WO2019236309A1 - Procédé d'identification et d'énumération de populations bactériennes spécifiques dans un échantillon liquide non enrichi - Google Patents

Procédé d'identification et d'énumération de populations bactériennes spécifiques dans un échantillon liquide non enrichi Download PDF

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Publication number
WO2019236309A1
WO2019236309A1 PCT/US2019/033648 US2019033648W WO2019236309A1 WO 2019236309 A1 WO2019236309 A1 WO 2019236309A1 US 2019033648 W US2019033648 W US 2019033648W WO 2019236309 A1 WO2019236309 A1 WO 2019236309A1
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Prior art keywords
sample
microorganisms
unenriched
growth
cfu
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PCT/US2019/033648
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English (en)
Inventor
Michael P. Perry
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Dupont Nutrition Biosciences Aps
E. I. Du Pont De Nemours And Company
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Publication of WO2019236309A1 publication Critical patent/WO2019236309A1/fr

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    • 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
    • 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

  • This invention relates to the field of microbiology and, in particular, to a method for identifying and enumerating specific bacterial populations in an unenriched liquid sample.
  • microorganisms The detection and enumeration of microorganisms is practiced in numerous settings such as in the food-processing industry where food is tested for contamination by microorganisms such as E. coli and S. aureus , the health care industry where patient samples and other clinical samples are tested for infection or contamination, environmental testing industry, pharmaceutical industry and the cosmetic industry.
  • MPN most probable number analysis
  • semi-solid nutrient media direct counting using, e.g ., agar petri dishes.
  • Microbial populations are typically enumerated using standard microbiology methods such as viable plate count, estimates of most probable number (MPN), and ATP assays, to name a few.
  • viable plate count method serial dilutions of the microbe-containing sample are spread onto a plate containing agar growth medium which may be specific and selective for the growth of a particular microbe. After incubation, the total number of bacterial colonies on the plate is counted and the number is then multiplied by the dilution factor. Since each colony arises from an individual viable cell, the colony count represents the number of bacterial cells present in the original sample. This number is converted to and expressed as CFU/ml (colony forming units/ml) or logio(CFU/ml).
  • Bacterial growth can also be determined by the turbidity of a growing culture of bacterial cells. Turbidity or optical density of the cell suspension can be determined
  • spectrophotometrically by measuring the absorbance of light at a particular wavelength passing through the cell suspension.
  • the absorbance is proportional to the number of cells in the suspension.
  • An optical density of 1 is generally equal to about 10 9 CFU/ml although this is a very approximate estimate and is influenced by a number of factors, including the size and shape of the cells, etc.
  • MPN Most probable number
  • ATP assays can also be used to measure the number of viable cells in culture medium based on the quantification of adenosine triphosphate (ATP). ATP is produced by living cells and can be measured indirectly by a luminescent chemical reaction (see, for example BacTiter- Glo assay from Promega, Madison, WI). The light emitted in the reaction is proportional to the concentration of ATP present in the culture which is, in turn, proportional to the number of viable cells present in the medium.
  • ATP adenosine triphosphate
  • the above methods can be used for pure cell cultures (containing a single strain of bacteria) or for determining the total number of cells in a sample that contains a mixed population of bacteria.
  • Selective growth media can be used to detect and enumerate specific microorganisms in biological and environmental samples containing a number of different bacterial species.
  • E. colt can be enriched for by plating the sample onto a specific E. colt- selective agar medium such as CHROMagarTM (from CHROMagar, Paris, France, Tel: +33 1 45 48 05 05). E. colt will appear blue while other Gram negative bacteria will be colorless on this medium. Gram positive bacteria will be inhibited.
  • CHROMagar also offers a product called AquaCHROMTM ECC. This product detects (but does not enumerate) the presence or absence of E. coli and coliforms in 100 ml water samples.
  • a method for identifying and enumerating one or more specific microorganisms in an unenriched liquid sample comprising:
