FR2889705A1 - BACTERIAL CULTURE MEDIUM IN A MINIMUM INORGANIC ENVIRONMENT COMPRISING GENTISATE AND / OR ONE OF ITS PRECURSORS, AND USE OF 3-HYDROXYBENZOATE IN SUCH A MEDIUM - Google Patents

Abstract

The present invention relates to a bacterial culture medium for at least one strain to be cultivated comprising, in a minimum inorganic medium, at least one gentisate precursor and / or gentisate or their derivatives, as a source of carbon and energy. It also relates to processes for enriching a bacterial strain or isolating such a strain in such a medium. It also relates to the combination of gentisate or its derivatives and / or at least one precursor of gentisate or their derivatives, in particular 3-hydroxybenzoate, as a source of carbon and energy, with at least one inhibitor, for the culture bacterial of at least one strain to be cultured, the inhibitor acting on the growth of bacteria different from the bacterial strain to cultivate and assimilating said source of carbon and energy, as well as the use of gentisate or its derivatives and / or at least one gentisate precursor or their derivatives, in particular 3-hydroxybenzoate, as a source of carbon and energy, under inhibiting conditions, for the bacterial culture of at least one strain to be cultured, said conditions acting on the growth of bacteria which are different from the bacterial strain to be cultivated and which assimilate said source of carbon and energy.

Description

The present invention relates to selective culture media

  bacterium comprising, in a minimum inorganic medium, gentisate or at least one gentisate precursor. It also concerns

  processes for enriching bacterial culture and / or isolating bacterial culture using such media, as well as the combination of at least one gentisate precursor and at least one inhibitor for enrichment and / or the isolation of bacterial culture, especially Salmonella, and the use of gentisate or one of its precursors, especially 3-hydroxybenzoate for this purpose.

  Many bacteria, including Salmonella, can be responsible for pathologies in humans, including foodborne illness, and are very common.

  There are many environments for the research of these bacteria in various samples (clinical, food, environmental). However, their isolation and identification remains a tedious job that requires enrichment (s), isolation, biochemical and antigenic tests. Marketed environments are usually sensitive or specific, but rarely both. In addition, none of them has been shown to be effective for Salmonella enumeration, in particular.

  The ideal selective medium should allow one bacterial cell of the strain to be grown or identified to donate one colony and must inhibit the other 30 bacterial species. In addition, if the inhibition of other bacteria is insufficient, a colored reaction in the medium may help distinguish the colonies from the strain to be grown or identify other colonies.

  To evaluate the interest of a selective medium, it is possible to evaluate the recovery rate (efficiency of plating), defined as the proportion between the number of colonies on the medium tested and the number of colonies on a reference rich medium (4). ). A medium that is useful according to the invention allows both a recovery rate close to 1 for the strain to be cultivated or identified, in particular Salmonella; and a recovery rate close to 0 for the different species of the strain to be cultivated or identified.

  The Applicant has now demonstrated that such a medium can be proposed, on the basis of a minimum inorganic medium, through a new source of carbon and energy: the gentisate or one of its precursors, especially 3-hydroxybenzoate (3-HB). Other advantages of these environments in particular with respect to known media, and for particular uses will become apparent on reading the description and examples which follow.

  Bacteria of the genus Salmonella are gram-negative bacilli belonging to the family Enterobacteriaceae. This genus is phylogenetically close to Escherichia and Citrobacter. It comprises two species, S. enterica, a habitual species (over 2200 serotypes), and S. bongori, a rare species. S. enterica is subdivided into several subspecies: enterica, salamae, arizonae, diarizonae, houtenae, and indica, which are further subdivided into serotypes. The serotypes mentioned here refer to Salmonella enterica subsp.

  Salmonella enterica subsp. enterica are intestinal parasites of warm-blooded animals and humans; the other subspecies preferentially contaminate cold-blooded animals (reptiles, amphibians). Within the enterica subspecies, strictly human serotypes (Typhi), strictly animal serotypes (Gallinarum) are distinguished, but most serotypes are ubiquitous (Typhimurium, Enteritidis ...) and contaminate humans, poultry, eggs, cattle ... We also find these bacteria in surface water and wastewater. Salmonella are responsible for several types of human diseases, including typhoid and paratyphoid fevers (Typhi and Paratyphi A, B, C serotypes), which in France are mainly cases imported from endemic areas; minor salmonellosis: gastroenteritis, sporadic or collective foodborne illness (TIAC), Salmonella being the most frequently isolated agent in foodborne illness; or extra-digestive manifestations in risky subjects such as immunocompromised individuals.

  Salmonellosis is acquired by ingestion of water and contaminated food. The most commonly implicated foods are eggs and egg preparations, deli meats, poultry, dairy products and meats. This contamination can be linked to defective hygienic practices: contaminated raw materials (intestinal carriage by warm-blooded animals), contaminating environment (equipment, personnel of collective restorations, manufacturing circuits), error in the manufacturing process, significant delay between preparation and consumption or breaking the cold chain, for example.

  As an indication, in 2003, 10472 Salmonella strains of human origin were reported and recorded at the National Reference Center for Salmonella.

  The search for germs, and especially these Salmonella germs in food and the environment (water) is therefore a public health issue, because Salmonella enterica is the main collective food poisoning agent (TIAC) in France.

  For example, Salmonella are isolated from stool cultures (minor and major salmonellosis), blood cultures or other biological fluids (typhoid fevers, bacteremia, invasive salmonellosis), environmental samples and products intended for human or animal consumption.

  On the other hand, most Salmonella are prototrophic but some serotypes, adapted to a particular host (Typhi, Paratyphi A, Gallinarum) are auxotrophic for one or more growth factors.

  In the case of monomicrobial samples (blood), ordinary agar plates such as trypto-casein-soya (TCS2) and Drigalski are sufficient for the isolation of Salmonella. Since the stool and food samples are polymicrobial (commensal flora of the digestive tract, environmental bacteria), their specific isolation requires the use of selective media containing inhibitors and substrates able respectively to inhibit the surrounding flora and to differentiate the surrounding flora. Salmonella from other germs, especially Citrobacter, Escherichia coli and Proteus.

  Usually, the search for Salmonella in a polymicrobial mixture requires several steps, namely pre-enrichment in non-selective medium (6 to 20h), carried out in peptone water; enrichment in selective liquid medium (24h); Müller-Kauffmann broth with tetrathionate, or selenite broth (with or without cystine or novobiocin), or Rappaport-Vassiliadis broth; isolation on agar-selective media from enriched broths (24-48h): Xylose-Lysine-Deoxycholate (XLD), Hektoen, for example, then confirmation by biochemical and antigenic identification, which generally requires at least 3 days of manipulation.

  Selective agar media such as Salmonella include Hektoen, Salmonella-Shigella (SS), xylose-lysinedesoxycholate (XLD), xylose-lysine-Tergitol 4 (XLT4), Novobiocin-bright-green-glucose (NBG) or Novobiocin media. brilliant-glycerol-lactose (NBGL) and Rambach. In addition, several chromogenic media have been developed more recently: SM-ID (Salmonella-Identification), CSE (Chromogenic Salmonella esterase), ABC, CHROMagar, SCM (Salmonella Chromogenic Medium).

  Some of these media exert an inhibitory effect on the commensal flora. Bile salts or sodium deoxycholate affect the growth of gram positive germs. Bright green (antiseptic dye), novobiocin and cefsulodine (antibiotics), and Tergitol 4 (detergent) inhibit Gram-positive flora and some Gram-negative bacteria. These products, possibly associated, oppose the invasion of the agar by Proteus spp and are usually used at doses of the order of 0.33 to 12.5 mg (brilliant green), 1 to 2 mg (Tergitol 4), mg (cefsulodine), 20 mg (novobiocin), 1 g (deoxycholate) and 5 to 9 mg (bile salts) per liter of medium.

  Moreover, in the case of Salmonella, the absence of acid production from certain sugars such as lactose, sucrose, glycerol, or salicin, is used in Hektoen, SS, XLD, XLT4, NBG, NBGL. Salmonella does not acidify the medium, have a distinct coloration of other enterobacteria capable of cultivating (Citrobacter, E. coli, Enterobacter, Shigella ...). In these same media, the combination of sodium thiosulfate and ammoniacal iron citrate gives a black color to the Salmonella colonies by production of hydrogen sulphide. The associated Salmonella-flora discrimination is therefore good, except for Proteus sp., Which also produces H2S. The perception of Salmonella lactose or sucrose + is not correct.

  These media, however, do not allow good isolation of Salmonella Typhi and Paratyphi A, producing little or no H2S. XLD is the least inhibitory and allows a high recovery rate of Salmonella.

  The Rambach medium, interesting for the isolation and enumeration of Salmonella in crustaceans, makes it possible to highlight two specific properties of the genus Salmonella: the production of acid from propylene glycol and the absence of (3-galactosidase (revealed by a chromogenic substrate, X-13-Gal or 5-bromo-4-chloro-3-p-indolyl-Dgalactopyranoside) Thus, colonies of nontyphic Salmonella enterica appear pink fuchsia, allowing rapid identification to the associated flora.

  However, the Typhi and Paratyphi A serotypes give colorless colonies that are difficult to detect, and some colonies of Citrobacter freundii, Pseudomonas aeruginosa and Acinetobacter baumannii may be red, with a similar color, which may lead to difficulties of assessment in the identification.

  Chromogenic media, more recent, allow a better discrimination between Salmonella and the associated flora. They contain chromogenic substrates that demonstrate Salmonella-specific enzymatic activities, including Typhi and Paratyphi A serotypes. Thus, SM-ID demonstrates the fermentation of glucuronate and the absence of R-galactosidase, giving colonies of Salmonella pink to red. CSE allows the detection of C8-esterase activity (through the use of SLPA-octanoate, chromogenic substrate), giving red-purplish colonies. ABC detects α-galactosidase activity of all Salmonella (blue colonies, X-α-Gal substrate) and non-almonella R-galactosidase activity (black colonies, 3,4-cyclohexeno-esculetin substrate or CHEGal). CHROMagar gives purple colonies of Salmonella (evidence of esterase activity), the other enterobacteria appear blue or colorless (activity 3-galactosidase) Finally, SCM detects the activity caprylatestérase of Salmonella, giving magenta colonies, thanks Magenta-cap or 5-bromo-4-chloro-3-indolyl caprylate The colonies of non-Salmonella appear blue (13-galactosidase activity, X- (3-GAL).

  These media can be used to search for certain bacteria, including Salmonella, in various clinical samples but also in foodstuffs, pharmaceuticals, environmental samples. They are most often recommended as isolation media after an enrichment phase, to improve the sensitivity of the method.

  So far, however, no medium of these known media has been used as a selective medium satisfactorily, and there is still a need for such a medium. Indeed, as indicated above, with the currently existing techniques, the isolation of Salmonella requires the use of several selective media containing inhibitors and substrates respectively capable of inhibiting the surrounding flora and differentiate Salmonella from other germs, especially Citrobacter, Escherichia and Proteus. On the other hand, the media according to the invention can fulfill the two goals of using a single selective medium and, in particular, to look for Salmonella without multiplying the steps.

  More specifically, the media of the invention use 3HB, its derivatives and / or other gentisate precursors. 3-HB is a non-nitrogen aromatic compound, cited as a carbon substrate in culture media, by Veron (1) among 146 substrates studied, without this 3-HB being used in the presence of inhibitor, the solutions described having 100 g / 1 of substrate.

