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WO2006079846A1 - Procédé de détection et d’identification des bactéries - Google Patents

Procédé de détection et d’identification des bactéries Download PDF

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Publication number
WO2006079846A1
WO2006079846A1 PCT/GB2006/000338 GB2006000338W WO2006079846A1 WO 2006079846 A1 WO2006079846 A1 WO 2006079846A1 GB 2006000338 W GB2006000338 W GB 2006000338W WO 2006079846 A1 WO2006079846 A1 WO 2006079846A1
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WO
WIPO (PCT)
Prior art keywords
bacteria
volatile
test sample
products
bacterial
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB2006/000338
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English (en)
Inventor
Gino Francesco
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GRAF INTERNATIONAL Ltd
Original Assignee
GRAF INTERNATIONAL Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB0501725A external-priority patent/GB0501725D0/en
Priority claimed from GB0511461A external-priority patent/GB0511461D0/en
Application filed by GRAF INTERNATIONAL Ltd filed Critical GRAF INTERNATIONAL Ltd
Priority to GB0716960A priority Critical patent/GB2438139A/en
Publication of WO2006079846A1 publication Critical patent/WO2006079846A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/497—Physical analysis of biological material of gaseous biological material, e.g. breath

Definitions

  • This invention relates to a method of detecting and identifying bacteria using gas chromatography. More particularly, the invention relates to the use of a gas chromatographic system employing a SAW detector for detecting and identifying bacteria.
  • Gas chromatography has also been used to identify bacteria through the detection of volatile short chain fatty acid methyl esters (FAME) formed by methylating characteristic fatty acids produced by the bacteria.
  • FAME volatile short chain fatty acid methyl esters
  • M. Sasser Technical Note #101 February 2001, Midi Inc., Newark, Delaware, US
  • more than 300 fatty acids and related compounds have been found in bacteria.
  • detectors are used to detect the separated chemical substances emerging from the gas chromatography column and these include flame ionisation (FID) detectors, thermal conductivity (TCD) detectors, electron capture (ECD) detectors, nitrogen-phosphorus detectors, flame photometric (FPD) detectors, photo-ionisation (PID) detectors and Hall electrolytic conductivity detectors.
  • FID flame ionisation
  • TCD thermal conductivity
  • ECD electron capture
  • FPD flame photometric
  • PID photo-ionisation
  • Hall electrolytic conductivity detectors Hall electrolytic conductivity detectors.
  • FPD flame ionisation detector
  • the effluent gas from the column is mixed with a combustible mixture of air and hydrogen and ignited, thereby pyrolysing any organic compounds present in the gas stream.
  • a large electric potential is applied at the tip of the burner and a collector electrode is positioned downstream of the burner tip, and any current resulting from pyrolysis of the organic compounds is measured.
  • a phenyl methyl silicone fused silica capillary column is used in conjunction with a flame ionization detector to detect the methyl esters of short chain fatty acids produced by the bacteria.
  • the biological samples suspected of containing the bacteria must first be subjected to a number of time-consuming procedures. Firstly, the biological sample is cultured on a standard growth medium and then a sample of about 40 mg of bacterial cells is harvested from the culture and subjected to saponification with sodium hydroxide in order to release fatty acids from lipids present in the sample. The free fatty acids are then derivatised by methylation in order to increase their volatility.
  • the invention provides a method of detecting and identifying bacteria, which method comprises: (a) taking a test sample suspected of containing the bacteria; (b) either (i) collecting volatile bacterial products directly from the test sample, or (ii) culturing the test sample in a bacterial growth medium for a period of no longer than 2 hours and then collecting volatile bacterial or products from the cultured test sample; (c) subjecting the volatile bacterial products to gas chromatography using a gas chromatography system employing a surface acoustic wave detector;
  • the invention provides a method of detecting and identifying bacteria, which method comprises collecting volatile bacterial secretion products from a test sample suspected of containing the bacteria without first culturing the bacteria, subjecting the secretion products to gas liquid chromatography using a gas chromatography system employing a surface acoustic wave detector; establishing a chromatographic profile for the test sample, interrogating a library of stored chromatographic profiles of volatile bacterial secretion products of individual species and/or strains of bacteria, comparing the chromatographic profile of the test sample with stored chromatographic profiles in the library and identifying the bacteria.
  • the invention also provides a method for the of diagnosis of a bacterial infection in a patient, the method comprising talcing a biological sample from the patient, placing the sample in a container so that there is a headspace above the sample in the container, collecting volatile bacterial products (e.g. volatile bacterial secretion products) from the headspace and subjecting them to gas chromatography using a gas chromatography system employing a surface acoustic wave detector, establishing a chromatographic profile for the test sample, interrogating a library of stored chromatographic profiles of volatile bacterial products (e.g. volatile bacterial secretion products) of individual species and/or strains of bacteria, comparing the chromatographic profile of the test sample with stored chromatographic profiles in the library and identifying the bacterial species responsible for the infection.
  • volatile bacterial products e.g. volatile bacterial secretion products
  • the method of the present invention differs from known methods of identifying bacteria using gas chromatography in several respects.
  • the test sample is tested without first employing a lengthy culturing stage for growing bacteria in the test sample.
  • a test sample is either tested directly or is cultured in a bacterial growth medium for only a short period of time (i.e. 2 hours or less) in order to bring the bacteria into an active state. More typically, the test sample is cultured for less than 1 hour, or less than 45 minutes, or less than 30 minutes, e.g. 20 minutes or less.
