiotics are penicillin-related drugs used to combat bacterial infections. Carbapanems are often thought of as a subset of beta- lactams, but for our purposes, beta-lactams refers to the cephems (cephalosporins or cephamycins), since the remaining beta-lactams are substantially not viable alternatives in terms of drug resistance. Recent increases in drug resistant bacteria strains have limited the suitability of this class of drugs in use against bacterial infections. Specifically, the carbapenemase and beta-lactamases produced by resistant bacteria degrade the drugs, resulting in nullification of bacteriacidal activity and drug ineffectiveness. Detection of Carbapenem-Resistant Enterobacteriaceae (CRE) is vital in treatment of patients with such bacterial infections (see "CDC Advisory CRE Technical Information 040313" for details). Detection of carbapenemase and or beta-lactamase activity can lead to differential treatment options for doctors in treatment of resistant bacterial infections. Thus, more and better methods of detecting such activity is desirable.
SUMMARY
[0004] Some embodiments provide a method for determining a drug resistance of a bacteria sample, comprising providing a bacteria cell suspension using the bacteria sample; providing a drug test mixture including carbapenem or beta-lactam drug;
combining the bacteria cell suspension and the drug test mixture in a reaction vessel to create a reaction mixture; monitoring an enzymatic degradation of the reaction mixture over time; and determining a level of carbapenemase or beta-lactamase activity in the bacteria sample based on the monitoring step. [0005] In some embodiments, the monitoring step includes reading a color change of the reaction mixture following incubation of the reaction mixture at 35°C.
[0006] In some embodiments, the step of preparing a drug test mixture includes adding a detection dye to the drug test mixture, and the color change results from a change in the detection dye. In some instances, the detection dye is resazurin.
[0007] In some embodiments, the reaction vessel is at least one well of a 96-well panel.
[0008] In some embodiments, the bacterial sample is not previously lysated. That is to say that a separate lysating step and separation are not required.
[0009] Some embodiments provide a test plate comprising at least one test well, defined by at least one wall; wherein the at least one well is coated with one or more of an antibiotic composition, a metal cation or salt, and a cation membrane disrupting surfactant.
[0010] In some embodiments, the at least one test well is coated with two or more of a beta-lactam antibiotic composition, a metal cation or salt, and a cation membrane disrupting surfactant.
[0011] In some embodiments, the beta-lactam antibiotic composition comprises a carbapanem or a cephem. In some embodiments, the antibiotic composition comprises an antibiotic selected from a carbapenem and a beta-lactam. In some embodiments, the antibiotic composition is a carbapenem selected from imipenem, meropenem, ertapenem, doripenem, panipenem, and biapenem. In some embodiments, the antibiotic composition is a beta-lactam selected from ceftazidime, cefotaxime, ceftriaxone, cefepime, cefazolin, ceftaroline, or aztreonam.
[0012] Some embodiments provide a composition for use in detecting beta-lactamase activity, the composition comprising a buffer; a metal cation; a protein solubilizing surfactant; a cation membrane disrupting surfactant; a dye; and a beta-lactam antibiotic drug. In some embodiments, the composition further comprises a beta-lactamase inhibitor. In some instances, the beta-lactamase inhibitor is phenyl boronic acid. In some instances, the beta-lactam antibiotic drug is a carbapanem. In some embodiments, the beta-lactam antibiotic drug is a cephem selected from ceftazidime, cefotaxime, ceftriaxone, cefepime, cefazolin, ceftaroline, or aztreonam.
[0013] In some embodiments, the buffer is present at about 0.06 to about 0.4 mg/mL of the test solution, the metal cation is present at about 0.01 mg/mL to about O.lmg/mL, the protein solubilizing surfactant is present at about 0.5 μg/mL to about 5μg/mL of the test solution, the cation membrane disrupting surfactant is present at about 1 μg/mL to about 10 μg/mL of test solution, and the dye is present at about 1.3 to about 8.33μg/mL of test solution.
[0014] In some embodiments, the buffer is tris(hydroxymethyl)aminomethane (TRIS), 4- (2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES), or 3-(N- morpholino)propanesulfonic acid (MOPS).