  In addition, the publication of Moscoso-Vizcarra (2) describes the metabolic pathways of 3-HB uptake by enterobacteria.

  Finally, US Pat. No. 5,824,552 describes a method for culturing animal cells, and in particular a medium stimulating the growth of cells whose carbon source is glucose. The method described in this document uses a culture medium containing benzoic compounds, especially derivatives of hydrobenzylic acid including 3-HB at concentrations of less than 50 tag / ml, cells cultured, exclusively animal. , not consuming this compound during their cultivation. Indeed, the derivative of hydrobenzylic acid is not a source of carbon and energy necessary for the maintenance of life and the multiplication of animal cells; in the examples, it is thus systematically added a carbon source, glucose.

  The notions of carbon source and growth factor are usually distinguished. A source of carbon and energy, in a culture medium, is catabolized by bacteria, or other living organism, which gives the necessary energy to syntheses as well as molecular materials. The mass of bacteria obtained at the end of growth is proportional to the amount of available carbon source. In addition, growth factors are substances that an organism can not synthesize, and the quantitative needs vary greatly from one strain to another. One distinguishes on the one hand the amino acids, incorporated as such in the proteins if the amino acid was source of carbon it would be catabolized and its carbons would be found anywhere and it is the same for the purines and the pyrimidines incorporated in the nucleic acids and on the other hand the vitamins which enter into the constitution of the coenzymes, and are recyclable, and of which very small quantities are necessary.

  The present invention relates to a bacterial culture medium for a strain to be cultivated comprising, in a minimum inorganic medium, at least one gentisate precursor and / or gentisate, as a source of carbon and energy.

  According to the invention, unlike the teaching of US Pat. No. 5,824,552, 3-HB, its precursors or derivatives and / or other precursors of gentisate or their derivative (s) constitute (s) a source of carbon and energy for bacteria to grow. Thus, according to the invention, the precursor concentration of gentisate and / or gentisate and / or their derivative (s) is such that it (s) constitute a source of carbon and effective energy. As such, and according to a variant of the present invention, 3-HB is used at a minimum final concentration of 0.1 g / l, preferably about 0.5 g / l, and more preferably 1 g About 1, i.e., about 1000 μg / ml in the medium. Such concentrations are therefore at least 2 times, preferably 10, and more preferably 20 times higher than the known concentration of the prior art. The media of the present invention also do not allow the culturing of animal cells.

  In a particular embodiment of the invention, the medium further comprises at least one inhibitor of the growth of bacteria different from the bacterial strain to be cultured and assimilated said carbon source.

  According to one form of the invention, and especially when the medium is in liquid form and / or for certain applications, it is not necessary to add an inhibitor so that the medium according to the invention is enriching.

  This is why the invention also relates, in one embodiment, to culture media in liquid form and without inhibitor.

  It also relates to culture media in semi-solid or gelled form and under inhibiting conditions, that is to say that the inhibition can be caused both by the presence of a growth inhibitor of bacteria different from the strain to cultivate and assimilating said source of carbon and energy only by salinity conditions, aerobic conditions, pH conditions and / or temperature conditions.

  The invention also relates to a process for enriching a bacterial strain in a medium according to the invention.

  It also relates to a method of isolating a bacterial strain in a medium according to the invention.

  It also relates to a method for culturing a bacterial strain in a medium according to the invention used under inhibiting conditions. These inhibitory conditions may be chosen from the presence of a growth inhibitor of bacteria different from the strain to be cultivated and assimilating said source of carbon and energy, the salinity conditions, the aerobic conditions, the control of the pH and / or the temperature control.

  Finally, the invention relates to the combination of gentisate or its derivatives and / or at least one gentisate precursor or their derivatives, as a source of carbon and energy, with at least one inhibitor, for the bacterial culture of at least one strain to be cultured, the inhibitor acting on the growth of bacteria different from the bacterial strain to be cultured and assimilating said source of carbon and energy.

  It also relates to the use of gentisate or its derivatives and / or at least one precursor of gentisate or their derivatives, as a source of carbon and energy, under inhibiting conditions, for the bacterial culture of at least one strain to cultivate, said conditions acting on the growth of bacteria that are different from the bacterial strain to be cultivated and which assimilate said source of carbon and energy.

  As appears from the examples, in a simpler way than by the methods used with the known media, the media according to the invention allow the culture and also the isolation of bacterial strains, and in particular Salmonella strains, including auxotrophic serotypes including Typhi, and Paratyphi, etc. The selectivity of these media is comparable to or better than that of known media. The media of the invention may allow a recovery rate of Salmonella often higher than that of the marketed environments, and this superiority is accentuated when the bacteria are stressed. The media of the invention also have the advantage of requiring smaller amounts of inhibitor (s) compared to the usual media. In addition, they specifically allow the detection of strains of Salmonella subjected to stress.

  As regards the detection of stressed bacteria, the recovery rate of Salmonella subjected to stress has proved, with the medium of the invention, at least equivalent to the TCS agar. The media of the invention are therefore of great interest in the search and enumeration of stressed Salmonella. In fact, bacteria are subjected to various stresses in their environment and in the materials they contaminate: temperature variations (freezing, heating, especially with food products), lack of nutrients, moisture content, oxidation stress, stress osmotic, for example, and, as for many Gram-negative bacteria, these unfavorable conditions can induce the passage of Salmonella especially in the state of non-culturable viable bacteria which makes them undetectable on standard culture media, except under certain conditions favorable that allow bacteria to be revived. Non-culturable viable bacteria are not detectable, but could retain and recover their infectivity under favorable conditions such as intestinal lumen.

  The ability of the medium according to the invention to revitalize certain bacteria, including Salmonella, viable non-cultivable is of particular interest for the search for said bacteria in food and water, by avoiding the pre-enrichment phase recommended by the standard NF EN ISO 6579.

  As a bacterial strain to be cultivated, mention may be made of any strain likely to use the source of carbon and energy according to the invention, that is to say any strain assimilating 3-HB and / or gentisate and / or their precursors and derivatives. Such strains include Citrobacter spp. including diversus and freundii, Enterobacter aerogenes, Klebsiella oxytoca, Salmonella sp. Salmonella enterica subsp indica, Salmonella enterica subsp. arizonae, Salmonella bongori, Escherichia coli, Salmonella enterica subsp. diarizonae, Salmonella enterica subsp., Salmonella enterica subsp. houtenae, Salmonella enterica salamae, Serratia marcescens or Serratia fonticola. In addition, Enterobacteriaceae and Pseudomonas spp and Burkholderia cepacia can be cited. Thus, particularly, according to the invention, the bacterium chosen from Salmonella sp., As bacterium to be cultured, is isolated or enriched. Salmonella enterica subsp indica, Salmonella enterica subsp arizonae, Salmonella bongori, Salmonella enterica subsp diarizonae, Salmonella enterica subspecita, Salmonella enterica subsp. houtenae, Salmonella enterica subsp salamae, Escherichia coli and other enterobacteriaceae According to the invention, Salmonella strains are more particularly targeted. According to the invention, it is more preferably still all serotypes or serovars of Salmonella enterica, and in particular the serotypes Typhimurium, Enteriditis, Typhi, Paratyphi A, Paratyphi B, Virchow, Hadar, Agona, for example, with the exception Gallinarum serotype.

  Minimum inorganic medium according to the invention means a medium containing no assimilable organic carbon feeds. In the absence of these foods, no bacteria can grow, and if a single source of carbon and energy is added, only the bacteria capable of assimilating it can grow.

  For example, the inorganic medium M70, used by Véron (1) to study the assimilation of various energy sources, is such a minimum medium. It consists of three inorganic solutions: a solution of trace elements, a Phosphorus-Calcium-Magnesium solution and the nitrogenous base and will be described below.

  However, any other inorganic minimal medium, containing substantially no organic carbon feed assimilable by the strain to be cultivated, is used according to the invention.

  As a source of carbon or energy that can be assimilated by the bacterial strain to be isolated, identified or counted, 3-hydroxybenzoate, its derivatives, or any other precursors or derivatives of gentisate or its derivatives as they are used, are used according to the invention. may be used in the following transformation route: 3-HB or other precursor of gentisate> gentisate methylpyruvate - * furmarate and gentisate pyruvate.

  Gentisate (2,5-dihydroxybenzoate), a compound resulting from the metabolism of 3-HB, is in fact also assimilable by certain bacterial strains such as Salmonella. Thus, gentisate can also be used as a source of carbon and energy, or as a mixture with a gentisate precursor. And, as a precursor of gentisate, mention may be made of 3HB and its derivatives.

  Among the derivatives, there may be mentioned any chemical form or substance likely to be in the form of gentisate, 3-HB, at the final pH of the medium, capable of spontaneously transforming or by means of a catalyst or a enzyme or physicochemical conditions or by bacterial transformation, gentisate or 3-HB. It is also possible to envisage any natural or synthetic product rich in 3-HB, for example a synthetic polymer degrading to 3-HB or gentisate.

  According to the invention, the gentisate and / or 3-HB concentrations are greater than 0.1 g / l, preferably about 0.5 g / l. Furthermore, they are preferably less than 5 g / l, preferably about 2 g / l. Thus, preferably of the order of 0.1 to 5 g / l of 3-HB, preferably they are of the order of 0.5 g / l to 2 g / l, more preferably of in the range of 0.8 to 1.2 g / I.

  In addition, the media of the invention may comprise one or more inhibitors. They make it possible to improve the selectivity of the medium. However, for the enrichment, for example, a liquid medium (without agar) according to the invention may not comprise an inhibitor.

  As an inhibitor, mention may be made of bile salts, sodium deoxycholate, detergents, antibiotics, certain thallium salts, including thallous sulphate, as well as dyes, and mixtures thereof. Examples of detergents that may be mentioned include Tergitol 4 and Tween 20. Antibiotics include novobiocin, cefsulodin and mixtures thereof.

  Examples of dyes that may be mentioned include dyes of the triphenylmethane type, especially diaminotriphenylmethane, and also in particular bright green and malachite green, gentian violet, acidic fuchsin, and mixtures thereof.

  However, other inhibitors may be suitable which inhibit the growth of undesired bacterial strains, particularly those which assimilate the carbon and energy source of the invention.

  The inhibitor can thus be chosen from inhibitors of gram-positive flora and / or gram-negative bacteria distinct from the bacterial strain to be cultured.

  A particular advantage of the media according to the invention lies in the fact that, given the nature of the minimum inorganic base medium, the doses of inhibitors used can be very small in certain cases compared to the doses useful in known media, for example in Kristensen's medium, bright green medium (usually 12.5 mg) and miliu novobiocin-bright green-glycerol-lactose (usually 7 mg / l).

  Among the preferred inhibitors, mention may be made of glossy green and thallous sulfate and their combinations. Thallium sulphate is intended to inhibit severe aerobic Gram-negative bacteria, such as Pseudomonas and Burkholderia, and bright green enterobacteria such as E. coli. Preferred doses are, for example, from about 1 to 3 mg / I brilliant green, preferably about 1.5 to 2.5 mg / I, and from about 0.02 to 0.002 g / I thallous sulfate, preferably 0.008 to 0.015 g / l, preferably about 0.01 g / l.

  In addition, according to one aspect of the invention, at least one growth factor can be added to the minimal medium. Growth factors accelerate revivification and are particularly useful for the growth of auxotrophic serotypes. This is particularly the case for certain auxotrophic bacterial strains, such as Salmonella Enteridis 64K, or Salmonella Typhi, Salmonella Paratyphi A or serotype Gallinarum. However, if it is desired to isolate only the strains transmitted by food, it is not essential to use such a growth factor. For example, simple pre-enrichment can be achieved without using a growth factor.

