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Rapid process for detecting pathogenic microorganisms

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Publication number
WO1989004372A1
WO1989004372A1 PCT/NO1988/000083 NO8800083W WO1989004372A1 WO 1989004372 A1 WO1989004372 A1 WO 1989004372A1 NO 8800083 W NO8800083 W NO 8800083W WO 1989004372 A1 WO1989004372 A1 WO 1989004372A1
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Prior art keywords
microorganisms
comprises
enzyme
fluorescent
fluorescence
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PCT/NO1988/000083
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French (fr)
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James D. Berg
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Berg James D
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/10Enterobacteria
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06Quantitative determination
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2334/00O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
    • C12Q2334/20Coumarin derivatives
    • C12Q2334/224-Methylumbelliferyl, i.e. beta-methylumbelliferone, 4MU
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/245Escherichia (G)
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/8215Microorganisms
    • Y10S435/822Microorganisms using bacteria or actinomycetales
    • Y10S435/848Escherichia
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/8215Microorganisms
    • Y10S435/822Microorganisms using bacteria or actinomycetales
    • Y10S435/848Escherichia
    • Y10S435/849Escherichia coli

Abstract

A rapid process for detecting pathogenic microorganisms in products for human consumption comprises contacting the microorganisms with a methylumbelliferone substrate. The substrate is hydrolyzed into methylumbelliferone by an enzyme given off by the microorganisms. Hydrolysis is accelerated by sodium lauryl sulfate, which renders the microorganisms more permeable to the substrate, the enzyme, or both. The methylumbelliferone is detected by its fluorescence, either in solution or on an agar medium supporting microcolonies formed from individual microorganisms.

Description

RAPID PROCESS FOR DETECTING PATHOGENIC MICROORGANISMS

This invention relates to processes for detecting pathogenic microorganisms, such as coliform bacteria and heterotrophic microorganisms, and more specifically has reference to processes for such detection in products for human consumption which operate in a sufficiently rapid time as to allow treatment of the product before such consumption if needed.

It has long been recognized that the quality of products for human consumption can be significantly affected by the presence of pathogenic microorganisms. Such products include drinking water, bathing water, food, food preparation equipment, and, in general, any vector which the microorganisms may use to invade the human body. If present in sufficient numbers, such microorganisms can cause product deterioration spoilage, disease, and significant economic loss. However, conventional processes for detecting such microorganisms in such products generally require twenty-four to seventy-two hours to complete. This elapsed time is generally too great to provide information about a decrease in product quality in time to take remedial measures. Three groups of microorganisms are commonly sought to be detected. Total coliform (TC) bacteria are those normally present in the colon or intestine of humans or animals. Fecal coliform (FC) bacteria are those TC bacteria which are generally present in the feces of humans or animals. Heterotrophic plate count (HPC) microorganisms are microorganisms which have an external source of nourishment. In this they are like animals which eat food, but are unlike plants which use photosynthesis to produce an internal source of nourishment from mere sunlight.

The two former groups (TC and FC) are indicators of sanitary quality. A process for their detection is conventionally considered to be "rapid" if it takes less than twenty-four hours to perform. The latter group (HPC) is an indicator of general microbiological quality. A process for its detection is conventionally considered to be "rapid" if it takes less than forty-eight hours to perform.

Recent development in rapid processes for use in water analyses are presented in Part 919, pp. 1031-1033, Standard Methods for the Examination of Water and Wastewater

(APIIA, 1985). They are generally not rapid enough to detect a hygienically significant concentration of microorganisms within the ordinary work shift of eight hours. The prior art includes processes for detecting microorganisms and quantifying the concentration thereof by detecting and quantifying the enzymes which the microorganis give off as they metabolize. The enzyme in turn is detected and quantified by its ability to cleave certain chemicals, known as fluorogenic substrates, into products. At least one of these products, the fluorescent portion, fluoresces; that is, it emits light at one wavelength when irradiated with light of a different wavelength. The fluorogenic substrate, however, does not fluoresce. The presence of fluorescence therefore indicates the presence of bacteria. See, e.g., Findl et al, U.S. Patent No. 4,242,447.

