DETECTION OF MICROORGANISMS USING GAS SENSORS

Abstract: A method is provided for assessing the contamination status of materials with respect to possible presence of microorganisms, particularly pathogenic microorganisms, by measuring the production of a specific gas or vapour evolved by a sample of the material when it is incubated with a bacterial enzyme substrate. More particularly the present invention relates to the selective detection of (Escherichia coli) organisms in the material sample and relating the presence and/or number of these to the presence and/or number of pathogenic organisms. The method is particularly applicable to testing foodstuffs for the likely presence of pathogens. Most preferred substrates comprise o-nitrophenyl-(beta)-D-glucuronidde and methylsalicyly-(beta)-D-glucuronide. Test kits for carrying out said method are also provided.

Description: DETECTION OF MICROORGANISMS USING GAS SENSORS. The present invention relates to a method of assessing the contamination status of materials with respect to possible presence of microorganisms, particularly pathogenic microorganisms, by measuring the production of a specific gas or vapour evolved by a sample of the material when it is incubated with a bacterial enzyme substrate. More particularly the present invention relates to the selective detection of Eschericia coli (E. coli) organisms in the material sample and relating the presence and/or number of these to the presence and/or number of pathogenic organisms. The method is particularly applicable to testing foodstuffs for the likely presence of pathogens. It is known to assess the bacteriological safety of foods by assessing the number of E.coli present and using that as an indication of the likely presence of pathogens. However, conventional methods are both time consuming and inaccurate (eg. see Ogden and Watt (199D Letters in Applied Microbiology, 13. 212-215), and there is a general need for more rapid and more automated methods. It is known to detect isolated E.coli by measuring the evolution of o-nitrophenol (ONP) from pure cultures to which o-nitrophenyl -β-D-galactoside (ONPG) has been added (Snyder et al. Anal. Chem. 1991. 63, 5 6-529). The endogenous β-galactoside enzyme in the E. coli is first stimulated by growing the culture overnight in a lactose medium, amounts of organism containing suspension are placed on filter disks with an equal volume of ONPG (2 mg/ml) and placed in sealed test tubes before incubating at 40 to 42°C. On sampling the seals are removed and the headspace vapour is analysed by use of an ion-mobility detector device of the type described in GB patents 1,274,634, 1,306,534 and 2,052,750. While this technique is useful for determination of quantities of E.coli in some applications, use of the β-galactosidase enzyme is not an option when monitoring pathogenic bacteria in foodstuffs as it is found in many harmless bacteria commonly associated with foodstuff manufacture and storage. A more specific method for detection and enumeration of E. coli in foodstuffs is required. The present inventors have now provided a method for the identification, detection and/or enumeration of organisms of species E. coli. which is specific enough to be used upon samples derived from foodstuffs such that false positive results are not obtained where only organisms associated with consumable food are present. The selected method is such that where false positives are obtained they result from organisms of genus Shigella and Salmonella, many of which are harmful and the presence of which would be an indication of contamination in themselves. The present invention provides a method for determining the presence and/or quantity of Escherichia coli organisms in a material comprising mixing a sample derived from said material with a solution containing a glucuronide conjugate, that comprising a glucuronate moiety and a moiety which is capable of being cleaved therefrom by the action of β-glucuronidase, said cleavage giving rise to a glucuronic acid and a gaseous or volatile compound; incubating the mixture and assessing the amount of any such gaseous or volatile compound evolved using a gas detector device and relating the presence and/or amount so evolved to the presence and/or quantity of organisms of species Escherichia coli. In its preferred form the present invention provides a method of assessing the contamination status of a foodstuff comprising treating a sample derived therefrom by the method described above wherein the detection of said evolved gaseous or volatile compound is related to the presence and/or amount of contamination with organisms of species E.coli or genus Shigella or Salmonella. In this way the presence of contaminating organisms and the likelihood of presence of further pathogens, other than Shigella and Salmonella, may be assessed and identification of foodstuffs not fit for human and/or animal consumption thus carried out in a manner that is relatively free from false