  The optimal concentration of a growth factor or the combination of several factors must be sufficiently small for bacterial growth to be insignificant in the absence of another source of carbon and usable energy, such as 3-HB or any other precursor of gentisate. Thus, the concentration is minimal to avoid that it constitutes a source of carbon, which would reduce the selectivity. It is according to the invention chosen to be less than the concentration allowing growth without other carbon source.

  As a growth factor, it is possible to use, for example, a growth factor chosen from amino acids, vitamins, purines, protein hydrolysates and yeast extracts, and mixtures thereof. Any product capable of supplying the medium with the necessary amino acids can be used. For example, a casein hydrolyzate, usually called casamino acid, may be used, but other protein hydrolysates or amino acid mixtures may also be used, optionally supplemented with cysteine, particularly L-cysteine.

  For example, from 0 to 200 mg / l of casamino acid medium, preferably up to about 100 mg / l, more preferably about 50 mg / l, may be used in the presence of 0 to 200 mg / l of L-formamide. cysteine HCl monohydrate, preferably up to about 100 mg / I, more preferably about 25 to 52 mg / I, ie 18 to 36 mg / I pure Cysteine medium.

  In addition, the media of the invention may comprise chromogenic substrates, which make it possible to demonstrate specific enzymatic activities of the cultivated or isolated strains. The addition of such a substrate which demonstrates a specific activity of the strain to be studied makes it possible to improve the Salmonella / non-Salmonella discrimination.

  As a chromogenic substrate, mention may be made of the substrates of the 4- [2- (4-octonoyloxy-3,5-dimethoxyphenyl) -vinyl] quinolinium-1-propan-3-yl-carboxylic acid bromide type ( SLPA-octanoate), 5-bromo-4-chloro-3-indoxyl-caprylate (X-caprylate), 5-bromo-4-chloro-3indoxylalpha-D-galactopyranoside (X-alpha-D-gal) or, to color non-Salmonella species, 5-bromo-4-chloro-3-indoxyl-beta-Dgalactopyranoside (X-beta-D-gal or X-gal), for example. The 5bromo-4-chloro-3-indoxyl radical (X, blue color after hydrolysis) can be replaced by 3-indoxyl (Y, another blue color after hydrolysis) or 5bromo-6-chloro-3-indoxyl (Magenta, magenta color after hydrolysis) or 6-chloro-indoxyl (Salmon, pink color after hydrolysis) or 5-iodo-3indoxyl (lodo, purple color after hydrolysis) or N-methylindoxyl (Green, green color after hydrolysis) or 4-methylumbelliferyl (4- MU, blue fluorescence after hydrolysis), indoxyl here being synonymous with indolyl. Thus, according to the invention, it is possible to use in particular a chromogenic substrate chosen from the X-caprylate, X-alpha-D-galactopyranoside, X-beta-D-galactopyranoside or X-galactopyranoside substrates, where X- represents the 5- bromo-4-chloro-3-indoxyl-, X- may be replaced by 3-indoxyl-, 5-bromo-6-chloro-3-indoxyl-, 6-chloro-3-indoxyl-, 5-iodo-3- indoxyl-, N-methylindoxyl- or 4-methylumbelliferyl-, and mixtures thereof.

  The substrates can be added to the agar or before autoclaving. Their concentration depends on the medium and the intensity of the desired coloring, the products and uses and the color detection modes generated. It can be chosen of the order of 100 mg / l. By way of example, for the chromogens comprising the 5-bromo-4-chloro-3-indoxyl group, the concentration may be of the order of 0.05 to 2 g / l of medium, preferably about 0.1 g / I.

  For substrates of the 5-bromo-4-chloro-3-indolylcaprylate and 5-bromo-4-chloro-3-indoxyl-α-D-galactopyranoside type, for example, the concentration can be chosen between approximately 0.1 and about 0.4 g / I.

  Of course, the concentrations in the various constituents mentioned above depend on the specific constituents, the conditions of use and implementation, and those skilled in the art will adapt.

  The media of the invention are prepared in the usual manner by dissolution and / or addition to a base medium defined above of the additives above. The way of incorporating the additives into the medium or base is not restrictive. The water-soluble additives are added in sterile aqueous solution, but the additives can also be added in solution in a suitable aqueous solvent.

  By way of example, mention may be made of a medium comprising, in Véron's medium, or any other minimal synthetic medium, requiring a source of carbon and energy, and in addition to 3 HB at a rate of 0.2 to 2 g / l. preferably 0.5 to 1.5 g / l, preferably about 1 g / l, casamino acids at 0 to 100 mg / l, preferably 50 to 100 mg / l, preferably about 50 mg / l. I, L-cysteine in hydrochloride monohydrate form at a rate of 0 to 100 mg / l, preferably 26 to 52 mg / l, preferably about 26 mg / l, 1 to 2 brilliant green AcBg-14; , 5 mg / l, preferably about 1.5 mg / l, and thallous sulfate in an amount of 0 to 0.025 g / l, preferably 0.01 to 0.025 g / l, preferably about 0.01 g / I.

  The culture media can be presented according to the invention in liquid form or in a solid form, in the case where a gelled vehicle is used, depending on their subsequent use. Thus, for the culture enrichment, the medium is usually in liquid form, whereas for isolation and identification, it is usually in solid or semi-solid form, usually on agar. However, any other conventional gelled vehicle is usable according to the invention.

  The media according to the invention can be used under the same conditions of pH and temperature as the usual media.

  These culture media are particularly useful for the specific isolation of Salmonella and other Gram-negative bacteria, and can be used to search for germs in many samples. By the choice of appropriate inhibitors, it is possible to envisage the inhibition of strict aerobic Gram-negative bacteria and enterobacteria such as E. coil. Without inhibitors of this bacterium, they may also be useful for the enrichment of certain bacterial strains, in particular E. coli, in addition to that of Salmonella.

  Other characteristics and advantages of the invention will appear on reading the examples below.

EXAMPLES

I Materials and Methods

STUDY STRAINS

  Bacterial strains were used: Salmonella enterica subsp. enterica, 13 non-Salmonella enterobacteria, and 8 non-enterobacteriaceae, but which are similar in their habitat and certain cultural and biochemical characteristics. They come from the collections of the Biodiversity Unit of Emerging Pathogenic Bacteria of the Pasteur Institute (BBPE), the National Salmonella Reference Center (CNR), or the WHO Collaborating Center for Salmonella (WHO CCOMS). Some of these are typical strains or historical strains of Kauffmann.

  The strains are stored in storage agar described below. After reseeding on Trypto-casein-Soy agar (TCS) or Drigalski, the identity and purity of the strains was confirmed by the appropriate tests. For the study, a parent culture of each of these strains is obtained on sloping TCS agar, kept at room temperature, and regular transplants are performed.

  In addition, for a portion of the strains, their ability to use 3-20-hydroxybenzoate (3-HB) as a carbon substrate was studied by inoculating Biotype-100 identification galleries (BioMérieux).

  The list of strains is detailed in Tables I, II and III below where also their provenance, their ability to use 3-HB, and the tests for which they were used are shown.

  A storage agar of the following composition was used: E2 meat casein peptone 10g Sodium chloride 3 g Meat extract 4 g Bacto agar 12 g NaOH pH = 7.2 Distilled water Qs 1 I Table I: Salmonella enterica strains subsp. enterica TESTS2 Use Use of 3-HB Evaltiona-medium Enteritidis 64K (ATCC 13076) CCOMS + + 297/67 CCOMS + + + 1281/04 CNR + + 1299/04 CNR + - + 1384/04 CNR + - + 1397/04 CNR + - + 1538/04 CNR + - + Typhimurium LT2 (ATCC 43971) BBPE + + + 58.58T (ATCC 13311) CCOMS + + + 1327/04 CNR + - + 1333/04 CNR + - + 1366 / 04 CNR + - + 1400/04 CNR + - + 1526/04 CNR + - + Virchow 41K CCOMS + - + 1396/04 CNR + - + Hadar 473K WHOC + - + 1368/04 CNR + - + Typhi Ty2 (ATCC 19430 ) CNR + + + 57K CCOMS + + - 2963/03 CNR + - + Newport 50K CCOMS + - + 1363/04 CNR + - + 1534/04 CNR + - + Infantis 158K CCOMS + - + Brandenburg 24K CCOMS + - + Derby 20K CCOMS + - + AÉona 1137/72 CCOMS + - + Heidelberg 16K CCOMS + + Napoli 173K CCOMS + + Dublin 65K CCOMS + + Indiana 573K CCOMS + + Serotype Strain Source Paratyphi B 5K Java CCOMS + - + Gallinarum 9727/03 CNR - - + 1 Symbols: +, use of 3-HB; -, not used.

  2 Symbols: +, use of the strain during the test; -, untested strain.

  Table II: Enterobacteria strains TEST2 Use Source of 3-HB1 midpoint Strain Escherichia coli 54.8T (ATCC 11775) E. col / 0103 H18 E. coli 0103 PMK1 E. coli 0103 PMK3 E. colt 0103 PMK4 Citrobacter freundii 57.32T Citrobacter koseri (variousus) 82.94T Enterobacter aerogenes 60.86T Enterobacter cloacae 60.85T Klebsiella pneumoniae SB107 Klebsiella oxytoca SB136 Proteus mirabilis PR14 Serratia marcescens 103235 BBPE - + BBPE - + BBPE + + BBPE + + BBPE + + BBPE + + BBPE + + BBPE + + BBPE - + -, strain no + + + +

BBPE

BBPE BBPE BBPE

  1 Symbols: +, use of 3-HB; -, not used.

  2 Symbols: +, use of the strain during the test; tested. + + +

  Table III: Strains of non-Enterobacteriaceae ESSAIS2 Source Use Evaluation of 3-HB1 point of the medium Staphylococcus aureus 65.8T BBPE ND3 + Enterococcus faecalis BBPE ND + 103015T Bacillus cereus 66.24T BBPE ND + Pseudomonas aeruginosa BBPE _ + LMG 1242 T Pseudomonas aeruginosa BBPE + Burkholderia cepacia LMG BBPE + + - 6889 Burkholderia cepacia 103277 BBPE + + + Symbols: +, use of 3-HB; - , not used.

  2 Symbols: +, use of the strain during the test; -, untested strain.

  ND: not determined A) BASE MEDIA: COMPOSITION, PREPARATION The M70 inorganic medium of Véron (1) was used as a base, 3-HB being added as the sole source of carbon and energy, and the yeast extract as a growth factor for auxotrophic strains.

  This study medium was prepared from three distinct solutions: micronutrient solution (0E): strain H3PO4 1.9600 g FeSO4, 7H2O 0.0556 g ZnSO4, 7H2O 0.0287 g MnSO4, 4H2O 0 , 0223 g CuSO4, 5H2O 0, 0025 g Co (NO3) 2, 6H2O 0.0030 g H3BO3 0.0062 g Sterile distilled water Qs 1000 ml The solution obtained by dissolution was sterilized by sterilizing filtration (0.22p filter) . It has been stored in the refrigerator at +4 C. For practical reasons, it is also possible to prepare this solution ten times more concentrated of the same components. This OE solution is included in the composition of solution A described below.

