MXPA97003314A - Means to detect enterococes in one sample - Google Patents

Abstract

The present invention relates to means for developing enterococci so that detection can be obtained within 24 hours.

Description

MEDIO Pfí fl DETECTOR EMTEROCOCOS IN ONE SAMPLE FIELD OF L INVENTION This invention is in the field of chemistry, biology and microbiology, and refers to novel means to detect the presence of target microbes in a sample of a possibly contaminated material.

BACKGROUND Microorganisms are ubiquitously present in biological samples and in environmental media suitable for their development. However, some prove to be harmful to higher organisms and it is important to have means to detect their presence in order to maintain public health. Many means are available to detect various types of microorganisms that offer various advantages with respect to speed and specificity. All microorganisms have certain requirements for their development and reproduction. In general, microorganisms require the presence of the following to develop: an energy source, which includes light and carbon compounds; and a source of raw materials that include carbon, nitrogen, sulfur and phosphorus, as well as oligomeric amounts of other minerals. In addition, the microorganisms must be present in a suitable environment, in which an appropriate temperature, pH, salinity and oxygen concentration is maintained. A common procedure used to detect the presence of microorganisms involves adding a specimen to a culture medium that contains all the necessary elements to allow its development. The sample can be natural or previously treated, for example. by membrane filtration, before being added to the culture medium; and the medium may or may not contain chemical substances such as antimetabolites or antibiotics, which are selectively active against microorganisms other than the target microorganisms. Usu- ally, these culture media have been sterilized to ensure that there is no interference by contaminating microbes and, usually, a rather long incubation step of 48 to 72 hours has been necessary to give proper detection of The entorococos. In addition, once development is detected in these procedures, the target microorganism must be confirmed using one or more of several tests, specific to a variety of physical and biochemical characteristics. Therefore, these procedures are very laborious and time consuming. Many efforts have been made to simplify and expedite detection procedures. Among these efforts have been attempts to measure specific byproducts of individual bacteria, electrical impedance analysis, ATP analysis and analysis with carbon-14-labeled substrate. Most have been shown not to be very effective. In addition, microbial development indicators that change color only after the microbe develops have been used. They usually react chemically with a rnebolic by-product produced by the target microbes. Chemicals that change color in the presence of pH changes associated with development, including phenol, bro- nocresol blue, and ro or neutral, have also been used. For example, Golber, in U.S. Patent No. 3,205,317, uses phenol red in the presence of an acid medium produced by the bacterial waste products. Berger and co-inventors, in U.S. Patent No. 3,496,066, describe the use of compounds that bacteria convert to food. Bochner, in U.S. Patent No. 4,129,483, discloses the use of a non-degradable substance that is reduced to produce a color change. In all these situations, the indicator is an additional substance and not one that also serves as a source of a required nutrient. Edberg, in U.S. Patent No., 925,78-., Describes the use of a nutrient indicator that serves not only as a source of nutrients, but also changes color when metabolized. The patent, incorporated herein by reference, provides a means that contains a nutrient indicator which, when etabolized by a target bacterium, releases a portion that imparts a color or other detectable change to the medium. The procedure takes advantage of the unique enzyme specificity for the particular species or particular groups of bacteria. It suggests the use of antibiotics to select the development of target microorganisms and provides specific examples of analysis based on fluids. Other previously used methods, such as Kilian and coauthors, Acta. Path. nicrobiol. Scand. , Sect. B paragraph 7 271-275 (1979) and Darnare co-authors, 3. Food Science. 50: 1735 (1985) report the use of agar-based media, without antibiotics. Enterococcus density is a predictor of public health risks associated with contaminated recreational waters. There are two accepted methods for the analysis of the density of enterococci in water samples; the multiple tube for the most probable number technique (MPN) and the membrane filter technique (MF) (Greenberg and co-authors, Standard methods for the evaluation of water and asteiater.) Eaton, AD (ed.) 18th edition, American Public Health Association (1992) and Mooney, K and coauthors, Testinq the waters: a national persoective on beach closin, Natural Resources Befesa Council, (1992)). Results based on the multiple tube technique may not be available for 72 hours, and the results of the membrane filter technique may not be available for 48 hours. The "MPN procedure involves a presumptive test of 24 to 48 hours in a series of azide and dextrose broth, followed by a 48-hour confirmatory test, using Enterococcus to selectively and 5.5% of brain infusion broth. The membrane technique involves the membrane filtration of water samples, followed by the incubation of a previously filtered sterile membrane in a medium selective for enterococci, and the selection means are E agar, followed by a substrate test ETA or rnEnterococcus aci. These methods can be tedious, laborious and time-consuming, which can lead to delays in notifying the public and, therefore, increase risks to public health.