  • the chemical transformation steps used in the prior art methods are not required in the method of the invention.
  • the test samples need not be subjected to a saponification step to release free fatty acids.
  • the method of the invention detects and uses in the identification method volatile substances that occur naturally in the bacteria or are released upon breakdown and destruction (e.g. pyrolysis) of the bacteria rather than substances that have been synthetically modified (e.g. derivatised), for example by esterification or methylation, to increase their volatility.
  • S AW detectors comprise a piezoelectric substrate formed from a material such as quartz or lithium tantalate to which electrodes are attached. A surface acoustic wave of a known frequency is created on the piezoelectric substrate. When an analyte from the gas chromatography column contacts the surface of the piezoelectric material, it alters one or more properties of the surface acoustic wave (e.g. the frequency) and the change in properties is detected by the electrodes, producing an electrical signal.
  • the piezoelectric substrate is coated with a polymer or other chemical having selective affinity for a particular analyte. Such devices have been used as electronic noses to detect specific substances or groups of substances.
  • the piezoelectric material is uncoated and is not intended to demonstrate specificity for any particular analyte.
  • SAW detectors Examples of SAW detectors and a detailed explanation of the construction and functioning of such detectors may be found in US patent number 5,289,715 and International patent application WO 97/35174, each of which is incorporated herein by reference in its entirety. The uses of SAW detectors are also discussed in the following articles:
  • SAW detectors for use in the method of the invention are those described in US patent number 5,289,715 and International patent application WO 97/35174.
  • gas chromatographs for use in the method of the invention are the "eNose” or “zNose” GC/SAW models available from Graf International of Tenterden, Kent, UK or from Electronic Sensor Technology of Newbury Park, California, US.
  • the method of the present invention may be used to detect and identify bacteria in a wide range of substrates including: • biological samples such as whole blood, plasma, serum, sputum, saliva, breath samples, sweat, semen, urine, interstitial fluid, faecal samples, cerebrospinal fluid, dialysate obtained in kidney dialysis, tears, mucus and amniotic fluid; • environmental samples such as soil, river water, sewage, drinking water, swimming pool water, swabs from surfaces in hospitals and other public buildings, samples from air filters, dust samples, samples from air- conditioning and ventilation systems, samples from restaurants and kitchens; and
  • the samples are analysed whilst they are still fresh.
  • the samples are not frozen before they are analysed.
  • the samples are held in a container (e.g. a sealed container), a headspace being left above the sample in which volatile products of the bacteria can collect.
  • the samples may be warmed or heated to a defined temperature to facilitate volatilization of volatile bacterial products and may be allowed to equilibrate at a particular temperature before analysis.
  • the gases and volatile components in the headspace above the sample are drawn off (e.g. by a pump) and are either concentrated and then injected into the gas chromatograph, or injected directly into the gas chromato graph.
  • a drying trap may be positioned between the sample container and the gas chroniatograph.
  • the headspace vapours collected from the test samples may be subjected to a pre-concentration stage, for example as described in International patent application number WO 97/35174 (Electronic Sensor Technology).
  • the volatile bacterial products can be collected from the head space of the container and injected directly into a gas chromatograph or they may be collected and preferably concentrated by a storage medium for later analysis by gas chromatography.
  • the volatile products can be withdrawn from the headspace of the container and adsorbed onto a temporary storage medium (pre- concentrator medium) such as Tenax tm TA or Tenax tm GC or another porous polymer resin such as a resin based on 2,6-diphenylene oxide.
  • pre-concentrator medium is contained within a sealable container which can then be connected to the gas chromatograph for desorption and analysis of the volatile bacterial products.
  • the pre-concentrator medium can take the form of a tube containing an adsorbent such as Tenax.
  • the tube typically has means at either end thereof to retain the adsorbent in the tube whilst allowing a gas or vapour sample to be sucked into the tube.
  • the tube can have a filter at either end thereof, the filter having a mesh size that allows vapours and gases to pass into the tube but retains the adsorbent in the tube.
  • One pre-concentrator of particular usefulness in the method of the present invention is the Model 3300 Remote Sampler Desorber available from Electronic Snesor Technology of Newbury Park, California, USA. This concentrator contains 100 mg of TenexTM adsorbent.
  • bacteria are identified by the characteristic profiles of the volatile bacterial products that they produce.
  • the volatile products can be products that are secreted by the bacteria or products that are produced by breakdown or destruction of the bacterial cell or the bacterial cell wall.
  • the volatile products may be substances that are released or produced upon thermal degradation (e.g. pyrolysis) of the bacteria.
  • the volatile products are substances that are either naturally present in or produced by the bacteria, or are formed during breakdown of the bacteria, but which are not synthetic chemical derivatives formed by reacting the bacterial components with one or more chemical reagents.
  • libraries of gas chromatographic profiles of each bacterial strain of interest can be built up by culturing the bacteria by methods well known to those skilled in the art of microbiology, introducing a sample from the culture into a sealed container, drawing off a sample of vapour from a headspace within the container; injecting the sample into the gas chromatograph, and recording the retention times of each component of the sample.
  • a comparison can be made with control chromatograms taken by sampling the nutrient media used to culture the bacteria and the GC peaks associated with the bacteria identified. Chromatograms can be recorded for a given bacterial species or strain when cultured under different conditions to identify those peaks in the chromatogram that remain constant.