  Solution A: CaCl2, 2H2O MgSO4, 7H2O KH2PO4 K2HPO4 EO Trace Element 3-hydroxybenzoic acid (Fluka) * Yeast extract (Difco) * Sterile distilled water * Not present in the original M70 medium.

  0.0147 g 0.123 g 0.680 g 2.610 g ml (or 1 ml of a ten-fold concentrated solution) 1.0 g 0.1 g Qsp 500 ml After complete dissolution of the various compounds, the pH of solution A was adjusted at 7.2. At this pH, 3-hydroxybenzoic acid is neutralized and is in the form of 3-hydroxybenzoate. The solution was then sterilized by sterilizing filtration on a 0.22 p filter or by autoclaving it for 20 minutes at 110 ° C. A turbid appearance of the solution at the outlet of the autoclave does not modify the quality of the medium.

  solution B: NaCl 7 g (NH4) 2SO4 1 g Agar (Difco) 15 g Distilled water Qs 500 ml The pH of this solution is adjusted to 7.2. It was then heated on a hot plate until the agar was completely dissolved. It was autoclaved for 20 minutes at 110 ° C.

  After sterilization, solutions A and B were maintained at a temperature of 50-60 ° C, avoiding agglomerating B agar solution. They were then mixed aseptically, thus giving one liter of medium. After homogenization, it was poured into petri dishes (20 ml in round boxes 9 mm in diameter, 70 or 80 ml in 12 * 12 cm square boxes). The boxes were stored at +4 C until use.

  To obtain the liquid form, the following are mixed: NaCl (NH 4) 2 SO 4 CaCl 2, 2H 2 O MgSO 4, 7H 2 O KH 2 PO 4 K 2 HPO 4 EO 3-hydroxybenzoate solution casamino acids Lcysteine HCl H 2 O bright green AcBg-14 20 25 7g 1 g 0.0147 g 0.1123 g 0.680 g 2,610 g ml (or 1 ml of concentrate x 10) 1g 0,05 g 0,026 g 0,0015 g thallous sulphate Distilled water 0,01 g qsp 1 liter pH 7,2 B) SELECTIVE MEDIUM OF SALMONELLA ACCORDING TO THE INVENTION Products incorporated in the above culture medium are added to solution A in a small volume of a sterile concentrated solution of these products.

  1.1 Inhibitors 1.1.1 Products The inhibitory activity of different products on bacteria listed above, has been tested in order to reduce the growth of non-Salmonella. The inhibitory effect was tested for increasing concentrations of product. These are the following products used in the respective concentration ranges in the final medium.

  Certified AcBg-14 Green Green marketed by Sigma (B-4014): 0.25 to 5 mg / I (and 5 to 25 mg / I) Thallous sulfate comm. by Fluka (88290): 0.05 to 1 g / I 1.1.2 Method of study The inhibitory power of the different products was determined by a gradient technique: the culture medium containing the inhibitor is poured into a Petri dish. square (12 * 12 cm) so as to create a concentration gradient within the agar.

  1.1.2.1 Preparation of media Solutions A and B are prepared as described above. In addition, stock solutions of the various inhibitors were prepared in distilled water and sterilized by filtration on a unit filter of 0.22 p (ex: 25 mg of brilliant green in 10 ml of water). To solution A, the inhibitor was added at a minimum volume. A solution A was thus prepared at the lowest concentration of inhibitor tested (eg 0.25 mg / l of brilliant green, ie 0.1 ml of the stock solution per 1 liter of medium) and another at the highest concentration. concentration (eg 5 mg / I brilliant green or 2 ml for 1 liter of medium).

  To reproduce the gradient, a first concentrated layer of agar (40 ml) was sloped. After drying, a diluted second layer (40m1) was poured over. The agar obtained was dried in a hood for about 30 minutes and sown within one hour.

  1.1.2.2 Inoculation method From a 24h culture on sloped TCS agar, a bacterial suspension of each strain tested was prepared in 5 ml of sterile distilled water. This suspension was calibrated at 0.5 MacFarland, using a densitometer.

  Seeding was done streaked with 1 μl of the most concentrated side to the most diluted. The dishes were then incubated at 37 C for 48h to 4 days.

  In parallel, two controls were inoculated: a TCS, culture control, and an M70 medium without inhibitor, to differentiate the effect of the inhibitor from that of the minimum medium. Seeding and incubation were carried out under the same conditions.

  The reading of the results was done at 18, 24, 48h and 4 days. The culture density (0, absence, +++, very good) and streak length from the diluted edge, from which the inhibitory concentration for each strain tested was deduced, was approximated.

  The results are given below (II) (1.1.).

  1.2 Growth factors 1.2.1 Products Three growth factors were tested, in addition to the yeast extract, at the following respective concentrations: L-cysteine (in the form of hydrochloride monohydrate, Prolabo): 18 and 36 mg / I of pure cysteine (ie 26 and 52 mg / l of cysteine, HCl, H2O) Casamino acids (Difco): 25, 50, 100, 200 and 300 mg / l (casein hydrolyzate).

  The effect of L-cysteine alone, and that of casamino acids alone, or associated with cysteine, have been studied.

  1.2.2 Method of study 1.2.2.1 Preparation of media Solutions A and B were prepared and sterilized as described above.

  Concentrated stock solutions of the two amino acids were prepared and sterilized by filtration on a 0.22 percent unit filter. They were kept at +4 C and protected from light. A small volume of these solutions was added to solution A (so as to obtain the desired final amount) then A was mixed with B. The casamino acids were directly incorporated in the desired amount in solution A, before sterilization.

  For each growth factor tested, 70 ml of the media thus constituted (35 ml of supplemented A + 35 ml of B) are poured into square Petri dishes 12 * 12 cm and then dried under a hood. These boxes were stored at +4 C until used.

  In addition, M70 media supplemented with growth factors (in the amounts tested) but lacking 3-HB were prepared. By comparing these cultures with those of 3-HB containing media, growth factor (s) concentrations were determined for which growth remained insignificant in the absence of a carbon source.

  1.2.2.2 Inoculation method From a 24h culture on sloped TCS agar, a bacterial suspension of each strain tested was prepared in 5 ml of sterile distilled water. This suspension was calibrated at 0.5 MacFarland, using a densitometer.

  Each suspension was seeded in a dial at 1 pI. A box divided into 6 dials allows the study of 6 strains per box.

  In parallel, controls were inoculated: a TCS (culture control), a control M70 / 3-HB without growth factor.

  The dishes were incubated at 37 C for 48h or even 4 days. The reading was performed at 24, 48 or even 4 days, noting the culture density (0, absence, +++, very good), compared to the controls.

  The results are given below (II) (1.2.).

  1.3 Concentrations of Useful Inhibitors For defined levels of growth factors, the inhibitor concentrations that can be used were determined by the evaluation of the recovery rate or efficiency of plating (eop) of the medium at the following concentrations: bright green: 1 1, 2.5 mg / I; thallous sulphate: 0.01 and 0.025 g / l; the two partners.

  This is a surface count method.

  1.3.1 Preparation of media The media were prepared as described above, supplemented with growth factors and inhibitors. They were cast in a round petri dish of 9 cm (20 ml per box). Controls without inhibitors were also prepared. The boxes were stored at +4 C until use and dried in a hood.

  1.3.2 Inoculation method From a 24h culture on sloped TCS agar, a TCS broth was inoculated with a cese and incubated for 18 h at 30 or 37 C. This broth was then diluted ten to ten in 9 ml of sterile distilled water to 10-7 dilution. To obtain the 10-1 dilution, dilute 1 ml of the initial broth in 9 ml of distilled water; to obtain the 10-2 dilution, 1 ml of the 10-1 dilution is diluted in 9 ml of distilled water, etc. This dilution in water is sufficient to avoid the supply of nutrients from the initial broth during the inoculation of the minimum medium.

  Dilutions 10-4 to 10-6 or 10-5 to 10 * To obtain the 10-1 dilution, dilute 1 ml of the initial broth in 9 ml of distilled water, to obtain the 10-2 dilution, 1 ml of The 10-1 dilution according to the strains was sown at 100 μl on the different media and controls TCS and M70 / 3-HB without inhibitors.These 100 μl were spread with a sterile rake over the entire surface of the box, until completely absorbed, for homogeneous seeding The operation was carried out in duplicate.

  The dishes were incubated for 48 hours at 37 ° C. The colony count was done after 24h and 48h. For each duplicate with a colony count between 15 and 300, the colony mean was calculated and compared to the mean obtained on TCS. The efficiency of plating (eop) was determined by calculating the ratio of the number of colonies counted on the medium tested to the number of colonies counted on TCS. In addition, the growth rate and the size of the colonies were noted.

  The results are given below (II) (1.3.).

  1.4 Selective medium of the invention and comparison with other media 1. 4.1 Medium M70 with 3-hydroxybenzoate (M70 / 3-HB) according to the invention This is the M70 / 3-HB base medium below to which the inhibitor (s), the growth factor (s), at the following concentrations has been added. This medium was prepared in round Petri dishes (9 cm in diameter or 20 ml of medium) and stored at +4 ° C. until use.

  The optimized M70 / 3-HB medium comprises, in addition to the base medium described in paragraph IA, casamino acids (0.050 g / l), L-cysteine (as hydrochloride monohydrate, 26 mg / l), brilliant green AcBg-14 (1.5 mg / I), and thallous sulfate (0.01 g / I).

  The casamino acids (50 mg of powder) and L-cysteine (5 ml of a stock solution containing 5.2 mg / ml of L-Cys, HCl, H 2 O) are added to 500 ml of non-sterile solution A ( qsp 1 liter of medium). Bright green (1.5 ml sterile 1 mg / ml stock solution) and thallous sulfate (1 ml sterile 0.01 g / ml stock solution) are added to the sterile solution A.

  The resulting agar is translucent and sky blue.

  1.4.2 Culture conditions For optimal growth in number and size of Salmonella, and inhibition of other species, the inoculated medium should be incubated for 48 hours at 37 ° C.

  Preparation A composition A solution is prepared: CaCl2, 2H2O 0.0147 g MgSO4, 7H2O 0.123 g KH2PO4 0.680 g K2HPO4 Trace element solution * 3-hydroxybenzoic acid (Fluka) Yeast extract (Difco) Casamino acids (Difco ) L-cysteine, HCl, H2O (Prolabo) Bright green AcBg-14 (Sigma) Thallous sulphate (Fluka) Sterile distilled water 2,610 g ml (or 1 ml of a ten-fold concentrated solution) 1.0 g 0.1 g 0.050 g 0.026 g or 5 ml of a 3.6 mg / ml 0.0015 g solution, ie 1.5 ml of a 1 mg / ml 0.010 g solution or 1 ml of a 0.01 g solution / ml Qsp 500 ml the solution of trace elements * having the same composition as that described above in the base medium.

  Solution B: This solution is identical to that described above for the basic medium.

  Under magnetic stirring the constituents of solution A were dissolved except brilliant green and thallous sulfate; the pH was adjusted to 7.2 0.2 to 25 ° C. The resulting solution was sterilized by filtration on a 0.22 μ Stericup filter. 1.5 ml of a 1.5 mg / ml sterile green gloss stock solution and 1 ml of a sterile thallium sulfate stock solution of 0.01 g / ml were added, these stock solutions having been prepared. separately and sterilized by filtration on unit filters of 0.22p. Solution B was prepared in a sterile 1-liter vial by dissolving the products with magnetic stirring and adjusting the pH to 7.2 to 25 ° C. The resulting solution was heated on a hot plate until that the agar is completely dissolved. This solution was sterilized by autoclaving for 20 minutes at 110 ° C. Maintained at a temperature of 50 ° to 60 ° C., 500 ml of A were mixed with the 500 ml of B. The cells were then poured into Petri dishes, the cast boxes obtained being blue and translucent. Before inoculation, the dishes were dried, by passing 15 to 30 minutes under a hood or in an oven. The boxes were stored and stored in the refrigerator (12h at room temperature).

