RU2460802C1 - Method for identifying yersinia enterocolitica - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: identifying a yersiniosis agent is enabled by using the intestinal yersinia pague FK-100 stripped on a double-layer grass lower layer of which represents an agar medium (pH 7.1) with added ampicillin in the concentration of 50 mcg/ml, while an upper layer of which is produced of the strain being tested cultivated on 1.5% Hottinger's agar at 28°C during one day and inoculated in 4 ml of Hottinger's broth, incubated at temperature 28°C to the final concentration n·10⁸ m.c./ml; thereafter 0.3 ml of the prepared culture is mixed with 4.5 ml of 0.7% Hottinger's agar melted and cooled to 45°C with 50 mcg/ml of ampicillin and poured as a second layer on the agar plates. The inoculation is incubated at 28°C for 20-24 hours. Yersinia enterocolitica in the sample being tested is identified from the other types of yiersinia by the presence of phagolysis on the growth culture.

EFFECT: method provides complete identification effectiveness.

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Description

The present invention relates to medical microbiology, in particular to laboratory diagnosis of the disease of yersiniosis caused by Yersinia enterocolitica.

In connection with the increasing role of Y. enterocolitica in human infectious pathology, great importance is attached to laboratory diagnosis of the pathogen, and, in particular, phagodiagnosis.

Given the similarity of many biological properties of plague, pseudotuberculosis, and intestinal Yersinia microbes, their distinctive feature is the unequal lizability of homo- and heterologous phages.

A diagnostic phage has not been developed to identify Y. enterocolitica. Unlike plague and pseudotuberculosis microbes, resistance to drugs (MPC 100-2500 μg / ml) of the penicillin series is a species trait in Y. enterocolitica. The bacteria Y. enterocolitica are resistant to ampicillin up to 1600 μg / ml.

The search for new methods in the bacteriological diagnosis of Y. enterocolitica is relevant and is primarily associated with the need to isolate the specific properties of the microorganism in an effective, simple and reliable way.

A known method for the detection of bacteria of the genus Yersinia and differentiation of pathogenic for humans species of Yersinia by the method of multiplex polymerase chain reaction (see US Pat. RU No. 2385941, CL C12Q 1/00, published 04/10/2010), which consists in using the method of multiplex PCR followed by gel electrophoresis analysis of the amplified fragment lengths, while differential diagnosis of non-pathogenic species of the genus Yersinia from pathogenic species of the genus is carried out using a set of primers [VF (SEQID NO: 1), YR (SEQ ID NO: 2), YP-R (SEQ ID NO: 5)], forming only one specific PCR- product, and in pathogenic species of Yersinia (Y. pestis, Y. pseudotuberculosis and Y. enterocolitica) two specific PCR products are formed.

The disadvantage of this method is its difficulty in implementation, since semantic specific gender-specific pairs of primers encoding the RLGFAGhK peptide and antisense species-specific primers having a homology of 95-100% with a nucleotide sequence are required.

For the prototype, we have chosen the method of identifying O1 cholera vibrios of two biovars closest in technical essence (see MUK 4.2.2 218-07 “Laboratory diagnosis of cholera.” Moscow, 2007, pp. 40-41), which consists in determining the sensitivity cholera vibrios to polymyxin B and diagnostic cholera phages (classic and eltor). At the same time, the cholerae biovar cholera vibrios are sensitive to polymyxin and do not grow on agar with the addition of 50 μg / ml polymyxin; eltor biovar cholera vibrios are resistant to it. Next, a biovar is determined on the basis of lucidity by one of the diagnostic phages (eltor or classic).

The disadvantage of this method is the impossibility of its use for the diagnosis of the causative agent of yersiniosis Y. enterocolitica, which is associated with the lack of growth on alkaline agar at pH 7.6-7.8, which requires Hottinger agar (pH 7.1-7.2); the optimum temperature for growing vibrios at 37 ° C, which does not correspond to the growth conditions of yersinia at 28 ° C; cholera phages specifically lyse cholera vibrios and are not active against bacteria Y. enterocolitica. The drug polymyxin (50 μg / ml) does not have a bacteriostatic effect on the cholera vibrios of the eltor biovar, while at the same time it inhibits the growth of yersinia at a concentration of 0.4-25.6 μg / ml.

Thus, the phagodiagnosis used in the known method requires for Y. enterocolitica a specific specific phage that is sensitive to this type of yersinia.

The technical task of the invention was to increase the identification efficiency of Y. enterocolitica by finding a simple method by using a bacteriophage that specifically lyses only bacteria of this type of yersinia on an environment with ampicillin.

