WO2019236309A1

WO2019236309A1 - Method for identifying and enumerating specific bacterial populations in an unenriched liquid sample

Info

Publication number: WO2019236309A1
Application number: PCT/US2019/033648
Authority: WO
Inventors: Michael P. Perry
Original assignee: Dupont Nutrition Biosciences Aps; E. I. Du Pont De Nemours And Company
Priority date: 2018-06-04
Filing date: 2019-05-23
Publication date: 2019-12-12

Abstract

A method for identifying and enumerating specific bacterial populations in an unenriched liquid sample is disclosed.

Description

TITLE

METHOD FOR IDENTIFYING AND ENUMERATING SPECIFIC BACTERIAL POPULATIONS IN AN UNENRICHED LIQUID SAMPLE

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 62/680,248, filed June 4, 2018 and to U.S. Provisional Patent Application No. 62/791,430, filed January 11, 2019, the disclosures of each of which are incorporated by reference in their entireties.

FIELD OF INVENTION

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.

BACKGROUND OF THE INVENTION

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.

Growth-based detection and enumeration of microorganisms is commonly practiced using either liquid nutrient media (most probable number analysis (MPN)) or 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. For the 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 10⁹ 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.

Most probable number (MPN) is a quantitative measurement of the concentration of microbes in a given medium. This method uses turbidity measurements of serial dilutions (to extinction) of a bacterial suspension to determine cell concentration. The last dilution where growth is observed is mathematically converted to the number of cells in the original sample.

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.

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. For example, E. colt can be enriched for by plating the sample onto a specific E. colt- selective agar medium such as CHROMagar™ (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 AquaCHROM™ ECC. This product detects (but does not enumerate) the presence or absence of E. coli and coliforms in 100 ml water samples.

There are cases, however, where plating onto this selective medium is not practical in the field and may take 24 to 48 hours for the colonies to appear on the agar plates. Therefore, there is a need for a simple, on-site procedure to identify and enumerate specific bacteria in a sample such as a biological, clinical or environmental sample such as water, soil, fecal material, surface swabs, etc. The procedure should be relatively simple and results should be available within one eight-hour work shift.

SUMMARY OF THE INVENTION

In one embodiment, there is disclosed a method for identifying and enumerating one or more specific microorganisms in an unenriched liquid sample comprising:

a) providing the unenriched sample; and

b) enriching the sample for growth of a specific genera or one or more species of microorganisms using microorganism-specific selective medium; and

c) 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).

In a second embodiment, there is disclosed a method for identifying and enumerating one or more specific microorganisms in an unenriched liquid sample comprising:

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 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).

In a third embodiment, the microorganisms that are identified and enumerated are selected from the group consisting of Escherichia, Enterobacter, Clostridia, Staphylococcus, Streptococcus, Bacilli, Salmonella, and Listeria.

In a fourth embodiment, the reporter system is a colorimetric reporter system. In a fifth embodiment, the microorganisms are identified and enumerated between about 4-9 hours following commencement of step (a).

In a sixth embodiment, the medium lacks agar or any other substance capable of solidifying under specific conditions.

BRIEF DESCRIPTION OF FIGURES OF THE INVENTION

Figure 1 sets forth absorbance spectra of E. coli grown with and without the modified CHROMagar™ 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 CHROM™ 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.

DETAILED DESCRIPTION

All patents, patent applications, and publications cited are incorporated herein by reference in their entirety.

In this disclosure, a number of terms and abbreviations are used. The following definitions apply unless specifically stated otherwise. The articles“a”,“an”, and“the” preceding an element or component are intended to be nonrestrictive regarding the number of instances (i.e., occurrences) of the element or component. Therefore“a”,“an”, and“the” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

The term“comprising” means the presence of the stated features, integers, steps, or components as referred to in the claims, but that it does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. The term“comprising” is intended to include embodiments encompassed by the terms“consisting essentially of’ and “consisting of’. Similarly, the term“consisting essentially of’ is intended to include embodiments encompassed by the term“consisting of’.

Where present, all ranges are inclusive and combinable. For example, when a range of“1 to 5” is recited, the recited range should be construed as including ranges“1 to 4”,“1 to 3”,“1-2”, “1-2 & 4-5”,“1-3 & 5”, and the like.