positive results while being easily automated and more rapid effected than those methods previously available. The glucuronide conjugate used with the method of the present invention may be any conjugate comprising a glucuronate moiety joined by a β-glucuronidase specific cleavable bond to a moiety which gives rise to a volatile compound on cleavage of said bond, provided that the volatile compound is specifically detectable using a gas detector device with respect to normal bacteriological metabolites. In its preferred form the method of the present invention the gas detector used is an ionisable gas detector device. Ionisable gas detector devices come in a number of forms but two in particular are suitable for specific detection of gases and volatile compounds against a background of interfering atmospheric gases or other compounds being evolved by a foodstuff sample. One such device is the ion mobility detector such as that used by the previously mentioned workers to detect ONP. These devices can be set up to ignore such molecules as ammonia and carbon dioxide and thus to specifically detect ONP evolved from the incubated sample by aspirating the headspace gases above it. The method for setting up such devices to detect specific classes of compound is well known in the gas detection art and may be achieved simply by following manufacturers operating instructions and carrying out various controls with likely interfering gases and volatiles. A further type of device that might be adapted to detect a specific gas or volatile compound is an ultraviolet light ionisable gas detector device. Such devices are available from HNU systems (see GB 1576474), Photovac or UVIC and use ultraviolet light to ionise aspirated gas or vapour in a sample and then measure the amount of gas or vapour so ionised using a collector electrode or electrodes. As ionisation of different compounds varies with the wavelength of UV light employed, it is possible to excite a selected class of compounds and thus selectively detect them. For example, comparing the ionisation potentials of various compounds: water (11.65 oeV) , ammonia (10.15oeV), nitrobenzene (9«82oeV) and phenol (8.5 oeV) , it can be seen that by choice of UV source power by selection of wavelength emitted that selective measurement is quickly yet reliably obtainable. By subtracting the amount of gas detected below wavelengths necessary to detect interfering compounds such as water and ammonia from that detected at wavelengths above these it is possible to identify the more complex salicylate or ONP evolution. A still further method of detection of the volatile moiety is by use of gas chromatography whereby vapour emitted by the mixture is aspirated into a collecting implement and then transferred to a gas chromatograph for analysis of its content. Any of the aforesaid devices might be employed to analyse the headspace gas above a sealed incubated sample. The number of organisms present in each case is conveniently assessed by reference to the amount of specific gas or volatile compound evolved per unit incubation time as compared to a reference curve derived from known levels of E. coli. Such time might be from a few minutes to many hours. It is preferred that the incubation time is from 10 minutes to 48 hours, more preferably from 30 minutes to 24 hours, most preferably overnight eg. 6 to 14 hours, preferably 6 to 8 hours. Suitable moieties giving rise to volatile ionisable compounds on cleavage of the conjugate bond by β-glucuronidase include nitrophenyl moieties, particularly o-nitrophenyl which gives rise to the relatively high vapour pressure sustaining o-nitrophenol, and methyl salicylate, but other such moieties will occur to those skilled in the art. The preferred conjugate o-nitrophenyl-β-D-glucuronide (ONPGluc), while relatively simple to prepare from its component parts glucuronic acid and o-nitrophenol, has not been commercially available previously but is now available from Grampian Enzymes, Arthrath, Ellon, Aberdeen, UK. Similarly preferred methylsalicylate-β-D-glucuronide is relatively simple to prepare using standard glucuronide preparation procedures (see review C A Marsh, 'D-Glucuronic acid and its glycosides' in 'Glucuronic acid. Free and Combined 1 (1966), Academic Press, Edited by G J Dutgon). In order to ensure that false positives derived from β-glucuronidase endogenous to the foodstuff are avoided the amount of conjugate added to a given amount of foodstuff derived sample needs to be controlled. These false positives particularly occur with liver derived materials. Whereas the previously described method for detecting E.coli in culture form utilises about 2 mg/ml ONPG the present inventors have found that the use of such large quantities of glucuronide conjugate gives rise to unacceptably unreliable results for foodstuffs screening. The particular limits for conjugate concentration for a given amount of sample varies from foodstuff to foodstuff and is best assessed by