This object is achieved by the use of enteric phage FC-100, which is applied as a track to a two-layer lawn, the lower layer of which is agar medium (pH 7.1) with the addition of ampicillin at a concentration of 50 μg / ml, and the upper one is obtained from the test strain, which is grown on 1.5% Hottinger agar at 28 ° C during the day and seeded in 4 ml of Hottinger broth, incubated for a day at a temperature of 28 ° C to a final concentration of n · 10 ⁸ MK./ ml, after which 0 , 3 ml of the obtained culture is mixed with 4.5 ml of molten and cooled up to 45 ° C, 0.7% Hottinger agar with 50 μg / ml ampicillin and the second layer is poured onto agar plates, after which the inoculum is incubated at 28 ° C for 20-24 hours and identified by the presence of the phagolysis zone on the grown culture, which confirms the presence of Y. enterocolitica in the test sample.

To carry out the method, the applicant used an enteric bacteriophage 1999, which was deposited in the collection of the laboratory of bacteriophages RostNIPCHI under the number FC-100.

Two types of Yersinia microorganisms are used as a control for checking the lytic effect of phage: phage-sensitive and ampicillin-resistant indicator strain Y. enterocolitica 2012 (deposited in the State Research and Design Clinical Hospital “Microbe” in Saratov under the number KM-206) and phage-resistant and ampicillin-sensitive strain Yu pse 103 deposited at the State Research and Design Bureau of Scientific Research, Scientific Research Institute of Microbiology "Microbe" of Saratov under the number KM-207).

The strain Yersinia enterocolitica 2012 (KM-206) was isolated from humans (Belgium), obtained in 1966 by the Museum of Living Cultures of RostNIPCHI and is characterized by the following features.

Cultural and morphological characters. Sticks mobile at 22 ° C have flagella, gram-negative. Spore and capsules do not form. On a dense nutrient medium (Hottinger agar, pH 7.2) forms small, convex, transparent colonies after 18-20 hours of cultivation at 25-28 ° C. In Hottinger broth, pH 7.2, gives a uniform turbidity.

Physiological and biochemical properties. The oxidase test is negative. It decomposes glucose, mannitol, sucrose, glycerin, xylose, sorbitol to acid without gas, and is not active against rhamnose, lactose, raffinose. Test for urease activity -, Christensen ++++. Nutritional needs - prototroph. Attitude to species diagnostic sera - biotype 3. Antigenic properties of the strain - serotype 3. Resistant to ampicillin (up to 1600 mg / ml).

Relation to homo- and heterologous phages. The strain is lysed by phages isolated from lysogenic cultures of Yersinia enterocolitica 1, 3, 12 serovars and is resistant to phages: diagnostic plague L-413C, diagnostic plague Pokrovskaya (P), diagnostic pseudotuberculous phage. The strain is resistant to penicillin 1000 units / ml; oxacilin 2500 units / ml; chloramphenicol 10 units / ml; tetracycline 5 units / ml. The strain is indicative for phages of the V morphogroup. The titers of phages propagated on the strain are n · 10 ⁷ -n · 10 ⁸ particles / ml. Strain KM 206 propagates in the broth and on Hottinger agar (pH 7.2), store it in a lyophilized state. It is able to detect and provide reproduction of moderate phages. Using strain KM 206 isolated phage 1998 - isolated from the lysogenic strain of Y. enterocolitica 1 serovar; phage 1999 - from a strain of Y. enterocolitica 12 of the serovar; phage 2010 - from a strain of Y. enterocolitica 3 serovar.

Strain Y. pseudotuberculosis 10368 (KM-207) was isolated from humans (Buryat Autonomous Soviet Socialist Republic) and is characterized by the following properties.

Cultural and morphological characters. Movable, short sticks with rounded ends, gram-negative. Spore and capsules do not form. On a dense nutrient medium (Hottinger agar pH 7.2) forms round, slightly convex colonies, grayish-yellow in color after 18-20 hours of cultivation at 25-28 ° C. In the Hottinger broth (pH 7.2) gives a uniform clouding of the medium.

Physico-biochemical properties. It decomposes glucose, maltose, mannitol, rhamnose, glycerol to acid without gas. Inactive for inositol, lactose, sucrose. Indole does not produce urease activity +. Sensitive to ampicillin (12.5 μg / ml). Nutritional needs - prototroph.

Relation to homo- and heterologous phages. The strain is resistant to the bacteriophage diagnostic plague L-413C, sensitive to the bacteriophage diagnostic plague Pokrovskaya (P), the diagnostic bacteriophage pseudotuberculosis. The titers of phages propagated on the strain are n · 10 ⁷ -n · 10 ⁸ particles / ml. Strain KM 207 propagates in broth and on Hottinger agar (pH 7.2). Store it in a lyophilized state.

The method is carried out on strains of Y. enterocolitica and Y. pseudotuberculosis.

