WO2006079846A1

WO2006079846A1 - A method of detecting and identifying bacteria

Info

Publication number: WO2006079846A1
Application number: PCT/GB2006/000338
Authority: WO
Inventors: Gino Francesco
Original assignee: Graf International Limited
Priority date: 2005-01-31
Filing date: 2006-01-31
Publication date: 2006-08-03
Also published as: GB2438139A; GB0716960D0

Abstract

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 products from the cultured test sample; (c) subjecting the volatile products to gas chromatography using a gas chromatography system employing a surface acoustic wave detector; (d) establishing a chromatographic profile for the test sample; (e) interrogating a library of stored chromatographic profiles of volatile bacterial products of individual species and/or strains of bacteria; and (f) comparing the chromatographic profile of the test sample with stored chromatographic profiles in the library and thereby identifying the bacteria.

Description

A METHOD OF DETECTING AND IDENTIFYING BACTERIA

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.

Background of the Invention

Methods of detecting, identifying and in some cases quantifying bacteria by detecting volatile chemicals secreted by the bacteria have been known for a number of years, and various instrumental techniques have been employed for this purpose. Examples of such techniques include gas chromatography and so-called "electronic noses".

Electronic noses typically comprise an array of separate sensor elements each capable of detecting, with varying degrees of selectivity, particular volatile test substances. In the case of bacteria, detection and identification of a particular bacterial species relies upon the detection of particular volatile chemicals secreted by the bacteria and which are characteristic for the bacterial species of interest. Bacteria secrete a wide range of volatile substances and examples are listed in the article by Gibson et al, Sensors and Actuators, B 44 (1997) 413-422) which describes the use of an electronic nose for the detection and identification of microrganisms. The use of an electronic nose to detect and identify bacteria or to detect and quantify the volatile products of bacteria is also described in Holmberg et al, Biotechnology Techniques, Vol. 12, No. 4, April 1998, 319-324, Brandgard et al, Biotechnology Letter, 23: 241-248, 2001, Schiffman et al, Seventh International Symposium on Olfaction and Electronic Noses, July 2000, Section 4: Medical/Microbial 173-179, and Stetter et al, Electrochemical Society Proceedings Volume 2— 1-15, 54-61.

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. According to 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. By identifying the fatty acids through detection of their volatile methyl esters and comparing the fatty acid profile of an unknown bacterial species with a database of bacterial profiles, the unknown species can be identified.

A method for identifying bacteria by GC detection of FAME compounds is also described in The Manual of Analytical Methods - Fourth Edition, produced by the US National Institute for Occupational Safety and Health (NIOSH) - see NIOSH Method 0801 - Aerobic Bacteria by GC-FAME - 15 January 1998). Method 0801, in common with the Sasser method described above, requires a methylation step in order to transform the fatty acids into the more volatile fatty acid methyl esters.

Gas chromatography is a long established and well known technique in which test substances are volatilised in a stream of an inert carrier gas (such as nitrogen, helium or argon) and are then separated as they pass through a column containing a liquid stationary phase. Separation of the components of the test substance usually takes place on the basis of molecular weight, the lower molecular weight substances passing through the column more quickly than components of higher molecular weight. The separated test substances are then detected by a detector as they emerge from the column, and the time taken for a given substance to pass through the column (the retention time) is recorded and matched against the retention times of known substances. In this way, the identity of each separated test substance can be identified.

There are two general types of gas chromatography column, namely packed columns and capillary columns. Packed columns contain a finely divided inert solid support material (such as diatomaceous earth) coated with a liquid stationary phase which is typically a high boiling liquid such as a silicone (e.g. methyl silicone) or a polyether such as a polyethylene glycol. Packed columns are usually between about 1.5 and 10 metres in length and typically have an internal diameter of 2 to 4 millimetres. Capillary columns, which are much smaller and have an internal diameter of a few tenths of a millimetre, can conveniently be divided into two types, namely wall- coated open tubular (WCOT) columns or support-coated open tubular (SCOT) columns. SCOT columns consist of a capillary tube in which the inner wall of the capillary is lined with a thin layer of support material such as diatomaceous earth onto which the liquid phase has been adsorbed. The more efficient WCOT columns consist of a capillary tube having walls that are directly coated by the liquid stationary phase. In one sub-type of WCOT column (the fused silica open tubular (FSOT) column, a fused silica capillary tube is enclosed within a polyimide coating and a liquid stationary phase is chemically bonded to the inner wall of the fused silica tube. The FSOT columns have much thinner walls than glass capillary columns and the additional strength provided by the polyimide layer gives flexibility to the column so that they can be wound into coils.