[0015] In some embodiments, the metal cation is provided as a metal salt selected from MgCl2, CaCl2, or ZnCl2.
[0016] In some embodiments, the protein solubilizing surfactant is Triton X-100, NP-40, or Tween 20.
[0017] In some embodiments, the cation membrane disrupting surfactant is benzalkonium chloride, cetyl trimethylammonium bromide, or dioctadecyldimethylammonium bromide.
[0018] In some embodiments, the dye is Resazurin, New Methylene Blue N, Indigo Carmine- b, Laurths Violet (thionin acetate), Trypan Blue, 2,3,5-Triphenyl-2H- Tetrazolium Chloride (TCC or tetrazolium red), Tetrazolium Blue Chloride, Tetrazolium Violet, 2,3-Diphenyl-5-Thienyl-(2) Tetrazolium Chloride (STC), Janus Green B, Orange Tetrazolium, Nile Blue A (Basic Blue B), or 2,6-Dichloroindophenol (DCIP).
[0019] In some embodiments, the antibiotic drug is a carbapenem selected from imipenem, meropenem, ertapenem, doripenem, panipenem, and biapenem or a beta- lactam selected from ceftazidime, cefotaxime, ceftriaxone, cefepime, cefazolin, ceftaroline, or aztreonam.
[0020] These and other embodiments will be understood by one of skill in the art upon reading this specification. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a graph depicting the evaluation of drug resistence in three samples.
EXEMPLARY EMBODIMENTS
[0022] Carbapenemase and beta-lactamase activity can be detected in bacteria using the Modified Hodge Test (an on-plate bacteria growth test) or via molecular markers (gene detection) using PCR or gene expression using RTPCR. Other growth tests include kits where different types of drug disks are added to bacteria growth media and bacteria exclusion zones are read for enzyme activity. Additionally there are bacteria lysis and enzymatic reaction tests developed as kits for such detection. This includes the detection outlined by Nordmann et. al. 2012 which uses lysis of bacteria followed by treatment of the lysate with Imipenem and a pH indicator to detect the degraded enzyme.
[0023] The methods disclosed herein include a rapid detection of the CRE
carbapenemase or beta-lactamase activity using a microbiology system, such as a product from Siemens' MicroScan product line. Specifically the methods described herein use carbapenem and/or beta-lactam drug degradation by resistant bacteria and the subsequent drug degradation product detection via change in color of vital dye or pH indicator dye, to detect carbapenemase or beta-lactamase activity. The proposed test uses bacteria cell suspensions made by customers or technicians with a bacteria test sample. The bacteria suspension is either dispensed into a well of well plate, such as a 96-well panel, containing the carbapenemase or beta-lactamase test mixture, or both the bacteria suspension and enzyme testing mixture are dispensed into the appropriate testing container, and the enzymatic degradation of the drug is read via color change following incubation at 35°C. The reaction rate depends on enzyme activity which in turn depends on the density of the bacteria suspension added. The density of the bacteria suspension can be increased by first growing the bacterial sample in or on a growth medium.
Detection can be as fast as 15 minutes but can be extended up to 4 hours depending on the detection dye used, enzyme activity, amount of enzyme produced by the bacteria (strain dependent) or concentration of bacteria used.
[0024] This test is unique in that no additional steps are required. No bacterial lysis and extraction of lysate is needed (as per Nordmann et. al. 2012), nor any specialized equipment such as a PCR or PCR based machine. The test is adaptable to various existing microbiology systems, such as the current MicroScan Panel, MicroScan AS-4 and MicroScan Walk-Away systems. For example, MicroScan customers can easily add this test to their current process as a presumptive early detection test (offline using the new invention, the carbapenemase test solution).
[0025] Additionally, as the assay can either use a Carabapenem or cephem the assay can be used to detect CREs as well as other beta-lactamase enzymes. In some embodiments, the system can be used to distinguish different classes of beta-lactimases, such as Metallo-beta-lactamases and Extended Spectrum Beta-lactimases.
