ENRICHMENT AND SELECTIVE CULTURE OF SALMONELLA AND SHIGELLA

Abstract

What is disclosed relates to the detection and identification of bacteria of the genera Salmonella and Shigella. It relates more precisely to the methods of microbiology and the culture media used for the detection, identification, isolation and/or analytical investigation of these bacteria. Relating to a method for enrichment and selective culture of bacteria of the genera Salmonella and/or Shigella contained in a biological sample. In the method, some or all of the sample is seeded in/on a culture medium including a nutrient component that favors the development and growth of the bacteria, and includes L-ornithine as a selective agent. It also covers a culture medium suitable for carrying out this method.

Claims

1. A method for enrichment and selective culture of bacteria of the genera Salmonella and/or Shigella contained in a biological sample, in which a part or the whole of said sample is seeded in/on a culture medium comprising a nutrient component that favors the development and growth of said bacteria, wherein the culture medium also comprises L-ornithine, as a selective agent.

2. The method as claimed in claim 1, wherein the L-ornithine concentration in said culture medium is above 10 g/L, and is less than or equal to 25 g/L.

3. The method as claimed in claim 1, wherein the culture medium comprises at least one additional selective agent selected from: sodium deoxycholate, Tergitol 4, bile salts, crystal violet and brilliant green.

4. The method as claimed in claim 1, wherein the culture medium comprises as selective agents: L-ornithine, at a concentration between about 15 g/L and about 20 g/L, and sodium deoxycholate, at a concentration between about 1 g/L and about 10 g/L.

5. The method as claimed in claim 4, wherein the culture medium also comprises, as selective agent, Tergitol 4, at a concentration between about 0.5 mL/L and about 5 mL/L.

6. The method as claimed in claim 1, wherein the culture medium is a discriminating medium.

7. The method as claimed in claim 6, wherein the culture medium comprises at least one chromogenic component selected from: a pH color indicator able to perform labeling of the colonies of microorganisms fermenting at least one of the carbohydrates present in the medium; a sodium thiosulfate/iron(III) citrate system, allowing detection of the H.sub.2S-producing bacteria; chromogenic and/or fluorogenic substrates allowing the detection of particular enzyme activities.

8. The method as claimed in claim 1, wherein the culture medium comprises a nutrient component repeating that of a culture medium selected from: XLD Agar, chromID Salmonella Elite Agar, Salmonella-Shigella Agar.

9. The method as claimed in claim 7, wherein the culture medium comprises a nutrient component and a selective component repeating those of XLD Agar, and is supplemented with L-ornithine, at a concentration between about 15 g/L and about 20 g/L.

10. A culture medium comprising a nutrient component that favors the development and growth of bacteria of the genera Salmonella and Shigella, wherein it also comprises L-ornithine, as a selective agent.

11. The culture medium as claimed in claim 10, wherein the concentration of L-ornithine is above 10 g/L, and is less than or equal to 25 g/L.

12. The culture medium as claimed in claim 10, wherein it comprises at least one additional selective agent selected from sodium deoxycholate, Tergitol 4, bile salts, crystal violet and brilliant green.

13. The culture medium as claimed in claim 10, wherein it comprises as selective agents: L-ornithine, at a concentration between about 15 g/L and about 20 g/L, and sodium deoxycholate, at a concentration between about 1 g/L and about 10 g/L.

14. The culture medium as claimed in claim 10, wherein it comprises a nutrient component repeating that of a culture medium selected from: XLD Agar, chromID Salmonella Elite Agar, and Salmonella-Shigella Agar.

15. The culture medium as claimed in claim 10, wherein it comprises a nutrient component and a selective component repeating those of XLD Agar, and is supplemented with L-ornithine, at a concentration between about 15 g/L and about 20 g/L.

Description

EXAMPLES: ELABORATION AND EVALUATION OF THE CULTURE MEDIA ACCORDING TO THE INVENTION

1/Selective Media According to the Invention, Prepared on the Basis of a Composition of XLD Agar

[0107] a) Preparation of Medium A1 and Medium A2

[0108] Starting from the composition of XLD Agar (bioMrieux, France), a selective isolation medium used conventionally for detecting Salmonella and Shigella, three improved culture media were prepared. They have the following compositions: [0109] Medium composition of XLD Agar with addition of an opacifier (TiO.sub.2; 0.5 g/L); [0110] Medium A1: composition of medium T, with addition of L-ornithine (5 g/L), [0111] Medium A2: composition of medium T, with addition of L-ornithine (10 g/L).