  Salmonella culture and inhibition of other species were carried out by incubation of the inoculated medium for 48 hours at 37 ° C.

  1.4.3 Other media The final medium assessment was performed by comparison with the TCS and the following known Salmonella selective media.

  Hektoen Enteric agar marketed by BioRad (64584) or Merck (1.11681) Salmonella-Shigella agar or SS Merck marketed by (1.07667) XyloseLysine-Deoxycholate agar or XLD by Merck (1.05287) Xylose-LysineTergitol 4 agar or XLT4 by Merck (1.13918) Rambach agar marketed by Merck (1.07500) Trypto-casein-soy (TCS) marketed by BioRad.

  These media were prepared according to the manufacturers instructions. They were poured into round Petri dishes (20 ml per dish) and kept at +4 ° C. until use, and the composition of the various media is as follows: Hektoen enteric agar Peptones 15 g Sucrose 14 g Lactose 14 g Salicine 2 g Sodium chloride 5 g Sodium thiosulfate 5 g Ferric citrate Ammonia (III) 1.5 g Bile salts 2 g Bromothymol blue 0.05 g Acidic fuchsin 0.08 g Agar 13.5 g Distilled water Qsp 1 I Salmonella Shigella agar (SS) Peptones 10g Lactose 10 g Desiccated beef bile 8.5 g Sodium citrate 10 g Sodium thiosulfate 8.5 g Ferric citrate 1 g Bright green 0.0003 g Neutral red 0.025 g Agar 12 g Distilled water Qsp 1 Xylose-Lysine-Deoxycholate (XLD) D-xylose 3.5 g L-lysine 5 g Lactose 7.5 g Sucrose 7.5 g Sodium chloride 5 g Yeast extract 3 g Sodium deoxycholate 2.5 g Sodium thiosulfate 6.8 g Ammonia iron citrate 0.8 g Phenol red 0.08 g Agar 13.5 g Distilled water Qsp 1 1 Xylose-Lysine-Tergitol 4 (XLT4) Prot ego peptone 1.6 g Yeast extract 3 g L-lysine 5 g D-xylose 3.75 g Lactose 7.5 g Sucrose 7.5 g Iron citrate III ammonia 0.8 g Sodium thiosulfate 6.8 g Chloride Sodium 5 g Phenol red 0.08 g Agar 18 g Tergitol supplement 4 4.6 ml Distilled water Qsp 1 I Rambach agar Peptone 8 g Sodium chloride 5 g Sodium deoxycholate 1 g Chromogenic mixture 1.5 g Propylene 10, 5 g Agar 15 g Distilled water Qsp 1 1 Tryptic casein-soya (TCS) Trypsic casein hydrolyzate 15g Soy peptone 5 g Sodium chloride 5 g Agar 15g Distilled water Qsp 11 1.5 Method of evaluation Inoculation The evaluation of the medium M70 / 3-HB was performed by comparing the efficiency of plating of this medium with that of other media, according to the enumeration method above. For a maximum of strains, from a broth of 18h, dilutions in sterile distilled water were carried out. Dilutions 10-4 to 10-7 are seeded by raking on the 7-ply, in duplicate. After 24 to 48 hours of incubation at 37 [deg.] C., the colonies on each of the plates were counted and the appearance and size of the colonies was recorded. The efficiency of plating was determined relative to TCS agar.

  1.6 Stressed bacteria test Two strains of Salmonella enterica subsp. enterica; Serotype Enteritidis 1299/04 and Serotype Typhimurium 1366/04 were subjected to stress as described below. The efficacy of the M70 / 3-HB medium was studied on these stressed strains by evaluating the efficiency of plating, compared to the various other media.

  1.6.1 Stress test A TCS broth was inoculated and incubated for 18 hours at 37 ° C. Under a hood, 50 μl of this broth was deposited on two glass slides, degreased, sterile (50 μl per slide). Both slides were dried under the flow of the hood until complete drying. The dried slides were put in 10 ml of sterile distilled water and the whole was then vortexed.

  1.6.2 Enumeration An enumeration of the suspension obtained above was carried out on the various media, starting from the 10-2 to 106 dilutions (carried out with the same technique as 3.3 and 4.3). The efficiency of plating of each medium relative to the TCS was calculated.

  The results are presented II (1.5) Results of recovery rates The following tables present the total number of colonies counted on the duplicates of the various media (number box 1 + number box 2), for the same volume of suspension inoculated.

  abieau iv: Denomorement aes kresuiiais at 4an / TCS INV. Hektoen SS XLD XLT4 Rambac h Entdis 297/67 176 186 87 160 267 128 140 Entdis 1281/04 180 205 218 210 209 23 85 338 336 415 364 388 256 331 Entdis 1299/04 185 216 172 159 194 48 106 Entdis 1384/04 205 232 - 217 228 73 167 Entdis 1397/04 220 194 - 193 201 20 132 Entdis 1538/04 198 175 - -239 57 118 266 265 245 262 247 229 233 Tm LT2 197 131 42 164 154 112 109 324 251 217 272 316 74 211 Tm 58.58T 151 106 3 0 29 9 2 Tm 1327/04 187 216 195 214 188 21 122 Tm 1333/04 168 205 171 157 184 8 90 296 267 293 251 288 219 231 Tm 1366/04 294 348 307 281 326 20 142 Tm 1400/04 202 237 213 169 218 11 102 Tm 1526/04 129 124 71 113 51 50 108 234 211 122 154 214 123 149 Virchow 41K 207 202 54 78 165 114 98 Virchow 1396/04 317 356 188 99 341 95 190 Hadar1368 / 04 308 331 218 306 329 256 331 316 402 338 232 315 298 284 445 284 17 232 871 69 22 TyphiTy2 524 76 111 411 70 350 131 Typhi 2963/03 796 799 90 650 404 354 6 108 91 29 88 91 36 66 Newport 1363/04 204 169 211 262 251 220 1 01 Newport 1534/04 231 299 50 280 310 241 219 Infantis 158K 248 280 7 116 176 204 260 Brandenburg 171 149 56 153 241 118 157 24K 856 547 49 99 498 56 135 Derby 20K 486 182 432 200 309 657 598 Agona 1137/72 210 127 14 43 268 40 59 406 223 9 53 353 17 96 Heidelberg 16K 306 340 547 49 306 320 416 764 676 48 78 86 261 311 Napoli173K 179 293 695 114 301 851 930 84 30 102 112 69 108 Dublin 65K 130 144 151 48 121 109 67 Indiana 573K 87 101 76 139 157 136 132 Paratyphi B 5K 87 58 16 50 53 72 77 Java Gallinarum 319 - 235 278 293 260 141 9727/03 Table IV: Non-Salmonella enumeration (results at 48h) TCS INV. Hektoen SS XLD XLT4 Rambach Citrobacter koseri 140 166 40 58 75 17 102 C.freundii 194 0 0 47 127 11 43 Escherichia con 136 0 2 17 132 0 112 PMK3 _ Enterobacter 302 125 14 0 44 0 0 Aerogenes Enterobacter 287 333 146 287 319 271 298 cloacae Klebsiella oxytoca 204 192 116 194 190 39 79 Serratia 169 0 58 126 108 5 65 marcescens Burkholderia 59 0 0 0 0 0 0 cepacia Table V: Enumeration of stressed bacteria (48h results) TCS INV. Hektoen SS XLD XLT4 Rambach Before stress 185 216 172 159 194 48 106 Entdis After stre0s) (dilution 50 58 36 38 34 6 20 1299/04 After stre0s) (dilution 541 566 269 342 402 38 208 Before stress 294 348 307 281 326 20 142 Tm After stress (dilution 94 104 50 15 54 1 35 1366/04 0 After stre0s) (dilution 829 774 466 370 569 26 449 Il Results 1. 1 Inhibitors 1.1.1 Bright green The activity of brilliant green was studied on strains: S. enterica serotypes Typhi Ty2, Typhi 57K, Typhimurium LT2, Typhimurium 58.58T, Enteritidis 64K, Enteritidis 297/67, Escherichia coli 54.8T, E. coli 0103 HI8, E. coli 0103 PMK1, E. coli 0103 PMK3 , E. coli 0103 PMK4, Citrobacter freundii 57. 32T, C. koseri 82.94T, Enterobacter aerogenes 60.86T, Pseudomnas aeruginosa LMG 1242T, Staphylococcus aureus 65.8T, Enterococcus faecalis 103015T, Bacillus cereus 66.24T.

  Two concentration gradients were prepared and tested: 0.25 to 5 mg / I and 5 to 25 mg / I.

  After 48h to 4 days of incubation, strains of serotype Enteritidis were found to be able to grow in the presence of bright green concentrations greater than 5 mg / l, whereas Escherichia coli 54.8T and HI8 were inhibited as early as 0.25. mg / I, Enterobacter aerogenes was inhibited above 3.8 mg / l. The strain of Pseudomonas aeruginosa, a non-user of 3-hydroxybenzoate, gave only microcolonies, unaffected by bright green.

  Finally, the inhibitory concentration is approximately 2.5 to 3 mg / l, for serotypes Typhimurium and Typhi 1.5 to 2 mg / l, for strains of Escherichia coli 0103 PMK1, PMK3, PMK4 3 to 3.5 mg / I, for Citrobacter strains.

  It has been concluded that the optimum bright green concentration is between 1 and 2.5 mg / I to allow inhibition of E. coli without affecting Salmonella.

  1.1.2 Thallium sulphate Thallium sulphate (thallous) has been studied to inhibit Pseudomonas spp. and Burkholderia cepacia. Its activity was tested on the following strains: S. enterica serotypes Typhi Ty2, Typhi 57K, Typhimurium LT2, Typhimurium 58.58T, Enteritidis 64K, Enteritidis 297/67; and Escherichia coli 0103 H18, E. coli 0103 PMK1, E. coli 0103 PMK3, E. coli 0103 PMK4, Citrobacter freundii 57.32T, C. koseri 82.94T, Enterobacter aerogenes 60.86T, Pseudomonas aeruginosa LMG 1242T, P. aeruginosa 9310577, Burkholderia cepacia LMG 6889, B. cepacia 103277, Staphylococcus aureus 65.8T, Enterococcus faecalis 103015T, Bacillus cereus 66.24T.

  Two gradients were prepared and tested: 0.05 to 1 g / l and 0 to 0.25 g / l.

  Pseudomonas and Burkholderia have been shown to be inhibited in the presence of thallium sulfate at any concentration and all other bacteria are inhibited at concentrations ranging from 0.1 g / I (Typhi, Escherichia coli 0103) to 0.5-0.6 g / I (Enterobacter aerogenes, Typhimurium and Enteritidis).

  Furthermore, Typhi Ty2 and Typhimurium LT2 were very well cultured at a concentration of 0.025 g / l and moderately well at 0.05 g / l (results obtained by inoculation of M70 / 3-HB agar plates supplemented with thallium 0.025 or 0.05 or 0.1 g / I). At these same concentrations, no culture was observable for Pseudomonas and Burkholderia.