An example of the aforementioned fluorogenic substrates are 4-methylumbelliferone (4-MU) compounds. The

4-methy lumbelliferone product fluoresces upon excitation with a light with a wavelength of about 365 nm to emit a light with a wavelength of about 465 nm. MU derivatives have been used in the prior art. Snyder et al., Appl. Environ. Microbiol., 51 (5): 969-977 (1986); Feng et al., Appl. Environ. Microbiol., 43 (6): 1320-1329 (1982); and Koumura et al., U.S. Patent No. 4,591,554. "Fluorescence" can be measured by measuring the amount of fluorescence after a given incubation time, or by measuring the incubation time needed to obtain a given amount of fluorescence. Processes which do this generally take enough time for the microorganisms to multiply exponentially through several generations. There is, therefore, an exponentially larger amount of fluorescence available to be measured at the end of the incubation period, whether that period is fixed or variable. "Fluorescence" can also be measured by frequently sampling the amount of fluorescence during the first part of the incubation period, and calculating therefrom the fluorescence velocity; that is, the time rate of change in the amount of fluorescence. Processes which do this generally do not take enough time for the microorganisms to multiply exponentially through several generations. Instead, such processes rely on the tendency of the microorganisms which are initially present to produce the same amount of enzyme during each sample period, thereby causing a linearly increasing amount of fluorescence. See Snyder et al., above.

Such rapid processes have many benefits. First, they are, by definition, more rapid than those which wait several generations. Second, their accuracy is good, for two reasons. One, they measure current activity of the initially present microorganisms, rather than having to make deductions from measurements of the activity of microorganisms several generations later. Two, they generate multiple data points, situation which lends itself well to linear regression analysis, rather than generating the single datum produced by the slower processes. Third, the linearity of the data points (or lack of it) is an indicator of the accuracy of the measurement.

Such rapid processes have a drawback as well. The microorganisms must be concentrated enough to produce a measurable amount of fluorescence velocity in less than a generation. When further concentration is not practical, means must be found which enhance the per-organism fluorescence velocity.

It is therefore an object of the present invention to provide a process which enhances the per-organism fluorescence velocity of an enzymatic measurement of a dilute sample of pathogenic microorganisms in a product for human consumption.

It is a further object of the present invention to enhance such velocity while using 4-methylumbelliferone as the fluorescent product. In accordance with one embodiment of the present invention, a process for assaying, in a brief period of time, a dilute concentration of living pathogenic microorganisms in a sample of product for human consumption, comprises six steps.

The first step is contacting the microorganisms of the sample with an actuating medium. The actuating medium comprises: (1) a nutrient which is capable of supporting metabolism of the microorganisms; (2) a production agent which is capable of inducing the production of an enzyme in said microorganisms when the microorganisms are metabolizing; (3) a fluorogenic substrate which is capable of reacting with the enzyme to release the fluorescent portion thereof; and (4) a permeability agent which is capable of increasing the permeability of the microorganisms to the enzyme, the fluorogenic substrate, or both.

The second step is incubating the resultant mixture in an environment which allows, during the period of incubation, metabolism of the microorganisms, production of the enzyme, contacting of the enzyme (whether inside or outside the microorganisms) with the fluorogenic substrate, and release of the fluorescent portion of the fluorogenic substrate.

The third step is irradiating, at intervals during the incubation period, the fluorescent portion with light of a wavelength sufficiently close to that of an excitation wavelength characteristic of the fluorescent portion, and sufficiently intense, as to cause the fluorescent portion to fluoresce. The remaining steps are measuring, at such intervals, the amount of emitted fluorescent light; calculating the velocity of emitted fluorescence; and determining the concentration of microorganisms from such velocity and a preestablished velocity-to-concentration correlation schedule.

In a preferred embodiment, the sample is liquid or has been liquefied, and the actuating medium is liquid. The preferred embodiment further comprises, before the contacting step, concentrating the sample by filtering it through a filter fine enough to retain the microorganisms, the subsequent contacting step being accomplished by placing the filter in the actuating medium.