BRIEF DESCRIPTION OF THE INVENTION The present invention incorporates a medium that allows the detection of entorococoe microbes in a liquefied, environmental or biological sample within just 24 hours. The present medium is different from previous media in which it takes about 48 to 72 hours to obtain an enterococcal test result. The medium also allows to quantify the amount of enterococci present in a sample and can be used * in rapid discriminatory methods. The medium contains an effective amount of the ingredients vitamin, amino acids, oligogenic elements and salt, operable to allow viability and reproduction of enterococci, in the presence of a nutrient indicator. The nutrient indicator is provided in an amount sufficient to allow a detectable characteristic signal to be produced in the medium, through the development of enterococci. The medium additionally contains effective amounts of selective agents that are active to prevent or inhibit the growth of non-target microorganisms (ie, which are not enterococci). Means that have been shown to be optimal in this invention, for the detection of enterococci in a sample, include (per liter) a biological regulator (eg, about 5.0 to 7.0 grams of N-tris (hydroxymethyl) methyl) -3-aninopropansul phonic, free acid (TAPS-free acid) and about 5.0 to 7.0 grams of the sodium salt of N-tris (hydroxymethyl) metii-3 ~ arninoprOpan-sulphonic acid, (TAPS-sodium) or about 4.0 to 5.0 grams of HEPES, free acid, and about 7.0 to 9.0 grams of HEPES-sodium salt)); and sodium bicarbonate (for example, about 1.5 to 2.5 grams). In addition, the following components are provided in the medium, approximately in the amounts indicated. Those who are experts in the field will understand that not all the components are necessary. You can also replace some components with other components with similar properties. The quantities of components can also be varied. Specifically, the medium contains (per liter) a total nitrogen content of about 1.0 to 1.7 grams (primarily from ammonium sulfate). The amino acids necessary for the development of the microorganisms that are going to be detected must also be provided. Not all amino acids must be provided, and the relative amount of each may vary. Amino acids can be supplied from a source vapedad. These can be supplied from natural sources (for example, extracts from whole organisms), co or mixtures or in purified form. Natural mixtures may contain varying amounts of said amino acids and vitamins. For general guidance, the specific amounts of said amino acids and vitamins are indicated below. These quantities are for guidance only and are not limitations to this invention. Those skilled in the art will recognize that many different combinations of amino acids and vitamins can be used in the media of this invention. The lists provided below exemplify only one of said examples. Normally only amino acids that can not be synthesized endogenously by the microorganisms to be detected should be provided. However, other amino acids can be provided in the medium of the invention. The amino acid contents preferably include at least the following, in approximately the following amounts (per liter): alanine (0.015 to 0.055 g), arginine (0.01 to 0.04 g), aspartic acid (0.04 to 0.10 g), stina (0.001 to 0.005 g), glutamic acid (0.05 to 0.15 g), glycine (0.01 to 0.03 g), histidine (0.010 to 0.06 g), isoleucm (0.01 to 0.10 g), leucma (0.03 to 0.08 g), lisma (0.03 to 0.07 g), ethionine (0.01 to 0.04 g), phenylalanine (0.01 to 0.04 g), proline (0.02 to 0.08 g), serine 0.01 to 0.05 g), threonine (0.01 to 0.04 g), tryptophan (0.002 to 0.00 * 6 g), tyrosine (0.01 to 0.02 g) and valine (0.02 to 0.05 g). Co-salts or an ion source can be provided by dissociation. Said salts may include potassium phosphate (e.g., about 0.5 to 1.5 g), copper sulfate (for example, about 40 to 50 μg), ammonium sulfate (for example, about 4.0 to 5.0 g), potassium iodide (for example, from 50.0 to 150.0 μg), ferric chloride (for example, about 150.0 to 250.0 ug), manganese sulfate (for example, about 300.0 to 500.0.0 g), sodium molybdate (for example, about 150.0 a). 250.0 μg, zinc sulfate (for example, around 300.0 to 500.0.0 g) and sodium chloride (for example, around 0.05 to 0.15 g) Other inorganic portions can be included to help the microbe develop. include the following (to the extent not yet provided in the previous sources of various chemical entities and described in quantities per liter): phosphorus (about 0.0005 g / 1), potassium (above 0.0004 g / 1), sodium ( about 0.03 to 0.05 g / 1) and oligomeric amounts of calcium, magnesium, aluminum, barium, chloride, cobalt, copper, iron, lead or, manganese, sulfate, sulfur, tin and zinc. The vitamins necessary for the development and reproduction of the microorganism to be detected can also be provided. They can be seen in a pure form or as part of more complex media. Vitamins may be present in approximately the following amounts (per gram of medium): biotin (about 0.15 to 0.40 μg / 1), pantothenic acid (about 45.0 to 55.0 μg / 1), p-oxine (about 6.0 to 9.0 μg / 1), riboflavin (around 10.0 to 20.0 μg / 1), folic acid (around 5.00 to 10.00 μg / 1), thiamine (around 10.0 to 20.0 μg / 1), vitamin B12 (around 0.20 to 0.30 μg / μl) and niacin (around 15.0 to 25.0 μg / 1) "Selective agents and, in particular, anti-iotics, which inhibit or prevent the development of non-target organisms, may also be provided . Many selective agents can be provided and the selective agents used depend on the target microorganism. Preferably, selective agents include one or more of the following at concentrations within the following ranges: amikacin sulfate (about 0.0045 to 0.0055 g / L), amphotericin B (about 0.00198 to 0.00242 g / 1), and bacitracin (about from 0.000475 to 0.00794 g / 1). Alternatively, it can be used as substitutes: thallium acetate, neoinicin sulfate, cyclohexirnide, tet racicline, colistin, ansiomycin or cydadamicin. Those who are experts in the field will recognize that carbon, nitrogen, organic elements, vitamins, amino acids and selective agents can be provided in many forms. In general, it is preferred to have a quantity of vitapunas, amino acids and selective agents in the scale of the amounts provided above.; but those skilled in the art will recognize that the actual properties of each ingredient may vary, so that the reduction in the amount of ingredient may be compensated with an increase in the amount of another. This is particularly relevant when you know the essential amino acids, the oligorneri e elements or the vitamins of the microorganism that you want to detect. Some ingredients may be provided in small amounts or may be omitted if they can be synthesized endogenously by the microorganism whose presence is to be determined. The nutrient indicator is present in the medium in an amount that is sufficient to support the development of the target microbe until a detectable signal is produced in the medium during development. 3 points, the ingredients: vitamin, amino acid, oligoneric element, salt and nutrient indicator, allow the sufficient development of the organism, so that a detectable change in the sample can be observed. The nutrient indicator alters a detectable characteristic of the sample only when it is metabolized by an organism. Preferably it alters a detectable characteristic only when it is inetabolized by the target microbe. Therefore, it can be used to confirm the presence or absence of the target microbe in the sample. It is preferable that the nutrient indicator is selected so as to be cleaved to release the indicator portion only by the target microbe present in the medium. That is, if other microbes pre-in the sample could divide the nutrient indicator, then the medium is formed so that we can not develop microbes substantially in the medium. Furthermore, although it refers to the nutrient indicator as the only source of the specific type of nutrient for the target microbe, the medium may contain other such sources; but in quantities that do not reduce the speci fi city of the medium. For example, the indicator-nutrient may be the only carbon source for this microorganism. Alternatively, other carbon sources (eg, amino acids) may be present that are not preferably used by the target microbe. If desired, a small amount of another carbon source may be present that could be used preferentially by the target microbe; but the quantity provided is such that it does not reduce the speci fi city of the medium without said carbon source. Most preferably, the nutrient indicator is 4-rnet? LurnbeLi fep 1-ß-D-glucopyranoside. Other nutrient indicators that can be used in the invention include the following: 5-bruno -? - chloro-3-? Ndox 1-ß-D-gl-copyranoside, on? Trofen? L-ß-D-glucop-ranoside, pm trofenil-ß-B-glucopyranoside, resofuran-β-B-glucopyranoside, 5-bromo-2-naphthyl-B-glucopyranoside, rose -.- B-glucopyranoside, VQM-Glc iodide (2-C2- C4- (D-gl? Copyranosyloxy) -3-methoxyphenyl-1-vinyl.i3-l-methyl-quinolin.io and iodide of VBzTM-Gl? C (2 ~ -C2-C) - (- B-gl? Copyranosyloxy) -3'-ethoxy-phenyl] -vi.nil &-3-methyl-benzothiazolium The term "enterococci" includes the following microorganisms: Enterococcus aviurn E. casseliflavus, E. E. columbae, E. dispar E. durans, F. faecalis, F. faeciu, E. qallinarum, E. hirae, E. rnalodoratus, E. rnundat.i, ..