Many different types of detector 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. In a flame ionization detector, the most commonly used type of 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.

In the system described in the Sasser article referred to above, 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. However, before the chromatographic analysis is undertaken, 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. Following methylation, the sample is subjected to solvent extraction and washing with sodium hydroxide solution before the resulting organic solvent extract is ready for analysis by gas chromatography. NIOSH Method 0801 described above similarly requires successive culturing, saponification and methylation steps. It will be appreciated that the foregoing procedures are complex, relatively expensive, time-consuming and require laboratory facilities. As such, they are not suitable for use in situations where rapid detection and identification of bacteria is required or in situations where no laboratory facilities are available.

Thus, at present, there remains a need for a method that enables the rapid detection and identification of bacteria directly from test samples without the need for lengthy culturing of the bacteria and/or chemical treatment of the bacteria as described above.

Summary of the Invention

The present invention provides an improved method of detecting and identifying bacteria by direct analysis of a biological sample to detect volatile bacterial products or components such as fatty acids using gas chromatography, rather than by analysis of synthetic chemical derivatives of bacterial secretion products.

Accordingly, in one aspect, 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;

(d) establishing a chromatographic profile for the test sample;

(e) interrogating a library of stored chromatographic profiles of volatile bacterial products of individual species and/or strains of bacteria; and

(f) comparing the chromatographic profile of the test sample with stored chromatographic profiles in the library and thereby identifying the bacteria.

In another aspect, 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.

The method of the present invention differs from known methods of identifying bacteria using gas chromatography in several respects. Firstly, in the present method, the test sample is tested without first employing a lengthy culturing stage for growing bacteria in the test sample. Thus, 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.

Secondly, the chemical transformation steps used in the prior art methods are not required in the method of the invention. Thus, the test samples need not be subjected to a saponification step to release free fatty acids. Furthermore, 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.

A further feature of the method of the invention is that the gas chromatography system used to identify the volatile substances makes use of a surface acoustic wave (SAW) detector rather than the flame ionisation detector.

Surface acoustic wave (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. In many prior art devices using a SAW detector, 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. However, in the method of the present invention, the piezoelectric material is uncoated and is not intended to demonstrate specificity for any particular analyte. 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:

G. W. Watson and E.J. Staples, "SAW Resonators as Vapor Sensors," Proceedings of the 1990 Ultrasonics Symposium, ρp.311-314, 90CH2938-9.

G.W. Watson, W. Horton, and E.J. Staples, "GAS Chromatography Utilizing SAW Sensors," Proceedings of the 1991 Ultrasonics Symposium, pp.305-309. E. J. Staples and G.W. Watson, "A GAS Chromatograph Incorporating an

Innovative New Surface Acoustic Wave (SAW) Detector," Pittcon Conference, 1-5 March 1998, New Orleans, Louisiana, Paper 1583CP.

E.J. Staples, T. Matsuda, and S Viswanathan, "Real Time Environmental Screening of Air, Water and Soil Matrices Using a novel Field Portable GC/SAW System," Environmental Strategies for the 21 st Century, Asia Pacific Conference, 8-10 April 1998, Singapore.

Particular 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.

Particular 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

• samples from food manufacturing and processing facilities and drinks manufacturing and processing facilities.

The biological samples can be analysed directly without first culturing the bacteria. Alternatively, in cases where the bacteria may be in a dormant state (e.g. as may be the case with some samples containing Mycobacterium tuberculosis or Clostridium difficile), or where the bacteria are capable of rapid multiplication (e.g. as in the case of MRSA which can undergo a first division in as little as 20 minutes), the sample may first be cultured in a suitable growth medium for a short period of time not exceeding 2 hours. By culturing the bacteria for a short period, they are induced into or maintained in an active state in which detection of the volatile products is rendered easier. In contrast to the conventional methods described in the introductory portion of this application, prolonged culturing of the bacteria- containing samples over 12 to 24 hours is not required since the method of the invention is sufficiently sensitive that it does not require substantial amplification of bacterial numbers in order for detection and identification to be possible.