performance of controls to determine levels that will not give a significant evolution of volatile compound in the absence of E. coli yet support such evolution when they are present. Using the preferred o-nitrophenyl-β-D-glucuronide (ONPGluc) conjugate of the invention the present inventors have determined that use of 0.5mg/ml conjugate is the upper limit that should be used for a 1 ml sample of 0.1 g/1 of minced beef in buffer while for chicken the upper limit is about O.lmg/ml. It should be realised that even very small numbers of E.coli may be detected if the sample and glucuronide conjugate mix are incubated for long enough, thus such controls should be set up such that long incubation does not give such false positive result. During incubation the microorganism derived β-glucuronidase activity increases, with exponential rate of increase of gas evolution, while background activity derived from foodstuff enzymes does not increase. The use of an ion mobility detector device of the Graseby 'Airborne Vapour Monitor' type provides capability for detection of o-nitrophenol vapour above a solution containing only nanogram/ml amounts. Typically the headspace to be sampled is that in a 5 to 10ml test-tube with a sample/substrate volume in the bottom, but any conventional incubation vessel volume might be used given a suitable incubation time to fill it with the evolved gas or vapour. To optimise for ONPGluc metabolism, either the incubation or a preincubation of the sample under investigation should be carried out using a selective E. coli medium in order to stimulate production of the β-glucuronidase enzyme in any E.coli present. Typically this should contain an amount of enzyme stimulant, eg. glucuronate eg. in the form of sodium glucuronate, and ideally this is used to replace other sugar or sugars in the selective medium used. A preferred selective medium for use in the invention is a modified lauryl sulphate tryptose broth (LST)-that being double strength and having lactose replaced therein by glucuronate; the composition of this is as shown below in Table 1. The final medium is sterilised, eg. by autoclaving at 121°C for 15 minutes, before use. The material to be sampled is conveniently suspended in a buffer solution prior to mixing with the selective medium. The buffer solution may be any that is compatible with the support of E. coli growth and is preferably one such as peptone phosphate buffer, a preferred buffer composition being given in Table 2 below. This is preferably used in chilled form for the homogenisation of material to be sampled, ie. the foodstuff, and is preferably sterilised before use. The glucuronide component is preferably filter sterilised. * The glucuronate component is filter sterilised and added to the basal medium after cooling. Incubations may be carried out at any temperature suitable for target organism growth, but preferably from 3 to 45°C, more preferably at about 37°C The present invention further provides test kits for the performance of the method of the invention; these comprising components specific to that method. In this regard the present invention also provides o-nitrophenyl-β-D-glucuronide per se, not previously available to the public or having industrial application. The method of the present invention will now be exemplified by way of illustration only by reference to the following non-limiting Example, further embodiments falling within the scope of the invention will occur to those skilled in the art in the light of the forgoing description and said Example and its associated Figure 1. Figure 1: shows the relationship between log BCIG plate counts obtained using the method of Ogden and Watt (see above) and the incubation time required to obtain a detectable amount of o-nitrophenol in the head space above a sample incubated according to the method of the invention carried out on the same sample. EXAMPLE 1. Foodstuff suspected of containing contaminating organisms (25 grams) was homogenised in 225 mis of chilled peptone phosphate buffer and 1ml of this was added to 1ml double strength LST broth and incubated at 37°C in a sealed standard sized test tube. Once preincubation was completed ONPGlucuronide was added aseptically to give a concentration of 0.05mg/ml. The headspace above the broth was sampled at hourly intervals using a Graseby Ionics Ltd (Watford UK) AVM-Ion Mobility Spectrometer device set to specifically detect ONP and the time taken for any such ONP to be evolved was recorded. To calibrate the system the number of E.coli in foodstuff samples was determined by counting colonies produced by seeding selective agar plates (Ogden and Watt (1991) Letters in Applied Microbiology, 13, 212-215) • The number of colonies found was plotted against time to detect ONP using the AVM device and the method of the invention for the same sample (See Figure 1). The major part of the scatter in the results in the table is due to the plate counting calibration technique and not the method.