E. psudoaviurn. E. ra ffinosus. E saecha ro lvti cus. E. seriousI i ci da, E,. so1 i.tarius v E. su.1 fureu. Among them, F _-_ aviurn. E. durans. E. faecalis. E. f ^ e iurn. E. aa11 inarurn v E. hi. rae are strains of fecal origin. The term is not limited to mean any given number of these species, and is not intended to exclude species that have not yet been discovered, but could be identified in the future and included in this genus, by those skilled in the art. The term "fecal streptococci" includes species of streptococcal bacteria present in the gastrointestinal tract of higher organisms. Includes such organisms as S. faecalis, S. faeciurn. S. aviurn v S. iallinarurn. S. faecalis. S. faecium, S. aviurn and S. gallinarurn K are what are commonly referred to as enterococci and are included within that term in the present. The term "24 hours" means the time necessary for about 95% of the liquid samples containing only about one to ten enterococci per 100 rnl to exhibit a detectable characteristic change. The temperature, amount and type of enzyme inducer present, the amount of nutrients provided in the medium and the relative health of the bacteria all affect the detection time. The amount of nutrients, such as amino acids, vitamins and oli.gomoric elements provided can affect the speed of development of the target microbe and, thus, the detection time. Balancing the sample thermally at an incubation temperature of about 35 ° C, after adding the medium, can decrease the time required for detection. The amount of enzyme inducer can also decrease the time for detection. The enzymatic inducers found in the medium are agents that act as an inducer of the enzyme that divides the nutrient indicator. The enzyme inducer, for example, may be a homologue for the nutrient indicator. Examples of such inductors are known in the art. The relative health of the microbe also affects the time needed for detection. The addition of agents such as pyruvate, which can help the recovery of damaged organisms, can therefore speed up detection. If a large number of bacteria are present in the sample, faster detection is also possible. In this invention, the provided means allow the detection of low amounts of target microbes (ie, less than 10/100 rnl) in the 24 hour time period, at least 95% of the time. You can use normal methods to determine this capacity.