Preferably the samples are analysed whilst they are still fresh. Preferably, also, the samples are not frozen before they are analysed. Typically 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. In order to avoid the swamping of signals by water vapour, a drying trap may be positioned between the sample container and the gas chroniatograph.

Prior to injection into the gas chromatograph, 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. For example, 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. The 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. In one embodiment, 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.

A pre-concentrator medium of this type can also be used to take breath samples. Thus, for example, a tube containing a pre-concentrator medium can be incorporated into a collection bag into which the patient breathes. Any volatile chemicals of bacterial origin can then be desorbed subsequently for analysis by GC.

An advantage of using a pre-concentrator medium is that sample vapours can be collected by drawing them through the medium over prolonged periods (e.g. up to 90 seconds in the case of the "Slickstick" described below), thereby facilitating concentration of the volatile components of the vapour in the medium and enabling the collection of larger quantities of volatile substances than is possible using more conventional collection methods where vapour is sampled for a much shorter period, e.g. several seconds. By using a pre-concentrator medium, the concentration of volatile bacterial products collected can be increased by up to ten times the concentrations collected using conventional methods, with the result that the sensitivity of the test method is greatly enhanced. The combination of the use of the pre-concentrator medium and the more sensitive surface acoustic wave detector means that volatile substances can be detected at concentrations down to as low as 1 part in 10¹².

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 Tenex™ adsorbent.

A suction device such as a vacuum cleaner may be used to collect bacteria from surfaces. Bacteria and other particulate matter can be collected in a collection container (e.g. a bag) in the vacuum cleaner and then emptied (e.g. by shaking) into a suitable container. The container can then be heated to release volatile components from the bacteria, and the head space in the container sampled in the normal manner. Small portable vacuum cleaners suitable for collecting bacteria are well known. Such vacuum cleaners typically contain filters of the High Efficiency Particulate Air (HEPA) filter type that will collect bacteria and particulate matter, but will allow gases to pass therethrough.

According to the invention, 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. For example, the volatile products may be substances that are released or produced upon thermal degradation (e.g. pyrolysis) of the bacteria. As stated above, 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.

The volatile products (e.g. secretion products) can be, for example, any one or more products selected from volatile fatty acids, esters, sulphur compounds, ketones, alcohols, amines, carboxylic acids, hydrocarbons such as alkenes, alkanes and aromatic compounds and compounds containing combinations of different functional groups such as the mycolic acids produced by Mycobacterium tuberculosis. Libraries can be established by talcing individual species and/or strains of bacteria and using the gas chromatography system of the invention to produce secretion profiles for each species or strain. More particularly, 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.

Examples of bacterial species that may be detected and identified by the method of the invention include Streptococcus pneumonia, Haemophilus influenza, Shigella flexneri, Staphylococcus aureus (e.g. methicillin resistant Staphylococcus aureus (MRSA) and in particular the strains methicillin-resistant coagulase-negative staphylococci and methicillin-sensitive Staphylococcus aureus), Pseudomonas aeruginosa, Salmonella enter idis, Enter ococcus faecάlis, Klebsiella pneumonia, Mycobacterium tuberculosis, Escherichia coli, Clostridium difficile and Salmonella typhimurium. Further bacterial species that may be detected include other mycobacterial species such as Mycobacterium bovis.

The bacterial samples taken may be subjected to pyrolysis to release volatile substances from the bacterial cells and also to kill the bacteria. Pyrolysis is particularly preferred when the bacteria are pathogenic bacteria such as MRSA and Mycobacterium tuberculosis. Pyrolysis may also be particularly useful with spore- forming bacteria such as Mycobacterium tuberculosis, Bacillus anthracis (the bacterium responsible for anthrax) and Clostridium difficile.

Pyrolysis is typically carried out by contacting the bacteria with a heater (e.g. a hotplate or heater block), or heating the enclosure in which the bacteria are contained. Heating is preferably carried out rapidly (flash-heating) so as to bring the heater to a very high temperature over a very short period, e.g. a few milliseconds, thereby vapourising any bacteria in contact with the heater. The pyrolysis products are then drawn off and subjected to GC analysis.

