Method of differentiating atoxigenic strains of cholera vibrios 01 and 0139 serogroups from toxigenic strains on hydrolase activity

FIELD: chemistry.

SUBSTANCE: atoxigenic strains of cholera vibrios 01 and 0139 serogroups are differentiated from toxigenic strains on presence of hydrolase activity. The substrate used is glycerin, which is introduced into molten Marten's agar at 2.5/100 ml of the medium with pH 7.8±0.1 and Nile blue sulphate indicator 1.5 mg/100 ml of medium, which is poured into petri dishes. Analysed broth cultures grown in a thermostat for 4 hours are deposited in volume of 20 mcl on a sector of developed agar and inoculations are incubated at 37°C for 36 to 48 hours. Strains are differentiated from their results of hydrolase activity: of the colonies grown on the medium are in form of soft semitransparent films, merging with the colour of the medium, then the analysed strain of cholera vibrio is toxigenic and epidermic. The colonies are a thick yellow or whitish deposit, then the strain is non-toxigenic haemolytic and is not epidemic.

EFFECT: method is disclosed for differentiating atoxigenic strains of cholera vibrios 01 an 0139 sergroups from toxigenic strains on hydrolase activity.

3 tbl, 3 ex

 

The present invention relates to Microbiology and can be used for differential diagnosis of hemolytic toxigenic and non-haemolytic toxigenic Vibrio cholerae.

Remains relevant today issue a comprehensive study of the properties of strains of Vibrio cholerae O1 and O serogroups.

The study of various enzymes causative agents of cholera, including: esterase, peptidases, triacylglycerols, amino acid sequences which are part of the Hly protein (Lip)-hly - the area responsible for the hemolytic properties of strains of Vibrio cholerae.

However, in this sequence contains amino acid motifs of α and β hydrolases, the phenotypic manifestation of which has not been studied. Many found short motifs gene hly To testify in favor of the fact that he is able to manifest itself in the phenotype depending on the nature and structure of the substrates in the environment.

A prototype of the selected method of differentiation atoxigenic strains of V. cholerae from toxigenic (see EN Pat. No. 2257415, C12Q 1/04, C12N 1/20, 27.07.2005, BI No. 21), namely, that as the nutrient medium used Martin agar with a pH of 9.0 with indicator - Nile blue sulfate in a concentration of 1.0-1.2 mg/100 ml of medium and substrate - emulsified chicken fat to the concentrations of 1.0-1.5 ml/100 ml, with further incubation for 48 h at 37°C, followed by staining micromason differentiate toxigenic strains of V. cholerae, having lipase activity from toxigenic without coloring micromason.

The disadvantage of this method is the use of non-standard substrate chicken fat, which helps to identify triacylglycerols activity of Vibrio cholerae, the latter include the true lipase, which is strictly substratespecific enzymes that require standard substrates, and chicken fat is not and requires biochemical characteristics, which complicates the process of differentiation.

In addition, the lipase activity in highly exposed to variability depending on the physiological state of the microorganism, which complicates its detection.

The task of the invention consisted in a simplified way differentiation atoxigenic strains of V. cholerae O1 and O serogroups from toxigenic on hydrolases activity.

This object is achieved in that in the method of differentiation atoxigenic strains of V. cholerae O1 and O serogroups from toxigenic on hydrolases activity, including the application of the studied culture on a nutrient medium with an indicator and a substrate with subsequent consideration of the MD results, as the substrate used glycerin, which is injected in molten agar Martin at the rate 2.5/100 ml of medium with a pH of 7.8±0.1 and indicator Nile blue sulphate 1.5 mg/100 ml of medium, pour in bowl Petri, then grown in an incubator for 4 hours studied broth culture is applied in a volume of 20 µl per sector designed agar and spend within 36-48 hours incubation crops at 37°C, then the results hydrolases activity strains spend their differentiation, if the environment has grown colonies are kind of delicate translucent films, merging with color environment, the analyzed strains of V. cholerae is toxigenic and epidemically significant, and if the colonies are dense yellowish or whitish plaque, we have nontoxigenic strain of hemolytic and not epidemically significant.

The method is as follows

Pre-training of the studied cultures in 1.0 ml of broth Martin pH 7.8±0.1 or 1% peptone water contribute strains of Vibrio cholerae, the minimum concentration of cells during seeding is 100 microbial cells. Crops incubated for four hours in a thermostat at 37°C.

Conduct design of differential nutrient medium: molten agar Martin pH 7.8 (±0,1) enter the glycerine - 2.5 ml/100 ml among the s and indicator Nile blue sulphate 1.5 mg/100 ml of medium. All manipulations should be carried out under aseptic conditions in order to avoid gains environment.