As used herein in connection with a numerical value, the term“about” refers to a range of +/- 0.5 of the numerical value, unless the term is otherwise specifically defined in context. For instance, 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.

It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

The term“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.

The term“unenriched sample” or“unenriched liquid sample” refers to the original sample prior to subjecting it to any means for enrichment.

The terms“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.

The terms“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).

The term“CFU” as used herein 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.

The term“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. Such 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. In addition, 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). However, depending on the means available, 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.

The term“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) providing the unenriched sample; and

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. There can be mentioned the 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, the 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. Preferably, the reporter system is a colorimetric reporter system. It also should be simple and fast to use in the field.

Non-limiting examples of compositions and methods disclosed herein include:

1. A method for identifying and enumerating one or more specific microorganisms in an unenriched liquid sample comprising:

a) providing the unenriched sample; and

2. A method for identifying and enumerating one or more specific microorganisms in an unenriched liquid sample comprising:

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).

3. The method of embodiment 1 or 2 wherein the microorganisms are selected from the group consisting of Escherichia, Enter obacter, Clostridia, Staphylococcus,

Streptococcus, Bacilli, Salmonella, Listeria.

4. The method of embodiment 1 or 2 or 3 wherein the reporter system is a colorimetric

reporter system.

5. The method of embodiment 1 or 2 or 3 or 4, wherein the microorganisms are identified and enumerated between about 4-9 hours following commencement of step (a). 6. The method of embodiment 1 or 2 or 3 or 4 or 5, wherein the medium lacks agar or any other substance capable of solidifying under specific conditions.

EXAMPLES

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al. , DICHONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY , 2D ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE

HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with a general dictionary of many of the terms used with this disclosure.

The disclosure is further defined in the following Examples. It should be understood that the Examples, while indicating certain embodiments, is given by way of illustration only. From the above discussion and the Examples, one skilled in the art can ascertain essential

characteristics of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt to various uses and conditions.

Additional abbreviations used in the Examples:

“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.

EXAMPLE 1

LIQUID CULTURE-BASED DETECTION OF GROWTH OF E COLI IN THE PRESENCE

OF MODIFIED CHROMAGAR™ MEDIUM

Modified CHROMagar™ medium (from CHROMagar™, 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 CHROMagar™. The CHROMagar™ 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

CHROMagar™ medium. The two cultures were grown overnight at 37 °C. The next day, only the CHROMagar™-containing culture turned blue. Both samples were scanned on a

spectrophotometer from 400-700 nm. An additional sample containing the CHROMagar™ and E. coli was spiked with blue food coloring dye in order to highlight the blue absorbance peak (circled line in Figure 1). Although the difference in the absorbance between the E. coli minus CHROM (triangled line) and the A. coli plus CHROM (squared line) is minimal (Figure 1), with better optimization and better measurements of the blue intensity, it is anticipated that this method can be used to quantify the amount of E. coli present in the sample.

EXAMPLE 2

LIQUID CULTURE-BASED DETECTION AND ENUMERATION OF GROWTH OF E COLI IN THE PRESENCE OF CHROMAGAR™ LIQUID ECC MEDIUM

To determine whether CHROM™ liquid ECC medium (from CHROMagar, Paris,

France) 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 mΐ or 50 mΐ of the overnight culture was added to a IX or a 2X concentration of the CHROM™ 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).

It is anticipated that these growth conditions could be optimized so that the color intensity after a certain point of optimal growth could be correlated to the number of cells in the original unenriched liquid sample. For example, known quantities of a microorganism, such as E. coli , (10 CFU/ml, 100 CFU/ml, 1000 CFU/ml) can be grown in the presence of a chromogenic reporter substrate for a finite period of time but still within the log phase of growth, e.g ., 6 hrs. The blue intensities of the solutions would then be measured and plate counts would be performed to count the actual number of E. coli in the solutions. Accounting for an approximate doubling time of 20 min for E. coli growth, the corresponding plate counts after 6 hours should be about 2 X 10⁶ CFU/ml, 2 X 10⁷ CFU/ml, and 2 X 10⁸ 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. These growth parameters would have to be developed experimentally to account for the lag phase of growth as well as different doubling times for different bacteria and would be standardized accordingly.