[0026] In accordance with the methods described herein, a biological sample is provided from a subject suspected of having a bacterial infection, and possibly a drug-resistant bacterial infection. In some instances, the biological sample may be incubated and grown in or on a growth medium, in others, it may be used directly. The biological sample is, without any prior lysis step, then contacted with a test solution which comprises a carbapanem or beta-lactam drug, a dye, and one or more of a buffer, a metal cation, a protein solubilizing surfactant, and a cation membrane disrupting surfactant to form a reaction mixture. The reaction mixture is then allowed to react, in some instances incubating for a period of time. During this period, the color of the reaction mixture is monitored. A color change indicates the presence of carbapanemase or beta-lactamase activity, thus indicating drug-resistance, to whichever drug was present in the test mixture. In some embodiments, the color change is detected by visual inspection. In other embodiments, the color change may be detected by instrumental analysis.
[0027] The biological sample may be any biological sample obtained from a subject. Examples of such "biological samples" include fluids, tissues, cell samples, etc.
Exemplary "biological samples" are whole blood, serum, plasma or urine.
[0028] As alluded to above, the biological sample may be worked up so as to provide a sufficient amount of sample for analysis, by incubating the sample in or on a growth medium as will be known in the art. In some cases, the biological sample may be tested directly without any workup.
[0029] In some embodiments, the biological sample is mixed with a test solution. The test solution comprises several component parts: the drug (carbapanem or beta-lactam), a dye, and one or more of a buffer, a metal cation, a protein solubilizing surfactant, and a cation membrane disrupting surfactant.
[0030] Any suitable buffer may be used. Exemplary buffers include
tris(hydroxymethyl)aminomethane (TRIS), 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES), or 3-(N-morpholino)propanesulfonic acid (MOPS). The buffer may be present at about O.Olmg/mL to about lmg/mL of the test solution. In some embodiments, the buffer is present at about 0.06 to about 0.4 mg/mL of the test solution. In some embodiments, the buffer is present at about 0.06mg/mL, about 0.08mg/mL, about O. lOmg/mL, about 0.2mg/mL, about 0.3mg/mL, about 0.4mg/mL or any value of range of values between any two of the recited values, including endpoints.
[0031] Metal cations are well known for use in enzymatic processes. Exemplary metal cations include, but are not limited to Magnesium, Calcium, and Zinc. The metal cation may be provided by a metal salt, for example MgCl2, CaCl2, or ZnCl2. The metal cation, in the form of a metal salt, may be present at about 0.01 mg/mL to about 0. lmg/mL. In some embodiments, the metal salt is present at about 0.013 to 0.085 mg/mL of the test solution. In some embodiments, the metal salt is present at about O.Olmg/mL,
0.013mg/mL, 0.015mg/mL, 0.018mg/mL, 0.02mg/mL, 0.03mg/mL, 0.04mg/mL, 0.05mg/mL, 0.06mg/mL, 0.07mg/mL, 0.08mg/mL, 0.085mg/mL, 0.09mg/mL, 0.
lmg/mL, or any value of range of values between any two of the recited values, including endpoints.
[0032] The protein solubilizing surfactant is a surfactant useful for solubilizing protein, such as Triton X-100, NP-40, or Tween 20. The protein solubilizing surfactant is present at about 0.5 μg/mL to about 5μg/mL of the test solution. The protein solubilizing surfactant may be present at about 0.64 to about 4.17μg/mL. In some embodiments, the protein solubilizing surfactant may be present at about 0.5 μg/mL, about 0.6 μg/mL, about 0.64 μg/mL, about 0.7 μg/mL, about 0.8 μg/mL, about 0.9 μg/mL, about 1.0 μg/mL, about 2.0 μg/mL, about 3.0 μg/mL, about 4.0 μg/mL, about 5.0 μg/mL, or any value of range of values between any two of the recited values, including endpoints.
