WO2006088381A1 - In vitro evaluation of micro-organisms and their antimicrobial agent susceptibilities - Google Patents
In vitro evaluation of micro-organisms and their antimicrobial agent susceptibilities Download PDFInfo
- Publication number
- WO2006088381A1 WO2006088381A1 PCT/NZ2006/000019 NZ2006000019W WO2006088381A1 WO 2006088381 A1 WO2006088381 A1 WO 2006088381A1 NZ 2006000019 W NZ2006000019 W NZ 2006000019W WO 2006088381 A1 WO2006088381 A1 WO 2006088381A1
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- micro
- culture
- cultures
- organism
- antimicrobial agent
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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
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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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
- SIFT-MS combines chemical ionization of analyte VCs using H 3 O+, NO+ or O 2 + with fast flow tube quadrupole-mediated identification and quantification in complex mixtures such as breath and ambient air regardless of the water vapor content in (near) real time. Product ions can then be identified with reference to a molecular-ion reaction and rate coefficient database.
- Bacterial infections are typically diagnosed by culturing samples of tissues or body fluids including blood.
- a common process is to use BactT/ALERT® bottles which contain small amounts of culture medium.
- the process of growing the microorganisms can be monitored through colour change and/or production of CO 2 .
- This system is fairly simple but lacks specificity and tends to be slow with typical times to obtain definitive positive or negative being 12-48 hours.
- Kiviranta et al demonstrated that the qualitative identification of three bacterial and two fungal species from culture headspace volatile organic compounds adsorbed on Tenax TA and analysed by GC-MS was highly dependent on the culture medium and the species - Kiviranta H, Tuomainen A, Reiman M, Laitinen S, Liesivuori J. Nevalainen A. Qualitative identification of volatile metabolites from two fungi and three bacteria species cultivated on two media. Cent Eur J Public Health 1998,6:296-299
- SPME solid phase micro extraction
- VFA volatile fatty acids
- NVFA non-volatile carboxylic acids
- Wang T, et al have recently expanded their SIFT-MS database to include the kinetic data for the reactions of several compounds related to the emissions produced by Pseudomonas species in vitro.
- Wang T, Smith D, Spanel P. Selected ion flow tube SIFT studies of the reactions of H 3 O+, NO+ and O 2 + with compounds released by Pseudomonas and related bacteria.
- lntl J Mass Spectrometry 2004; 233: 245-251 This article also includes a report on VCs produced by Pseudomonas species of bacteria.
- IMS ion mobility spectrometry
- Shnayderman et al describe the use of micromachined differential (ion) mobility spectrometry to measure headspace gases from bacteria growing in liquid culture. They applied pattern discovery/recognition algorithms (ProteomeQuest) to identify the VC profiles of four species including Escherichia coli, Bacillus subtilis, Bacillus thuringiensis and Mycobacterium smegmatis. They conclude that their combination of technology and bioinformatics data analysis has potential for diagnosis of bacterial infections.
- Lechner et al used proton transfer reaction mass spectrometry (PTR-MS) to measure the liquid culture headspace VCs of Escherichia coli, Klebsiella, Citrobacter, Pseudomonas aeruginosa, Staphylococcus aureus and Helicobacter pylori.
- the patterns of VCs detected differed in quantity and composition for each species tested.
- the authors erroneously conclude that they were the first to describe the headspace screening of bacterial cultures as a potential microbiological diagnostic approach.
- the invention relates to a method of determining the metabolic consequences of an antimicrobial agent for a micro-organism, comprising establishing the presence of a micro-organism(s) by sampling by means of
- SIFT-MS the VCs produced by the bacterium or micro-organism, identifying the micro-organism(s) analysing the VCs produced by the micro-organism(s) and determining the antimicrobial susceptibility of the micro-organism(s).