  • a selection of GC peaks that are not "culture sensitive" or "culture specific” may then be selected to provide a characteristic profile for the bacterial species or strain in question.
  • a sample e.g. dust or a biological sample containing bacteria
  • a heater for example an electrically powered hotplate
  • the heater e.g. hotplate
  • the volatile components are withdrawn from the container (e.g. by suction) for analysis by the GC method of the invention.
  • a test sample is introduced into a pyrolysis unit arranged in-line with the gas chromatograph so that the pyrolysis products are carried directly into the gas chromatograph.
  • a communications link allowing transfer of information between each remote location and the databank; whereby data defining a chromatographic profile for a test sample generated by a gas liquid chromatography apparatus at a remote location can be transmitted along the Communications link to the databank and compared with a library of stored chromatographic profiles of volatile bacterial products of individual species and/or strains of bacteria in the databank thereby to identify the bacteria, and wherein information regarding the identity of the bacteria can be transmitted along the communications link to the remote location.
  • the method of the invention may be used to detect and identify each bacterial species or strain in a particular sample or it may be used to screen for a predetermined number of bacteria of relevance to the context in which it is used.
  • means e.g. software
  • the defined group of chromatographic profiles could be, for example, the profiles of pathogenic bacteria associated with common bacterial infections and diseases, and could consist of, for example, up to fifty profile, e.g. up to forty profiles, and more particularly up to thirty five chromatographic profiles.
  • the volatile components of the blood sample are separated and exit the column at different times whereupon they are detected by the surface acoustic wave (SAW) detector 16, which measures the concentration of each component.
  • SAW detector comprises a piezoelectric crystal with an electrode on one end that generates 500-megahertz ultrasound waves on the surface of the crystal. An electrode on the other end picks up these waves.
  • the separated volatile components from the chromatography column impinge upon and are adsorbed by the surface of the detector crystal, causing a small change in the frequency of the surface acoustic wave and hence a small change in the tone arriving at the detector electrode. The difference in tone indicates how much of the volatile component is present.
  • both the concentration of each volatile component and the retention time of the component are measured and are recorded in the form of a chromatogram.
  • the chromatogram is then compared with a library of chromatograms for various bacterial species. Where a significant number of the key characteristic chromatographic peaks in a bacterial standard are found in the chromatograph of the blood sample, then it can be inferred that the bacterial species in question is present in the blood sample.
  • the “SlickStick” (available from Electronic Sensor Technology of Newbury Park, California, US) is a glass or metal tube of approximately 115 mm in length and 6.5 mm in diameter containing an adsorbent such as the "Tenex” adsorbent described above. The ends of the tube are closed by a filter which retains the "Tenax" within the tube but allows gases and vapours to pass through. A cap may be used to prevent movement of gases or vapours in or out of the tube after sampling.
  • “SlickStick” is connected to a mobile sample unit (available from Electronic Sensor Technology) that comprises a pump that can be used to suck a defined volume of gas (e.g. about 30 ml) through the "SlickStick". Volatile chemicals from the bacteria in the filter are therefore drawn into the tube and are adsorbed on the "Tenax" adsorbent. The tube may then be sealed with a cap and transferred to a location where there is a gas chromatograph of the type described in relation to Figure 1.
  • a mobile sample unit available from Electronic Sensor Technology
  • a pump that can be used to suck a defined volume of gas (e.g. about 30 ml) through the "SlickStick”. Volatile chemicals from the bacteria in the filter are therefore drawn into the tube and are adsorbed on the "Tenax" adsorbent.
  • the tube may then be sealed with a cap and transferred to a location where there is a gas chromatograph of the type described in relation to Figure 1.
  • the method of the invention may be used for the rapid analysis and detection of M. tuberculosis strains.
  • a sample of sputum from a person suspected of being infected with M. tuberculosis is sealed into a container and the headspace above the sputum is subjected to analysis using the GC apparatus described above.
  • the resulting chromatograni is then compared with a library of chromatograms for various strains of M. tuberculosis and the presence or absence of a pathogenic strain is confirmed.
  • the sample suspected of containing M. tuberculosis can be subjected to pyrolysis in the apparatus shown in Figure 2.
  • a sputum sample can be deposited on the heater plate 206 in the container 202 and the lid 204 replaced to give a closed sealed container.
  • the heater 206 is then switched on to heat the sputum sample rapidly to a high temperature to bring about pyrolysis of the sputum sample and release volatile substances into the headspace above the sample.
  • the volatile substances are then sucked into the "Slickstick" 210 and analysed as described above.
  • MRSA methicillin-resistant Staphylococcus aureus
  • MRSA infections are typically transmitted by contact with a person who has an infection or is colonized with the bacteria.
  • the bacteria can be spread by direct contact of an infected person or carrier with a non-infected person, or by means of an intermediary such as a medical professional or other carer who has touched an infected person and has then come into contact with another patient before washing his or her hands.
  • a small vacuum cleaner device employing an internal bacterial filter
  • samples are taken from flat surfaces such as floors and walls, or from other inanimate objects such as fabrics and furnishings.
  • the bacteria are thus collected within the filter of the vacuum cleaner.
  • the collected bacteria can then be analysed either by direct sampling of the atmosphere within the filter using the apparatus described above in relation to Figure 1 or by collecting vapour samples from the environment with the filter using a temporary storage medium such as a "SlickStick".
  • MRSA typically divide about every 20 minutes