[0033] The cation membrane disrupting surfactant can be benzalkonium chloride, cetyl trimethylammonium bromide, or dioctadecyldimethylammonium bromide. The cation membrane disrupting surfactant may be present at about 1 μg/mL to about 10 μg/mL. The cation membrane disrupting surfactant may be present at about 0.26 to about 1.67μg/mL
[0034] The dye may be any suitable dye, such as a pH indicator, a vital dye, a metabolic indicator, a redox indicator, or the like. Exemplary dyes include, but are not limited to Resazurin, New Methylene Blue N, Indigo Carmine- b, Laurths Violet (thionin acetate), Trypan Blue, 2,3,5-Triphenyl-2H-Tetrazolium Chloride (TCC or tetrazolium red), Tetrazolium Blue Chloride, Tetrazolium Violet, 2,3-Diphenyl-5-Thienyl-(2) Tetrazolium Chloride (STC), Janus Green B, Orange Tetrazolium, Nile Blue A (Basic Blue B), 2,6- Dichloroindophenol (DCIP). The dye may be present at about 1.3 to about 8.33μg/mL of test solution. Resazurin is a metabolic indicator and a redox indicator that is particularly well-suited for the methods and systems described herein.
[0035] The drug may be any carbapanem or beta-lactam. In embodiments where cabapenem is used, the carbapanem is selected from imipenem, meropenem, ertapenem, doripenem, panipenem, and biapenem. In some embodiments, imipenem is used. In embodiments where a beta-lactam is used, the beta-lactam may be a cephalosporin or a monobactam. In some embodiments, the beta-lactam is selected from ceftazidime, cefotaxime, ceftriaxone, cefepime, cefazolin, ceftaroline, or aztreonam. In some embodiments, the beta-lactam is selected from ceftazidime, cefotaxime, and ceftriaxone.
[0036] In some instances, additional tests may be desirable for specific class of carbapenemases or beta-lactamases. This can be achieved by running a parallel sample with the addition of a carbapenemase or beta-lactamase inhibitor. For detection of Metallo B-lactamase class carbapenemases, inhibition of the reaction with addition of ImM EDTA will give a negative carbapenemase test result (positive result for presence of Metallo-B-lactamase). To detect beta-lactamase inhibitor sensitivity, phenyl boronic acid or another beta-lactamase inhibitor, such as Clavulanic Acid, Tazobactam,
Avibactam, may be used. In each case, a first sample is treated with the basic test solution described herein with either the carbapenem drug or the beta-lactam drug. A second aliquot of the same biological sample is treated in parallel with the basic test solution plus an addition of the inhibitor. Where a color change occurs in the first (inhibitor- free) test, but not in the second (inhibitor containing) test, the test reveals inhibitor sensitivity. [0037] In the case of testing for metallo-beta-lactamase activity, the inhibitor is ethylenediaminetetraacetic acid (EDTA). A color change in the inhibitor free test accompanied by lack of color change in the inhibitor containing test, indicates the presence of metallo-beta-lactamase. Such indication can provide valuable information to a trating physician as to which drugs he should or should not use to treat the patient.
[0038] In the case of beta- lactamase inhibitor sensitivity, the inhibitor to be added is phenyl-boronic acid or a beta-lactamase inhibitor such as Clavulanic Acid, Tazobactam, Avibactam, or the like. As above, color change in an inhibitor-free test sample with a simultaneous lack of color change in the inhibitor-containing test sample indicates inhibitor sensitivity, giving the treating physician more information on the bacteria to be treated.
[0039] In some embodiments, the test solution may be a single solution or multiple component parts which come together to complete the test solution. Thus, in some embodiments, the test solution is complete and awaits addition of the biological sample to begin the test reaction. In other embodiments, one or more component parts may be added in preparing the biological sample and then combined with the remaining component parts to initiate the test reaction. In other embodiments, one or more component of the test solution may be dried in a sample vessel, such as a microtitre well, and reconstituted by adding the remaining components of the test solution.
[0040] Detecting Drug Resistance Activity.
[0041] When drug resistance activity is detected, the treating physician can be alerted to the fact that the particular bacteria involved is resistant to a particular drug. The physician is then free to tailor his treatment, avoiding the resistance. In most instances, it is therefore desirable to test for both carbapenemase and beta-lactamase activity. Thus, in some instances, side by side tests will be performed, in separate vessels or wells analyzing both carbapenemase and beta-lactamase activity. Where one or the other activity is detected, the physician can be alerted to tailor the treatment towards the appropriate drug.