[0112] As L-ornithine, we may notably mention that produced and marketed by the company SIGMA-ALDRICH, USA (ref. O2375).

[0113] b) Evaluation of the Media for Detection of Salmonella and Shigella

[0114] Different species of Salmonella (eight serotypes including S. enteritidis, S. Typhimurium, S. Paratyphi A, B and C, S. Gallinartim, S. Virchow, S. Derby), of Shigella (four species including S. sonnei, S. boydii, S. flexneri, S. dysenteriae), as well as 15 other strains of Gram-negative bacteria (in this case strains of S, coil, and of other enterobacteria of the genera Proteus, Citrobacter, Enterobacter and Pseudomonas, Acinetobacter), all from the applicant's collection, were used.

[0115] The bacteria were suspended in physiological saline solution, and then seeded on the various media, according to the 4 quadrants technique. The colonies formed on the agars were examined visually after 18-24 hours of incubation at 37 C.

[0116] The results obtained are summarized in Table 1 below. The inhibitions observed relate either to the size of the colonies, or the density of growth of the strains on the agars.

TABLE-US-00001 TABLE 1 non-Salmonello, Salmonella-Shigella: non-Shigella strains: Medium strains detected/strains tested strains inhibited/strains tested T 16/16 0/15 A1 16/16 7/15 A2 16/16 7/15

[0117] These results show good sensitivity for detection of the strains of Salmonella and Shigella by all the media. However, only Medium A1 and Medium A2 are able to reduce the growth of Gram-negative bacteria other than Salmonella and Shigella.

2/Optimization of the Concentration of L-Ornithine of the Selective Media According to the Invention, Prepared on the Basis of a Composition of XLD Medium

[0118] a) Preparation of Medium A3

[0119] In view of the results presented previously, a new medium, designated Medium A3, was prepared. Its composition repeats that of Medium A2, but with double the concentration of L-ornithine (20 g/L).

[0120] b) Evaluation of Medium A3 for Detecting Salmonella and Shigella

[0121] Medium A3 was tested for detecting Salmonella and Shigella, and compared against Medium T and Medium A2. For this purpose, different species of Salmonella (serotypes including S. enteritidis, S. Typhimurium, S. Paratyphi B and C, S. Gallinarum, S. Virchow, S. Derby) of Shigella (species including S. sonnei, S. boydii, S. flexneri, S. dysenteriae), various strains of Escherichia coli (8 strains), as well as other Gram-negative bacteria (Proteus, Enterobacter and Pseudomonas), all from the applicant's collection, were used.

[0122] The bacteria were suspended in physiological saline solution, and then seeded on the media, by the 4 quadrants technique. The colonies formed on the agars were examined visually after 18-24 hours of incubation at 37 C.

[0123] The results obtained are summarized in Table 2 below. The inhibitions observed relate either to the size of the colonies, or to the density of growth of the strains on the agars.

TABLE-US-00002 TABLE 2 non-Salmonella, non-Shigella, Salmonella-Shigella: Escherichia coli: non-E. coli strains strains strains: detected/strains inhibited/strains strains inhibited/strains Medium tested tested tested T 14/14 0/8 0/14 A2 14/14 5/8 6/14 A3 14/14 7/8 8/14

[0124] These results show excellent sensitivity of all the media for detecting the target strains of Salmonella and Shigella, with detection of all fourteen strains for the three formulas.

[0125] However, only Medium A2 and Medium A3 are able to reduce the growth of bacterial genera other than Salmonella and Shigella, and notably for the species E. coli. The best selectivity is obtained at an L-ornithine concentration of 20 g/L.

3/Selective Media According to the Invention, Prepared on the Basis of a Composition of chromID Salmonella Elite Agar

[0126] a) Preparation of Medium B

[0127] Medium B was prepared from the composition of chromID Salmonella Elite Agar (bioMrieux, France), to which L-ornithine was added to obtain a final concentration of 15 g/L. chromID Salmonella Elite Agar is a chromogenic medium originally designed for selective isolation and identification of Salmonella, from samples of human origin.