In one embodiment, bacteria are introduced into a pyrolysis chamber and are contacted with a metal plate with an electrical heating element attached. The metal plate is rapidly heated to a temperature which effectively fries the bacteria causing them to vaporise in a matter of a few milli-seconds. This rapid form of heating serves to localise the heat on one surface within the pyrolysis chamber allowing subsequent heat dissipation to the other colder surfaces of the chamber.

In another embodiment, a sample (e.g. dust or a biological sample containing bacteria) is introduced into a sealed container in which there is a heater (for example an electrically powered hotplate), the heater (e.g. hotplate) is activated to flash-heat the interior of the container to destroy any bacteria present and to vaporise characteristic volatile components of the bacterial cell or cell wall, and the volatile components are withdrawn from the container (e.g. by suction) for analysis by the GC method of the invention. In a further embodiment, 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.

The method of the invention may be performed at a single location or over several locations. For example, samples may be collected using a temporary storage medium as described above and the samples from a number of locations taken to a gas chromatograph at a central point. The comparison of the chromatogram of the test sample with a library of chromatograms may be carried out at the same location as the gas chromatograph or at a different (e.g. remote) location. Where the comparison is carried out at the same location, a computer or other processor can be United to the gas chromatograph, the computer or other processor containing a library of stored GC profiles and software enabling comparison of the test sample profile and the library profiles. Alternatively, a databank of stored GC profiles may be provided at a remote location and the comparison of the test sample profile with a library of stored profiles carried out by exchange of data along a communications link such as a telecommunications link.

In another embodiment of the invention, a gas chromatograph may be located in a general doctor's surgery so that samples can be taken from patients visiting the surgery, or taken by a doctor visiting patients at home or at other locations and then brought back to the surgery, and then analysed using the gas chromatograph. The resulting GC profile can then be transmitted along a communications link to a central databank for comparison with a library of stored GC profiles and identification of the bacteria. The identity of the bacteria can then be transmitted back along the communications link to the general practitioner's surgery. In addition to transmitting the identity of the bacteria, the databank may contain a library of antimicrobial products linked to the stored GC profiles so that a prescription for a particular antimicrobial drug (s) can be transmitted to the GP 's surgery along with the diagnosis and identification of the bacteria. In a further refinement of this approach, the sample for the patient may be screened not only for bacterial species and strains responsible for symptoms displayed by the patient but also for any other infections or illnesses where the symptoms may be masked or not otherwise apparent. A major advantage of the approach set out above is that it removes from the General Practitioner the burden of identifying the cause of the symptoms displayed by a patient suffering from a bacterial infection.

Accordingly, in another aspect, the invention provides a method of detecting and identifying bacteria, which method comprises collecting volatile bacterial products (e.g. volatile bacterial secretion products) from a test sample suspected of containing the bacteria, subjecting the volatile bacterial products to gas liquid chromatography using a gas liquid chromatography system employing a surface acoustic wave detector; establishing a chromatographic profile for the test sample, sending data defining the chromatographic profile for the test sample along a communications link to a database contained within a computer or other processor at a remote location, interrogating a library of stored chromatographic profiles of volatile bacterial products of individual species and/or strains of bacteria in the database at the remote location, comparing the chromatographic profile of the test sample with stored chromatographic profiles in the library and identifying the bacteria.

In a further aspect, the invention provides a distributed system for detecting and identifying bacterial infections in patients in a plurality of remote locations, the distributed system comprising:

(i) a plurality of gas liquid chromatography apparatuses at different remote locations (e.g. wherein each of the gas chromatography apparatuses comprises a surface acoustic wave detector);

(ii) a central databank, the databank containing a library of stored chromatographic profiles of volatile bacterial products (e.g. bacterial secretion products) of individual species and/or strains of bacteria;

(iii) 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. For example, means (e.g. software) may be employed so that the chromatographic profile of a sample is compared against a defined group of chromatographic profiles in a library. 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 chromatograph, or a computer or other data processing means linked to the chromatograph, may be programmed to activate an alarm when a particular bacterial species or strain within the defined group is detected and identified. Such an alarm could be audible or visual or a combination of the two.

The invention will now be illustrated in more detail, but not limited, by reference to the embodiments shown in the drawings and described in the specific examples.

