# Patent

## Title
Vibrio bacteria detection and differentiation

## Abstract

The invention concerns a culture medium for isolating Vibrio bacteria, characterised in that it comprises, in a Vibrio culture medium, at least a chromogenic agent selected among the β-glucosidase substrates and the β-galactosidase substrates.

## Background
The present invention relates to a chromogenic culture medium intended to reveal bacteria of the Vibrio genus.
The Vibrio genus is associated with cholera (V. cholerae) and with several other clinical pathologies. In particular, V. parahaemolyticus is responsible for certain types of acute gastroenteritis, in particular in Japan, observed after ingesting contaminated food (generally products from the sea, fish, shellfish, crustacia or molluscs which are raw or only slightly cooked).
Transmission of the cholera vibrio generally takes place from human to human (vomit, fecal matter, sweat), or via contaminated foods. Moreover, vibrios are naturally found in the microbial flora of coastal and estuary waters. Included among their natural hosts are molluscs, crustacia, fish, echinoderms and plankton.
Vibrio infections pose a public health problem, and it is therefore important to have a reliable and rapid test for detecting food contaminations by these bacteria.
There exist today reasonably selective culture media which make it possible to detect the presence of vibrios in samples. In particular, use is made of TCBS agar (sodium Thiosulfate—sodium Citrate—bovine Bile—Sucrose) (Diagnostics Pasteur, ref. 69456; Oxoid, ref. CM 333; Merck, ref. 10263). The vast majority of enterobacteria and enterococci grow very little, or not at all, on this medium compared to vibrios. 
TTG agar (Taurocholate—Tellurite—Gelatin) is also used, but is relatively tedious to prepare, requiring regular controls and rigorous dosing of the potassium tellurite.
One of the drawbacks of the TCBS medium is that it does not generally make it possible to differentiate between the various species of vibrios. It would in fact be advisable to be able to identify the species of Vibrio present in the sample analyzed. In particular, it would be advantageous to be able to identify the presence of and to differentiate V. cholerae, V. parahaemolyticus, and V. alginolyticus. The latter species is only rarely associated with diarrhoea producing pathologies, but is probably the entity most frequently isolated in countries with a temperate climate, in a large diversity of clinical samples.
In order to improve the health risk evaluation, it is therefore advisable to be able to differentiate the various types of vibrios which may be present in samples (food samples, feces, etc.), more finely than that which was obtained with the preceding media, which could only differentiate sucrose+ bacteria (such as V. alginolyticus or V. cholerae) for sucrose− bacteria (such as V. parahaemolyticus).
The present invention proposes to provide a novel culture medium for detecting and/or differentiating bacteria of the Vibrio genus, characterized in that it comprises, in a Vibrio culture medium, at least one chromogenic agent chosen from β-glucosidase substrates and β-galactosidase substrates.
The chromogenic agent which is a substrate for one of the enzymes mentioned above comprises a chromophore, which is released by hydrolysis of the substrate by its enzyme. Thus, the bacterial colony becomes colored according to the chromophore released.
Said chromophore is preferably chosen from indoxyl, haloindoxyl (bromoindoxyl, chloroindoxyl, fluoroindoxyl, iodoindoxyl, dichloroindoxyl, chlorobromoindoxyl, trichloroindoxyl), methylindoxyl or hydroxyquinoline derivatives. Preferred derivatives are in particular chosen from the following derivatives: 6-chloroindoxyl, 5-bromoindoxyl, 3-bromoindoxyl, 6-fluoroindoxyl, 5-iodoindoxyl, 4,6-dichloroindoxyl, 6,7-dichloroindoxyl, 5-bromo-4-chloroindoxyl, 5-bromo-6-chloroindoxyl, 4,6,7-trichloroindoxyl, N-methylindoxyl and 8-hydroxyquinoline.
