CULTURE MEDIUM FOR THE IDENTIFICATION OF SALMONELLA AND PROCESS FOR ITS USE

Abstract: The present invention relates to a culture medium intended for the identification of Salmonella bacteria, comprising, in addition to a culture medium for Salmonella, as chromogenic agents, a combination of : a chromogenic compound linked to a C7-C10 fatty acid, and an appropriate detergent which promotes the liberation of the chromogenic compound. The present invention also relates to a process for detecting Salmonella.

Description: Culture medium for the identification of Salmonella and process for its use The present invention relates to an isolation medium permitting the identifi¬ cation of bacteria of the Salmonella species. The identification of the bacterium Salmonella, which is pathogenic for man, is a major problem in medical bacteriology and in the monitoring of food hygiene. Thus, in the case of epidemics transmitted by chicken farms, the birds infested in the intestinal tract are not ill but constitute a reservoir of Salmonella. The latter can be propagated, especially by eggs, in the diet following these epidemics. In fact, Salmonella is a bacterium whose notification is obligatory. Nowadays, it is becoming essential to provide for the large scale detection of infected sites and more particularly of farms infected with the Salmonella bacterium in order to reduce these epidemics. Indeed, Salmonella are generally to be recognised among other microorgan¬ isms such as the commensal species Escherichia coli and Proteus. The detection of Salmonella is normally carried out on agar medium for the selective isolation of enterobacteria which permits the differentiation of pathogenic enterobacteria and the detection of suspicious Salmonella colo¬ nies. An ideal isolation medium should permit the growth of enterobacteria, the differentiation of the various species present so as to permit subsequent identification of a colony of each type, and the detection of suspicious Salmonella colonies. The object of the present invention is to make it possible to carry out highly simplified routine tests for Salmonella, for example methods for monitoring the microbiological quality of water using soaking methods, or the monitor- ύ g of the microbiological quality of food factories using contact methods, the diagnosis or alternatively the detection of pathogenic microorganisms using traditional microbiological Petri dish methods on solid medium or on semi-solid selective medium, by means of media according to the invention. The object of the present invention is to permit the detection and differen¬ tiation of Salmonella by revealing an activity specific to these bacteria. The Salmonella bacterium has among its range of enzymes esterases which cleave in particular caprylate derivatives such as nitrophenyl caprylate. It could therefore be envisaged to use in an isolation medium a chromogen attached to the caprylate and its derivatives, a substrate for esterases, which would be liberated into the medium under the action of these enzymes, allowing the identification of Salmonella colonies. However, tests on chromogens such as 5-bromo-4-chloro-3-indolyl caprylate in a conventional culture medium for Salmonella have not made it possible to identify colorations specific for the inoculated colonies. It has been shown, surprisingly, that the addition of a detergent to the culture medium at an appropriate concentration, and which is compatible with a high Salmonella growth, made it possible to obtain a coloration of the Salmonella colonies. The present invention therefore relates to a culture medium intended for the identification of Salmonella bacteria, comprising, in addition to a culture medium for Salmonella, as chromogenic agents, a combination of: a chromogenic compound linked to a C 7 -C 10 fatty acid, and an appropriate detergent which promotes the liberation of the chro¬ mogenic compound. Preferably, the chromogenic compound is chosen from caprylic acid esters, advantageously chosen from derivatives of indolyl caprylate and their salts, in particular 5-bromo-4-chloro-3-indolyl caprylate. Preferably, the 5-bromo-4-chloro-3-indolyl caprylate will be used at a con¬ centration of between 40 and 200 mg/1. The appropriate detergent will be advantageously chosen from fused poly- cyclic detergents, in particular salts of carboxylic acid derivatives-cholane polyols. Preferably, the appropriate detergent will be chosen from the alkali metal salts of cholic acid or its derivatives, in particular sodium deoxycholate. The sodium deoxycholate may be used at a concentration greater than 1 g/1, preferably of between 1.5 and 5 g/1, a concentration which is perfectly com¬ patible with a high Salmonella growth. Advantageously, the chromogenic compound/appropriate detergent weight ratio is between 1/10 and 1/100. It should be noted that the present invention does not consist in a mere addi¬ tion of detergent to an identification medium, and that it is necessary to consider the chromogenic compound/appropriate detergent pair. Thus, for the indolyl caprylate derivatives, only the fused polycyclic deter¬ gents, derivatives of cholane, make it possible to obtain a coloration of the Salmonella colonies, whereas its replacement with a detergent such as the standard anionic detergents (especially the detergents marketed under the trade mark TERGITOL by the company UNION CARBIDE Corp.) does not make it possible to identify a coloration. It is also particularly important to note that the chromogenic agent of the medium according to the invention does not result in coloration with many other enterobacteria even if they are allowed to incubate for several weeks. A person skilled in the art will of course know how to supplement the medium according to the present invention so as to eliminate exceptional non-Salmonella bacteria, especially by identifying other Salmonella-specific properties, in particular the properties relating to cleavage or metabolism of beta-galactosides and beta-glucosides. Advantageously, the medium according to the present invention will also contain beta-glucosides and/or beta-galactosides. Thus, a coloured Salmonella colony will be easily identified on a microbio- logical device even in the presence of thousands of other colonies, or on a semi-solid medium in order to increase the specificity of detection. A deep blue coloration characteristic of the presence of Salmonella is gen¬ erally obtained. However, other colorations may be envisaged depending on the chromogenic compound used. Moreover, the detection will be facilitated by the fact that it necessitates neither the use of a non-viable coloured indicator nor that of a specific illumination. The present invention also relates to a process for detecting strains of Sal¬ monella in any sample, for which the culture medium as described above is inoculated with the said sample and in that the coloration characteris,tic of the presence of Salmonella is at least identified. Depending on the form and presentation of the medium and depending on the test carried out, the medium is preferably inoculated by contact, by soaking, by surface inoculation or by inoculation within the mass. Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The preferred specific embodiments are, therefore, to construed as merely illustrative, and not limitative of the disclosure in any way whatso¬ ever. The entire disclosures of all applications, patents, and publications cited above and below, and of corresponding application French 92-12 533, filed October 20, 1992, are hereby incorporated by reference. The examples below are intended to illustrate other characteristics and advantages of the present invention. EXAMPLE: Detection of Salmonella strains Various concentrations of sodium deoxycholate are added to the medium S (in g/1: meat extract 1; yeast extract 2; peptone 5; sodium chloride 5; agar 15; 5-bromo-4-chloro-3-indolyl caprylate 0.2). The dishes are incubated at 37°C and the blue coloration of the colonies of the bacterium Salmonella AR3001 and of the control bacterium E. coli AR2901 is examined after incubating for 24 hours at 37°C. Salmonella is identified only with the chromogenic substrate and deoxycho¬ late combination. Salmonella AR3001 E. coli AR2901 Deoxycholate 0.5 g 1 colourless colourless Deoxycholate 1.0 g/1 light blue colourless Deoxycholate 1.5 g/1 blue colourless Deoxycholate 2.0 g/1 darkish blue colourless Deoxycholate 2.5 g/1 dark blue colourless Deoxycholate 3.0 g/1 dark blue colourless Various enterobacteria strains are isolated on the medium T (in g/1): meat extract 1; yeast extract 2; peptone 5; sodium chloride 5; agar 15; neutral red 0.03; Cellobiose 10; sodium deoxycholate 2.5; 5-bromo-4-chloro-3-indolyl caprylate 0.2). The dishes are incubated at 37°C and the coloration of the colonies is exam¬ ined after incubating for 24 hours. The coloration of the colonies makes it possible to distinguish Salmonella from the other enterobacteria. Salmonella AR3001 blue Salmonella AR3002 blue Salmonella AR3003 blue Salmonella AR3004 blue Salmonella AR3004 blue Citrobacter AR30033 pink Citrobacter AR3437 pink Citrobacter D9316 pink Citrobacter D9352 pink Serratia AR3493 pink Enterobacter AR3494 pink Klebsiella B2497 pink Klebsiella B2593 pink