The term "medium" means a solid mixture, powder or liquid, that contains all or substantially all of the nutrients necessary to allow a microbe to develop and reproduce. This invention includes both the media that is sterilized and the non-sterile media. The term "liquefied" means substantially in liquid form, although it also means that it includes pulverized or oven-dried samples of solid substances which have at least 10% liquid content. The phrase is intended to exclude a gelled medium, such as that found in agar. The terms "environmental" and "biological" mean tornadoes from or from a substance capable of supporting one or more life forms, including algae, yeast and bacteria. Examples include, but are not limited to: recreational waters, marine waters, drinking water, wastewater effluents and food substances. The term "inoculation" means at or near the time when the liquefied, environmental or biological sample is mixed with the medium of this invention. It means that it is the moment in which the two substances are substantially mixed together. The term "effective amount" is an amount within the scale that allows or promotes the development and reproduction of a target microorganism. That is to say, an amount that allows microbes or other organisms to adapt to the environment, synthesize the necessary constituents for reproduction and, subsequently, reproduce. It is not intended to be specific and may vary depending on factors such as the size of the sample and the concentration of the microorganisms. In general, the term indicates the amount required for detection of 100 target microbes per 1 ml of sample; most preferably, less than 100 microbes per 100 rnl of sample, or even 1 microbe per 100 rnl of sample. The terms "vitamins", "amino acids", "oligomeric elements" and "salts" are intended to include all the molecules, compounds and substances classified in each category by experts in the field, whether organic or inorganic, and It pretends that the categories exclude any substance that may be necessary for or that leads to the maintenance of life. The term "nutrient indicator" means a molecule or substance that contains a portion that is a source for an essential nutrient and a portion that causes or produces a characteristic change observable in the medium or in the sample. A nutrient indicator includes sources of nutrients bound to or conjugated with chromogens or fluorogens. Sources of nutrients can provide essential ingredients: vitamin, mineral elements or amino acids, or carbon. Normally, as a microorganism progresses from the stage in which nutrients accumulate for reproduction (waiting phase) to the phase during which reproduction actually occurs at a relatively rapid rate (registration phase) the nutpc onal requirements increase. Consequently, increased amounts of the nutrient indicator are inetabolized and a detectable and characteristic change occurs. Preferably the nutrient indicator includes a nutrient portion and a chromogen or a fluorogen. Croinogens include any portions that produce a color change observable on the visible scale. The fluorogens include any portions that fluoresce when exposed to an excitatory light source. Examples include, but are not limited to: orthofi trophenyl, phenol phthalein and 4-rnet l urnbeliferone portions. While the nutrient indicator may provide the only source of an essential nutrient, other sources of such nutrients may be provided, as long as adequate selectivity and adequate sensitivity of the medium is maintained. The term "detectable characteristic signal" includes any change in a sample that may be perceived by one or more of the human senses. The term includes examples such as a color change in the scales of visible or non-visible wavelength, a change of state, such as between solid, liquid and gas, a gas emission or a change of smell. The term "target microbe" means the microorganism whose presence or absence is to be detected. It preferably includes enterococcal species and is fecal retinococcus, which can live in the gastrointestinal tract of higher organisms. In a preferred embodiment, the nutrient indicator alters the color of the specific medium for the microbe. The color change may be apparent on the visible wavelength scale or it may be a fluorescence that is apparent on the visible wavelength scale when exposed to a light source of excitation. This is achieved by dividing a crooked portion or a fluorescent portion. A chromogenic portion is a portion that changes the color of the sample on the visible scale when it is in unconjugated form, ie, it is no longer conjugated to or bound to a nutrient portion. A fluorescent portion is a portion that changes the color of the sample on the non-visible scale when it is in unconjugated form, ie, it is no longer conjugated to or bound to a nutrient portion. Examples of the crinogenic portions that can be conjugated to a nutrient portion include, but are not limited to: orthotrophenyl portions that produce a yellow color when released from the nutrient indicator; and the phenol-talein portions that produce a red color when released from the nutrient indicator. Examples of fluorescent portions include the netilurnbeli portions that become fluorescent at about 355 n, when released from the nutrient indicator.; and the bromo-chloro-indole portions, which turn blue when released from the nutrient indicator. Most preferably, the medium uses the fluorescent portion 4-rnetilurnbeli-feryl-3-B-glucopyranoside. Preferably the medium also contains one or more selective agents, such as antibiotics that prevent or inhibit that the microbes different from the target microbe, nebolize the nutrient indicator. That is, preferably, the medium contains agents that are specific for microbes other than enterococci or fecal streptococci and effectively prevent or inhibit the development of at least some of those microbes. The term is intended to include agents such as sodium azide, thallium acetate, naldixic acid, neopucine sulfate, gentamicin sulfate, bile salt, sodium chloride, lylohexirnide, tetracycline, colistin. , ansiomycin, clinda icma and polyacin B. Be preferred includes: amicacin sulfate (e.g., about 0.0045 to 0.0055 g / liter), amphotenne B (e.g., about 0.00198 to 0.00242 g / liter) and bacitracin (for example, around 0.000475 to 0.000794 g / liter). The term "specific medium for the microbe" means a means that allows the substantial development of only the target microbe. This includes media containing one or more specific antibiotics to inhibit the growth of microorganisms other than the target microbe and includes media containing alternatively or additionally one or more nutrient indicators that, preferably, are not metabolized by microorganisms other than the target microbe in any substantial degree. The term "substantial", when used in this context, means that the medium still allows for specific detection (ie, at least 95% or even 98% accuracy) and sensibility (ie, at least 95% or up to 98% of detection levels) of the target microbe, when measured by normal procedures. In another preferred embodiment, the medium contains an agent that acts as an inducer of the enzyme dividing the nutrient indicator. This agent, for example, can be a counterpart of the nutrient indicator. Such inducers include:? So? Rop? L-B-galactoside (IPTG) which induces the activity of β-galactosidase, and et? L-β-Dt? Og3ucos? Da, which induces the activity of? glucosidase Preferably the medium allows the detection of enteroccocci (including fecal streptococci) within 24 hours. Preferably the med is also in the form of a water-soluble, non-sterile powder, to allow easy addition to liquid samples. In another aspect, the invention incorporates a method to detect the presence or absence of enterococci and fecal str-eptococci in a liquefied sample, by contacting the sample with the medium described above, incubating the sample mixture and means to detect it. nar- the presence or absence of the detectable characteristic signal. Incubation can be carried out at a variety of temperatures, but preferably between 35 ° C and 45 ° C. Preferably, the detectable characteristic signal can be observed within 24 hours. The invention incorporates providing samples preferably from a water source, which includes fresh water, sea water, drinking water supplies or wastewater. Another aspect of the invention is a method for detecting the presence or absence of a target microbe in an environmental or biological liquid sample, which preferably includes the step of heating the sample to the incubation temperature in a liquid incubator, after adding the specific medium for the microbe. Most preferably, the incubation temperature is around 35 ° C. The term "liquid incubator" means a Liquid heated at a specified temperature or at a specified temperature scale. This can include any form of water baths, for example. Said incubator is advantageous with respect to previously used air incubators, since the medium reaches the proper incubation temperature more rapidly. In another aspect, the invention incorporates a method to quantify the quantity or number of enterococci present in a liquid sample, by contacting a liquid sample with the medium described above, placing the liquid sample, which includes the medium, in the Containers, incubate the sample and medium liquid mixture, observe the quantity and quality of a detectable characteristic signal and compare the quantity and quality of a detectable characteristic signal with the probable number values. This quantification procedure allows comparing the quantity and quality of the characteristic that has been altered, preferably a color change, with the probable number values, obtained from samples where the concentration and the characteristic change have been correlated. with samples for which the concentration of enterococci or bacteria is known. See, for example, Comoendiurn or Methods for the Microbiological Exammation of Foods. 3a. edition, edited by Vanderzant and Split + s. Oesser, 1992. The most probable number technique allows the estimation of bacterial concentrations that are below the detectable levels of all others. In the preferred embodiments, the invention uses the apparatus described by Naqui in the US patent application Serial No. 08 / 201,110, incorporated herein by reference. The step of quantification involves providing a sample of an environmental or biological sample in a liquid form, placing or supplying the sample in the sample container bag described by Naqui, mixing the sample with a medium to allow and promote the development of viable bacteria. , incubate the sample, detect the quantity and quality of the color change and compare the quantity and quality with the results obtained for a series of samples for which the concentration of bacteria is known. It is preferred that the incubation step be carried out at 41 ° C for a period of 24 hours, using the medium described above. The invention provides the optimum means to determine the presence of enterococcal microorganisms (including fecal streptococci). Enterococci can be detected much earlier in this medium than in those currently available. Consequently, the results of the test are available quickly. Fast results save both money and time in the lab. L-speed also decreases the threat to public health, by allowing early warnings and remedial measures to cope with the presence of some microorganisms in places such as drinking water supplies and rocreational waters. In addition, the method of this invention generally does not require confirmatory tests, since specific nutrient indicators can be used for the microorganism. On the other hand, the invention does not require the use of a sterile medium, as many other methods require. Other aspects and advantages of the invention will be apparent from the following description of its preferred embodiments and the claims.

