Method for intraspecies differentiation of vibrio cholerae o139

FIELD: virology, medicinal microbiology.

SUBSTANCE: method involves using phage-sensitive strains of Vibrio cholerae eltor KM-199 and Vibrio cholerae O139 KM-152 as test-strains, and differentiation is carried out for two steps. At the first step Vibrio cholerae eltor KM-199 is used as the strain-indicator and this strain is incubated with analyzed culture for 20-24 h. Then prepared mixture of microorganisms is heated at temperature 55-57°C for 40-60 min and inoculated on lawn with the strain-indicator Vibrio cholerae eltor KM-199 that is inoculated preliminary in layer 0.7% of agar on plate with 1.5% Marten's agar. After culturing for 24 h at 37°C phagolysis is recorded. The second differentiation step is carried out by using phage obtained from lysogenic strain of analyzed culture by its applying on the strain-indicator Vibrio cholerae O139 KM-152 that is used for detection of filamentous phages only by carrying out the direct phagotyping. Using the proposed method allows carrying out phagotyping with high precision and obtaining additional indices of choleraic vibrios isolating from clinical material. Invention can be used in laboratory diagnosis of cholera.

EFFECT: improved and valuable method for differentiation.

1 tbl, 2 ex

 

The present invention relates to medical Microbiology and can be used in the laboratory diagnosis of cholera.

Feature of strains of Vibrio cholerae O139 "Bengal", received in Rostov NPCI from different countries (India, Japan, France, Kyrgyzstan), and other dedicated in Russia was lysogeny. Detection of the specific label in the form of lithogenetic by phages of different Northrup and serological types, it was important for the identification of toxigenic Vibrio cholerae O139 serogroup. Cholera diagnostic bacteriophages used to indicate the causative agent of cholera, as well as special methods of differentiation of Vibrio cholerae according to certain characteristics proved to be unsuitable for Vibrio cholerae O139 in connection with favoritestest last. In this regard, and has the problem of development through phage methods, namely propagational and specific lysis by the phage, intraspecific differentiation of strains of Vibrio cholerae O139.

There is a method of faguibine Vibrio cholerae O139 (see India, Chakrabati and others, journal J. Clin Environ, 2000, Jan. 38 (1): 44-9), namely, that through the five phages selected in the epidemic, 500: strains of V. cholerae O139 serogroup were divided into 10 pagation.

However, despite the fact that the proposed method gives the possibility to differentiate the strains of a new serotype, ispolzovanie in Russia is not possible, because in our country diagnostic and teruya phages for V. cholerae O139 serogroup missing.

A prototype of the selected method of differentiation of epidemic and neèpidemičeskaâ vibrios species Vibrio eltor type of immunity of temperate phages (see EN Pat. No. 1767879, C 12 Q 1/04, 1994 (01.30), which is used as a test system with two indicator cultures of Vibrio eltor 570/KM-114/ and 570/222 N/KM-117/, when this strain KM-114-sensitive V temperate phages of all known immunotypes and its subculture strain KM-117 is resistant to the lytic activity of phage III immunotype, identifying by means of this test system strains of V. eltor carrying phages III immunotype V morphography, the latter are regarded as suspicious belonging to an epidemic.

The disadvantage of this method of differentiation is that it is impossible to carry out the intraspecific differentiation of Vibrio cholerae O139 serogroup, which contain different morphology and antigenic properties of the subjects on the phage lysis, as new strains of serogroup common carrier phages two Northrup I and V, or I, or V. This fact confirms the lack of accuracy of this method.

The task of the invention was to develop a method of intraspecific differentiation . cholerae O139, which allows high accuracy to distinguish strains marker phage attribute.

This object is achieved in that in the known method intraspecific differentiation V.cholerae O139, including joint incubation of the test culture with indicator test strain with subsequent favoritemovie, as the test strains used focustitle strains of V.cholerae eltor KM-199 and V.cholerae O139 KM-152, and the differentiation is carried out in two stages, the first stage as the indicator strain used V. cholerae eltor KM-199, incubated it with the test culture for 20-24 hours, then the mixture of microorganisms at a temperature 55-57°warm up With 40-60 minutes and plated on lawns of indicator strain V. cholerae eltor KM-199, which is pre-seeded with a layer of 0.7% agar on the plate 1.5% agar Martin, and through days of cultivation at 37°To conduct the registration of paralysis, while the second stage of the differentiation is carried out, using the phage obtained from Lisovenko strain of the test culture by drawing on the indicator strain of V. cholerae O139 KM-152, which reveal only filamentosa phages, conducting direct favoritemovie.