- the invention also relates to a method of identifying a micro-organism(s) and the metabolic consequences of an antimicrobial agent on the micro-organism comprising the steps of inserting a sample containing the micro-organism(s) into a primary culture container, allowing micro-organism(s) within the primary culture of the sample to multiply in the container dividing the primary culture into a plurality of secondary cultures and charging separate containers with individual secondary cultures, analysing the VCs in the headspace above the secondary cultures by means of SIFT-MS to ascertain whether micro-organisms exist in the secondary culture determining the type of micro-organism, splitting the secondary cultures into a plurality of tertiary cultures and inserting the tertiary cultures into separate containers with each container including a specific antimicrobial agent at a specific concentration, analysing the VCs in the headspace above each tertiary culture to determine the antimicrobial susceptibility of the micro-organism.
- the primary culture is divided into the secondary cultures after a period of rest or incubation from the commencement of the test.
- the primary culture is divided into a plurality of secondary cultures with one of the secondary cultures being retained as a control culture to enable a positive or negative report to be generated as to the presence of an infectious bacterium or micro-organism and to enable the infection to be identified.
- the primary culture is divided into three secondary cultures.
- the secondary culture is tested after a period of rest from the commencement of the test and if the test provides a positive result, the secondary culture is divided into the plurality of tertiary cultures.
- the secondary culture is divided into a plurality of tertiary cultures which are each combined with a specific antibiotic or antimicrobial substance at a specific concentration.
- the tertiary cultures are tested after a period of rest from the commencement of the test to determine the susceptibility of the bacterium or microorganism to the antibiotic or antimicrobial substance.
- the susceptibility of the bacterium or micro-organism to the antimicrobial agent is determined by the presence of either a high concentration of the chosen antibiotic or antimicrobial substance or a medium concentration of the antibiotic or antimicrobial substance.
- the invention in another aspect relates to a method of identifying an infectious micro-organism and the metabolic consequences of an antimicrobial agent in a blood culture comprising the steps of inserting a primary blood culture into a container, allowing bacteria or organisms within the primary blood culture to multiply in the container dividing the culture into a plurality of secondary cultures and charging separate containers with individual secondary cultures, analysing the VCs in the headspace above the secondary cultures by means of SIFT-MS to ascertain whether an micro-organism(s) exists in the secondary blood culture and to determine the type of micro-organism, splitting the secondary cultures into a plurality of tertiary cultures and inserting the tertiary cultures into separate containers with each container including a specific antimicrobial agent at a specific concentration, analysing the VCs in the head space above each tertiary culture to determine whether the micro-organism in the tertiary culture has grown and to ascertain whether the antimicrobial agent has been inhibitory to the growth of the microorganism.
- a report is generated in which the micro-organism is identified and the antimicrobial susceptibility of the micro-organism is displayed.
- SIFT-MS involves the generation of precursor ions (e.g. H 3 O+, O 2 +, NO+) from a discharge source that are mass selected by an "upstream" quadrupole mass filter.
- precursor ions e.g. H 3 O+, O 2 +, NO+
- the selected ion species is then injected into the flow tube by a fast flowing stream of inert carrier gas, eg helium.
- headspace atmospheres are introduced above conventional Biomerieux BacT/ALERT® aerobic or anaerobic blood cultures at a controlled rate into the flow tube precursor ion stream.
- the count rates of the resulting product ions are then to be determined by a "downstream" quadrupole and particle multiplier detector.
- the detector quadrupole is scanned over a predetermined m/z range to obtain a spectrum of product ions.
- SIMscan selected ion mode
- the count rate of selected product ions is determined as the downstream spectrophotometer is switched to and remains on selected m/zs.
- Bacterial blood culture Standard Biomerieux (Durham, NC, USA) BacT/ALERT® SA, FA and SN disposable, plastic culture bottles containing 40 ml of culture medium were used throughout the experiments. 9ml of healthy, uninfected human blood and 1 ml aliquots of antibiotics prepared in sterile water were added (in the relevant experiments) as required to each bottle prior to the addition of 1 ml of bacterial species suspended in sterile physiological saline.