  • step (b) using a reporter system that is capable of identifying and enumerating the one or more enriched-for microorganisms in the unenriched liquid sample wherein the reporter system is added before, during or after step (b).
  • a method for identifying and enumerating one or more specific microorganisms in an unenriched liquid sample comprising:
  • step (b) identifying and enumerating the enriched specific genera or one or more species of microorganisms in the unenriched liquid sample using a reporter system, wherein the reporter system is added before, during or after step (a).
  • the microorganisms that are identified and enumerated are selected from the group consisting of Escherichia, Enterobacter, Clostridia, Staphylococcus, Streptococcus, Bacilli, Salmonella, and Listeria.
  • the reporter system is a colorimetric reporter system.
  • the microorganisms are identified and enumerated between about 4-9 hours following commencement of step (a).
  • the medium lacks agar or any other substance capable of solidifying under specific conditions.
  • Figure 1 sets forth absorbance spectra of E. coli grown with and without the modified CHROMagarTM medium. An additional sample containing the CHROMagar and E. coli (circles) was spiked with blue food coloring dye in order to highlight the blue absorbance peak.
  • Figure 2 depicts the growth of E. coli in IX or 2X CHROMTM Liquid ECC medium.
  • Figure 3 is a graph depicting the growth of the culture expressed as logio(MPN) and the blue intensity at 650 nm.
  • Figure 4 is a graph depicting the absorbance at 610 nm for each of the starting concentrations of E. coli in CFU/mL.
  • Figure 5 is a graph depicting the growth of the culture expressed as logio(MPN) and blue intensity (at 610 nm) for 100 CFU/ml starting concentration.
  • Figure 6 is a graph depicting the growth of the culture expressed as logio(MPN) and blue intensity (at 610 nm) for 1000 CFU/ml starting concentration.
  • Figure 7 is a graph depicting the growth of the culture expressed as logio(MPN) and blue intensity (at 610 nm) for 10000 CFU/ml starting concentration.
  • Figure 8 is a graph depicting the growth of the culture expressed as logio(MPN) and blue intensity (at 610 nm) for 100000 CFU/ml starting concentration.
  • the term“about” refers to a range of +/- 0.5 of the numerical value, unless the term is otherwise specifically defined in context.
  • the phrase a“pH value of about 6” refers to pH values of from 5.5 to 6.5, unless the pH value is specifically defined otherwise.
  • sample means any sample containing or suspected to contain populations of microorganisms. This includes, but is not limited to, food, water, sediments, wastewater, soil, sand, sludge, plant or animal samples such as animal feed, tissue samples, blood, urine, fecal material, breast milk (such as human or cow milk), a clinical sample such as blood or urine or mucus, a pharmaceutical sample, a cosmetic sample or surfaces that may contain microbial populations.
  • unenriched sample or“unenriched liquid sample” refers to the original sample prior to subjecting it to any means for enrichment.
  • microorganism and“microbe” are used interchangeably herein and refers to a microscopic organism which may exist in its single-celled form or in a colony of cells. This term includes both eukaryotes and prokaryotes. There can be mentioned bacteria, fungi, yeasts, algae and the like.
  • microorganism selective medium and“microorganism selective media” are used interchangeably herein and refer to media that is selective, that is, formulated to grow only certain microbes while inhibiting the growth of other microbes. Accordingly, the growth of cells in a selective media indicates the presence of certain types of cells within a sample (such as a liquid sample).
  • CFU means“colony forming units” and is a measure of viable cells in which a colony represents an aggregate of cells derived from a single progenitor cell.
  • MPN or“most probable number” is a quantitative measurement of the concentration of microbes in a given medium. It is expressed as CFU/ml (colony forming units/ml), logio(CFU/ml) or logio(MPN).
  • Enrichment or“enriching for” mean growing a specific microbe under suitable conditions for promoting growth of said specific microbe while suppressing or inhibiting growth of other non-target microbes that may be present in the biological or environmental sample.
  • the suitable conditions also include growth and medium compositions that are favorable to producing compounds or molecules that can be detected by specific reporter systems.
  • reporter systems can detect the production and/or modification of molecules, especially those extracellular molecules related to redox and electron transfer as well as extracellular proteins and enzymes.
  • Typical media compositions include enriched media containing diverse nutrient sources such as peptone, yeast extract, or casamino acids, for example.
  • media may include minimal media such as SL10, or similar, supplemented with an electron donor (lactate, acetate, etc) and electron acceptor (nitrate, fumarate, etc).