In this embodiment, the production agent comprises lactose, the fluorescent portion comprises

4-methylumbelliferone, the permeability agent comprises sodium lauryl sulfate, and the fluorescent portion is caused to fluoresce with emitted light of a wavelength of about 465 nm by being excited with light of a wavelength of about 365 nm.

When it is desired to measure heterotrophic plate count mi cro o rganisms in a concentration of at least 500 microorganisms per 100 milliliters in at least 15 minutes, the enzyme comprises lipase or esterase, the fluorogenic s u b s t ra t e comprises 4-methylumbelliferone-heptanoate, and the environment comprises a temperature of about 20°C. When it is desired to measure coliform bacteria in a concentration of at least 60 coliforms per 100 milliliters in at least 15 minutes, the enzyme comprises B-D-galactosidase, and the f luorescent substrate comprises 4-methylumbelliferone-B-D-galactoside. For total coliforms the environment comprises a temperature of about 35°C. For fecal coliforms, the environment comprises a temperature of abou t 41.5°C.

In accordance with a second embodiment of the present invention, a process for assaying living pathogenic microorganisms in a liquid or liquefied sample of product for human consumption in a concentration of at least 1 microorganism per 100 milliliters, and in a time of at least six hours, comprises five steps.

The first step is filtering the sample through a filter fine enough to retain the microorganisms. The second step is placing the filter and microorganisms against a solid or at least semi-solid substance. The substance comprises the same constituents as are comprised in the actuating medium of the first embodiment of the present invention.

The third step is incubating the substance, filter, and microorganisms in an environment which allows, during the period of incubation, metabolism and reproduction of the microorganisms, production of the enzyme, contacting of the enzyme (whether inside or outside the microorganisms) with the fluorogenic substrate, and release of sufficient of the fluorescent portion of the fluorogenic substrate from each single microorganism and its descendants to form a visible microcolony under fluorescent conditions.

The fourth step is irradiating the microcolonies with light of a wavelength sufficiently close to that of an excitation wavelength characteristic of the fluorescent portion, and sufficiently intense, as to cause the microcolonies to fluoresce. Finally, one simply counts the number of fluorescent microcolonies.

In a preferred embodiment an alkaline solution is added to the microcolonies at the end of the incubation period, thereby enhancing fluorescence and facilitating counting; and the substance against which the filter and microorganisms is placed comprises agar. The remainder of the preferred embodiment of this second embodiment is the same as the remainder of the preferred embodiment of the first embodiment, and detects the same microorganisms.

The above and other important features of the present invention will be better understood from the following detailed description, made with reference to the following drawings, in which: FIG. 1 is a graph showing fluorescence production by coliform bacteria in drinking water contaminated with raw sewage (solid circles), with the addition of lactose (open circles), and with the addition of sodium lauryl sulfate (triangles);

FIG. 2 is a graph correlating fluorescence velocity with total coliform concentration, with error bars indicating the range obtained when replicate samples (open circles) are incubated from one original sample; FIG. 3 is a graph similar to FIG. 2, except that it correlates fluorescence velocity with fecal coliform concentration rather than with total coliform concentration;

FIG. 4 is a photograph of the agar of the second embodiment, showing individually fluorescing microcolonies; FIG. 5 is a graph correlating measurements obtained from the agar process with comparable measurements obtained from the conventional M 7h FC process;

FIG. 6 is a graph similar to FIG. 1, except that it shows heterotrophic plate count (HPC) activity rather than coliform activity;

FIG. 7 is a graph similar to FIGS. 2 and 3, except that it correlates fluorescence velocity with HPC activity rather than total coliforms, and omits replicate samples; and

FIG. 8 is a graph similar to FIG. 7, except that, instead, of drinking water, it shows results from water sampled from a fish farm (open circles during start up; solid circles during operation) and a nearby river (dashed line). The present invention makes use of the enzyme activity living microorganisms, particularly bacteria but including the fungi, to detect their presence and to quantify them. Enzymes unique to a group of microorganisms can be further used to identify the group to the exclusion of others present in the sample.

Specifically, the present invention uses enzyme activity as determined by the detection of MU-derived enzyme products to detect and quantify two groups of microorganisms, the coliform group of bacteria and heterotrophic microorganisms.