Claims: SL_ _______. 1. A method for determining the presence and/or quantity of microorganisms of species Escherichia * coli in a material comprising mixing a sample derived from said material with a solution containing a glucuronide conjugate, that conjugate comprising a glucuronate moiety and a moiety which is capable of being cleaved therefrom by the action of β-glucuronidase, said cleavage giving rise to a glucuronic acid and a gaseous or volatile compound; incubating the mixture and assessing the amount of any such gaseous or volatile compound evolved using a gas detector device and relating the presence and/or amount so evolved with the presence and/or quantity of Escherichia coli. 2. A method as claimed in claim 1 wherein the material is a foodstuff and the presence and/or amount of Escherichia coli is related to the likelihood of presence of pathogens. 3. A method as claimed in claim 1 or claim 2 wherein the glucuronide conjugate comprises a glucuronate moiety joined by a β-glucuronidase specific cleavable bond to a moiety which gives rise to a gas or volatile compound on cleavage of said bond, wherein the gas or volatile compound is specifically detectable using a gas detector device. 4. A method as claimed in claim 3 wherein the detector device is capable of specific detection of volatile compounds against a background of interfering atmospheric gases or other compounds being evolved by a foodstuff sample. 5. A method as claimed in claim 3 or 4 wherein the gas or volatile compound is ionisable. 6. A method as claimed in claim 5 wherein the detector device is an ion mobility detector, an ultraviolet light ionisable gas detector or a gas chromatograph. 7. A method as claimed in any one of claims 1 to 6 wherein the conjugate is o-nitrophenyl-β-D-glucuronide or methylsalicylyl -β-D-glucuronide. 8. A method a claimed in any one of the preceding claims wherein the incubation of the mixture evolves o-nitrophenol or methylsalicylic acid when targeted microorganisms are present. 9. A method as claimed in any one of the preceding claims wherein the incubation is carried out for between 10 minutes and 48 hours. 10. A method as claimed in claim 9 wherein the incubation is carried out for between 30 minutes and 24 hours. 11. A method as claimed in claim 10 wherein the incubation is carried out for between 6 and 14 hours. 12. A method as claimed in any one of the preceding claims wherein the concentration of conjugate used is below that which produces the gas or volatile compound when incubated with a sterilised sample of the material. 13. A method as claimed in claim 7 wherein the conjugate is o-nitrophenyl-β-D-glucuronide and its concentration in the incubation mixture is 1.5mg/ml or less. 14. A method as claimed in claim 8 wherein conjugate concentration in the incubation is 0.5mg/ml or less. 15. A method as claimed in claim 8 wherein conjugate concentration in the incubation is O.lmg/ l or less. 16. A method as claimed in any one of the preceding claims wherein the conjugate concentration is between 0.01 and 0.05mg/ml. 17. A method as claimed in any one of the preceding claims wherein the incubation or a preincubation of the sample under investigation is carried out using an E. coli selective medium whereby production of β-glucronidase is stimulated. 18. A method as claimed in claim 17 wherein the E. cnli selective medium contains a glucuronate. 19. A method as claimed in claim 17 wherein the glucuronate is sodium glucuronate. 20. A method as claimed in claim 17 wherein the medium is a modified double strength lauryl sulphate tryptose broth having lactose replaced therein by a glucuronate. 21. A method as claimed in any one of the preceding claims substantially as described in Example 1. 22. A test kit for determining the presence and/or quantity of microorganisms of species Escherichia coli in a material characterised in that it contains a gluconuride conjugate; that conjugate comprising a glucuronate moiety and a moiety which is capable of being cleaved therefrom by the action of β-glucuronidase, said cleavage giving rise to a glucuronic acid and a gaseous or volatile compound. 23. A test kit as claimed in claim 22 wherein the gas or volatile compound is ionisable. 24. A test kit as claimed in claim 23 wherein the gas is detectable by use of an ion mobility detector, an ultraviolet light ionisable gas detector or a gas chromatograph. 25. A test kit as claimed in claim 22 wherein the conjugate is o-nitrophenyl-β-D-glucuronide or methylsalicylyl-β-D-glucuronide. 26. A test kit as claimed in claim 22 further comprising an an L. coli selective medium capable of stimulated production of E.coli β-glucuronidase. 27. A test kit as claimed in claim 26 wherein the E.coli selective medium contains a glucuronate. 28. A test kit as claimed in claim 27 wherein the glucuronate is sodium glucuronate. 29. A test kit as claimed in claim 28 wherein the medium is a modified double strength lauryl sulphate tryptose broth having lactose replaced therein by a glucuronate. 30. A test kit for assessing the likelihood of contamination of a material with pathogenic microorganisms comprising a test kit as claimed in any one of claims 22 to 29. 31. ortho-Nitrophenyl-β-D-glucuronide.

Inventors: OGDEN IAIN DEREK, STRACHAN NORVAL JAMES COLIN, OGDEN, IAIN, DEREK, STRACHAN, NORVAL, JAMES, COLIN
Applicants: MINI AGRICULTURE & FISHERIES, OGDEN IAIN DEREK, STRACHAN NORVAL JAMES COLIN, THE MINISTER OF AGRICULTURE FISHERIES AND FOOD IN 
Application year: 1993
Main classification: C12Q1
IPC codes: C07H15, C12Q1, G01N33