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Abstract

L’invention concerne un procédé de détection et d’identification des bactéries, lequel consiste : (a) à prélever un échantillon de test suspecté de contenir les bactéries ; (b) soit (i) à recueillir des produits bactériens volatiles directement à partir de l’échantillon de test, soit (ii) à mettre en culture l’échantillon de test dans un milieu de croissance bactérienne pour une période ne dépassant pas 2 heures, puis à recueillir des produits bactériens volatiles à partir de l’échantillon de test mis en culture ; (c) à soumettre les produits volatiles à une chromatographie gazeuse à l’aide d’un système de chromatographie gazeuse reposant sur un détecteur d’ondes acoustiques superficielles ; (d) à établir un profil chromatographique pour l’échantillon de test ; (e) à interroger une bibliothèque de profils chromatographiques stockés de produits bactériens volatiles d’espèces individuelles et/ou de souches de bactéries ; et (f) à comparer le profil chromatographique de l’échantillon de test avec des profils chromatographiques stockés dans la bibliothèque et ainsi à identifier les bactéries.
PCT/GB2006/000338 2005-01-31 2006-01-31 Procédé de détection et d’identification des bactéries Ceased WO2006079846A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
GB0716960A GB2438139A (en) 2005-01-31 2006-01-31 A method of detecting and identifying bacteria

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB0501725.6 2005-01-31
GB0501725A GB0501725D0 (en) 2005-01-31 2005-01-31 A method of analysis
GB0511461.6 2005-06-06
GB0511461A GB0511461D0 (en) 2005-06-06 2005-06-06 A method of analysis

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Cited By (16)