BRIEF DESCRIPTION OF THE PREFERRED MODALITIES In the following description reference will be made to various methodologies known to the experts in the chemical, biological and microbiological fields. The publications and other materials that establish said known methodologies, to which reference is made, are incorporated here as reference in s? whole, if they had been fully exposed here. The compositions, methods and products of this invention are applicable to biological and environmental samples and are useful in the chemical, biological and microbiological fields for the detection of microorganisms.

DETECTION OF MICROORGANISMS BASED ON ENZYMPHYTIC SPECIFICITY Specific microorganisms derive their nutrients from a variety of sources; however, some sources may be unique to a particular microorganism or a particular group of microorganisms. Families, groups or species of microorganisms can share the speci fi city for certain nutrients that are lacking in other crops. By taking advantage of the metabolic characteristics of specific microorganisms, it is possible to prove their presence. Many enzymes have been identified that are specific to particular groups or species and probably others will be 3 identified in the future. The group of bacteria called enterococci, contains a unique constituent enzyme, ß-glucosidase (Littel and coauthors, OPDJ, Fnviron, Microbio! .. 45: 522-627 (1983) .Catalyza The hydrolysis of suitable chlorogenic or fluorogenic substrates. or appropriate selective environments, which results in a color or fluorescent signal that can be perceived visually or espetrically, a substratum of specific ß-giucosi dasa can serve as the nutrient indicator in media designed to sense enterococci and provides a source The main carbon substrate in the formulation of the medium Several nutrient indicator substrates are available, however, the substrate used to detect enterococci is the substrate fluorogen 4-rnet? lurnbel? fepl-ß-D-giucopirans. In the presence of viable enterococcal bacteria in a sample, the nutrient indicator is metabolized, thereby dividing the indicator portion which, when divided, becomes fl uous. rescente The released glucose portion is then used by enterococcal bacteria to promote development.

THE ENTEROCOCOS The group of fecal enterococci and streptococci is a valuable bacterial indicator to determine the oi-; degree of fecal contamination in recreational surface waters (Greenberg and co-authors, Standard Methods for the Examination of Water and Uastewater, 18th edition, Eaton, A. D. (ed.) American Public Health Association (1992)). The genus Enterococcus now contains 18 species: Enterococcus avium, Enterococcus casseliflavu. Enterococcus cecoruin. Enterococcus coiumbae, Enterococcus dispar, Enterococcus durans, Enterococcus faeca is, Enterococcus faecium, Fnterococc? S gallmarum. Enterococcus hirae, Fnterococcus rnalodoratus, Enterococcus rnundati i. Enterococcus pseudoav urn. Fnterococcus ra i nosus. Enterococcus saccharolvticu. Enterococcus ser-iolici a. Enterococcus solitarius and Enterococcus sulfureus.

Among them, Enterococcus aviurn. Enterococcus durans. Enterococcus faecal s. Enterococcus faeciurn, Enterococcus g llmarurn and Enterococcus hirae are the strains of fecal origin (Hernández and coauthors, Uat Res., 27: 597-505 (1993)). These bactepas survive much more than other indicators in marine environments. Enterococci are also resistant to wastewater treatment, including chlorination, and are thus sensitive indicators of the survival of enteric pathogens and viruses in water samples. It has been shown that there is a direct correlation between the concentration of enterococci in marine waters and the risks of gastroenteritis associated with swimming (Cabelli, Uat.Sci. Tech., 21: 13-21 (1989)).; and Cabelli and coauthors, 3ournal WPCF (1983)). The United States Environmental Protection Agency (U.S. EPA) has recommended that the group of bacteria called enterococci be used as a bacterial indicator for freshwater and marine waters. The current safety guidelines for recreational waters, based on the density of enterococci, are 33 entorococci per 100 inl of fresh water and 35 enterococci per 100 inl of seawater, respectively. The current normal techniques for measuring the densities of enterococci in recreational marine and sweet waters are hard and laborious and time consuming. The results, based on MPN, require a minimum of 72 hours and MF requires 48 hours. While enterococci are more sensitive bacterial indicators for health risks associated with swimming, the time required for interpretation of the results has hampered their acceptance for adequate surveillance. The diagnostic system based on ß-gl ucosidase allows the early detection of enterococcal species in water samples, within 24 hours. The quick results allow to accelerate the answers on the part of the government to put into practice measures such as the closing of beaches to protect public health.