[0042] With the exception of the drug used, the test solution is substantially the same whether testing for carbapenemase or beta-lactamase activity. In some embodiments, therefore, the test solution is prepared as separate components in a kit, where the drug is separate from one or more of the other components. These can be separate solutions, or the drug may be present, dried or wet, in the sample vessel or well.
[0043] In some embodiments, a previously prepared well plate may contain at least one well containing carbapenem and at least one well containing a beta-lactam. Each well may be in in certain location designated for evaluating drug resistance. For example, a standard 96-well plate could have two wells assigned for evaluation of carbapenemase and beta-lactamase activity. The remaining cells could be assigned for other common tests or analyses of the same biological sample, or could contain the same tests for other biological samples.
[0044] In some examples, a microtiter well plate may contain at least two wells. A first of the two well is designated for carbapenemase evaluation and a second of the two wells is designated for beta-lactamase evaluation. The first well is provided with a quantity of a carbapenem drug, such as imipenem. The second well is provided with a quantity of a beta-lactam drug, such as cefotaxime or ceftazidime. In both wells, the drug may be dried for ease of storage until use. In some embodiments, one or more of the other test solution components may also be present along with the drug. In particular, the cation, in the form of a metal salt is particularly well suited for drying in a well plate. Additionally, the cation membrane disrupting surfactant is also well suited for drying in a well plate. Thus, each of the first and second wells may contain the drug to be evaluated, the metal cation (in the form of a salt), and/or the cation membrane disrupting surfactant. In such embodiments, a test solution containing the buffer, dye, and protein solubilizing surfactant is provided separately. The test solution is added to the two wells, along with the biological sample. Once the test solution and biological samples are added, the wells are incubated at 35 °C and color change is monitored. Color change indicates the presence of a drug resistant bacteria.
[0045] FIG. 1 illustrates some exemplary results. Three biological samples were tested with imipenem. An E. coli sample and two K. pneumonia CREs were evaluated to show how susceptible strains (the E. coli) and resistant strains change over time. The Y-axis shows values that come off the instrument. In this instance, negative 4,000 is baseline at time point of zero. In this case the instrument acts like a spectrophotometer, the 560 wave length in 30 minute intervals. The substantially horizontal line represents a E. coli strain known not to be resistant to carbapenems. The other two lines represent K. pneumoniae strains known to resistant to carbapenems. It is expected, that the K.
pneumoniae would degrade imipenem and as shown between 60 and 90 minutes there is a steep and continuous decline representative of a change from the dye going from blue to purple to pink, indicating the breakdown of the imipnem by the carbapenemase containing bacteria. Thus, it is clear from this data, a treating physician could be notified within about 60 to 90 minutes that the bacteria in question is carbapenem resistant, and the patient's treatment tailored accordingly. In most instances, the resistant nature can be readily identified with about 4 hours. Compared to current techniques which take overnight, this is a significant improvement.
[0046] Depending upon sample preparation, carbapenemase activity will yield steeper and faster drop offs. By accumulating additional data, it will be possible to detect and predict the activity of a particular bacteria, to better advise treating physicians on which drugs to use and perhaps in what concentrations.
[0047] An exemplary test solution includes 20mM Tris, 0.5mM magnesium chloride, 1% Triton X-100, 0.4mg/L benzalkonium chloride (Zephiran) and a dye. Dyes include resazurin at 0.02g/L or phenol red at 0.04g/L (these are minimal dye concentrations). Test Solution has final pH of 7.8. In some embodiments, the drug is either 2.5mg/mL
Imipenem or cefotaxime or ceftazidime. For detection of B-lactamase, cefotaxime or ceftazidime drugs are particularly well suited for the reaction. For detection of carbapenemase, Imipenem drug is particularly well suited for the reaction.
[0048] For detection of KPC and other serine active site carbapenemases, competitive inhibition of the reaction with addition of Phenylboronic Acid (1.5mg) will give a negative carbapenemase test result (positive for presence of KPC or other serine active site carbapenemase).