As noted above, the coliform group includes the total coliforms (TC) and the fecal coliforms (FC). These are considered indicators of the sanitary quality of food, water, or process equipment; that is, their presence is generally thought to indicate contamination by fecal material. The coliform group constituents possess the enzyme

B-D-galactosidase. The present invention detects this enzyme by the use of 4-MU-B-D-galactoside in the presence of known selective ingredients to exclude the activity and presence of any other B-D-galactosidase positive non-coliforms. TC and FC are differentiated on the basis of temperature: TC are detected at 35°C; and FC are detected at 41.5°C. Heterotrophic activity is commonly measured by detecting heterotrophic bacteria or fungi as colonies appearing on various solid media (e.g. Nutrient agar) or as positive growth in liquid media (e.g. MPN Tube test) after an incubation period of typically twenty-four to seventy-two hours for mesotrophic heterotrophs. For bacteria the result is a heterotrophic plate count (HPC) value which, as noted above, gives a general indication of the microbiological quality of the food, water, or equipment in question. The lipase/esterase group of enzymes is common to the heterotrophs during aerobic or anaerobic conditions of growth. The p resen t invention therefore employs lipase/esterase activity, detecting it with 4-MU-heptanoate, in a similar manner as described for the coliforms herein.

The general procedure for the detection of TC, FC, or heterotrophic activity in the present invention is as follows:

(a) the sample is concentrated by passing it through a membrane filter (0.2 urn to 0.80 urn pore size);

(b) the microorganisms which are retained with the filter are aseptically placed in contact with a sterile medium containing the appropriate 4-MU-substrate; and

(c) the resulting fluorescence is measured and utilized as

(1) the rate of production of fluorescent product in the liquid medium associated with the sample determined at regular intervals over about fifteen minutes using a fluorescence detecting meter, or

(2) the direct observation by eye of fluorescent product associated with colonies resulting from growth (division) of the bacteria originally retained on the filter during the concentration step.

TC are detected in the manner described above by incubation of the sample plus 4-MU-B-D-galactoside at 35°C, whereas FC are detected by like incubation at 41.5°.

Heterotrophic activity is detected in the manner described above by incubation of the sample plus 4-MU-heptanoate at 20°C.

The invention is illustrated by the following examples

EXAMPLE 1

Total coliform (TC) bacteria were analyzed in the following manner. Natural populations of coliforms were obtained from a river (Nidelven, Trondheim, Norway) receiving sewage effluent and from fresh raw domestic sewage. Samples were collected daily for experiments and diluted in tap water in varying concentrations to model drinking water with fecal coliform (FC) and nonspecific heterotrophic plate count (HPC) bacterial contamination. Concentration of coliforms ranged from 1/100 ml to 1000/100 ml.

500 ml of a water sample were filtered through a 0.45 um pore size 47 mm diameter membrane filter (Millipore) and aseptically placed in a 250 ml flask containing 18 ml of sterile buffered actuating medium. (The 500 ml sample size was chosen as the maximum volume of surface water of turbidity < 5 NTU that could be filtered in a short period of time). The medium consisted of phosphate buffered saline (PBS; pll 7.3), 0.02% sodium lauryl sulfate (Sigma Chemical Co., St. Louis, Missouri), 0.56% nutrient broth (Difco Laboratories, Detroit, Michigan), and 0.35% lactose. One of the fluorogenic substrates was added to each flask and to a sterile control flask containing the sterile medium, then placed in a shaking water bath. 4-methylumbelliferone-B-D-galactoside (4-MU-B-D-galactoside) was added at a concentration equal to 0.05 mg/ml. The samples were incubated at 35°C for determining TC activity, and at 41.5°C for determining FC activity. Flasks were removed from the bath and placed in a fluorimeter (Turner Model 111) every five minutes for thirty minutes to measure the initial fluorescence velocity. The excitation and emission wavelengths for methylumbelliferone are about 365 and 465 nm using light filters 7-60 and 2A+47B respectively. Fluorescen velocity was determined by least squares linear regression.