- CFU colony forming units
- SIMscan Selected Ion Mode
- Time to positive (TTP) result was recorded for all bottles remaining on the blood culture system. Bottles intended for SIFT-MS analysis that flagged positive prior to the projected testing time were removed from the machine, and incubation continued without agitating at 37 0 C. The mean concentration of each VC metabolite listed above was measured for triplicate negative controls as for other cultures. The standard deviation for each analyte for negative controls at each assay time (ie 8 and 24 hours) was calculated. A threshold, or negative cutoff value for each analyte was determined as the mean of the negative control values plus 2 standard deviations from that mean. A bottle was recorded as positive if a level above the threshold was measured for at least one analyte (Tables 4 and 5).
- Table 2 Final plate-count confirmed dilutions for time to positive result bacterial blood cultures.
- Table 3 Bacterial blood culture time to positive versus VC evaluation scheme
- Table 4 Conventional blood culture vs SIFT-MS- time to positive result-Aerobic.
- Table 5 Conventional blood culture vs SIFT-MS- time to positive result-Anaerobic.
- Table 6 VCs produced at statistically significant levels at 8 hours by five bacteria under aerobic and anaerobic growth conditions
- Table 7 VCs produced at statistically significant levels at 24 hours by five bacteria under aerobic and anaerobic growth conditions
- E. coli or S. aureus were inoculated into Biomerieux BacT/ALERT® SA bottles including 9ml of fresh uninfected blood. Each bacterial species was incubated in triplicate either alone or in the presence of antibiotics above or below their predetermined minimal inhibitory concentrations (MIC). Gentamicin was added to E. coli cultures at either 2 ⁇ g/ml or 0.05 ⁇ g/ml (MIC 0.25- 1 ⁇ g/m)l. Flucloxacillin was added to S. aureus cultures at 2 ⁇ g/ml or 0.05 ⁇ g/ml (MIC 0.12-0.5 ⁇ g/ml) .
- MIC minimal inhibitory concentration
- Table 1 illustrates the characteristic VC patterns for each test species.
- the headspaces of Pseudomonas aeruginosa cultures had relatively high absolute concentrations of acetic acid and acetone, compared to other analytes (p ⁇ 0.001), and an absence of methanethiol.
- Streptococcus pneumoniae metabolic VCs were marked by high acetaldehyde, acetone, ethanol and dimethyl sulfide compared with intermediate acetic acid, ammonia and dimethyldisulfide (p ⁇ 0.001).
- the dimethyldisulfide concentration (180 ppb v/v) significantly exceed low hydrogen sulfide and methanethiol (p ⁇ 0.001).
- Neisseria meningitidis cultures demonstrated very high acetone production compared with low acetaldehyde, acetic acid, ethanol dimethyl sulfide and dimethyldisulfide (p ⁇ 0.001) and no detectable hydrogen sulfide or methanethiol.
- SIMscan VC concentrations were measured in triplicate E. coli or S. aureus test and control culture headspaces following six or 22 hours of incubation in the presence or absence of gentamicin or flucloxicillin above or below their MIC.
- the VC analyte concentrations of uninoculated control media (containing blood) did not change significantly ( ⁇ 20% for any analyte) from background levels between six and 22 hour measurements.
- the mean, minima and maxima integrated headspace analyte concentration measurements obtained from E. coli and S. aureus cultures are compared in Tables 8 and 9.
- VCs consistent with concentration-dependent discrimination include acetic acid, 2-aminoacetophenone, dimethyldisulfide, ethanol, hydrogen sulfide, methanethiol, pentanols and propanol.