  The optimum concentration of thallium sulfate is less than 0.025 g / I, to inhibit Pseudomonas and Burkholderia and not affect the growth of Salmonella.

  1.2 Growth factors The influence of cysteine and / or casamino acid supplementation has been tested, with cysteine studied on Typhi Ty2, Typhi 57K, Enteritidis 64K and Enteritidis 297/67 (the latter is not auxotrophic), alone and in combination, at the following concentrations: L-cysteine, HCl, H2O: 26 and 52 mg / I (18 and 36 mg / l cysteine). It has been found that cysteine promotes the growth of Typhi, but has no influence on Enteritidis 64K, and that the effect is identical for 18 and 36 mg / I (Table VI).

  Table VI: Amino Acid Test: Results at 48h Cys Control 18 Cys 36 M70 Typhi Ty2 - ++ ++ Typhi 57K - + + Entendis + + + 64K - - Hearend ++ ++ ++ 297/67 Culture: - very weak; low; + average; ++ good.

  Furthermore, the influence of the casamino acids was tested for concentrations of 25, 50, 100, 200, 300 mg / l and in combination with 26 mg / l of L-cysteine, HCI, H2O. The strains used were: S. enterica serotypes Typhi Ty2, Typhi 57K, Enteritidis 64K and Enteritidis 297/67, Typhimurium LT2; and Enterobacter aerogenes, Citrobacter freundii 57.32T, C. koseri 82.94T, Escherichia co / i 54.8T, E. coli H18, E. coli 0103 PMK3, Klebsiella pneumoniae SB107. This test was performed on M70 medium with and without 3-HB. The results at 48 hours of incubation are presented below. (Table VIII) Table VII: Casamino Acid Tests: Results at 48 hrs (a) L [) M70 (and 3HB ni -) - - - - - - - - - - CAA) CAA25 - - - - - - - - - - - - CAA25 + 3HB - ++ ++ ufc * - ++ _ _ ++ - CAA50 - - - / - / - / - - - - - - / CAA50 + 3HB - - ++ ++ 0 - ++ ++ - CAA100 - / - / '- / - - _ -PI: - kt - / - / CAA100 + 3HB _ + + ++ ufc * - / +++ - / - / ++ - / CAA200 - / - / - / CAA200 + 3HB - / + ++ ++ ufc * +++ - / - / ++ CAA300 _ / ' , _ / _ / CAA300 + 3HB + _ / + ++ ++ 0 +++ - / - / ++ CAA50 + Cys + 3HB + - / + ++ _ / ++ ++ - / CAA100 + + + + + ++ ++ _ / + Cys + 3HB 3HB: 3-hydroxybenzoate, CAA: casamino-acids, Cys: L-cysteine, HCI, H2O Culture: 0 none - very weak, weak, + average, ++ good; + ++ very good. * Ufc: colony-forming unit It was concluded that the addition of cysteine at 50 mg / l to casamino acids allows for a good culture of strains, including Typhi, without favoring that of non-strains. users of 3-HB.

  1.3 Medium M70 with 3-hydroxybenzoate (M70 / 3-HB) The M70 medium containing two inhibitors: brilliant green and thallium sulfate was studied in the presence of the following growth factors: casamino acids at 50 mg / I associated with 26 mg / I L-cysteine hydrochloride monohydrate (18 mg / I L-cysteine).

  The recovery rate of Salmonella was evaluated according to the different concentrations of brilliant green, with or without thallium sulfate, by counting the bacteria that can be cultivated on the medium in comparison with TCS agar.

  The following strains using 3-HB: Typhimurium LT2, Typhi Ty2, Citrobacter freundii 57.32T, Escherichia coli 0103 PMK3, Enterobacter aerogenes 60.86T, and Burkholderia cepacia 103277 were used.

  Glossy green has been studied alone at different concentrations 1; 1.5; 2 and 2.5 mg / I. This test confirms the inhibitory activity of this dye on Escherichia coli, starting at 1.5 mg / l. The averages of the number of colonies counted (duplicate assay), the efficiency of plating, and the size of the colonies are given in the table below. (Table VIII)

  Above 1.5 mg / I, the number and size of colonies counted decreased for Salmonella and Enterobacter aerogenes, indicating an inhibitory effect. Citrobacter freundii cultivated with difficulty, giving microcolonies of diameter much less than 1 mm, and its culture was considered negative. The same has been true for Burkholderia cepacia, which can only be counted up to 1.5 mg / I.

  Table VIII: Recovery Rates for Increasing Concentrations of Bright Green (BV) (Results at 48h) TCS Control VB 1 VB 1.5 VB 2 VB 2.5 M70 / 3 mg / 1 mg / 1 mg / 1 mg / I

HB

  1 to ND d 0.89 0.89 0.92 0.63 Typhimurium (101) b (90) (89.5) (93) (64) LT2 2-5 mm 1-3mm 1-3mm <_1 mm <1 mm 1 ND 0.65 0.60 0.56 0.45 (72, 5) Typhi Ty2 (120.5) (78) (67.5) (54.5) _ 5mm - 1-2mm 1- 2 mm _ 1 mm _ 1 mm Escherichia (64,5) ND 0 * 0 0 0 co / i PMK3 <5 mm - 1 mm - - - 1 0 * 0 * 0 * 0 * 0 * Citrobacter (112,5) freundii <1 <1 <1 3-5 mm 1 mm 1 mm mm mm mm Citrobacter 1 0.59 1.06 0.82 0.81 0.82 koseri (113) (66.5) (120) (93) ( 91.5) (93) 2-3 mm 5 2mm <_1 mm 3-7 mm _ 2 mm _ 2 mm 1 0.86 0.64 0 * 0 * 0 * Enterobacter (230) (198.5) (147) , 5) aerogenes 1 <1 <1 3-5 mm <_ 1 mm <1 mm mm mm mm 1 0.63 0.96 0 * 0 0 Burkholderia (96) (60.5) (92.5) cepacia 1 2 mm 1 mm <_1 mm Invisible mm a: Recovery rate compared with TCS b: Average number of colonies counted on duplicates 5 C: Diameter of colonies in mm d: Not determined *: microcolonies The association of 0 , 01 or 0.025 g / 1 of bright green thallium sulphate was studied according to the same method (Tab water IX). The addition of thallium sulfate at these low concentrations does not affect the culture of Salmonella tested. Strains of Escherichia coli and Burkholderia cepacia were totally inhibited in the presence of 1.5 mg / I of bright green and 0.01 g / l of thallium sulfate. The results observed for Citrobacter freundii and C. koseri remained unchanged.

  A concentration of thallium sulphate of 0.01 g / l, added to 1.5 mg / l of brilliant green, therefore seems sufficient to make the M70 / 3-HB medium even more selective.

  Table IX: Media recovery rates in the presence of a combination of green-gloss (VB) and thallium sulphate (ST) (results at 48h) ST ST indicator VB 1.5 VB 1.5 TCS M70 / 3- VB 1 , 5 + ST + ST HB 0.01 0.025 0.01 0.025 0.96 0.91 1.03 0.93 0.76 0.84 Typhimurium (11b'S) (110) (104) (117.5) ( 107) (86.5) (96.5) LT2 3-6c 2-3 mm <_ 2 mm 1-3 mm 1-3 <2 mm <_ 2 mm mm 1 1.11 0.88 1.14 0.87 0.99 0.92 Typhi Ty2 (236) (262) (208.5) (275) (206) (233.5) (215.5) <2 <4 mm <2 mm < 2 mm <_ 2 mm mm <_ 1 mm <_ 1 mm Escherichia (74,5) ND d 0 * ND ND 0 ND coli PMK3 3-5 _ 1 _ _ _ _ mm mm 1 ND 0 * ND ND 0 * ND Citrobacter (70.5) freundii 3-5 1 1 mm mm 1 1.02 0.94 1.22 1.1 0.99 0.9 Citrobacter (141.5) 144) (132.5) (172) , 5) (155) (140) (128) koseri <2 7mm _ 3mm _ 3mm _ 3mm _ 2mm _ 2mm mm 1 1 0.98 0.79 0 0 0 0 Burkholderia (28.5) (28) (22) , 5) cepacia <2 <1 1 mm _ - - - mm mm IO a: Recovery rate compared with TCS b: Average number of colonies counted on duplicates C: Colony diameter in mm d: Not determined *: microcolonies Note: There was no difference in appearance between colonies of the different strains tested. They appeared all round, regular, opaque, beige to green.

  1.4 Evaluation of the Final Medium Comparison with known media The effectiveness of the medium to demonstrate the bacteria was compared with that of 5 commercialized media: Hektoen, Salmonella-Shigella, XLD, XLT4, and Rambach.

  The results obtained at 48h for Salmonella are shown in Table X. The averages of recovery rates on M70 / 3-NB varied between strains and serotypes from 0.46 (Typhi Ty2) to 1.30 (Heidelberg) with an average of 0.94 and a median of 0.96. The diameter of the colonies after 48h ranged from 1 to 2.5 mm.

  For the other media, the mean and median (and maximum) coverage recoveries are 0.28 and 0.21 (0.02 to 0.87) on Hektoen BioRad, 0.92 and 0.98, respectively (0). , 21 to 1.23) on Hektoen Merck, 0.72 and 0.84 (0 to 1.60) on SS, 0.97 and 0.98 (0.11 to 1.96) on XLD 0.44 and 0.36 (0.04 to 1.56) on XLT4 and 0.63 and 0.60 (0.01 to 1.52) on Rambach.

  The diameter of the colonies on these different media varied from 2 to 5 mm after 48 hours of incubation.

  In Table X, the confidence intervals at 95 (ie 5% error) of the recovery rates of each strain tested are plotted on each of the media. These were calculated according to the formula developed by Williams in 1968 to estimate the confidence interval of the efficiency of plating (oep) (5). The confidence interval for 5% error is defined as such: eop 1,966, where 6 is the standard deviation (standard error). The standard error corresponds to the square root of the variance, defined by the following formula: = X1 (X1 + X2) / X23 X1: the total number of bacteria counted on medium 1 (sum of the bacteria counted on the two boxes of the duplicate) X2: the total number of bacteria counted on the medium 2 Always taking X1 <X2 for the calculation.

  The values of X are reported in the appendix (Table, Table IV, Table V).

  M70 / 3-HB medium was the most effective for 22 trials out of 43 (18 strains out of 31): all strains of Typhimurium, 2 strains of Virchow, 2 strains of Hadar, 1 Typhi, 1 Newport, Infantis , Derby, and Napoli. Salmonella Gallinarum was not cultivable.

  By comparing the averages of the recovery rates, the difference between the medium M70 / 3-HB and TCS is not significant (Normal law 5%: u = 1.86, p> 0.05). Similarly, XLD shows an equal number of Salmonella (u = 0.45, p> 0.05, NS), as well as Hektoen Merck (Student's 5% test: t = 1.392, p> 0, 05, NS). Other media have significantly lower recovery rates than M70 / 3-HB and TCS (p <0.05).

  Salmonella colonies showed a black color on Hektoen media, SS, XLD, XLT4 (24h production of H2S) and Rambach roses (use of propylene glycol). Both strains of Typhi and the Gallinarum strain appear colorless on all media. Hadar 173K and Napoli 473K also gave colorless colonies on Hektoen, SS, XLD, and XLT4 and roses on Rambach.