[0049] For detection of Metallo B-lactamase class carbapenemases, inhibition of the reaction with addition of ImM EDTA will give a negative carbapenemase test result (positive result for presence of Metallo-B-lactamase).
[0050] This invention fills a gap in the current offerings and adds to the testing options offered by current microbiology systems. The system can be used in the microtiter and/or 96-well microtiter formats. [0051] Examples of Rapid Carbapenemase testing. [0052] Example 1 : Use of panel as vessel only. [0053] Test solution containing the following: [0054] 0.4mg/mL Tris, [0055] 0.085mg/mL MgCl2, [0056] 4.^g/mL Triton X-100, [0057] 1.67μg/mL Benzalkonium Chloride, [0058] 8.33μg/mL resazurin.
[0059] Solution was pH 7.8, and a 2X concentrate created.
[0060] A 2 McFarland standard inoculum of bacteria is added to the equal amounts to the 2X test solution.
[0061] Imipenem is added to a final concentration of 0.75mg/mL
[0062] Reaction solution was blue in color and opaque when transferred to a 35C incubator.
[0063] Following 2-4hrs of incubation the reaction was read for color change (pink color formation).
[0064] In strains with previously observed Carbapenemase activity a pink color formation was observed.
[0065] Example 2:
[0066] Above testing (Test 1) was duplicated with Ceftazidime and Cefotaxime drugs tested with ESBL (Extended Spectrum Beta-lactamase) bacteria. A positive reaction (pink color change) was observed with ESBL containing bacteria.
[0067] Example 3: [0068] A modification of the above reaction (Test 1), where EDTA
(Ethylenediaminetetraacetic acid) was added at a final concentration of 0.3mg/mL was able to repress the carbapenemase activity of a metallo-beta-lactamase version of the carbapenemase (NDM strain). As the EDTA chelates metal ions and the metallo-beta- lactamases require metal ions for activity, EDTA can be used to distinguish that class of carbapenemases. At least two wells will be required. One containing the standard setup as in Test 1, another well with Test 1+ EDTA. Where the blue to purple color change happens in Test 1 but not in Test 1 + EDTA it will indicate metallo-beta-lactamase is present.
[0069] Example 4:
[0070] A modification to the above reaction (Test 1), where phenyl-boronic acid or a beta-lactamase inhibitor (Clavulanic Acid, Tazobactam, Avibactam, etc..) will be added can be used to distinguish the classes of carbapenemases or beta-lactamases that are sensitive to beta-lactamase inhibitor drugs. At least two wells will be required. One containing the standard setup as in Test 1 , another well with Test 1 + phenyl-boronic acid or a beta-lactamase inhibitor. Where the blue to pink color change happens in Test 1 but not in Test 1 + phenyl-boronic acid or a beta-lactamase inhibitor, it will indicate a beta- lactamase inhibitor sensitive carbapenemase or beta-lactamase.
[0071] Example 5: In panel testing.
[0072] Test solution containing the following:
[0073] 0.06 - 0.4mg/mL Tris
[0074] 0.013 - 0.085mg/mL MgC12
[0075] 0.64 - 4. ^g/mL Triton X-100
[0076] 0.26 - 1.67μg/mL Benzalkonium Chloride
[0077] 1.3 - 8.33μg/mL resazurin
[0078] Solution was pH 7.8, and a 25X concentrate created.
[0079] IX concentrate is added to a 0.5 McFarland standard inoculum of bacteria. [0080] The test solution was added to series of wells on a custom Siemens MicroScan panel with the following drug dilutions:
[0081] Ertapenem 0.25 - 32μg/mL
[0082] Imipenem 0.25 - 64μg/mL [0083] Ceftriaxone 0.12 - 32 μg/mL [0084] Ceftazidime 0.12 - 32 μg/mL [0085] Cefotaxime 0.12 - 32 μg/mL [0086] Aztreonam 0.25 - 64 μg/mL
[0087] (Note: Ertapenem, Imipenem and Meropenem are Carbapanems; Ceftriaxone, Ceftazidime and Cefotaxime are Cephalosporins; and Aztreonam is a monobactam beta- lactam drug)
[0088] Panels are then incubated and read via a Siemens MicroScan Walk-Away instrument at 30 minute increments starting at time zero up to 6 hours.