For implementing the method using two strains of V. cholerae as a test system V. cholerae eltor and V. cholerae SG-36 (O139 serogroup). Strain V.choleae eltor (P-13169) deposited under number KM-199 in GCPRICE "Microbe", Saratov and is characterized by the following features.

Cultural and morphological characteristics.

Motile, slightly curved sticks with a length of 1.2-2.7 μm, a diameter of 0.2-0.4 µm with a single polar located flagellum. Spores and capsules are not formed. Gram-negative.

In dense environments - Martin agar and agar of Hottinger, pH 7,6±0,2 - forms a translucent colonies with a bluish tint, 1,5±0.5 mm in diameter.

In liquid media, broth Martin, and broth of Hottinger, 1%peptone water, pH 7.8±0,2 forms a uniform haze, a thin film on the surface environment.

Biochemical properties.

Decomposes to acid without gas mannose, sucrose, glucose, lures, does not break the arabinose, oxidizes glucose in the medium Hugh Leifson in aerobic and anaerobic conditions (in 4 days), decarboxylase lysine, not breaks down arginine, does not produce hydrogen sulfide. Strain resistant to 30 μg/ml polymyxin b.

Serological properties.

Attitudes towards Homo-heterologous phages: strain literoitca diagnostic phages El tor circulation, ctx+, ctx-, phage V classic "With" not literoitca. The ratio of strain to the species diagnostic sera: strain agglutinated Of-cholera serum and serum Ogawa, but not agglutinated sera Inaba, RO.

Strain V. cholerae SG-36 (O139 serogroup) deposited under number KM-152 and characterizes the I following attributes.

Cultural and morphological characteristics.

In smears from agar and broth cultures, gram stained, the cells are gram-negative species, polymorphic, slightly curved and straight rods. Mobile, when grown in 0.3% alkaline agar is observed turbidity of the column of agar. When viewed in the electron microscope - monotachi.

Growth on solid nutrient media on alkaline agar (pH of 7.6 to 7.8), vibrios form after 18 hours of growth at 37°With colony correct form, with a diameter of 1.5-2 mm, with wet shiny surface, translucent in transmitted light.

Growth in liquid nutrient media: alkaline broth produced with a gentle film.

The biochemical activity of strain: strain decomposes glucose under aerobic and anaerobic conditions, mannose, sucrose, glucose, mannitol, starch with the formation of acid without gas, does not produce acetylaminophenol, indole, decarboxylase lysine, not breaks down arginine, arabinose, does not produce hydrogen sulfide. Strain resistant to 30 μg/ml polymyxin b.

Attitudes towards Homo-heterologous phages: strain not literoitca diagnostic phages: holonymy classical El tor circulation, TAPV 1-7, literoitca phage I morphography 16488 isolated from V. cholerae O139-16488. The ratio of strain to the species diagnostic sera: strain agglutinated serum O139 serogroup produced in RPCI; not as glutenfreeda Of-cholera serum and typespecification sera Inaba, Ogawa, RO. Strain O139 serogroup virulent for rabbits-suckers in the dose of 107M.K.

Genetic strain: lysogeny, ctx+tcp-. The strain is not able to cleave the synthetic substrate for neuraminidase 2-(4-methylumbelliferyl)-N-acetyl-L-D-Narimanovo acid.

The product synthesized by the strain: the producer of temperate phage, serologically related phages of Vibrio cholerae El tor circulation, serotype II, III immune category.

Activity (productivity) strain: produces a temperate phage in title 107/ml.

Example 1.

Prepare a suspension of cell suspension focustitle indicator strain of Vibrio cholerae eltor KM-199 and studied strain of Vibrio cholerae O139 (R-16488)such that 1 loop per diem agar cultures contribute in one tube with 2 ml of broth Martin (pH of 7.6-7.8)to conduct incubation for 20 hours at a temperature of 37°C. the mixture is Then heated to a temperature 55-57°within an hour and put a drop of suspension on a lawn of strain Vibrio eltor KM-199, pre-planted in a layer of 0.7% agar on the plate 1.5% agar Martin.

Accounting is carried out after 24 hours of cultivation at 37°With, in place of applying drops of a zone of enlightenment lawn. In the studied strain of V. cholerae O139 (R-16488) is lysogeny and highlights the phage.

The test strain of V. cholerae eltor KM-199 (P-13169) is a universal sensitivity is determined as being on all types of phages O139, thus allowing to carry out the differentiation of strains of V.cholerae O139 their lithogenetic and nelinejnosti (see table).