Lfl SELECTIVITY In general, the activity of β-glucosidase is a specific characteristic to detect enter-ococos and also fecal estr-eptococci. However, other bacteria also possess such enzymatic activity. These include the genera of the family Enterobact-eriaceae (Enterobac r aerogenes, E. colacae), Klebsiella pneu oniae, Serratia marcescens, Listeria Qnocvto enes and Fragile Bacterioides. Certain selective factor (s) can be used to inhibit the development of those other ß-glucoside-positive bacteria (ie, different from the enterococcal species). A selective medium for specific microorganisms can be produced by enhancing a combination of specificity to the enzyme and selective environments. For example, in a medium of this invention, microbes that are not target, which do not possess β-glucoeidase activity and can not digest the nutrient indicator, are deleted. Heterotrophic bacteria or other non-target microbes that possess ß-glucosidase are selectively suppressed during the test period by the combination of specifically formulated antibiotic / chemical reagents and other physical parameters (pH and temperaure). Typical selective agents that can be used in the medium of this invention include sodium azide, thallium acetate, nalixidic acid, neomycin sulfate, gentanymic sulfate, bile salt, sodium chloride and polyrnicine. B (Hernández and co-authors, Wat. Res. 27: 597-505 (1993); Knuntson and co-authors, Appl. Environ. Microbiol., 59: 935-938 (1993); and Littel and co-authors, Appl. Environ. Microbiol. , 45: 522-527 (1983)). twenty The combination of specificity to the enzyme and antibiotic selectivity provides multiple obstacles that prevent microbes that are not enterococcal, competitive, from being detected within the test period of 2? hours. This avoids a single highly toxic element that can not only inhibit microorganisms that are not target, but also suppresses target microbes.

THE NUTRIENT INDICATOR In enterococci, ß-glucosidase catalyzes the conversion of the fluorescent substrate, 4-rnet i lurnbe i i fer il-ß-D-glucoprosarnide (MUD) to 4 r rnet? Lu boletin and glucose. The fluorophore, 4-rnet? Lurnbel íferona, emits blue fluorescence when it is. excites at 355 nn (which can be seen through a lamp UV of 365 n). The portion of sugar released, glucose, serves as a main source of carbon to support the development of enterococci. An increased level of nutrient indicator can provide better microbial development and stronger fluorescence; however, high levels of nutrient indicator can also cause cytotoxicity in the cells and a higher level of background fluorescence. K ~ methyluinbelifepl-β-B-glucopyranoside (MUB) at a level of 25 rng / 1 does not inhibit the development of the enter-ococos species.

THE ENZYME INDUCERS The majority of glycoid hydrolases (ß-glucosidase, ß-galactosidase and ß-glucuronidase are inducible.) The induction of ß-galactos idasa activity by means of isopropyl-β-D-thiogalactoside (IPTG) is a classic example. Ethyl-β-thioglucoside, which works in a similar way, is an ß-glucosidase inducer.

THE DEVELOPMENT STIMULATORS NaHCOa (2 g / liter), Tween-80 (0.75 rnl / 1) and KH ^ PO * (5 g / 1) stimulate the development of fecal str-eptococcal species isolated from waters (see, for example, Lachica and coauthors, 3. Appl. Bactepol., 31: 151-156 (1968)). Other oligomeric elements, such as one or more specific amino acids (glutarnic acid), one or more vitamins (lipoic acid) and one or more nucleotides (quinet i n-pbosi da) may have developmental activities for the enter species. -ococos. A medium that has been shown to be optimal for detecting enterococci is described in Table 1.

TABLE 1 CQ? PQNEIMTE IT, IS6F IENTE SOURCE AMOUNT Nitrogen Nitrogen of amine 0.02 to 0.05 g / 1 Amino acids alanine 0.015 to 0.055 g / 1 to gi ia 0.01 to 0.04 g / 1 aspartic acid 0.04 to 0.1 g / 1 cyst at 0.001 a 0.005 g / 1 glutamic acid 0.05 to 0.15 g / 1 glycine 0.01 to 0.03 g / 1 histidine 0.01 to 0.05 g / 1 isoleucine 0.01 to 0.10 g / 1 leucine 0.03 to 0.08 g / 1 lysine 0.03 to 0.07 g / 1 rnethionine 0.01 a 0.04 g / 1 phenylalanine 0.01 to 0.04 g / 1 proline 0.02 to 0.08 g / 1 seriña 0.01 to 0, .05 g / 1 threonine 0.01 to 0.04 g / 1 tripto anus 0.002 to 0.006 g / 1 ti rosin 0.01 to 0..02 g / 1 valine 0.02 to 0..05 g / 1 Elements: Calcio vestigios-aclorur-o vestiges cobalt vestiges copper vestiges TABLE i continuaci n INGREDIENT SOURCE AMOUNT iron vestiges piorno vestiges manganese vestiges phosphate 0.0005 to 0.005 g / 1 potassium 0.0004 to 0.004 g / 1 sodium 0.03 to 0.06 g / 1 sulfate vesti ios sulfur vestiges tin vestiges zinc vestiges Vitamins biotin 0.15 to 0.4 μg / 1 choline 5.0 to 10..0 μg / 1 pyridoxine 6.0 to 7.5 μg / 1 riboflavin 10.0 to 20.0 μg / 1 thiamine 20.0 to 20.0 μg / 1 vitamin B1.2 0.2 at 0.3 μg / 1 niacin 15.0 to 25.0 μg / 1 pantothenic acid 45.0 to 65.0 μg / 1 COMPONENT II HEPES, free acid 4,032-4,928 g / 1 HEPES, sodium salt 7,301-8,933 g / 1 Nitrogen base of modified yeast 4,635-5,565 g / 1 TABLE I (continued) sodium bicarbonate 1.8-2.2 g / l potassium phosphate 0.1-1.0 g / 1 ß-ETG 0.009-0.011 g / 1 (ethyl-ß-D-thioglucoside) MUD 0.02-0.03 g / 1 (4-met i 1 urnbel i f eri 1 -ß-D-gl ucopiranos i d) arnicacin sulfate 0.0045-0.0055 g / 1 amphotericin B 0.00198-0.00242 g / 1 bacitracin 0.000476-0.000794 g / 1 * Vestigios = less than 0.001 g / liter PS ETHODS AND USE The medium of this invention can be used in three different formats. First, it can be used to detect the presence or absence of enterococci. Second, it can be used to quantify the amount of enterococci present in a sample. In third, it can be used in a discriminant format to relate the time to obtain a positive test result, with the concentration of the enter-ococos in a sample.