  Table X: Salmonella recovery rates (48h results) MHOB / 3 Hektoen SS XLD XLT4 Ra hbac Entdis 0.86-0.36-0.62 0.72-1.39-0.56-0.620.98 297 / 67 1.26 0.49 0.13 1.10 1.65 0.90 0.80 0.18 1.06 0.20b 0.91 0.19 1.52 0.13 0.73 0.17 0.96-1.32 1.05-1.0- 0.99%; Entdis ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 1,1 , 17 1281/04 0 84-1 14 1,11-0,94- 1,02- 0, 64- 0 83-1,13 1.22 1.28 0.88 '1.35 * 099 0.15 1.23 0.12 1.08 0.14 1.15 0.13 0.76 0.12 0.98 0.15 Entdis 1.00-1.34 0.74-1.12 0.68-0 , 86- 0.18-0.43-0, 71 1299/04 1.17 0.17 0.93 0.19 * 0.8600.118 1, 5.20,19 0.26 0.08 0, 57 0.14 Entdis 0.96-1.30 0.88-0.94-0.26 -0.64_0.98 * 1384/04 113 017 1.24 1.28 0.46 081 017 1.06 0 , 18 1.11 0.17 0.36 0.10 Entdis 0.71-1.05.71-0.74-0.70-0.73 0 _ *% I53 1397/04 0.88 0.17 '' '0.60 0.13-0.88 0.17 0.91 + 0.17 0.09 + 0.04 0.70-1.06 * 0.93-0.18-0.40-0, 68,088,018 - "1.25 0.34 0.54 0.14 Entdis 1.09 0.16 0.26 0.08 1538/04 0.83-1.17 0.76-1.08 0.81 - 0.77- 0.710.73-1.03 1.00 0.17 0 92 0 16 * 1.15 1.09 1.0 1 0.88 0.15 0.98 0.17 0.93 0, 16 0.86 0.15 0.51-0.81 0.14-0.28 0.66-0.62-0.44 - 0.42-0.68 0.66 0.15 0, 21 0.07 1.00 0.94 0.70 0.55 0.13 MT LT2 0.83 0.17 0.78 0.16 0 , 57 0.13 0, 64-0.90 0.55-0.79 00.70- 0.83-0.17-0.54-0.76 0.77 0.13 0.67 0.12 *, 98 1; 13 0.29 0.65 0.11 0.84 0.14 0.98 0.15 0.23 0.06 Tm 0.53-0.87 0-0.04 0.080.02-0-0.03 58.58T 0.70 0.17 0.02 0.02 0 07 0.0610.04 0.01 0.02 0.16-0.88 Tm 0.99-1.33 0.85-1.23 0 , 97-0.80-0.06-0 50-0.80 1327/04 '1.16 0.17 1.04 0.19 * 1.31 1.20 0.16 0.65 0.15 1 , 14 0.17 1.00 0.20 0.11 + 0.05 0.73-0.90-0.02-1.05-1.39 0.81-1.23 1.13 0.40 -0.68 1 1.22 0.17 1.02 + 0.21 * 1.28 0.54 0.14 0 0.07 Tm 0.93 0.20 1.09 0.19 0.05 0, 03 1333/04 0.75-1.05 0.831.15 0.71-0.81-0.61-0 65-0.91 0.90 0.15 0.99 0.16 * 0.8590.14 0.97.10.16 0.74 0.13 0.78 0.13 MHOB / 3 Hektoen SS XLD XLT4 Ra hbac Tm 1.05-1.31 0.891.19 0.80-0.97-0.04 -010 0.38-0.58 1366/04 1.18 0.13 1.04 0.15 * 0.9610,16 1.11,? 0.14 0.07 0.03 0.48 0.10 Tm, 1.01-1.33 0.87-1.23 0.67-0.90 0.020.38-0.62 1.01 1.6 08 1400/04 1.17 0.16 1.05 0.18 * 0.84 0.17 1.08? 0.18 0, 0500.03 0.50 0.12 0.72-1.20 0.39-0.71 0.66-0.27 0.26-0.65-1.05 0.2, Tm 0.96 0.24 0.55 0.16 * 0, 810.22 0, 050.13 0.3950.13 0.84 0.21 1526/04 0.530.74-0.42-0.73-1.07 0.41-0.63 0.51-0.77 0.79 1.08 0.64 0.90 0.17 0.5210, 11 0.66 0.13 0.91-10.17 0.53 0.11 0.64 013 Virchow 0.79-1.17 0.18-0.0 , 34% 88 040.36-0.58 41 K 0.98 0 19 0.26 0.08 0.96 0.68 0.47-10.11 0.38 0.10 0, 80 0.16 0 , 55 0.13 Virchow 0.98-1.26 0.48-0.70 Ï 0.24-0.94-% 230 49-0 71 1396/04 1.112.014 0.59 0.11 0.330, 07 1.08? 0.14 0.30 0.07 0.60 0.11 Hadar 0, 60-1.02 0-0.08 0.10-0.11-0.24-0 35-0 65 473K 081 021 0.04 0.04 0.18-0.88 0.19-0.08 0.3640.12 0.50 0.15 0.93-1.21 0.59-0.83 0.760 84-0.69- 0, 93-1.21 Hadar 1.08 0.83.90.14 1.07 0.14 1.07 0.14 0.71 0.12 0.92 0.16 0, 9910.15 1368/04 1.15-1.39 '0.93-1.21% 15 1.16 1.09 0 76-1.04 1.27 0.12 1, 07 0.14 * 0, 90 0.14 0.73 0.12 1.00 0.16 0.94 0.15 0.54-0.74 0.02-0.06 0.44-1.90-0.12-0, 03-0.07 Typhi 0.64 0.10 0.04 0.02 0.260.08 1.600.06 111 0.05 0.02 Ty2 0.42-0.50 0.73-% 268 0.23 0, 27 0.06 0.6 0.1.37 0.85 0.12 0, 32 0.06, 1.27 0.10 0.18 0.05 Typhi 10.90-1.10 0.09-0 , 13 0.74-0.45-% 350-0, 02 2963/03 1.00 0.10 0.11 0.02 0.8290.08 0.550.06 0.60.06 0.01 0.01 0.60- 1.08 0.16-0.38 0.15 04- 01,6107- 00,20460,42-0,80 Newport 084 0,24 0, 27 0,11 0,81, 0,231I0,84 0,23 0,33 0,13 061 0,19 1363/04 0.79-1.13 0.69-0.99% 340.66-0.96 0.96 0.17 0.84 0.15 * 0 09 0.890 14 n 65.70 19 0.81 0.15 1.21-0.87-I 0.77-1.13 1534/04 1.29 0.13 0.22 0.07 1.35 1.43 1.21 0.95 0.18 Newport 1.16-1.42 0.15-0.29 1.07- MHOB / 3 Hektoen SS XLD XLT4 Ra hbac 1, 21 0.14 1.34 0.13 1.04 0.17 Infantis 0.98 1.28 0.01-0.05 0.37-% '80 88-1 22 158K 1.13 015 003 0.02 50.97 1.05 0.17 0.47 0.10 0.71 0, 14 0.82 0.15 Branden 0 68-1 06 0 23-0 43 0.69-1.27-0.53-0.72_1.12 burg 24K 0, 87 0.19 0.33 0, 10 1.09.55 0.85 092 020 0.89 0.20 1.41 0.14 0.69 0.16 0.57-0.71 0.04-0.08% '0.013 -0.19 0.64 0.07 0.06 0 02.140.64 0.09 0.16 0.03 Derby 0.12 0.02 0.58 0.06 0.07 0.02 20K 0.81- 1.01 0.65-0.83 0.23-0.58-0.25-0.41-0.53 03.04 0.91 0.10 0.74 0.09 *! 0, 8,30.05 0.66 '0.08' 0.030, 05 0.47 0.06 0.13-1.14 -0.13 Agona 0.47-0.73 0.03-0, 0.20-0.36 0.27 1, 42 0.25 0.28 0.08 1137/72 0.60 0.13 0.07 0.04 0.20 0.07 1.28 0.14 0.19 0.06 0.46-0.64 0.01-0.03 0.09-0.75-0.02-0 19-0.29 0.07 0.99 0.06 Heidelbe 0, 0.09 0.02 + 0.01 0.13 0.04 0.87 0.12 0.04 0.02 0.24 0.05 rg 16K 0.810.92-1.63-0.10-1 0 I 19-1,41,81-1,11 1,11 * 1,20 1,79 0.20 130 0.11 0, 96 0.15 1.06 0.14 1.71 0.08 0, 0.05 0.96 0.15% 82.0 90.79-0.97 0.04-0.88 0.36-0.46 14.039 0.88 0.09 0.06 0 , 02 0.41 0.05 Napoli 0.10 0.02 0, 11 0.03 0.34 0.05 173K 0.97-1.15 0.18 *% 99 0.0.30-, 040 106 009,, 0.90 0.16 0.35 0.05 0.21 0.03 0.34 0.05 0.82 0.08 0.13 0.03 0.42-0.74 0.13 0,29 0, 52- 0,58-0,33- 0,55_0,93 0,88 0,96 0,61 074 0,19 0,58 0,16 0,21 0,08 Dublin 0,70 0 , 18 0.77 0.19 0.47 0.14 65K 0.42-0.80 0.98-1.40 1.13-1.210.29 -0.88-1.34 0.61 0.19 1 , 19 0.21 * 1.250.19 1.39 0.18 0.404'50.15 1, 11 0.23 Indiana 0.91-1.41 0.60-1.14 1.43-1.65 -1 , 39- 1 34-1 70 d 77 AnG A 70. ff 573K 1.16 0.25 0.87 0.27 1.52 0.18 1.60 0.17 1.80 0.15 1, 56.0, 17 Paratyphi 0.45-0.89 0.08-0.28 0.37-0 , 40- 0.57- '0.62-1.16 B 5K 0, 67 0.22 0.18 0.10 0.77 0.82 1.09 0.89 0.27 Java 0.57 0, 0.61 0.21 0.86 0.26 Gallina-0.77-0.77-0.69-0.62-0.86 .-, A7 0.35-0.43 rum 0 ** 0, 74 0.12 0.95 0.44 0.09 9727/03 0.87 0.14 0.92 0.15 0.82 0.13 a: Confidence interval (5% error) of I 'efficiency of plating b: Average recovery rate 95 ° confidence difference: Hektoen Merck **: Gallinarum microcolonies on M70 / 3-HB (diameter <1 mm): culture considered null -: not determinable Strain tests Non-Salmonella (Table XI) showed that the M70 / 3-HB medium did not allow the cultivation of either Citrobacter freundii, Escherichia coli, Serratia marcescens and Proteus mirabilis (microcolonies), or Burkholderia cepacia (no recovery rate). ). Citrobacter koseri and Klebsiella oxytoca showed good coverage on M70 / 3-HB, with colonies 2 mm in diameter. On the other hand, there was no difference in appearance between Salmonella colonies and non-Salmonella colonies.

  The other media are more or less inhibitory with respect to these strains, with low recovery rates (variable according to strains and media); they give 48h colonies whose diameter varies from 3 to 5 mm. The colonies observed differ from colonies of Salmonella by their coloring. In fact, Hektoen, SS, XLD, and XLT4 make it possible to differentiate black colonies from H2S-producing bacteria (Salmonella, Proteus) from colonies of non-producing H2S strains that appear colorless to yellow. On Rambach, a chromogenic medium, non-Salmonella colonies appear blue-violet by the presence of beta-galactosidase, whereas Salmonella colonies are pink to red.