The rapid fluorescence processes were compared with the conventional enumeration techniques described below. Heterotrophic plate count (HPC) bacteria were recovered on SPC (Difco) or R2A agar and counted after three days incubation at 20°C. Total coliform (TC) bacteria were assayed with the membrane filtration technique using m-ENDO agar (Difco) and incubated for twenty-four hours at 35°C. The seven h o u r and standard twenty-four hour membrane filtration processes (APHA, 1985) were used to recover fecal coliform (FC) at 41.5°C and TC at 35°C respectively. Peptone (0.1%) was used for all serial dilutions. All assays were done according to Standard Methods (APIIA, 1985). Production of methylumbelliferone from 4-MU-B-D-galactoside by coliform bacteria in drinking water contaminated with raw sewage (solid circles) is shown in FIG. 1. Addition of lactose enhanced induction of R-D-galactosidase (open circles). Addition of sodium lauryl sulfate, a common selective ingredient for coliforms, further enhanced activity

(triangles). Enzyme assay temperature was 35°C.

The addition of lactose and sodium lauryl sulfate to the medium enhanced galactosidase activity. The specificity of MU-glactosicle for members of the coliform group was tested by pretreating water samples with common inhibitors employed in coliform media (Standard Methods, 1985). Most of the activity was retained after pretreatment suggesting that the galactosidase activity was attributable to the coliform group as defined by the conditions of the assay. Replacement of sodium lauryl sulfate by the selective agents used in mT7h medium, polyethylene ether WU-1 and tergitol 7 did not enhan activity (data not shown). Linearity of the rate of product formation was generally high, r = .99 for most assays. Therefore the duration of the assay in the remaining experiments was decreased from 120 minutes to 30 minutes, although 15 minutes was sufficient. FIG. 2 shows the correlation between initial rate of hydrolysis of 4-MU-B-galactoside, as measured by initial fluorescence velocity, and the concentration of total coliform bacteria derived from raw sewage and mixed in drinking water. Enzyme assay temperature was 35°C. Error bars, associated with open circles represent the range of fluorescence velocity obtained when replicate samples (5 < n < 8) are incubated from one original sample.

EXAMPLE 2

Fecal coliform (FC) bacteria were determined in the same manner as in Example 1 except that an incubation temperature equal to 41.5°C was used.

The results are shown in FIG. 3. Initial rate of hydrolysis of 4-MU-B-D-galactoside by fecal coliform bacteria derived from raw sewage and mixed in drinking water, as measured by initial fluorescence velocity, is compared with fecal coliform bacterial concentration. Enzyme assay temperature was 41.5°C. Error bars as socia ted with open circles have the same meaning as in FIG. 2. EXAMPLE 3

This example demonstrates direct counting of FC. Concentrations of FC < 100/100 ml were analyzed by direct counting. Water samples were filtered and placed on a solid or at least semi-solid substance, namely M 7h FC agar (Standard Methods, 1985) lacking the pll indicators and d-mannitol but containing 4-MU-B-D-galactoside. Fluorescent galactosidase positive microcolonies were visible after six hours incubation at 41.5°C with a 366 nm long wave ultraviolet light source (UVL-56, Ultraviolet Products, San Gabriel, California). The addition of 0.2 ml of an alkaline solution, namely 0.1 N NaOH, after the incubation period increased the fluorescence and facilitated counting.

FIG. 4 shows an example of the appearance of the microcolonies after six hours of incubation. FIG. 5 compares concentrations as measured by the agar direct counting process with concentrations measured by the conventional M 7h FC process. Pluses represent raw sewage contaminated drinking water stored at 15°C and sampled after twenty-four to seventy-two hours for coliforms, and solid circles represent river water samples. The dashed line represents the ideal correlation, and is not fit to the data. The average ratio of MU 6h MF to M 7h FC was 1.21 to 1 for thirty-four samples. Sixty-two fluorescing colonies randomly selected from three experiments showed 100% confirmation as FC by gas production in lauryl tryptose broth incubated at 35°C for twenty-four hours and in EC broth incubated at 44°C for twenty-four hours. In all experiments HPC concentrations ranged from 300/ml to 50,000/ml.