Brief Description of the Drawings

Figure 1 is a schematic illustration of a gas chromatography system suitable for use in the method of the invention.

Figure 2 is a side sectional view of a pyrolysis container according to a further embodiment of the invention.

Detailed Description of the Preferred Embodiments As shown in Figure 1, a gas chromatographic (GC) system comprises an inlet 2 and sampling pump 4 leading to a thermally controlled rotary valve 8. A water trap (not shown) containing a Nafion® dryer is connected to the inlet 2 to remove water vapour. Rotary valve 8 is connected to a loop 10 containing a Tenax^tm filled trap, a source 12 of carrier gas (e.g. helium), and a capillary column 14. Downstream of the capillary column 14 is a SAW sensor 16.

By way of example, the gas chromatograph (GC) may be a model 7100 bench top GC/SAW electronic nose as described above. The GC in this embodiment has a fused quartz DB-624 column containing a 6% cyanopropyl-phenyl 94% dimethyl polysiloxane stationary phase available from Agilent Technologies.

A biological sample (for example whole blood) is taken from a patient suspected of having a bacterial infection and is stored in a container sealed by a septum. The container is warmed for a defined period of time to allow volatile components of the test sample to escape into the headspace of the container. A sampling needle connected to the inlet 2 is then inserted through the septum in the container and the gases in the headspace are drawn into the inlet and pumped through the Tenax filled trap 10 for a pre-selected time. The volatile components of the blood sample are thereby adsorbed onto the Tenax and hence are concentrated. The volatile components are subsequently desorbed by electro-capacitative heating and are directed via the rotary valve 8 to the capillary column 14.

As they pass though the capillary column, 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. The 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. Thus 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 advantage of the analytical method of the invention is that it enables bacteria in a wide variety of sample to be detected and analysed very rapidly without the need to grow up bacterial cultures from the samples and without the need for the chemical modifications such as saponification and methylation required by some prior art methods.

Prior to GC analysis, a test sample may be subjected to pyrolysis, which serves the twin purposes of releasing characteristic volatile substances as the bacterial cell is broken down and vapourised and also killing the bacteria and thereby avoiding the problem of disposing of the sample subsequent to the test. An apparatus for collecting and pyrolysing bacterial test samples is shown in Figure 2.

A portable vacuum cleaner containing a HEPA filter is first used to remove dust or other samples containing bacteria from a surface. The filter is then emptied out into the container 202 shown in Figure 2, and the lid 204 fitted to provide a sealed container. The container 202 has an electrically operated heater 206 mounted on its base. The container 202 has a port 208 in its side wall in which is mounted a "Slick Stick" pre-concentrator 210.

Once a sample of dust or a particulate matter containing bacteria has been collected, and the container sealed, the contents of the container are subjected to pyrolysis by flash-heating. Thus the heater is rapidly (e.g. over a period of a few milliseconds) heated up to a suitable temperature to vapourise and destroy the bacteria and release characteristic volatile substances into the head space of the container. The volatile substances are then sucked through the "Slick Stick" by means of a pump (not shown) so that they are adsorbed on to the adsorbent in the "Slick Stick". After heating for a sufficiently long time to kill the bacteria and ensure that all of the volatile materials have been collected, the "Slick Stick" is removed and subjected to desorption-gas chromatography as described below.

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. The

"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. The "SlickStick" is then connected to the gas chromatograph inlet by means of a luer connector and the adsorbed volatile chemicals are desorbed by heating the tube to about 200 ⁰C using a heater surrounding the "SlickStick" and drawing the desorbed volatile chemicals into the into the gas chromatograph by suction where they are analysed in the usual manner.

EXAMPLE 1

Detection of Mycobacterium tuberculosis Infection

The warning signs indicating that a patient is suffering from active tuberculosis include coughing, and confirmation of the nature of the disease is typically achieved by taking a sample of sputum and analysing the sample for the presence of the causative bacterium M. tuberculosis. At present, detection and identification of the offending bacterial species can be carried out in several ways, for example by culturing the sample and identifying the bacteria in the culture by classical methods, or by means of a procedure making use of the polymerase chain reaction (PCR) to amplify and identify characteristic bacterial DNA in the sample.

The method of the invention may be used for the rapid analysis and detection of M. tuberculosis strains. Thus, 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.