  • the enrichment step can be performed at a temperature of about 30° C to 43° C, such as any of about 30°C, 3 l°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 4l°C, 42°C, or 43 °C for a period of about 4 to about 16 hours, such as about 4 to about 9 hours, such as any of about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 hours (inclusive of all times in between these values).
  • a person skilled in the art may adapt the temperature and the duration of this incubation step in view of the general knowledge present in the art.
  • the term“enumeration” as used herein refers to determining or estimating or quantifying the number of microorganism in a sample, culture, liquid medium, etc.
  • reporter system refers to any means by which the presence of a cell or protein can be detected. Depending on the type of reporter system used, emitted signals can be detected optically by (bioluminescence, fluorescence or colorimetry), electrochemically, or enzymatically.
  • a method for identifying and enumerating one or more specific microorganisms in an unenriched liquid sample comprising:
  • step (b) using a reporter system that is capable of identifying and enumerating the one or more enriched-for microorganisms in the unenriched liquid sample wherein the reporter system is added before, during or after step (b).
  • Microorganism suitable for identification and enumeration by the method disclosed herein include but are not limited to Escherichia, Enterobacter, Clostridia, Staphylococcus, Streptococcus, Bacilli, Salmonella, and Listeria.
  • Samples can be obtained from any source as was noted above.
  • food-processing industry where food is tested by contamination by microorganisms such as E. coli and S. aureus , animal feed, animal care where animal samples such as urine, blood, feces, milk (such as, but not limited to, cow, goat, or sheep milk), and the like are tested for infection or contamination
  • animal care where animal samples such as urine, blood, feces, milk (such as, but not limited to, cow, goat, or sheep milk), and the like are tested for infection or contamination
  • health care industry where patient samples and other clinical samples such as blood, serum, urine, feces, mucus, tears, semen, vaginal secretions, and the like are tested for infection or contamination
  • environmental testing industry where samples such as soil and water are evaluated, pharmaceutical industry and the cosmetic industry.
  • any reporter system useful for identifying and enumerating one or more microorganisms in a sample can be used.
  • the reporter system is a colorimetric reporter system. It also should be simple and fast to use in the field.
  • compositions and methods disclosed herein include:
  • a method for identifying and enumerating one or more specific microorganisms in an unenriched liquid sample comprising:
  • step (b) using a reporter system that is capable of identifying and enumerating the one or more enriched-for microorganisms in the unenriched liquid sample wherein the reporter system is added before, during or after step (b).
  • a method for identifying and enumerating one or more specific microorganisms in an unenriched liquid sample comprising:
  • step (a) enriching the unenriched sample for growth of a specific genera or one or more species of microorganisms using a microorganism-specific selective medium; and b) identifying and enumerating the e enriched specific genera or one or more species of microorganisms in the unenriched liquid sample using a reporter system, wherein the reporter system is added before, during or after step (a).
  • microorganisms are selected from the group consisting of Escherichia, Enter obacter, Clostridia, Staphylococcus,
  • Streptococcus Bacilli, Salmonella, Listeria.
  • “hr” means hour(s);“mL” means milliliter;“°C” means degrees Celsius;“mg” means milligram(s);“mm” means millimeter;“g” means gram(s);“mM” means millimolar;“%” means percent;“CFU/mL” means colony forming unit per milliliter;“min” means minute(s);“NIC” means non-inoculated control (negative controls in microbial culture experiments);“pg/L” means microgram per liter;“nM” means nanomolar;“mM” means micromolar.
  • Modified CHROMagarTM medium (from CHROMagarTM, Paris, France) was evaluated for its usefulness in identifying the presence of E. coli in liquid culture rather than as colonies on plates or filter membranes.
  • E. coli was grown in Tryptic Soy Broth (TSB) medium and a modified version of CHROMagarTM.
  • the CHROMagarTM was prepared according to the supplier’s protocol but was not autoclaved in order to allow for the agar to settle out. The upper phase was then pipetted off the top and sterilized by autoclaving. The resulting medium will not solidify without the presence of the agar. It could then be used as a true liquid medium. Twenty microliters of an overnight culture of E. coli was added to 2 ml of TSB medium and 2 ml of the modified
