Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
  • C07H15/20—Carbocyclic rings
  • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
• • 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
• • 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
  • C12Q1/10—Enterobacteria
• • 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
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
• • 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
  • C12Q2304/00—Chemical means of detecting microorganisms
  • C12Q2304/40—Detection of gases
• • 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
  • C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
• • 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
  • C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
  • C12Q2334/10—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases p-Nitrophenol derivatives
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
  • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
  • G01N2333/245—Escherichia (G)

Definitions

• 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.
• E. coli Eschericia coli
• 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 ⁇ -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.
• 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.
• 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 ⁇ -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.
• 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.
• 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.
• 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 ⁇ -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- ⁇ -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.
• methylsalicylate- ⁇ -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).
• 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.
• OPGluc o-nitrophenyl- ⁇ -D-glucuronide
• 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.
• 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.
• 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 ⁇ -glucuronidase enzyme in any E.coli present.
• a selective E. coli medium in order to stimulate production of the ⁇ -glucuronidase enzyme in any E.coli present.
• 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.
• the present invention also provides o-nitrophenyl- ⁇ -D-glucuronide per se, not previously available to the public or having industrial application.
• 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.

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• 1993
  • 1993-08-20 WO PCT/GB1993/001778 patent/WO1994004705A1/en not_active Application Discontinuation
  • 1993-08-20 EP EP93919459A patent/EP0656066A1/de not_active Withdrawn
  • 1993-08-20 AU AU49674/93A patent/AU4967493A/en not_active Abandoned

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