The next (second stage) method is the use of indicator strain V. cholerae KM-152 (P-16373) for phage V. cholerae O139 I morphography. For this 0.5-1 ml of 4-hour broth culture of strain KM-152 contribute in 4 ml of 0.7% agar Martin and poured agar plate, then on the lawn culture is applied in the form of a "track" drop phage isolated from V. cholerae O139 (R-16488) FB. On the lawn of strain KM-152 phage BF forms a turbid zone of lysis. Strain KM-152 identifies only filamentosa phages to which the strain KM-152 sensitive and resistant to other temperate phages III immunotype having a head and a bone.

In addition, the experiments revealed that the phage I morphography V. cholerae O139 differ from phage V morphography high sensitivity to the chloroform. As a result of exposure to phages chloroform 1:10 phages I morphography were killed and phages V morphography remained viable. Therefore, inactivation of the cells in the mixed culture of the indicator strain lysogeny culture) when selecting phage using heat at a temperature 55-57°C for 40-60 minutes, while phages I and V morphography remain viable.

Example 2.

Perform as described in example 1, use the Zuya mixture V. cholerae eltor KM-199 and V. cholerae O139 (R-17625). The mixture is heated at a temperature of 56°C for 40 minutes, and then put a drop of suspension in the form of a "track" on the lawn of strain V. eltor KM-199, pre-planted in a layer of 0.7% agar on the plate 1.5% agar Martin. Accounting is carried out after 24 hours of cultivation at 37°With, in place of applying the drops form a zone of lysis of the culture.

Therefore, the studied strain of V. cholerae O139 (R-17625) nerisovannye and the phage does not emit.

The proposed method intraspecific differentiation was developed based on the study of new epidemic strains of toxigenic V. cholerae O139 serogroup specialists Rostov NPCI. Was studied 50 strains, of which 25 ctx+tcp+strains isolated from cholera patients and 25 ctx-tcp-cultures isolated from environmental samples (see table).

According to the method, the strains were divided into lysogeny (25 strains ctx+tcp+and nasogenian (25 strains ctx-tcp-). Phage III immunotype contains 6 strains.

In the proposed method pageproducts indicator KM-199 was detected in 25 ctx+tcp+strains, 25 ctx-tcp-the strains did not contain the phage in the supernatant. Phage III immunotype detected in 6 strains, he didn't lizirovat V. cholerae O139 KM-152. However, pageproducts observed in 19 strains on V. cholerae O139 KM-152, providing additional WPPT is erential this group and increasing effectiveness 3 times.

The practical value represents the allocation of the proposed method of phage from strain V. cholerae O139-P16488 that provides the diagnostic preparation of phage V. cholerae O139.

Phage selected on the strain KM-199, analyzes 13 strains ctx+tcp+V. cholerae O139, which improves laboratory diagnosis of Vibrio cholerae new serogroup.

The table presents the results of increasing the number of differentiated strains of the proposed method.

The use of the proposed method intraspecific differentiation of V. cholerae O139 allows the indicator strains KM-199 KM-152 in certain technological sequence with high accuracy to favoritemovie, while receiving additional characteristics of V. cholerae allocated from clinical material.

In addition, paraproducts and sensitivity to phage isolated from Lisovenko strain, allow direct favoritemovie and give sensitive method of laboratory diagnostics V.cholerae O139 serogroup, as differentiated strains of vibrios between a marker phage and simplified primary characteristics of the studied strains on pageType, which significantly reduces the time necessary to perform rapid analyses and allows you to quickly identify sources of infection, territory, region of its distribution, the rst is their differentiation foci.

Table
Number of studied strains of V.cholerae O139Differentiation of Vibrio cholerae O139 serogroup
The proposed methodAccording to the method
PageproductsImmunity KM-152Lysis by phage from V.cholerae O139 (16488)PageproductsImmunity
KM-199KM-152KM-114KM-117
25 ctx+tcp+2519613256
25 ctx-tcp-25000250

How intraspecific differentiation of Vibrio cholerae O, including joint incubation of the test culture with indicator test strain with subsequent favoritemovie, characterized in that as the test strains used focustitle strains of Vibrio cholerae eltor KM-199 and Vibrio cholerae O KM-152, and the differentiation is carried out in two stages, the first stage as the indicator strain used Vibrio cholerae KM-199, incubated with the studied cool is dependent within 20-24 h, then the mixture of microorganisms at a temperature 55-57°warm up With 40-60 min and plated on lawns of indicator strain of Vibrio cholerae eltor KM-199, which is pre-seeded with a layer of 0.7% agar on the plate 1.5% agar Martin, and through days of cultivation at 37°To conduct the registration of paralysis, while the second stage of the differentiation is carried out, using the phage obtained from Lisovenko strain of the test culture by drawing on the indicator strain of Vibrio cholerae O KM-152, which reveal only filamentosa phages, conducting direct favoritemovie.



 

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