Agar is poured into glass Petri dishes. After that grown in thermostat broth culture in a volume of 20 µl automatic pipette dosing is applied on the sector designed agar. Incubation of crops is carried out in a thermostat at 37°C for 36-48 hours.

As a result, the glycerin, which is added to the nutrient medium, is subjected to hydrolysis, and various strains of V. cholerae with different speeds. While glycerin in neeterificirovannah state becomes a substrate, a directional hydrolysis which occurs in α and β positions.

Thus, gidrolizny activity of V. cholerae considered as a differential characteristic.

Records of the results carry through 36-48 hours. If the sectors of the agar culture was grown in the form of an opaque dense micromason whitish-yellow color, the color of the environment does not change, we conclude that the studied strain of hemolytic atoxigenic and has no epidemiological significance. If the environment has grown colonies have views of the blue semi-transparent films, blending with the color of the environment, the analyzed strains of V. cholerae is toxigenic, meaning epidemically significant.

It is noted that during incubation of cultures within 72 hours of the mi is rogatory as hemolytic atoxigenic strains (ctx -Hly+)toxigenic and non-haemolytic (ctx+Hly-) take the same view: micromason toxigenic variants compacted yellowish tint, similar to hemolytic strains. This suggests that gidroliznaja activity specific for all V. cholerae, but hemolytic strains possess more pronounced hydrolases activity, which manifests itself in them after 36-48 hours on medium with glycerol, which allows for the differentiation of Vibrio cholerae inside view on this topic.

Example 1

As the studied strains used culture V. cholerae eltor No. 14863 (ctx-Hly+and 5879 (ctx+Hly-); V.cholerae O139 "Bengal" No. 16077, R-16131 (ctx+Hly-and 17918 (ctx-Hly+); V.cholerae classica No. 569 In and 251 (ctx+Hly-As the substrate used glycerin in a concentration of 0.5-1.0%. Accounting results after 48 hours of cultivation (see table 1).

From table 1 it is seen that when the concentration in the environment of 0.5-1.0% of glycerol impossible to identify gidrolizny microbial activity and conduct differentiation of toxigenic from non-haemolytic nontoxigenic hemolytic variants, since the substrate concentration in the environment is not sufficient for its development and visualization. This is evidenced by equal growth in all variants of V. cholerae with the formation of the translucent blue colonies without active hydrolysis of glycerol.

Example 2

As the studied strains used culture V. cholerae eltor No. 14863 (ctx-Hly+and 5879 (ctx+Hly-); V.cholerae O139 "Bengal" No. 16077, P-16131 (ctx+Hly-and 17918 (ctx-Hly+); V.cholerae classica No. 569 In and 251 (ctx+Hly-). As the substrate used glycerin in a concentration of 2.5%. Accounting results after 48 hours of cultivation (see table 2).

Table 2 shows that when the concentration in the environment of 2.5% glycerol observe the differentiation of hemolytic nontoxigenic (ctx-Hly+) toxigenic and non-haemolytic (ctx+Hly-) variants of Vibrio cholerae on hydrolases activity. This hemolytic strains of El tor No. 14863 and O139 serogroup No. 17918 grow by sectors of the agar in the form of a dense opaque micromason whitish-yellow color, the color of the environment does not change. These strains are classified as hemolytic, no epidemiological significance. The obtained results confirm the methods for determining the epidemiological significance of Vibrio cholerae, is given in MU 4.21097-02 "Laboratory diagnosis of cholera". - M., 2002. - 95 C., namely, in the sample of Greig, there is complete hemolysis of sheep red blood cells, and the results of PCR diagnostics indicate the absence of ctx AB-gene.

The toxigenic strains (ctx+Hly-) cholerae O1 El tor circulation No. 5879, classical biovars the No. V, 251, A serogroup No. 16077, R-16131, growing form on agar translucent blue, blending with the color of the environment, microgate. Active hydrolysis was not observed, which indicates that their toxigenicity. In the sample of Greig and PCR these cultures are characterized as toxigenic non-haemolytic strains.

Thus, the nature of growth on medium containing 2.5% glycerol strains of V. cholerae can be concluded about demolitionist and oxygenate strains that determines their pijnacker.