Example 1. Lysis of enteric Yersinia bacteriophage of strain Y. enterocolitica on medium with ampicillin (50 μg / ml)

Strain Y. enterocolitica 2012 (KM-206) is grown on 1.5% Hottinger agar in a Petri dish at 28 ° C for 24 hours. One loop of the obtained daily agar culture is inoculated in 4 ml of Hottinger broth and grown for a day at a temperature of 28 ° C to a final concentration of n · 10 ⁸ m.k. / ml. Then 0.3 ml of broth culture and 0.1 ml of ampicillin at a concentration of 50 μg / ml are added to 4 ml of 0.7% Hottinger agar, previously melted and cooled to 45-47 ° C.

The resulting mixture was poured in a second layer on the surface of a plate of 1.5% Hottinger agar, prepared in advance and already containing 50 μg / ml ampicillin (pH 7.1-7.2). After the top layer has hardened, the enteric Yersinia phage FC-100 is applied to the latter in the form of a “track” on the lawn of the seeded culture.

After this, the seeding is incubated in an incubator at 28 ° C for 20-24 hours and the result is taken into account.

Thus, we observe the growth of the culture and the presence of the lysis zone of the culture at the site of application of the phage, which confirms the presence of strain Y. enterocolitica.

Example 2. Inoculation of strain Y. pseudotuberculosis on agar with the addition of ampicillin

Inoculation of strain Y. pseudotuberculosis 10368 (KM-207) is grown on 1.5% Hottinger agar in a Petri dish at 28 ° C for 24 hours and all subsequent steps are carried out as described in example 1, but without phage and using ampicillin .

Inoculation is incubated in an incubator at 28 ° C for 20-24 hours and the result is taken into account.

We observe the absence of growth of the microbe Y. pseudotuberculosis in a medium with ampicillin, that is, the microbe of the species Y. enterocolitica is absent in the sample.

Example 3. Sowing the strain of Y. pseudotuberculosis on medium without antibiotic and conducting a sample with enteric phage

Sowing of the strain Y. pseudotuberculosis on Hottinger agar and a sample with enteric phage phage is carried out, as in example 2, without the addition of ampicillin. The presence of the growth of the Y. pseudotuberculosis strain and the absence of a lytic spot, indicating the phage resistance of bacteria, is regarded in favor of the microbe Y. pseudotuberculosis.

The applicant identified 75 strains of Y. enterocolitica on a medium with ampicillin (see table), and 80 strains of Y. pseudotuberculosis were also included in the test. The strains of Y. pseudotuberculosis did not grow on an agar medium with an antibiotic, and on Hottinger agar without ampicillin addition, they were resistant to enteric phage. The table shows the results of the effective use of the method in all identified strains of the species Y. enterocolitica.

Thus, representatives of the genus Yersinia Y. enterocolitica and Y. pseudotuberculosis differ in sensitivity to intestinal Yersinia phage and the antibiotic ampicillin. The strains of Y. pseudotuberculosis are not able to grow on a medium with an antibiotic and are resistant to bacteriophage.

Based on the foregoing, the use of phage FC-100 lucidity on a double layer of culture medium with enhanced inhibitory effect of ampicillin gives 100% identification efficiency of Y. enterocolitica.

Using the proposed method allows its effective use in laboratory diagnostics: since the pronounced dependence of the sensitivity to phage FC-100 on the species in the strains on which it is cultivated, the growing conditions in the presence of an antibiotic in the medium allows it to be used as an additional test for the differentiation of pathogens of intestinal yersiniosis, which can be used in laboratory diagnostics in order to:

- to differentiate the phagosensitive strains of Y. enterocolitica from the resistant strains of Y. pseudotuberculosis resistant to enteric phage;

- apply a sign of ampicillin resistance to detect Y. enterocolitica in mixed samples with Y. pseudotuberculosis;

- carry out typing of Y. enterocolitica with phage FC-100 strains of various O-serovars.

Claims (1)

A method for identifying Yersinia enterocohlitica by using microbial lucidity with a diagnostic phage on an agar medium and its resistance to an antibiotic, characterized in that an enteric-siny phage FC-100 is used, which is applied as a track to a two-layer lawn, the lower layer of which is an agar medium (pH 7, 1) with the addition of ampicillin at a concentration of 50 μg / ml, and the upper one is obtained from the studied strain, which is grown on 1.5% Hottinger agar at 28 ° C for 24 hours and seeded in 4 ml of Hottinger broth, the day is incubated at a temperature of 28 ° C to a final concentration of n · 10 ⁸ MK / ml, after which 0.3 ml of the obtained culture is mixed with 4.5 ml of molten and cooled to 45 ° C 0.7% Hottinger agar with 50 μg / ml of ampicillin and the second layer is poured onto agar plates, after which the inoculation is incubated at 28 ° C for 20-24 hours and identification is made by the presence of a phagolysis zone in the grown culture, which confirms the presence of Y. enterocolitica in the test sample.