Claims: CLAIMS 1. Culture medium intended for the identification of Salmonella bacteria, characterised in that it comprises, in addition to a culture medium for Salmonella, as chromogenic agents, a combination of: a chromogenic compound linked to a C7-C10 fatty acid, and an appropriate detergent which promotes the liberation of the chromogenic compound. 2. Culture medium according to Claim 1, characterised in that the chro¬ mogenic compound is chosen from caprylic acid esters. 3. Culture medium according to Claim 2, characterised in that the chro¬ mogenic compound is chosen from derivatives of indolyl caprylate and their salts. 4. Culture medium according to Claim 3, characterised in that the chro- mogenic compound is 5-bromo-4-chloro-3 -indolyl caprylate. 5. Culture medium according to Claim 4, characterised in that the 5-bromo-4-chloro-3-indolyl caprylate is used at a concentration of between 40 and 200 mg/1. 6. Culture medium according to one of Claims 1 to 5, characterised in that the appropriate detergent is chosen from fused polycyclic deter¬ gents, more particularly from the salts of carboxylic acid derivatives- cholane polyols. 7. Culture medium according to Claim 6, characterised in that the appro¬ priate detergent is chosen from the alkali metal salts of cholic acid or its derivatives. 8. Culture medium according to Claim 7, characterised in that the appro¬ priate detergent is sodium deoxycholate. 9. Culture medium according to Claim 8, characterised in that the sodium deoxycholate is used at a concentration greater than 1 g/1, preferably of between 1.5 g/1 and 5 g/1. 10. Culture medium according to one of Claims 1 to 9, characterised in that the chromogenic compound/appropriate detergent weight ratio is between 1/10 and 1/100. 11. Culture medium according to one of Claims 1 to 10, characterised in that it is presented in a liquid, semi-liquid or solid form. 12. Process for detecting Salmonella strains in any sample, characterised in that the culture medium according to any one of Claims 1 to 11 is inoculated with the said sample, and in that the coloration characteris- tic of the presence of Salmonella is identified. 13. Detection process according to Claim 12, characterised in that the cul¬ ture medium is inoculated by contact, by soaking, by surface inocula¬ tion or deposition or by inoculation within the mass.

Inventors: RAMBACH ALAIN, RAMBACH, ALAIN
Applicants: MERCK PATENT GMBH, RAMBACH ALAIN, MERCK PATENT GESELLSCHAFT MIT BESCHRAENKTER HAFTUNG
Application year: 1993
Main classification: C12Q1
IPC codes: C12N1, C12Q1