  Table XI: Non-Salmonella Recovery Rates (48h Results) M70 / 3 Hektoen * SS XLD XLT4 Rambach

HB

  C.koseri 1.00-1 -0 0.28-0.54 0.39-0.69 0.06-0.18 0 92 0> 19> 39 54-0, 38 1.19 0.19b 0 , 29 0.10 0.41 0.13 0.54 0.15 0.12 0.06 0.73 0.19 freundii 0 ** 0 0.16-0.32 0.49-0.69 0, 0.3-0.09 0.15-0.29 C.freundii 0.24 0.08 0.64 0, 0.06 0.03 0.22 0.07 E.coli PMK3 0 ** 0-0, 03 0.07-0.19 0.64-1.20 0.61-1.0 0.01 0.02 0.13 0.06 0.97 0.23 0.82 0.21 E.aerogenes 0 , 32-0.50 0.03-0, 07 0 0.10-0.20 0 0 0.41 0.09 0.05 0.02 0.15 0.05 E.cloacae 1.02-1, 30 0.410.61 0.84-1.16 16.97-1.25 0.78-1.10 0.88-1.20 1.16 0.14 0.51 0.10 1.00 0, 16 1,11 0,14 0,94 0,16 1,04 0,16 K.oxytoca F 0,75-1,13 0,44-0,70 0.79-1, 11 0.75-1.11 0.12-0.26 0.29-0.49 0.94 0.19 0.57 0.13 0.95 0.19 0.93 0.18 0.19 0.07 0.39 0.10 S.marcescens 0 ** 0.24-0.44 0.58-0.92 0.49-0.89 0-0.06 0.27-0.49 0.34 0.10 0.75 0, 17 0.64 0.15 0.03 0.03 0.38 0.11 B.cepacia 0 0 0 0 0 0: Confidence interval (5% error) of the efficiency of plating b: Average recovery rate 95 * confidence difference: Hektoen BioRad **: Microcolonies on M70 / 3-HB (diametric) re <1 mm): culture considered null 5 -: not detachable 1.5 The effectiveness of the medium M70 / 3-HB was also evaluated on two strains of Salmonella enterica previously subjected to stress: Enteritidis 1299/04 and Typhimurium 1366 / 04. The results of this test are given in Table XII. The recovery rate on M70 / 3-HB has been slightly reduced after stress, and remains greater than or equal to 1, ie at least equivalent to the TCS. Rates were down on Hektoen, SS and XLD, and remained equivalent on XLT4 and Rambach. Stunting on different media

  Table XII: Salmonella Recovery Rates Previously Stressed (Results at 48h)

SS

XLD

M70 / 3-

HB

  Hektoen * XLT4 Rambach Entdis Before 1.00- 0.68- 0.86- 1.34h 0.74-1.02 1.04 1.24 1299/04 Stress 1.17 0.17 0.93 0.19 0.86 0.18 1.05 0.19 0.18-0.34 0, 26 0.08 0.43-0.71 0.57 0.14 After 044-038-002- stress, 0.83 -1.49 0.41-1, 03 019-0.61 '(dilution 1.16 0.33 0.72 0.31 1.08 0.98 0.22 0.40 0.21 4) 0, 76 0.32 0.68 0.30 0.12 0.10 After 0.54-0.64-0.05 stress 0.94-1.16 0.430.57 0.32-0.44 0.72 0.84 0.09 (dilution 1.05 0.11 0.50 0.07 0.38 0.06 103) 0.63 0.09 0.74 0.01 0.07 0.02 0.80 0.97- 0.04-Before 1.05-1.31 0.89-1, 19 0.38-0.58 1.12 1.25 0.10 Stress 1.18 0.13 1.04 0 0.48 0.10 0.96 0.16 0.11 0.14 0.07 0.03 After 0.07-0.38 Tm stress 0.86-1.36 0.35-0 71 0, 25 0.76 0-0.03 0.23-0.51 1366/04 (dilution of n 1.11 0.25 0.53 0.18 0.01 0.09 0.57 0.19 0.01 0.02 0.37 0.14 After 0.40-0.62-0.02 stress 0.841.02 0.50-0.62 0.48-0.60 0 50 0'76 0, 04 (dilution 0.93 0.09 0.56 0.06 0, 54 0.06 0 3) 0.45 0.05 0.69 0.07 0.03 0.01 a: Previous results (Table X) b: Confidence interval (5% of e r) of the efficiency of plating C: Average recovery rate 95% confidence interval *: Hektoen Merck 54 BIBLIOGRAPHIC REFERENCES (1). Véron M. Nutrition and Taxonomy of Enterobacteriaceae and Related Bacteria. I. 5 Method of study of auxanograms.

  Ann. Microbiol. Inst. Pastor 1975; 126A: 267-274.

  (2). Véron M., Le Minor L. Nutrition and Taxonomy of Enterobacteriaceae and Related Bacteria. II. Overall results and classification III. Nutritional characteristics and differentiation of taxonomic groups Ann. Microbiol. Inst. Pastor 1975; 126B: 111-147.

  (3). Moscoso-Vizcarra M., Popoff M. Pathways of dissimilarity of benzoic acids by enterobacteria. Ann. Microbiol. Inst. Pastor 1977; 128A: 199-204.

  (4). Williams T. Interval estimates for effiency of plating. J. Gen. Virol. 1968 2: 13-18.

Claims (34)

  Bacterial culture medium for at least one strain to be cultivated comprising, in a minimum inorganic medium, at least one precursor of gentisate and / or gentisate or their derivatives, as a source of carbon and energy.   2. Culture medium according to claim 2, characterized in that the bacterial strain to be cultured is selected from Salmonella sp. of which Salmonella enterica subsp indica, Salmonella enterica subsp arizonae, Salmonella bongori, Salmonella enterica subsp diarizonae, Salmonella enterica subsp., Salmonella enterica subsp. houtenae, Salmonella enterica salamae, Escherichia col / and other enterobacteria.   3. Culture medium according to claim 1 or 2, characterized in that the bacterial strain to be cultivated is selected from the serotypes or serovars of Salmonella enterica and in particular the serotypes Typhimurium, Enteriditis, Typhi, Paratyphi A, Paratyphi B, Virchow, Hadar and Agona.   4. Medium according to one of claims 1 to 3, characterized in that the bacterial strain is a strain of Salmonella, including a strain of stressed Salmonella.   5. Medium according to one of claims 1 to 4, wherein the gentisate precursor is selected from 3-hydroxybenzoate and / or their derivatives and mixtures thereof.   6. Medium according to one of claims 1 to 5, wherein the gentisate precursor is 3-hydroxybenzoate.   7. Culture medium according to one of claims 1 to 6, characterized in that the precursor concentration of gentisate and / or gentisate or their derivative (s) is greater than 0.1 g / l.   8. Culture medium according to one of claims 1 to 7, characterized in that the precursor concentration of gentisate and / or gentisate or their derivative (s) is greater than 0.5 g / l.   9. Culture medium according to one of claims 1 to 8, characterized in that the precursor concentration of gentisate and / or gentisate or their derivative (s) is less than 5 g / l.   10. Culture medium according to one of claims 1 to 9, characterized in that the precursor concentration of gentisate and / or gentisate or their derivative (s) is less than 2g / l.   11. Culture medium according to one of claims 1 to 10, characterized in that the precursor concentration of gentisate and / or gentisate or their derivative (s) is between 0.8 and 1.2 g / ml. I.   12. Culture medium according to one of claims 1 to 11, characterized in that it further comprises at least one inhibitor of the growth of bacteria different from the bacterial strain to cultivate and assimilating said source of carbon and energy .   13. Medium according to one of claims 1 to 12, wherein the inhibitor is selected from Gram-positive flora inhibitors and / or gram-negative bacteria distinct from the bacterial strain to be cultured.   14. Medium according to one of claims 1 to 13, wherein the inhibitor or inhibitors are selected from bile salts, sodium deoxycholate, detergents, antibiotics, dyes and mixtures thereof.   15. Medium according to one of claims 1 to 14, wherein the inhibitor or inhibitors are chosen from dyes of the triphenylmethane type, especially diaminotriphenylmethane, brilliant green, malachite green, gentian violet, acid fuchsin and mixtures thereof. .   16. Medium according to one of claims 1 to 15, wherein the inhibitor or inhibitors are selected from thallous sulfate and brilliant green and mixtures thereof.   17. Culture medium according to one of claims 1 to 16 in gelled form.   18. Culture medium according to one of claims 1 to 16 in liquid form.   19. Culture medium according to one of claims 1 to 11 in liquid form without inhibitor.   20. Medium according to one of claims 1 to 19 further comprising a growth factor selected from amino acids, vitamins, purines, protein hydrolysates and yeast extracts, and mixtures thereof.   21. Culture medium according to one of claims 1 to 20, wherein the strain to be grown is auxotrophic and the medium comprises a growth factor distinct from a carbon strain.   22. Medium according to one of claims 1 to 21 further comprising a growth factor is a protein hydrolyzate, including a casein hydrolyzate, optionally supplemented with L-cysteine.   23. Culture medium according to one of claims 1 to 22, wherein the concentration of growth factor is less than the concentration for the growth of said strain without other carbon source.   24. Culture medium according to one of claims 1 to 23, further comprising at least one chromogenic substrate selected from the substrates Xcaprylate, X-alpha-D-galactopyranoside, X-beta-D-galactopyranoside or Xgalactopyranoside, where X- represents 5-bromo-4-chloro-3-indoxyl-, X- which can be replaced by 3-indoxyl-, 5-bromo-6-chloro-3-indoxyl-, 6-chloro-3-indoxyl-, 5-iodo 3-indoxyl-, N-methylindoxyl- or 4-methylumbelliferyl-, and mixtures thereof.   25. Medium according to one of claims 1 to 24, wherein the chromogenic substrate is chosen from 5-bromo-4-chloro-3-indolylcaprylate and 5-bromo-4-chloro-3-indoxyl-α-D-galactopyranoside. and their mixtures.   26. Culture medium according to one of claims 1 to 25, wherein the chromogenic substrate concentration is 0.1 to 0.4 g / l.   27. Process for enriching a bacterial strain in a medium according to one of claims 1 to 26.   28. The method of claim 27, wherein the strain is of the Salmonella or E. coli type.   29. A method of isolating a bacterial strain in a medium according to one of claims 1 to 26.   30. The method of claim 29, wherein the strain is of the Salmonella type, including a stressed strain of Salmonella.   31. A method of culturing a bacterial strain in a medium according to one of claims 1 to 26 carried out under inhibiting conditions.   32. The method of culturing a bacterial strain according to claim 31, wherein the inhibiting conditions are chosen from the presence of a growth inhibitor of bacteria different from the strain to be cultivated and assimilating said source of carbon and energy. salinity conditions, aerobic conditions, pH control and / or temperature control.   33. Association of gentisate or its derivatives and / or at least one precursor of gentisate or their derivatives, in particular 3-hydroxybenzoate, as a source of carbon and energy, with at least one inhibitor, for the bacterial culture of at least one strain to be cultured, the inhibitor acting on the growth of bacteria different from the bacterial strain to be cultured and assimilating said source of carbon and energy.   34. Use of gentisate or its derivatives and / or at least one precursor of gentisate or their derivatives, in particular 3-hydroxybenzoate, as a source of carbon and energy, under inhibiting conditions, for the bacterial culture of least one strain to be cultivated, said conditions acting on the growth of bacteria which are different from the bacterial strain to be cultivated and which assimilate said source of carbon and energy.