Presence / absence The procedure to detect the presence or absence of enterococcus species in water samples is described below. 1.- First one or more water samples are collected using sterile pre-calibrated containers. The sample volume can be of varying amounts, including 100 mi, 10 rnl or 1 rnl, depending on the application. The medium of this invention can be added in a powder form to the collected water sample. Alternatively the medium may be present in powder form in the containers, before the sample is collected. 2.- Then heat equilibrium The sample vessels (that is, it is brought to the temperature of incubation in a water bath) preferably at 35 ° C or 4L ° C ± 1 ° C, for 20 minutes. . 3. A blue fluorescence is indicative of the presence of enterococcal species in the water sample tested when 4-methylutnbeli epl-β-B-glucopyranoside is the indicator-nutrient. The fluorescence can be visually seen using a UV lamp of 365 nm. Alternatively, the signal can be monitored by means of a fluorescence otometer spectrum using an excitation wavelength of 365 nrn and an emission wavelength of 440-450 n. A positive reaction can occur at any time within 24 hours, if viable bacteria are present in the sample. That is, approximately 95% of the samples containing 10 cfu (colony forming units) / 100 rnl will exhibit a detectable characteristic change within 24 hours. The time for detection, which varies from about 12 to 24 hours, varies with the concentration and presence of different species or strains of enterococci.

Quantification The same or medium can be used to quantify the target molecules. The analysis can be carried out with the regular MPN format of 5 or 10 tubes or with the "Quanti-tray" MPN format of 50 to 100 concavities (see Naq? I, US patent application Serial No. 08 / 201,110). The procedure for the quantification of enterococci using the medium of Table I is described below. 1.- A sample of water is collected using one or more precalibrated sterile containers. The volume of the sample can be in varying amounts, including 100 ml, 10 ml or 1 lj depending on the application. The invention can be added in a powder form to the sample of collected water. Alternatively, the medium of this invention may be present in powder form in the container, before the sample is collected. 2.- Then the sample is poured into a "Quanti-tray" and thermally sealed to create 50 or 100 MPN concavities. Alternatively, sample aliquots can be distributed in 5 or 10 MPN tubes. 3. - The sample containers are incubated, preferably at 35 ° C or 41 ° C i? ° c. 4.- The fluorescence can be read visually using a UV lamp of 355 nm. A blue fluorescence is indicative of a positive reaction when 4-rnetil urnbel i fepl-ß-B-glucopyranoside is used as the nutrient indicator. The density of enter-ococos in the water samples can be correlated directly with the number of positive concavities of the "Quanti-t ray" or with the number of positive tubes, using the calculation formula MPN, MPN = Nln (N / NX ), where N r-epr-represents the total number of tubes or concavities tested and X represents the total number of tubes or positive concavities. The maximum time for detection is 24 hours.

Rapid discrimination The means described above can also be used for the application of rapid approval / disapproval discrimination. This format involves the use of a linear direct relationship between the density of enterococci in a sample of water tested with the time of detection of positive results. The procedure for rapid approval / disapproval discrimination test, using the same formula, is as follows: 3.- Collect a sample of water using sterile containers previously calibrated. The sample volume can be in varying amounts, including 100 l, 10 inl or 1 rnl, depending on the application. The medium of this invention can be added in powder form to the collected water sample. Alternatively, the medium of this invention can be present as a powder in the container, before the sample is collected. 2.- Preferably incubate the sample vessels at 35 ° C or 41 ° C ± 1 ° C. 3.- You can read the luorescence visually or fluorospectrofoto electrically. A blue fluorescence is indicative of a positive reaction. The time to obtain a positive result is used as a indicator to determine if the sample of water tested is at the level of "danger", "concern" or "approval".

EXPERIMENT l This experiment was carried out to determine the nutrient indicator that provides the fastest detection.