Once the growth parameters are standardized and the blue intensity of the medium is optimized to correspond to a certain amount of E. coli in the samples, it is believed that this technology would be useful in detecting and enumerating a microorganism such as E. coli in a variety of unenriched liquid samples. For example, 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.

Enumeration can be demonstrated as follows:

A. Correlate intensity of chromogenic reporter with the number of cells.

Grow and enrich a culture of a microorganism, such as E. coli , using a microorganism- specific medium and a chromogenic reporter under standardized conditions.

Measure CFU/ml (by plate counts) every hour while also measuring the intensity of the chromogenic reporter at the same hourly timepoints. For the plate counts, 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. After incubation of the plates (for example, 24 hr at 37 °C.), 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.

Accordingly, an overnight culture of E. coli (in Tryptic Soy Broth, TSB) was diluted to 100 CFU/ml, in 50 ml of IX CHROMagar™ 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. The cell-free supernatants were then scanned from 500 to 700 nm in a BioTek Synergy MX microplate reader (BioTek, Winooski, VT). Two maximum peaks were noted in the blue-green spectrum at 610 nm and 650 nm. Figure 3 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 10⁹ CFU/ml and continued increasing in intensity the longer the cells grew but started to plateau at 24 hours of growth.

B. Correlating the intensity of chromogenic reporter back to starting CFU/ml

concentration.

Known quantities ( e.g ., 10 CFU/ml, 100 CFU/ml, 1000 CFU/ml) of a microorganism, such as E. coli , are grown using 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.

Accordingly, to determine that the color intensity after optimal growth could be correlated to the number of cells in the original unenriched liquid sample, known quantities of E. coli , were grown in IX CHROMagar™ Liquid ECC medium. Briefly, 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. Cell counts were determined at each timepoint by the MPN method. The 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. The cell-free supernatants were then scanned from 500 to 700 nm in a BioTek Synergy MX microplate reader. Two maximum peaks were noted in the blue-green spectrum at 610 nm and 650 nm. Figure 4 shows the absorbance peak at 610 nm for each of the starting concentrations. Figures 5-8 show the results of both the growth and blue intensity (at 610 nm) for each of the individual starting concentrations. For the 100 CFU/ml starting concentration, the culture started turning blue after about 8 hours. For the 1000 CFU/ml starting concentration, blueness was observed after about 7 hours. For the 10000 CFU/ml starting concentration, blueness of the medium was observed after about 6 hours. And for the 100000 CFU/ml starting concentration, blueness was observed after about 5 hours. Therefore, to quantify the amount of E. coli in an original unenriched liquid sample, cultures should be grown and the blue intensity monitored hourly after 4 hours of growth. If samples start turning blue from 5 to 6 hours, there was approximately 100000 CFU/ml E. coli in the original sample. If samples start turning blue from 6 to 7 hours, there was approximately 10000 CFU/ml in the original sample. If samples start turning blue from 7 to 8 hours, there was approximately 1000 CFU/ml in the original sample. And, if samples start turning blue from 8 to 9 hours, there was approximately 100 CFU/ml in the original sample.

C. Test 8-10 different isolates of a microorganism such as E. coli . to determine if the assay is useful in enumerating a microorganism in an unenriched liquid sample.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method for identifying and enumerating one or more specific microorganisms in an unenriched liquid sample comprising: a) providing the unenriched sample; and b) enriching the sample for growth of a specific genera or one or more species of microorganisms using microorganism-specific selective medium; and c) 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).

2. A method for identifying and enumerating one or more specific microorganisms in an unenriched liquid sample comprising: a) enriching the unenriched sample for growth of a genera or one or more species of microorganisms using a microorganism-specific selective medium; and 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)

3. The method of claim 1 or 2, wherein the microorganisms are selected from the group consisting of Escherichia, Enterobacter, Clostridia, Staphylococcus, Streptococcus, Bacilli, Salmonella, and Listeria.

4. The method of any one of claims 1-3, wherein the reporter system is a colorimetric reporter system.

5. The method of any one of claims 1-4, wherein the microorganisms are identified and enumerated between about 4-9 hours following commencement of step (a).

6. The method of any one of claims 1-5, wherein the medium lacks agar or any other substance capable of solidifying under specific conditions.