[0128] b) Evaluation of Medium B for Detecting Salmonella and Shigella

[0129] Medium B was tested for detecting Salmonella and Shigella, and compared with Medium 1, as described above, as well as with Medium A4. The composition of the latter repeats that of Medium T, to which L-ornithine was added to obtain a final concentration of 15 g/L.

[0130] For this evaluation, different serotypes of Salmonella including S. enteritidis, S. Typhimurium, S. Paratyphi A, B and C, S. Gallinarum, S. Virchow, S. Derby), of Shigella including the species S. sonnei, S. boydii, S. flexneri, S. dysenteriae), different strains of Escherichia coli (6 strains), as well as of other Gram-negative bacteria Proteus, Enterobacter, Citrobacter, Pseudomonas), Gram-positive bacteria (staphylococci, enterococci) and yeasts (Candida), all from the applicant's collection, were used.

[0131] The bacteria were suspended in physiological saline solution, and then seeded on the media, by the 4 quadrants technique. The colonies formed on the agars were examined visually after 18-24 hours of incubation at 37 C.

[0132] The results obtained are summarized in Table 3 below. The inhibitions observed relate either to the size of the colonies, or to the rate of growth of the strains on the agars.

TABLE-US-00003 TABLE 3 non-Salmonella, non-Shigella, Salmonella-Shigella: Escherichia coli: non-E. coli strains: strains detected/strains strains inhibited/ strains inhibited/ Medium tested strains tested strains tested T 16/16 0/6 4/19 A4 16/16 5/6 10/19 B 13/16 6/6 17/19

[0133] These results show excellent sensitivity of Medium T and of Medium A4 for detecting the strains of Salmonella and Shigella, with detection of all sixteen strains tested. Medium B makes it possible to detect thirteen strains out of the sixteen tested and shows imperfect selectivity with respect to Salmonella and Shigella.

[0134] However, only Medium A4 and Medium B are able to reduce the growth of microorganisms other than Salmonella and Shigella, notably the growth of the species coll.

4/Optimization of the Discriminating and Chromogenic Capacity of the Selective Media According to the Invention

[0135] a) Preparation of Medium C

[0136] Medium C was prepared from the composition of XLD Agar (bioMrieux, France), to which the following were added: an pacifier (TiO.sub.2; 0.5 g/L), L-ornithine (15 g/L) and a chromogenic substrate, X-beta-ribofuranoside (0.06 g/L).

[0137] b) Evaluation of Medium C for Detecting Salmonella and Shigella

[0138] Medium C was tested for detecting Salmonella and Shigella, and compared with XLD Agar.

[0139] For this evaluation, thirty strains of Salmonella (serotypes including S. enteritidis, S. Typhimurium, S. Paratyphi A, B and C, S. Gallinarum, S. Virchow, S. Derby, S. Dublin, S. Choleraesuis, S. Infantis, S. Arizonae, S. Cubana), forty strains of Shigella (species including S. sonnei, S. boydii, S flexneri, S dysenteriae), and fifteen other Gram-negative bacteria (Proteus, Citrobacter, Enterobacter, E. coli and Pseudomonas, Acinetobacter), all from the applicant's collection, were suspended in physiological saline solution, and then seeded on the media, by the 4 quadrants technique. The colonies formed on the agars were examined visually after 18-24 hours of incubation at 37 C.

[0140] The observations made are summarized in Table 4 below.

TABLE-US-00004 TABLE 4 Strains XLD Agar Medium C (number of strains tested) (strains detected/strains tested) (strains detected/strains tested) Salmonella (30) 30/30 colonies from 30/30 colonies from (incl. 28 colorless with 2 to 4 mm (incl. 28 colorless with 1.5 to 3 mm black center) black center) Shigella (40) 40/40 colonies from 40/40 colonies from (colorless) 1.5 to 5 mm (colored 1 to 4 mm gray-blue) (4 strains with a lower density of growth) Other Gram-negative 15/15 colonies from 10/15 Observation of the strains (15) (high density of growth) 2 to 5 mm inhibitions, based on the size of the colonies and the density of growth

[0141] These results show excellent sensitivity of XLD Agar and Medium C, for detecting the strains of Salmonella and for Shigella, with detection of all seventy strains.