EXAMPLE 4

Heterotrophic plate count (HPC) activity was determined in water samples from a river and a drinking water tap. 500 ml of sample was filtered through a 0.45 um membrane filter and placed in a flask containing phosphate buffered saline

(pll 7.3).

The flasks were placed in a shaking water bath at 20°C and 4-MU-heptanoate was added at a concentration equal to 0.05 mg/ml. Fluorescence production was measured in the same manner as in Example 1. The rate of production of methylumbelliferone is shown in FIG. 6, and is compared with HPC enumeration in

FIG. 7.

EXAMPLE 5

Another source of heterotrophic plate count activity was chosen, namely, the water in a recirculation fish farm. Fluorescence velocity is compared with heterotrophic activity in FIG. 8. The samples were taken during a start-up period in 1986 (open circles) and several months later in 1987 (solid circles) when the bacterial population composition had shifted as a function of the changing aquatic environment. The fish farm results are compared with the heterotrophic plate count activity from a nearby river, marked as dashes in FIG. 8 and labeled "Nidelva". The samples were handled in the same manner as described in Example 4.

Claims

P A T E N T C L A I M S
A process for assaying, in a brief period of time, a dilute concentration of living patliogenic microorganisms in a sample of product for human consumption, the proces comprising:
(a) contacting the microorganisms of the sample with an actuating medium comprising:
(1) a nutrient which is capable of supporting metabolism of the microorganisms:
(2) a production agent which is capable of inducing the production of an enzyme in said microorganisms when the microorganisms are metabolizing;
(3) a fluorogenic substrate which is capable of reacting with the enzyme to release the fluorescent portion thereof; and
(4) a permeability agent which is capable of increasing the permeability of the microorganisms to the enzyme, the fluorescent substrate, or both; (b) incubating the resultant mixture in an environment which allows, during the period of incubation:
(1) metabolism of the microorganisms;
(2) production of the enzyme;
(3) contacting of the enzyme (whether inside or outside the microorganisms) with the fluorescent substrate; and
(4) release of the fluorescent portion of the fluorescent conjugate;
(c) irradiating, at intervals during the incubation period, the fluorescent portion with light of a wavelength sufficiently close to that of an excitation wavelength characteristic of the fluorescent portion, and sufficiently intense, as to cause the fluorescent portion to fluoresce;
(d) measuring, at such intervals, the amount of emitted fluorescence;
(e) calculating the velocity of emitted fluorescence;
(f) determining the concentration of microorganisms from such velocity and a preestablished velocity-to- concentration correlation schedule.
2. The process of Claim 1, wherein the sample is liquid or has been liquefied, and wherein the actuating medium is liquid; the method further comprising, before the contacting step, concentrating the sample by filtering it through a filter fine enough to retain the microorganisms, the subsequent contacting step being accomplished by placing the filter in the actuating medium.
The process of Claim 2, wherein:
(a) the production agent comprises lactose;
(b) the fluorescent portion comprises 4-methylumbelliferone;
(c) the permeability agent comprises sodium lauryl sulfate; and
(d) the fluorescent portion is caused to fluoresce with emitted light of a wavelength of about 465 nm by being excited with light of a wavelength of about 365 nm.
4. The process of Claim 3, wherein:
(a) the enzyme comprises lipase or esterase;
(b) the fluorogenic substrate comprises 4-methylumbelliferone-heptanoate;
(c) the microorganisms comprise heterotrophic plate count microorganisms;
(d) the environment comprises a temperature of about 20°C; and
(e) the concentration is at least 500 microorganisms per 100 milliliters, and the period of time is at least 15 minutes.
5. The process of Claim 3, wherein:
(a) the enzyme comprises B-D-galactosidase;
(b) the fluorogenic substrate comprises 4-methylumbelliferone-B-D-galactoside;
(c) the microorganisms comprise coliform bacteria; and
(d) the concentration is at least 60 microorganisms per 100 milliliters, and the period of time is at least 15 minutes.
The process of Claim 5, wherein:
(a) the microorganisms comprise total coliforms; and
(b) the environment comprises a temperature of about 35°C.
7. The process of Claim 5, wherein:
(a) the microorganisms comprise fecal coliforms; and
(b) the environment comprises a temperature of about 41.5°C.
8. A process for assaying living pathogenic microorganisms a liquid or liquefied sample of product for human consumption in a concentration of at least 1 microorgan per 100 milliliters, and in a time of at least six hours, the process comprising:
(a) filtering the sample through a filter fine enough to retain the microorganisms;
(b) placing the filter and microorganisms against a solid or at least semi-solid substance comprising:
(1) a nutrient which is capable of supporting metabolism and reproduction of the microorganisms;
(2) a production agent which is capable of inducing the production of an enzyme in said microorganisms when the microorganisms are metabolizing;
(3) a fluorogenic substrate which is capable of reacting with the enzyme to release the fluorescent portion thereof; and
(4) a permeability agent which is capable of increasing the permeability of the microorganisms to the enzyme, the fluorogenic substrate, or both; (c) incubating the substance, filter, and microorganism in an environment which allows, during the period of incubation:
(1) metabolism and reproduction of the microorganisms;
(2) production of the enzyme;
(3) contacting of the enzyme (whether inside or outside the microorganisms) with the fluorogenic substrate; and
(4) release of sufficient of the fluorescent portion of the fluorogenic substrate from each single microorganism and its descendants to form a visible microcolony under fluorescent conditions;
(d) irradiating the microcolonies with light of a wavelength sufficiently close to that of an excitation wavelength characteristic of the fluorescent portion, and sufficiently intense, as to cause the microcolonies to fluoresce; and
(e) counting the number of fluorescent microcolonies.
The process of Claim 8, further comprising adding an alkaline solution to the microcolonies at the end of the incubation period, whereby fluorescence is enhanced and counting is facilitated.
10. The process of Claim 9, wherein:
(a) the production agent comprises lactose;
(b) the fluorescent portion comprises 4-methylumbelliferone;
(c) the permeability agent comprises sodium lauryl sulfate;
(d) the enzyme comprises lipase or esterase;
(e) the substance against which the filter and microorganisms is placed comprises agar; and
(f) the fluorescent portion is caused to fluoresce with emitted light of a wavelength of about 465 nm by being excited with light of a wavelength of about 365 nm.
11. The process of Claim 10, wherein:
(a) the enzyme comprises lipase or esterase;
(b) the fluorogenic substrate comprises 4-methylumbelliferone-heptanoate;
(c) the microorganisms comprise heterotrophic plate co microorganisms; and
(d) the environment comprises a temperature of about 20°C.
12. The process of Claim 10, wherein:
(a) the enzyme comprises B-D-galactosidase;
(b) the fluorogenic substrate comprises 4-methylumbellif erone-B-D-galac tosid e; and
(c) the microorganisms comprise coliform bacteria.
13. The process of Claim 12, wherein:
(a) the microorganisms comprise total coliforms; and
(b) the environment comprises a temperature of about 35°C.
14. The process of Claim 12, wherein:
(a) the microorganisms comprise fecal coliforms; and
(b) the environment comprises a temperature of about 41.5°C.
PCT/NO1988/000083 1987-11-05 1988-11-03 Rapid process for detecting pathogenic microorganisms WO1989004372A1 (en)

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EP19880909391 EP0386051B1 (en) 1987-11-05 1988-11-03 Rapid process for detecting coliform microorganisms
DE19883852825 DE3852825T2 (en) 1987-11-05 1988-11-03 Quick method for detecting coliform microorganisms.
DE19883852825 DE3852825D1 (en) 1987-11-05 1988-11-03 Quick method for detecting coliform microorganisms.

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DE3852825T2 (en) 1995-05-18 grant
EP0386051A1 (en) 1990-09-12 application
EP0386051B1 (en) 1995-01-18 grant
DE3855762D1 (en) 1997-02-27 grant
US5292644A (en) 1994-03-08 grant
DE3852825D1 (en) 1995-03-02 grant
DE3855762T2 (en) 1997-05-07 grant
EP0574977A1 (en) 1993-12-22 application
EP0574977B1 (en) 1997-01-15 grant

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