[0089] Active degradation of each drug is documented (change of color from blue to pink). Algorithms for detection and optimization are to be calculated.
[0090] Example 6:
[0091] Addition of 0.3mg/mL EDTA (final concentration) to the reaction (Test 5) will allow for detection of metallo-beta-lactamases via removal of the metal ion required by those enzymes for degradation of carbapenems. At least two wells will be required. One containing the standard setup as in Test 1, another well with Test 1+ EDTA. Where the blue to purple color change happens in Test 1 but not in Test 1 + EDTA it will indicate metallo-beta-lactamase is present.
[0092] Example 7:
[0093] Addition of phenyl-boronic acid or a beta-lactamase inhibitor (Clavulanic Acid, Tazobactam, Avibactam, etc..) will allow for detection of those carbapenemases and beta- lactamases that are sensitive to the beta-lactamase inhibitors. At least two wells will be required. One containing the standard setup as in Test 5, another well with Test 5 + phenyl-boronic acid or a beta-lactamase inhibitor. Where the blue to pink color change happens in Test 1 but not in Test 5 + phenyl-boronic acid or a beta-lactamase inhibitor, it will indicate a beta-lactamase inhibitor sensitive carbapenemase or beta-lactamase.
[0094] The description herein is illustrative in nature only. Those of skill in the art will recognize variations without departing from the spirit or scope of this disclosure.

Claims

CLAIMS We claim:
1. A method for determining a drug resistance of a bacteria sample, comprising: providing a bacteria cell suspension using the bacteria sample; providing a drug test mixture including carbapenem or beta-lactam drug; combining the bacteria cell suspension and the drug test mixture in a reaction vessel to create a reaction mixture; monitoring an enzymatic degradation of the reaction mixture over time; and determining a level of carbapenemase or beta-lactamase activity in the bacteria sample based on the monitoring step.
2. The method of claim 1, wherein the monitoring step includes reading a color change of the reaction mixture following incubation of the reaction mixture at 35°C.
3. The method of claim 2, wherein, the step of preparing a drug test mixture includes adding a detection dye to the drug test mixture, and the color change results from a change in the detection dye.
4. The method of claim 3, wherein the detection dye is resazurin.
5. The method of claim 1, wherein the reaction vessel is at least one well of a 96- well panel.
6. The method of claim 1 , wherein the bacterial sample is not previously lysated.
7. A test plate comprising: at least one test well, defined by at least one wall; wherein the at least one well is coated with one or more of an antibiotic composition, a metal cation or salt, and a cation membrane disrupting surfactant.
8. The test plate of claim 7 wherein the at least one test well is coated with two or more of a beta-lactam antibiotic composition, a metal cation or salt, and a cation membrane disrupting surfactant.
9. The test plate of claim 7, wherein the beta-lactam antibiotic composition comprises a carbapanem or a cephem.
10 The test plate of claim 7, wherein the antibiotic composition comprises an antibiotic selected from a carbapenem and a beta-lactam.
11. The test plate of claim 7, wherien the antibiotic composition is a carbapenem selected from imipenem, meropenem, ertapenem, doripenem, panipenem, and biapenem.
12. The test plate of claim 7, wherein the antibiotic composition is a beta-lactam selected from ceftazidime, cefotaxime, ceftriaxone, cefepime, cefazolin, ceftaroline, or aztreonam.
13. A composition for use in detecting beta- lactamase activity, the composition comprising: a buffer; a metal cation; a protein solubilizing surfactant; a cation membrane disrupting surfactant; a dye; and a beta-lactam antibiotic drug.