- S. aureus growth at six hours of incubation is indicated in Table 9 by the production of ammonia and dimethylsulfide. Production of these analytes was inhibited to uninoculated, antibiotic-containing control levels by flucloxacillin above and below its MIC for this organism. After 22 hours of in vitro blood culture acetaldehyde, 2-aminoacetophenone, ethanol, formaldehyde and indol concentrations above uninoculated controls indicated growth. Those VCs that discriminated between the metabolic effects of flucloxacillin concentrations in a concentration-dependent manner (p ⁇ 0.05), recorded in Table 9, include 2- aminoacetophenone, ethanol and formaldehyde. Table 9: Effect of flucloxacillin on S. aureus VC production
- Flucloxacillin above and below its demonstrated MIC, significantly reduced (p ⁇ 0.01) the production of ammonia and dimethylsulfide by S. aureus at six hours incubation.
- the inhibition of aminoacetophenone, ethanol and formaldehyde was notable (p ⁇ 0.01) at 22 hours with Flucloxacillin above its MIC while the lower antibiotic concentration of was not inhibitory (Table 9).
- Figure 1 illustrates a schematic form of the steps of the present invention which are involved in the detection and determination of the presence of microorganisms in a blood culture using SIFT-MS to analyse the VCs in the headspace above the blood culture under investigation.
- a sample of blood for instance 10 ml
- the bottle has a piercable septum top and in a highly preferred form the bottle may be that known under the trade name Biomerieux BacTALERT®.
- T1 which may typically be three hours from the start of the test
- the primary culture is divided into secondary bottles 2, 3 and 4. While in the form illustrated the primary culture is divided into three secondary BacT/ALERT® bottles, it will be understood the number of divisions of the primary culture will be at the option of the operator and of the circumstances.
- the secondary cultures are tested by SIFT-MS for growth of micro-organisms. If the test is positive, a report can be provided that the patient has a blood infection. The blood culture in the secondary bottle 4 is retained as the control culture.
- the blood cultures are split into small quantities as indicated at 5, for instance 5ml aliquots which are separately put into bottles which contain high and medium concentrations of different antimicrobial agents as illustrated as A through E.
- the VCs are tested by SIFT- MS to identify whether the micro-organisms have grown in the presence of each antimicrobial agent and the susceptibility of the micro-organisms to the antimicrobial agent.
- the tertiary blood culture identified as C indicates a positive sensitivity to both a medium concentration and a high concentration of the antimicrobial agent.
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Abstract
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP06716796A EP1851327A4 (en) | 2005-02-15 | 2006-02-14 | In vitro evaluation of micro-organisms and their antimicrobial agent susceptibilities |
US11/884,410 US20080261263A1 (en) | 2005-02-15 | 2006-02-14 | In Vitro Evaluation of Micro-Organisms and Their Antimicrobial Agent Susceptibilities |
AU2006214871A AU2006214871A1 (en) | 2005-02-15 | 2006-02-14 | In vitro evaluation of micro-organisms and their antimicrobial agent susceptibilities |
CA002597591A CA2597591A1 (en) | 2005-02-15 | 2006-02-14 | In vitro evaluation of micro-organisms and their antimicrobial agent susceptibilities |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NZ538235A NZ538235A (en) | 2005-02-15 | 2005-02-15 | Evaluation of bacterial growth and antibiotic sensitivity in blood cultures using selected ion flow tube mass spectrometry |
NZ538235 | 2005-02-15 |
Publications (1)
Publication Number | Publication Date |
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WO2006088381A1 true WO2006088381A1 (en) | 2006-08-24 |
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ID=36916707