In identifying M. tuberculosis, the GC profile may be formed from short chain fatty acids and/or the higher molecular weight mycolic acids and/or other volatile compounds. An advantage of the present invention is that analysis for mycolic acids is carried out without the need to subject the sample to heat treatment to bring about thermal cleavage of the mycolic acids from the bacterial cell wall. If necessary, however, a heating step can be employed to increase the detectable levels of mycolic acids and/or other volatile compounds.

In one embodiment of the invention, the sample suspected of containing M. tuberculosis can be subjected to pyrolysis in the apparatus shown in Figure 2. Thus, 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.

An advantage of this method is that the pathogenic bacteria are killed during the test and hence there are no residual dangerous biological samples that require disposal.

The pyrolysis method is also advantageous in the case of bacteria, and in particular spore-forming bacteria, that can exist in a dormant phase, where active secretion of characteristic volatile substances may not be taking place. EXAMPLE 2

Detection of Methicillin Resistant Staphylococcus aureus (^"MRSA) Infection

Staphylococcus aureus is a species of bacteria commonly carried on the skin or in the throats or noses of many people. Staphylococcus aureus can cause relatively minor infections (such as pimples and boils) that can be treated without antibiotics in many cases but can also cause more serious infections (such as surgical wound infections and pneumonia). In the past, serious Staphylococcus aureus infections have been treated with penicillin-related antibiotics but, over the past few decades, the treatment of Staphylococcus aureus infections has become more difficult because of the widespread development of bacterial resistance to antibiotics, including the commonly used penicillin-related antibiotics. The strains of Staphylococcus aureus that are resistant to penicillin related antibiotics such as methicillin are referred to generically as methicillin-resistant Staphylococcus aureus, or MRSA. There are many different strains of MRSA, with differing degrees of immunity to the effects of various antibiotics, and the spread of MRSA, particularly in hospitals, has become a severe problem. 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.

One of the difficulties in combating MRSA it that the bacteria can survive for prolonged periods not only on biological substrates but also on inanimate objects or surfaces such as walls, floors and other surfaces, sinks, linen and bedding, furnishings, clothes and even implements such as mops that are used for cleaning. Eradicating harmful strains of the bacteria a given environment is therefore difficult.

The method of the present invention may be used to detect MRSA.

Detection of MRSA on Non-Biological Surfaces Using 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".

The resulting chromatogram is compared with a library containing chromatograms of various MRSA strains and any MRSA strains in the samples are therefore identified.

The advantage of using the "Slick Stick" method is that samples can be taken rapidly from a large number of locations (e.g. within a hospital) with a separate "Slick Stick" being used to collect a sample at each location. The samples can then be analysed rapidly using the gas chromatographic method of the invention to build up a picture of the extent of the problem of MRSA contamination at each location.

As an alternative to using the "SlicJk Stick" method, swabs can be taken from surfaces such as door handles and the swabs subjected to "head space" analysis using the GC method of the invention.

Detection of MRSA on Patients

MRSA on patients can be samples by taking swabs from parts of the body such as the throat, nose and skin where MRSA may typically be expected to be found.

The swabs can then be held within a sealed container and the headspace above the swab in the container sampled and subjected to GC analysis for the presence of the MRSA.

Since MRSA bacteria are also found in urine, urine samples may also be taken and analysed as described above. The samples suspected of containing MRSA can be subjected to pyrolysis using the apparatus shown in Figure 2 and described above.

Although the method of the invention allows for direct analysis of the test samples, it may be advantageous in the case of rapidly dividing bacteria such as MRSA (MRSA typically divide about every 20 minutes) to culture the bacteria for a short while prior to carrying out the GC analysis.

For example, a swab taken from a body surface such as the nasal cavity can be used to inoculate a culture medium such as an agar plate which is then incubated in standard fashion at 35 ⁰C to 37 ⁰C for about 20 minutes. A sample from the culture medium is then subjected to GC analysis using the apparatus described above. By culturing the test sample in this way, it is possible to ensure that the bacteria are in an "active" or "energetic" state rather than a dormant state, and it is envisaged that this should facilitate production and detection of the characteristic volatile substances in the bacteria.

Equivalents

It will readily be apparent that numerous modifications and alterations may be made to the specific embodiments of the invention described above without departing from the principles underlying the invention. All such modifications and alterations are intended to be embraced by this application.