14. The composition of claim 13, further comprising a beta-lactamase inhibitor.
15. The composition of claim 14, wherein the beta-lactamase inhibitor is phenyl boronic acid.
16. The composition of claim 14, wherein the beta-lactam antibiotic drug is a carbapanem.
17. The composition of claim 14, wherein the beta-lactam antibiotic drug is a cephem selected from ceftazidime, cefotaxime, ceftriaxone, cefepime, cefazolin, ceftaroline, or aztreonam.
18. The composition of claim 14, wherein: the buffer is present at about 0.06 to about 0.4 mg/mL of the test solution, the metal cation is present at about 0.01 mg/mL to about 0.1 mg/mL, the protein solubilizing surfactant is present at about 0.5 μg/mL to about 5μg/mL of the test solution, the cation membrane disrupting surfactant is present at about 1 μg/mL to about 10 μg/mL of test solution, and the dye is present at about 1.3 to about 8.33μg/mL of test solution.
19. The composition of claim 14, wherein the buffer is
tris(hydroxymethyl)aminomethane (TRIS), 4-(2-hydroxyethyl)- 1 -piperazineethanesulfonic acid (HEPES), or 3-(N-morpholino)propanesulfonic acid (MOPS).
20. The composition of claim 14, wherein the metal cation is provided as a metal salt selected from MgCl2, CaCl2, or ZnCl2.
21. The composition of claim 14, wherein the protein solubilizing surfactant is Triton X-100, NP-40, or Tween 20.
22. The composition of claim 14, wherein the cation membrane disrupting surfactant is benzalkonium chloride, cetyl trimethylammonium bromide, or
dioctadecyldimethylammonium bromide.
23. The composition of claim 14, wherein the dye is Resazurin, New Methylene Blue N, Indigo Carmine- b, Laurths Violet (thionin acetate), Trypan Blue, 2,3,5-Triphenyl- 2H-Tetrazolium Chloride (TCC or tetrazolium red), Tetrazolium Blue Chloride, Tetrazolium Violet, 2,3-Diphenyl-5-Thienyl-(2) Tetrazolium Chloride (STC), Janus Green B, Orange Tetrazolium, Nile Blue A (Basic Blue B), or 2,6-Dichloroindophenol (DCIP).
24. The composition of claim 14, wherein the antibiotic drug is a carbapenem selected from imipenem, meropenem, ertapenem, doripenem, panipenem, and biapenem or a beta-lactam selected from ceftazidime, cefotaxime, ceftriaxone, cefepime, cefazolin, ceftaroline, or aztreonam.
25. The composition of claim 14, wherein: the buffer is tris(hydroxymethyl)aminomethane (TRIS), 4-(2-hy droxy ethyl)- 1- piperazineethanesulfonic acid (HEPES), or 3-(N-morpholino)propanesulfonic acid (MOPS); wherein the metal cation is provided as a metal salt selected from MgCl2, CaCl2, or
ZnCl2; the protein solubilizing surfactant is Triton X-100, NP-40, or Tween 20; the cation membrane disrupting surfactant is benzalkonium chloride, cetyl trimethylammonium bromide, or dioctadecyldimethylammonium bromide; the dye is Resazurin, New Methylene Blue N, Indigo Carmine- b, Laurths Violet (thionin acetate), Trypan Blue, 2,3,5-Triphenyl-2H-Tetrazolium Chloride (TCC or tetrazolium red), Tetrazolium Blue Chloride, Tetrazolium Violet, 2,3-Diphenyl-5-Thienyl-(2) Tetrazolium Chloride (STC), Janus Green B, Orange Tetrazolium, Nile Blue A (Basic Blue B), or 2,6-Dichloroindophenol (DCIP); and the antibiotic drug is a carbapenem selected from imipenem, meropenem, ertapenem, doripenem, panipenem, and biapenem or a beta-lactam selected from ceftazidime, cefotaxime, ceftriaxone, cefepime, cefazolin, ceftaroline, or aztreonam.
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CN106467924A (en) * 2016-09-20 2017-03-01 安徽理工大学 A kind of test method evaluating antibiotic substance antibacterial activity in vitro
CN106467924B (en) * 2016-09-20 2019-12-20 安徽理工大学 Test method for evaluating in-vitro antibacterial activity of antibacterial substance

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