[0142] However, only Medium C is able to reduce the growth of the other Gram-negative bacteria.

5/Optimization of the Selectivity of the Selective Media According to the Invention

[0143] a) Preparation of Medium D

[0144] Medium D was prepared from the composition of XLD Agar (bioMrieux, France), to which the following were added: an opacifier (TiO.sub.2; 0.5 g/L), L-ornithine (15 g/L), a chromogenic substrate, X-beta-ribofuranoside (0.06 g/L) and Tergitol 4 (1.5 mL/L).

[0145] The Tergitol 4 used may notably be that manufactured and marketed by SIGMA-ALDRICH, USA (ref. Niaproof 4 N1404).

[0146] b) Evaluation of Medium D for Detecting Salmonella and Shigella

[0147] Medium D was tested for detecting Salmonella and Shigella, and compared with XLD Agar.

[0148] The following were used for this evaluation: [0149] 19 strains of Salmonella (3 of S, Typhimurium, 2 of S. enteritidis, 1 of S. Virchow, 3 of S. Paratyphi A, 3 of S. Paratyphi B, 2 of S. Paratyphi C, 1 of S. Cubana, 1 of S. Dublin, 1 of S. Arizonae, 1 of S. Gallinarum, 1 of S. Choleraesuis); [0150] 29 strains of Shigella (4 of S. boydii, 13 of S, flexneri, 12 of S. sonnei), [0151] 15 strains of Gram-negative bacteria, neither Salmonella nor Shigella (4 of E. coli, 2 of P. vulgaris, 1 of P. mirabilis 1 of E. cloacae, 1 of E. aerogenes, 1 of P. aeruginosa, 1 of A. baumanii, 1 of E. hoshinae, 2 of C. braakii, 1 of C. youngae).

[0152] These strains, from the applicant's collection, were suspended in physiological saline solution, and then seeded on the media, by the 4 quadrants technique. The colonies formed on the agars were examined visually after 18-24 hours of incubation at 37 C.

[0153] Table 5 below presents the observations made for cultures of pure strains.

TABLE-US-00005 TABLE 5 Strains XLD Agar Medium C (number of strains tested) (strains detected/strains tested) (strains detected/strains tested) Salmonella (19) 19/19 15 strains forming colonies 19/19 4 strains forming colonies of (colorless with of 2 mm or more; (17 colorless with 2 mm or more; black center) 4 strains forming black center, 2 15 strains forming colonies of less gray-blue) colonies of less than 2 mm than 2 mm Shigella (29) 29/29 20 strains forming colonies 29/29 10 strains forming colonies of (all colorless) of 2 mm or more; (28 colored 2 mm or more; 9 strains forming colonies gray-blue, 1 19 strains forming colonies of of less than 2 mm colorless) less than 2 mm Other Gram-negative 15/15 9 strains forming colonies 15/15 12 strains for which a notable strains (15) of 2 mm or more; reduction of the size of the 9 strains forming colonies colonies is observed of less than 2 mm

[0154] Table 6 below presents the observations made for culture of mixed strains.

TABLE-US-00006 TABLE 6 XLD Agar Medium C Fertility of Density of the medium Size Enzyme activity growth Size Enzyme activity S. typhimurium 2.2 2 mm colorless colonies 2.2 1.5 mm colorless colonies (ATCC 14028) + with black center with black center C. braakii 2.2 1.5 mm yellow colonies 2.2 1.5 mm yellowish-green (ATCC15580) colonies S. enteritidis 2.3 2.5 mm colorless colonies 2.3 1.5 mm colorless colonies (IM195) + with black center with black center Sh. boydii 2.1 0.75 mm colorless colonies 2.3 0.5-0.75 mm gray-blue colonies (ATCC 8700) S. paratyphi B 2.3 2.5 mm colorless colonies 2.3 2 colorless colonies (ATCC 10719) + with black center with black center E. coli 2.1 1-1.5 mm yellow 1.1 very fine colorless colonies (ATCC 25922) colonies S. paratyphi C 2.1 1.5-2 mm colorless colonies 3.1 1.5 mm colorless colonies (ATCC 13428) + with black center with black center E. coli 2.2 2.5-3 mm colonies somewhat 3.1 2 mm beige colonies (ATCC 12453) beige