  • CHROMagarTM medium The two cultures were grown overnight at 37 °C. The next day, only the CHROMagarTM-containing culture turned blue. Both samples were scanned on a
  • E. coli can be used to detect the presence of E. coli in culture rather than as colonies on plates or filter membranes, growth of E. coli was tested in IX and 2X concentrations of the medium.
  • An initial culture of E. coli was grown overnight in TSB medium. The next day, 5 m ⁇ or 50 m ⁇ of the overnight culture was added to a IX or a 2X concentration of the CHROMTM liquid ECC medium, prepared following the supplier’s protocol. Samples were grown overnight at 37 °C.
  • the blue/green intensity of the culture medium after 24 hr growth of E. coli is shown in Figure 2.
  • the samples grown in the 2X medium concentration appear more intensely colored. It should be noted that the blue/green color of the media increased in intensity as the cultures grew (data not shown).
  • the corresponding plate counts after 6 hours should be about 2 X 10 6 CFU/ml, 2 X 10 7 CFU/ml, and 2 X 10 8 CFU/ml for the original respective 10 CFU/ml, 100 CFU/ml, 1000 CFU/ml starting concentrations.
  • the blue intensities of the solutions would also vary among the three samples and would correlate to the number of cells present. The measurements would then be used to calculate the number of cells in the original sample.
  • the blue intensity of the medium is optimized to correspond to a certain amount of E. coli in the samples.
  • this technology would be useful in detecting and enumerating a microorganism such as E. coli in a variety of unenriched liquid samples.
  • a gram of fecal material can be added to E. coli enrichment medium containing a colorimetric reporter to enrich for the microorganism.
  • the E. coli is then grown under standardized conditions and time and the blue intensity of the reporter is measured spectrophotometrically. Based on the standardized growth calculations, the intensity of the color should then correlate to the amount of E. coli in the original unenriched fecal material.
  • CFU/ml by plate counts) every hour while also measuring the intensity of the chromogenic reporter at the same hourly timepoints.
  • serial dilutions of the microbe-containing enrichment sample are spread onto a plate containing agar growth medium which is selective for the growth of the microbe.
  • the total number of bacterial colonies on the plates is counted and the number is then multiplied by the dilution factor.
  • the colony count represents the number of bacterial cells present in the sample. This number is converted to and expressed as CFU/ml (colony forming units/ml) or logio(CFU/ml).
  • the blue intensity of the sample is also measured by centrifuging the sample at each time point to remove the cells which may interfere with the absorbance reading at 610 nm wavelength (the exact wavelength to be experimentally determined).
  • the blue intensity of the cell-free supernatant is then measured at 610 nm by using a spectrophotometer.
  • the colony counts from the plates and the blue intensity of the cell-free supernatant at each time point are compared to each other.
  • an overnight culture of E. coli (in Tryptic Soy Broth, TSB) was diluted to 100 CFU/ml, in 50 ml of IX CHROMagarTM Liquid ECC medium. Cell concentration was confirmed by the MPN microtiter plate method. The sample was then grown at 37 °C for 24 hours. A 1 ml sample of the culture was collected hourly for 9 hours and then at 24 hours of growth. Cell counts at each timepoint were determined by the MPN method. Each 1 ml sample was then centrifuged for 3 min at maximum speed to remove the cells which would interfere with the absorbance measurements at 610 nm.
  • FIG. 1 shows the growth of the culture expressed as logio(MPN) and the blue intensity at 650 nm.
  • the culture medium started turning blue when the cell concentration reached about 1 x 10 9 CFU/ml and continued increasing in intensity the longer the cells grew but started to plateau at 24 hours of growth.
  • Known quantities e.g ., 10 CFU/ml, 100 CFU/ml, 1000 CFU/ml
  • a microorganism such as E. coli
  • a microorganism-specific medium under standardized conditions in the presence of a chromogenic reporter for about 6 hours. Plate counts and the blue intensity of the medium at the about 6 hour timepoint are measured as described above. Plate counts and the blue intensity of the medium correlate with each other and correspond to the starting 10 CFU/ml, 100 CFU/ml, 1000 CFU/ml concentrations.
  • E. coli E. coli .
  • IX CHROMagarTM Liquid ECC medium an overnight culture of E. coli (in Tryptic Soy Broth, TSB) was diluted to 100 CFU/ml, 1000 CFU/ml, 10000 CFU/ml, and 100000 CFU/ml in 50 ml of IX CHROM Liquid ECC medium. Cell concentrations were confirmed by the MPN microtiter plate method. Each sample was then grown at 37 °C. A 1 ml sample of each culture was collected hourly after 4 hours of growth for up to 9 hours.