Materials and methods The strains of enterococci used on agar plates with TSAI blood were developed. The following microorganisms were developed: E. faecalis ATCC 19433, ATCC 33012, ATCC 33186, ATCC 35550, ATC 29212; E. faecium ATCC 19434, ATCC 35567; E. d? Rane ATCC 6056, ATCC 11576, ATCC 19432; E. aviurn ATCC 14025, ATCC 35665; E. gallinaru ATCC 49573; Streptococcus bovis ATCC 9809, ATCC 35034 and S. eq? Inus ATCC 9812. Cell suspensions were prepared by turning Lae cells from plates using a sterile cotton swab (pre-churned) and resuspending in 50 mM of regulator HEPES, pH 7.5, to a turbidity equivalent to the MacFarland standard. The following enzyme substrates were tested for sensitivity to ß-glucosidase: o-nitrofem 1 -ß ~ B-glucop? ranoside (ONPD) and 4-rnet? l? nbel I-ferone-ß-D-gl? copiranoside (MUB). The data showed that the glycopyranoside was the substrate most sensitive to the β-glucosidase activity of enterococci. This substrate, when excited at 315 μrn (monitor with UV lamp), gives blue fluorescence by hydrolysis by β-glucosidase.

EXPERIMENT 2 A study was carried out using the nutrient formula provided in Table 1. It allowed the detection of enter-ococci at 1-2 cfu / LOO rnl in the period of 16 to 22 hours (see the following table). Samples of water were collected and added to the medium described above. The sample vessels were then incubated at 4 1 ° C ± 1 ° C. The sample vessels were monitored for the appearance of a blue fluorescence. The results are shown below and indicate the sensitive detection of enterococci in a medium of this invention.

DETECTION OF ENTEROCOCOS WITH A PROTOTICO PROCEDURE OF ENTEROCOCOS Time Cepa; Inoculum 15 r 18 hr 20 hr- 22 hr E. faecalis ATCC 33186 26 cfu / 100 inl 2.6 cu / 100 rnl E. faecium ATCC 35667 15 cfu / 100 rnl 1.5 cfu / 100 rnl E. aviurn ATCC 35665 20 cfu / LOO rnl B 2 cfu / 100 ml E. durans ATCC 6056 11 cfu / 100 rnl B 1.1 cf? / 100 ml D "+" positive; "-" negative; "D" weak fluorescence. Other embodiments are within the scope of the following claims.

Claims (1)

NOVELTY OF THE INVENTION R IVI DIC CIO ES

1 - . 1 - - A means for detecting the presence or absence of enterococci in a liquefied sample, in the term of 24 hours, characterized in that it comprises: (a) an effective amount of vitamins, amino acids, oligomer elements and salts, operable to allow viability and the reproduction of enterococci; (b) an effective amount of one or more nutrient indicators, provided in an amount sufficient to give a detectable characteristic signal in the medium during the development of the enteroc; said nutrient indicator releasing a nutrient portion in the presence of β-glucosidase; and (c) an effective amount of one or more selective agents to prevent or inhibit the growth of microorganisms other than enterococci. 2. The medium according to claim 1, further characterized in that the nutrient indicator is metabolized to a portion serving as a source of nutrition and a portion that alters an observable characteristic of the medium. 3. The medium according to claim 2, further characterized in that the nutrient indicator alters the color of the medium in the visible wavelength scale, by means of the action of the β-glucosidase activity. 4. The medium according to claim 2, further characterized in that the nutrient indicator alters the color of the medium on a non-visible wavelength scale, by means of the action of the β-glucosidase activity. 5. The medium according to claim L, further characterized in that the nutrient indicator is 4-rnetilumbelifepl-β-B-glucopyranoside. 6. The medium according to claim 1, further characterized by comprising:? N regulator, from 4.0 to 6.0 g / liter of ammonium sulfate, a source of carbon dioxide and phosphorus ions, an effective amount of an Inductor -f-glucosidase, an effective amount of an indicator-nutrient, an effective amount of antibiotics to inhibit the development of fungi and bacteria gratn-poeitivas and grarn-negativvas, different from Enterococci; and enough amino acids, vitamins, trace elements and minerals to support the development of enterococci at a rate that allows detection within 24 hours. 7. The medium according to claim 6, further characterized in that it comprises: a r-egulador, from 4,000 to 6,000 g / 1 of Yeast nitrogen base, modified; from 1.0 to 2.5 g / 1 of baking soda; from 0.1 to L.O g / 1 potassium phosphate; from 0.009 to 0.031 g / 1 of ß-ETG, from 0.02 to 0.03 g / 1 of MUB, from 0-0045 to 0.0055 g / 1 of amikacin sulfate, from 0.00198 to 0.00242 g / liter of amphotericin B and from 0.00476 a 0.00794 g / 1 of bacitracin; and enough amino acids, 4. 1 vitamins, oligomeric and mineral elements to support the development of enter-ococos at a speed that allows detection within 24 hours. 8. Method for detecting the presence or absence of enter-ococos in a liquefied sample, characterized in that it comprises the steps of: (a) contacting a liquid sample with the medium of claim 1; (b) incubating the liquid sample and the medium; and (c) monitoring the liquid sample to determine the presence of a detectable change in a. physical characteristic. 9. The method according to claim 8, further characterized in that the monitoring step takes a maximum of 24 hours. 10. The method according to claim 8, further characterized in that the liquid sample is made from a source of fresh water or seawater. 11. The method according to claim 8, further characterized in that the liquid sample is taken from a wastewater source. 12. The method according to the claim 8, further characterized in that the liquid sample is taken from a source of drinking water. 13. - The method according to claim 8, further characterized in that the detectable physical characteristic change is a color change. 14. The method according to claim 8, further characterized in that the change in detectable physical characteristic is a change in color visible in the presence of a light source of excitation. 15. The method according to claim 8, further characterized in that the incubation step is carried out at 35 ° C to 45 ° C. 16. Method for quantifying the quantity or number of enterococci present in a liquid sample, characterized in that it comprises the steps of: (a) contacting a liquid sample with the medium of claim 1; (b) place the liquid sample and the medium in containers; (c) incubating the liquid sample mixture and medium; (d) observe the quantity and quality of a detectable change in a physical characteristic; and (e) compare the quality and quantity of a detectable change in said physical characteristic, with the most probable number values.