Example 3

As the studied strains used culture V.cholerae eltor No. 14863 (ctx-Hly+and 5879 (ctx+Hly-); V.cholerae O139 "Bengal" No. 16077, R-16131 (ctx+Hly-and 17918 (ctx-Hly+); V.cholerae classica No. 569 In and 251 (ctx+Hly-As the substrate used glycerin in a concentration of 2.5% (see table 3). Analysis of the reaction produced through 36, 48, 72 and 96 hours of cultivation on the environment.

From table 3 it can be seen that under cultivation within 36 hours hemolytic strains of V.cholerae eltor No. 14863 (ctx-Hly+) grows on agar in the form of a dense opaque micromason whitish-yellow color, the color of the environment does not change. However, hemolytic strain O139 serogroup No. 17918 (ctx-Hly+) did not show hydrolases activity and formed a blue of translucent colonies. Toxigenic in the ways cholerae was grown in a semi-transparent film blue color.

Under cultivation of strains within 48 hours hemolytic variants (ctx-Hly+) Ol and O139 serogroups form microgate whitish-yellow, while all toxigenic variants of Vibrio cholerae are also blue translucent film. From these data we can conclude that the differentiation of toxigenic non-haemolytic (ctx+Hly-and nontoxigenic hemolytic (ctx-Hly+) strains of V. cholerae possible at the stage of cultivation of strains on a nutrient medium with glycerol for 48 hours.

When growing strains within 72 hours on agar with 2.5% glycerol culture as nontoxigenic hemolytic and toxigenic non-haemolytic strains acquire dense whitish tint and become indistinguishable. Culture strains of classical biovars retain the transparency. After 96 hours of cultivation all strains formed on the sectors of agar dense whitish, with a waxy film colony. At this stage we see the manifestation of hydrolases activity of all strains of Vibrio cholerae, and differentiation in this case is impossible.

Thus, it is established that differentiation of Vibrio cholerae on hydrolases activity on solid nutrient medium with the addition of glycerol is most evident when substrate concentration of 2.5 ml/100 ml of medium is under cultivation of strains for 48 hours at 37°C.

From the experiments conducted by the staff of Rostov NPCI found that gidrolizny activity of V. cholerae can be identified by a bacteriological method on solid nutrient medium with the addition of glycerol as a standard substrate in an optimal concentration of 2.5 ml/100 ml of medium. At the same time manifests intraspecific differentiation of toxigenic and non-haemolytic nontoxigenic hemolytic strains of O1 serogroup of biovars of classical and El tor circulation and cholerae O139 serogroup "Bengal", allowing to determine the epidemiological significance of the studied strain.

The use of the present invention allows for the expense of glycerol as a substrate to identify hydrolases activity of V. cholerae O1 and O139 serogroup "Bengal" to perform a simple way differentiation atoxigenic strains O1 and O139 serogroups from toxigenic.

While glycerin is standard and available organic compound that does not require additional features, which are many domestic producers.

Table 1
Biovariant serovariantsno strainCharacterization by PCR, etc. of Greig ctx/Hl Characteristic growthThe definition of epidemiological significance
14863ctx-Hly+Growth without active hydrolysis of glycerol (education translucent blue colonies)Hydrolytic activity was not detected, all strains have worked equally, differentiation is impossible, pijnacker not installed
V.cholerae eltor5879ctx+Hly-Growth without active hydrolysis of glycerol (education translucent blue colonies)
V.cholerae classicaWctx+Hly-Growth without active hydrolysis of glycerol (education translucent blue colonies)
251
16077Growth without active hydrolysis of glycerol (education translucent blue colonies)
V.cholerae A "Bengal"R-16131 ctx+Hly-
17918ctx-Hly+Growth without active hydrolysis of glycerol (education translucent blue colonies)

The method of differentiation atoxigenic strains of V. cholerae O1 and O139 serogroups from toxigenic on enzymatic activity, including the application of the studied culture on a nutrient medium with an indicator and a substrate, incubation of crops at 37°C and differentiation of strains by staining colonies, characterized in that the differentiation is carried out on hydrolases activity, as the substrate used glycerin, which is injected in molten agar Martin at the rate 2.5/100 ml of medium with a pH of 7.8±0.1 and indicator Nile blue sulphate 1.5 mg/100 ml of medium, the medium is poured into Petri dishes, then grown in thermostat for 4-h studied broth culture is applied in a volume of 20 µl per sector designed agar, incubation of crops is carried out within 36-48 h and when grown on medium colonies in the form of a blue semi-transparent films, blending with the color of the environment, differentiate the studied strain as toxigenic and epidemically significant, and in the presence of colonies in the form of dense yellowish or whitish nale is and differentiate the studied strain as nontoxigenic hemolytic and not epidemically significant.



 

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