Claims

1. 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 products from the cultured test sample;

(c) subjecting the volatile products to gas chromatography using a gas chromatography system employing a surface acoustic wave detector; (d) establishing a chromatographic profile for the test sample;

2. A method according to claim 1 wherein the volatile bacterial products are collected directly from the test sample without first culturing the test sample.

3. A method according to claim 1 wherein the test sample is cultured in a bacterial growth medium for a period of no longer than 2 hours and then the volatile bacterial products are collected from the cultured test sample

4. A method according to claim 2 for 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 liquid 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.

5. A method according to any one of the preceding claims wherein the test sample is contained within a sealed container so that there is a headspace above the sample in the container, and volatile bacterial products (e.g. bacterial secretion products) are collected from the headspace and subjected to gas liquid chromatography.

6. A method according to any one of the preceding claims wherein the test sample or the cultured test sample are subjected to pyrolysis prior to gas chromatography step (c).

7. A method of diagnosis of a bacterial infection in a patient, the method comprising taking 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 liquid chromatography using a gas liquid 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 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.

8. A method according to any one of claims 1 to 4 wherein the test sample or biological sample, as the case may be, is selected from:

• biological samples such as whole blood, plasma, serum, sputum, saliva, sweat, semen, urine, interstitial fluid, faecal samples, breath 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

9. A method according to claim 8 wherein the biological sample is blood, sputum or saliva.

10. A method according to any one of the preceding claims wherein the volatile bacterial products (e.g. volatile bacterial secretion products) from the test sample or biological sample are collected (and preferably concentrated) by a temporary storage medium for subsequent analysis by the gas liquid chromatography system.

11. A method according to claim 10 wherein the temporary storage medium comprises an adsorbent onto which the volatile bacterial products (e.g. volatile bacterial secretion products) are adsorbed.

12. A method according to claim 11 wherein the temporary storage medium is in the form of a tube containing the adsorbent and through which the volatile bacterial products (e.g. volatile bacterial secretion products) can be drawn by suction.

13. A method according to any one of the preceding claims wherein the bacteria are selected from Streptococcus pneumonia, Haemophilus influenza, Shegella flexneri, Staphylococcus aureus (e.g. methicillin resistant Staphylococcus aureus (MRSA)) Pseudomonas aeruginosa, Salmonella enteridis, Enterococcus faecalis, Klebsiella pneumonia, Mycobacterium tuberculosis, Escherichia coli and Salmonella typhimurium; and are optionally further selected from other Mycobacterium species such as Mycobacterium bovis.

14. A method according to any one of the preceding claims wherein the chromatographic profile is defined by volatile bacterial products (e.g. volatile bacterial secretion products) selected from volatile fatty acids, esters, sulphur compounds, ketones, alcohols, amines, carboxylic acids, hydrocarbons such as alkenes, alkanes and aromatic compounds.

15. A method of detecting and identifying bacteria, which method comprises collecting volatile bacterial products (e.g. volatile bacterial secretion products) from a test sample suspected of containing the bacteria, subjecting the volatile bacterial products (e.g. volatile bacterial secretion products) products to gas liquid chromatography using a gas liquid chromatography system employing a surface acoustic wave detector; establishing a chromatographic profile for the test sample, sending data defining the chromatographic profile for the test sample along a communications link to a database contained within a computer or other processor at a remote location, interrogating a library of stored chromatographic profiles of volatile bacterial volatile bacterial products (e.g. volatile bacterial secretion products) of individual species and/or strains of bacteria in the database at the remote location, comparing the chromatographic profile of the test sample with stored chromatographic profiles in the library and identifying the bacteria.

16. A distributed system for detecting and identifying bacterial infections in patients in a plurality of remote locations, the distributed system comprising:

(i) a plurality of gas liquid chromatography apparatuses at different remote locations (e.g. wherein each of the gas chromatography apparatuses comprises a surface acoustic wave detector); (ii) a central databank, the databank containing a library of stored chromatographic profiles of volatile bacterial products (e.g. volatile bacterial secretion products) of individual species and/or strains of bacteria; (iii) 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 (e.g. volatile bacterial secretion 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.

17. A method of detecting and identifying bacteria substantially as described herein with reference to the accompanying drawing and examples.