Thus, preferably the β-glucosidase substrate is an indoxyl glucoside, and/or the β-galactosidase substrate is an indoxyl galactoside.
The concentration of each chromogenic agent in the medium is between approximately 0.02 and 0.5 g/l. A preferred concentration is 0.1 g/l.
V. parahaemolyticus is described as being negative for the β-glucosidase characteristic (Rapid 20E identification gallery from the company BioMérieux, Marcy l'Etoile, France), and as being positive for the β-galactosidase characteristic (API 20 NE identification gallery from the company BioMérieux mentioned above).
Surprisingly, the invention shows that V. parahaemolyticus can exhibit a reverse phenotype for these two characteristics on solid medium. Thus, the use of a chromogenic agent which is a substrate for β-glucosidase makes it possible to color the colonies on solid medium, whereas they remain colorless when a chromogenic agent which is a substrate for β-galactosidase is used.
It is, however, difficult to distinguish and differentiate V. parahaemolyticus from V. alginolyticus, which are often found in the same samples of marine origin, since these two species have many common characteristics. Thus, the two species are both positive for the β-glucosidase characteristic. Surprisingly, the addition of sucrose to the medium according to the invention, in particular at a high concentration, makes it possible to cause the β-glucosidase activity of V. alginolyticus to disappear, while at the same time allowing it to remain in V. parahaemolyticus. Thus, the medium according to the invention makes it possible to readily distinguish between V. parahaemolyticus and V. alginolyticus, by simple coloration or lack of coloration of the colonies present on the culture medium.
A high concentration of sucrose should be considered to be a sucrose concentration greater than 5 g/l, preferably between 5 g/l and 35 g/l. An effective sucrose concentration is 20 g/l.
The present invention also makes it possible to detect V. cholerae, and to differentiate it with respect to the other two species mentioned above. Specifically, V. cholerae exhibits β-galactosidase activity without exhibiting β-glucosidase activity. Thus, the addition of a chromogenic agent which is a substrate for β-galactosidase, to a Vibrio culture medium, makes it possible to distinguish V. cholerae from the other vibrios, by coloration of the colonies formed.
In order to be able to differentiate the various bacteria in a single step, it is therefore advantageous to use a culture medium which comprises both a chromogenic agent which is a substrate for β-galactosidase and a chromogenic agent which is a substrate for β-glucosidase.
In fact, addition of sucrose to the culture medium does not cause the β-galactosidase characteristic of V. cholerae to disappear. This result is all the more surprising since an inhibition is observed for V. alginolyticus, which exhibits the same sucrose+ characteristic as V. cholerae. 
Thus, the use of a culture medium comprising both a chromogenic agent which is the substrate for β-galactosidase and a chromogenic agent which is a substrate for β-glucosidase makes it possible to differentiate V. parahaemolyticus (mauve when 5-bromo-6-chloro-3-indoxyl-β-glucoside is used), V. cholerae (blue when 5-bromo-4-chloro-3-indoxyl-β-galactoside is used) and V. alginolyticus (colorless). Such a medium according to the invention therefore makes it possible to detect and differentiate three different strains of vibrios, whereas the media of the prior art only make it possible to obtain a two-category differentiation (sucrose+ and sucrose−).
The present invention is also directed toward a method for detecting and/or differentiating bacteria of the Vibrio genus in a sample, characterized in that it comprises the following steps:

    a. inoculating a culture medium according to the invention with said sample or an inoculum derived from the sample,
    b. detecting the presence of bacteria of the Vibrio genus on said culture medium,
    c. optionally, differentiating V. cholerae, V. parahaemolyticus and V. alginolyticus present on said culture medium.


The use of these media is generally preceded by an enrichment step, using methods known to those skilled in the art. Alkaline peptone water containing 1 g % of NaCl (pH 8.6) or alkaline peptone water containing 3 g % of NaCl may in particular be used.
Diverse selective factors for Vibrio, which are well known to those skilled in the art, may be added to the medium of the present invention. Thus, the presence of an alkaline pH, of thiosulfate, of citrate, of bile, or biliary salt, of magnesium, of calcium, of teepol, of SDS, of Polyximine, of colistine, of tellurite, etc. makes it possible to improve the properties of the medium according to the invention, and to isolate the Vibrios in a satisfactory manner.


EXAMPLES
Example 1
A preferred medium for use of the invention comprises (per liter):
















Agar
  15 g



Peptone
  15 g



Yeast extract
  3 g



NaCl
  10 g



5-Bromo-6-chloro-3-indoxyl-β-glucoside
 0.1 g



5-Bromo-4-chloro-3-indoxyl-β-galactoside
 0.1 g



Sucrose
  20 g



Glucose
  1 g



Lactose
 0.1 g



IPTG
0.04 g



Trizma base
  5 g



Sodium citrate
  10 g



Sodium thiosulfate
  10 g



Sodium cholate
  3 g










Example 2
Plating bacteria out on various media gave the following results.
















Medium of the





invention



TCBS
BTB-Teepol
(Example 1)







V. cholerae
Yellow
Yellow
Blue


V. alginolyticus
Yellow
Yellow
Colorless


V. parahaemolyticus
Green
Blue-green
Mauve









The use of the medium according to the invention thus makes it possible to differentiate the three species of vibrios, compared to the media of the prior art.

## Claims

1. A culture medium comprising (per liter);
















Agar
  15 g



Peptone
  15 g



Yeast extract
  3 g



NaCl
  10 g



5-Bromo-6-chloro-3-indoxyl-β-glucoside
 0.1 g



5-Bromo-4-chloro-3-indoxyl-β-galactoside
 0.1 g



Sucrose
  20 g



Glucose
  1 g



Lactose
 0.1 g



IPTG
0.04 g



Trizma base
  5 g



Sodium citrate
  10 g



Sodium thiosulfate
  10 g



Sodium cholate
   3 g.

2. A method for differentiating V. parahaemolyticus from V. alginolyticus in a sample of bacteria of the Vibrio genus, comprising the following steps:
a. inoculating a Vibrio culture medium comprising at least one chromogenic agent that is a substrate for β-glucosidase and sucrose at a concentration of greater than 5 g/l with a sample of bacteria of the Vibrio genus or an inoculum derived from said sample, and
b. detecting enzyme activity for V. parahaemolyticus and an absence of β-glucosidase activity for V. alginolyticus in said culture medium, thereby differentiating V. parahaemolyticus from V. alginolyticus.

3. The method of claim 2, wherein in said Vibrio culture medium, said at least one chromogenic agent releases, by hydrolysis, a chromophore selected from the group consisting of an indoxyl, haloindoxyl, methylindoxyl and hydroxyquinoline.

4. The method of claim 2, wherein in said Vibrio culture medium said β-glucosidase substrate is indoxyl glucoside.

5. The method of claim 4, wherein said β-glucosidase substrate is 5-bromo-6-chloro-3-indoxyl-β-glucoside.

6. The method of claim 2, wherein said Vibrio culture medium further comprises a β-galactosidase substrate that is indoxyl galactoside.

7. The method of claim 2, wherein said Vibrio culture medium further comprises a β-galactosidase substrate that is 5-bromo-4-chloro-3-indoxyl-β-galactoside.

8. The method of claim 3, wherein said haloindoxyl is selected from the group consisting of bromoindoxyl, chloroindoxyl, fluoroindoxyl, iodoindoxyl, dichloroindoxyl, chlorobromoindoxyl, and trichloroindoxyl.

9. The method of claim 8, wherein said haloindoxyl is selected from the group consisting of: 6-chloroindoxyl, 5-bromoindoxyl, 3-bromoindoxyl, 6-fluoroindoxyl, 5-iodoindoxyl, 4,6-dichloroindoxyl, 6,7-dichloroindoxyl, 5-bromo-4-chloroindoxyl, 5-bromo-6-chloroindoxyl and 4,6,7-trichloroindoxyl.

10. The method of claim 3, wherein said hydroxyquinoline is 8-hydroxyquinoline.

11. The method of claim 3, wherein said methylindoxyl is N-methylindoxyl.