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Abstract

L'invention concerne un procédé d'identification et d'énumération de populations bactériennes spécifiques dans un échantillon liquide non enrichi.
PCT/US2019/033648 2018-06-04 2019-05-23 Procédé d'identification et d'énumération de populations bactériennes spécifiques dans un échantillon liquide non enrichi WO2019236309A1 (fr)

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US201862680248P 2018-06-04 2018-06-04
US62/680,248 2018-06-04
US201962791430P 2019-01-11 2019-01-11
US62/791,430 2019-01-11

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US5610029A (en) * 1994-11-04 1997-03-11 Idexx Laboratories, Inc. Medium for detecting target microbes in a sample
WO1998045406A1 (fr) * 1997-04-09 1998-10-15 Minnesota Mining And Manufacturing Company Procede et dispositifs pour diviser des liquides d'echantillons biologiques en microvolumes
JP2000000100A (ja) * 1998-06-15 2000-01-07 San Kagaku Kk 大腸菌群検出用試験シート
JP2004236574A (ja) * 2003-02-05 2004-08-26 Meiji Yakuhin Kk 細菌性膣症診断用液体培地
US20080124754A1 (en) * 2001-12-14 2008-05-29 Grete Lorentzen Rapid method of detection and enumeration of sulfide- producing bacteria in food products
KR20150114313A (ko) * 2014-04-01 2015-10-12 건국대학교 산학협력단 증균 단계에서 스크리닝이 가능한 캠필로박터 특이 크로모제닉 액체배지 및 그 제조방법
CN106556599A (zh) * 2016-10-25 2017-04-05 上海美凯纯生物科技有限公司 一种微生物快速检测方法

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WO1998045406A1 (fr) * 1997-04-09 1998-10-15 Minnesota Mining And Manufacturing Company Procede et dispositifs pour diviser des liquides d'echantillons biologiques en microvolumes
JP2000000100A (ja) * 1998-06-15 2000-01-07 San Kagaku Kk 大腸菌群検出用試験シート
US20080124754A1 (en) * 2001-12-14 2008-05-29 Grete Lorentzen Rapid method of detection and enumeration of sulfide- producing bacteria in food products
JP2004236574A (ja) * 2003-02-05 2004-08-26 Meiji Yakuhin Kk 細菌性膣症診断用液体培地
KR20150114313A (ko) * 2014-04-01 2015-10-12 건국대학교 산학협력단 증균 단계에서 스크리닝이 가능한 캠필로박터 특이 크로모제닉 액체배지 및 그 제조방법
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