EP0656066A1 - Detection de micro-organismes a l'aide de capteurs de gaz - Google Patents

Detection de micro-organismes a l'aide de capteurs de gaz

Info

Publication number
EP0656066A1
EP0656066A1 EP93919459A EP93919459A EP0656066A1 EP 0656066 A1 EP0656066 A1 EP 0656066A1 EP 93919459 A EP93919459 A EP 93919459A EP 93919459 A EP93919459 A EP 93919459A EP 0656066 A1 EP0656066 A1 EP 0656066A1
Authority
EP
European Patent Office
Prior art keywords
conjugate
glucuronate
gas
glucuronide
test kit
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP93919459A
Other languages
German (de)
English (en)
Inventor
Norval James Colin Strachan
Iain Derek Ogden
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Minister of Agriculture Fisheries and Food UK
Original Assignee
Minister of Agriculture Fisheries and Food UK
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB929217843A external-priority patent/GB9217843D0/en
Application filed by Minister of Agriculture Fisheries and Food UK filed Critical Minister of Agriculture Fisheries and Food UK
Publication of EP0656066A1 publication Critical patent/EP0656066A1/fr
Withdrawn legal-status Critical Current

Links

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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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Toxicology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

On décrit un procédé qui permet d'évaluer l'état de contamination de matériaux par la présence d'éventuels micro-organismes, pathogènes notamment, par la mesure de la production d'un gaz ou d'une vapeur spécifique provenant d'un échantillon de ce matériau lorsqu'on le fait incuber avec un substrat porteur d'une enzyme bactérienne. Plus précisément, l'invention concerne la détection sélective d'organismes d'Escherichia coli dans cet échantillon de matériau ainsi que la corrélation entre la présence et/ou le nombre de ces organismes et la présence et/ou le nombre d'organismes pathogènes. Ce procédé s'applique particulièrement aux examens concernant la détection dans des produits alimentaires de la présence probable d'organismes pathogènes. Les substrats préférés incluent O-nitrophényle-β-D-glycuroconjugué et méthylsalicyly-β-D-glycuroconjugué. On décrit également des trousses d'examen permettant d'appliquer ce procédé.
EP93919459A 1992-08-21 1993-08-20 Detection de micro-organismes a l'aide de capteurs de gaz Withdrawn EP0656066A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
GB9217843 1992-08-21
GB929217843A GB9217843D0 (en) 1992-08-21 1992-08-21 Detection of microorganisms using gas sensors
GB929219150A GB9219150D0 (en) 1992-08-21 1992-09-10 Detection of microorganisms using gas sensors
GB9219150 1992-09-10
PCT/GB1993/001778 WO1994004705A1 (fr) 1992-08-21 1993-08-20 Detection de micro-organismes a l'aide de capteurs de gaz

Publications (1)

Publication Number Publication Date
EP0656066A1 true EP0656066A1 (fr) 1995-06-07

Family

ID=26301476

Family Applications (1)

Application Number Title Priority Date Filing Date
EP93919459A Withdrawn EP0656066A1 (fr) 1992-08-21 1993-08-20 Detection de micro-organismes a l'aide de capteurs de gaz

Country Status (3)

Country Link
EP (1) EP0656066A1 (fr)
AU (1) AU4967493A (fr)
WO (1) WO1994004705A1 (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9411515D0 (en) * 1994-06-09 1994-08-03 Aromascan Plc Detecting bacteria
WO1997008337A1 (fr) * 1995-08-25 1997-03-06 Unipath Limited Procedes et appareil de detection de micro-organismes
GB9700012D0 (en) 1997-01-02 1997-02-19 Aromascan Plc Improvements in the detection of bacteria
GB9704676D0 (en) 1997-03-06 1997-04-23 Aromascan Plc Condition indicator
GB9704627D0 (en) * 1997-03-06 1997-04-23 Aromascan Plc Detection of antigenic species
US8518663B2 (en) 2009-04-27 2013-08-27 The Charles Stark Draper Laboratory, Inc. Rapid detection of volatile organic compounds for identification of Mycobacterium tuberculosis in a sample
FR2976952A1 (fr) 2011-06-27 2012-12-28 Commissariat Energie Atomique Procede pour caracteriser la presence ou l'absence d'un microorganisme dans un milieu biologique
WO2014180974A1 (fr) 2013-05-09 2014-11-13 Ramem, S.A. Procédé de diagnostic de la narcolepsie à base de composés organiques volatils
US10835185B2 (en) 2018-03-08 2020-11-17 General Electric Company System and method for detecting ventilator-associated pneumonia (VAP)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9404705A1 *

Also Published As

Publication number Publication date
WO1994004705A1 (fr) 1994-03-03
AU4967493A (en) 1994-03-15

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