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EP1978087A1 (fr) * 2007-04-02 2008-10-08 Consultatie Implementatie Technisch Beheer B.V. Système et procédé de détection de micro-organismes
WO2009054913A1 (fr) * 2007-10-19 2009-04-30 The Charles Stark Draper Laboratory, Inc. Détection rapide de composés organiques volatils pour identifier des bactéries dans un échantillon
WO2012143901A1 (fr) 2011-04-21 2012-10-26 North-West University Procédé pour permettre la distinction entre différents pathogènes
US8518663B2 (en) 2009-04-27 2013-08-27 The Charles Stark Draper Laboratory, Inc. Rapid detection of volatile organic compounds for identification of Mycobacterium tuberculosis in a sample
US20150099694A1 (en) * 2012-04-27 2015-04-09 Specific Technologies Llc Identification and susceptibility of microorganisms by species and strain
US20160002696A1 (en) * 2013-02-20 2016-01-07 Alifax Holding Spa Method to identify bacterial species by means of gas chromatography/mass spectrometry in biological samples
ITUB20155975A1 (it) * 2015-11-27 2017-05-27 Alifax Srl Procedimento per la rilevazione di attivita' batterica in un campione biologico e relativa unita' di rilevazione
CN110763785A (zh) * 2019-11-13 2020-02-07 中国科学院声学研究所 一种毒品测定方法
CN110785661A (zh) * 2017-04-21 2020-02-11 英索特有限公司 用于检测含油果实、种子和坚果中的酸败的方法
CN112180004A (zh) * 2020-08-31 2021-01-05 四川省中医药科学院 一种利用声表面波气相色谱仪现场鉴别高挥发性中药材的方法
CN112180002A (zh) * 2020-08-31 2021-01-05 四川省中医药科学院 一种利用声表面波气相色谱仪现场鉴别低挥发性中药材的方法
CN113466402A (zh) * 2021-03-16 2021-10-01 伊诺司生技股份有限公司 用于检测妇女疾病的气体检测系统及其检测方法
CN114874895A (zh) * 2022-05-25 2022-08-09 佳木斯大学 一种细菌培养实验系统
CN115308409A (zh) * 2022-08-31 2022-11-08 中国科学院合肥物质科学研究院 一种基于挥发物检测的细菌鉴别装置及方法
WO2023031568A1 (fr) 2021-09-06 2023-03-09 Aryballe Utilisation d'au moins deux précurseurs de composés volatils pour caractériser la susceptibilité des microorganismes contenus dans un échantillon biologique à émettre des composés volatils
EP4477756A1 (fr) * 2023-06-15 2024-12-18 Commissariat à l'Energie Atomique et aux Energies Alternatives Procédé de détection et d'identification de micro-organismes dans un échantillon par fragmentation de l'échantillon

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Cited By (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1978088A3 (fr) * 2007-04-02 2008-12-10 Consultatie Implementatie Technisch Beheer B.V. Procédé et dispositif d'identification d'un processus dans lequel des substances gazeuses sont libérées ou consommées
WO2008120987A3 (fr) * 2007-04-02 2008-12-24 Consultatie Implementatie Tech Système et procédé de détection de micro-organismes
WO2008120986A3 (fr) * 2007-04-02 2009-02-26 Consultatie Implementatie Tech Procédé et dispositif d'identification d'un processus dans lequel sont libérées ou consommées des substances gazeuses
EP1978087A1 (fr) * 2007-04-02 2008-10-08 Consultatie Implementatie Technisch Beheer B.V. Système et procédé de détection de micro-organismes
WO2009054913A1 (fr) * 2007-10-19 2009-04-30 The Charles Stark Draper Laboratory, Inc. Détection rapide de composés organiques volatils pour identifier des bactéries dans un échantillon
WO2009091375A3 (fr) * 2007-10-19 2009-11-05 The Charles Stark Draper Laboratory, Inc. Détection rapide de composés organiques volatils pour l'identification de bactéries dans un échantillon
US8518663B2 (en) 2009-04-27 2013-08-27 The Charles Stark Draper Laboratory, Inc. Rapid detection of volatile organic compounds for identification of Mycobacterium tuberculosis in a sample
WO2012143901A1 (fr) 2011-04-21 2012-10-26 North-West University Procédé pour permettre la distinction entre différents pathogènes
US20150099694A1 (en) * 2012-04-27 2015-04-09 Specific Technologies Llc Identification and susceptibility of microorganisms by species and strain
US9862985B2 (en) * 2012-04-27 2018-01-09 Specific Technologies Llc Identification and susceptibility of microorganisms by species and strain
US20160002696A1 (en) * 2013-02-20 2016-01-07 Alifax Holding Spa Method to identify bacterial species by means of gas chromatography/mass spectrometry in biological samples
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