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Application Number | Title | Priority Date | Filing Date |
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PCT/NZ2006/000019 WO2006088381A1 (en) | 2005-02-15 | 2006-02-14 | In vitro evaluation of micro-organisms and their antimicrobial agent susceptibilities |
Country Status (6)
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US (1) | US20080261263A1 (en) |
EP (1) | EP1851327A4 (en) |
AU (1) | AU2006214871A1 (en) |
CA (1) | CA2597591A1 (en) |
NZ (1) | NZ538235A (en) |
WO (1) | WO2006088381A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2013529458A (en) * | 2010-06-11 | 2013-07-22 | ブルーカー ダルトニック ゲーエムベーハー | Mass spectrometric measurement of β-lactamase resistance |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2430460B1 (en) * | 2009-05-15 | 2018-07-11 | Biomerieux, Inc | Automated transfer mechanism for microbial detection apparatus |
AU2010248809B2 (en) | 2009-05-15 | 2015-07-09 | Biomerieux, Inc. | System and methods for rapid identification and/or characterization of a microbial agent in a sample |
JP2012010623A (en) * | 2010-06-30 | 2012-01-19 | Hitachi High-Technologies Corp | Method of testing microorganism and test system |
JP6018808B2 (en) * | 2012-06-12 | 2016-11-02 | 株式会社日立ハイテクノロジーズ | Microorganism test method and test system |
CA3176865A1 (en) * | 2020-04-27 | 2021-11-04 | Livia SCHIAVINATO EBERLIN | Identifying and classifying microorganisms |
CN113913348B (en) * | 2021-11-25 | 2023-06-30 | 云南中烟工业有限责任公司 | Klebsiella grignard SA34 and application thereof |
-
2005
- 2005-02-15 NZ NZ538235A patent/NZ538235A/en not_active IP Right Cessation
-
2006
- 2006-02-14 AU AU2006214871A patent/AU2006214871A1/en not_active Abandoned
- 2006-02-14 WO PCT/NZ2006/000019 patent/WO2006088381A1/en active Application Filing
- 2006-02-14 EP EP06716796A patent/EP1851327A4/en not_active Withdrawn
- 2006-02-14 CA CA002597591A patent/CA2597591A1/en not_active Abandoned
- 2006-02-14 US US11/884,410 patent/US20080261263A1/en not_active Abandoned
Non-Patent Citations (6)
Title |
---|
ALLARDYCE R.A. ET AL.: "Detection of volatile metabolites produced by bacterial growth in blood culture media by selected ion flow tube mass spectrometry (SIFT-MS)", JOURNAL OF MICROBIOLOGICAL METHODS, vol. 65, 2006, pages 361 - 365, XP005387703 * |
CARROLL W. ET AL.: "Detection of volatile compounds emitted by Pseudomonas aeruginosa using selected ion flow tube mass spectrometry", PEDIATRIC PULMONOLOGY, vol. 39, May 2005 (2005-05-01), pages 452 - 456, XP008120928 * |
SCOTTER J.M. ET AL.: "Real-time detection of common microbial volatile organic compounds from medically important fungi by selected ion flow tube mass spectrometry (SIFT-MS)", JOURNAL OF MICROBIOLOGICAL METHODS, vol. 63, November 2005 (2005-11-01), pages 127 - 134, XP005113147 * |
See also references of EP1851327A4 * |
SMITH D. ET AL.: "Selected ion flow mass spectrometry of urine headspace", RAPID COMMUNICATIONS IN MASS SPECTROMETRY, vol. 13, 1999, pages 724 - 729, XP008121086 * |
WANG T., SMITH D., SPANEL P.: "Selected ion flow tube, SIFT, studies of the reactions of H3O+, NO+ and O2+ with compounds released by Pseudomonas and related bacteria", INTERNATIONAL JOURNAL OF MASS SPECTROMETRY, vol. 233, 2004, pages 245 - 251, XP004505185 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2013529458A (en) * | 2010-06-11 | 2013-07-22 | ブルーカー ダルトニック ゲーエムベーハー | Mass spectrometric measurement of β-lactamase resistance |
JP2016039814A (en) * | 2010-06-11 | 2016-03-24 | ブルーカー ダルトニック ゲーエムベーハーBruker Daltonik GmbH | MASS SPECTROMETRIC MEASUREMENT OF β-LACTAMASE RESISTANCES |
Also Published As
Publication number | Publication date |
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CA2597591A1 (en) | 2006-08-24 |
EP1851327A4 (en) | 2009-01-14 |
NZ538235A (en) | 2008-07-31 |
US20080261263A1 (en) | 2008-10-23 |
EP1851327A1 (en) | 2007-11-07 |
AU2006214871A1 (en) | 2006-08-24 |
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