RU2531236C1 - Method of detecting microorganism of species vibrio parahaemolyticus - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method comprises inoculation of the test strain on the lawn of the culture, applying a drop of diagnostic phage preparation in centre of the cup, holding at the temperature of 37°C for 20-24 hours and confirmation of its belonging to the microorganisms of the species V. parahaemolyticus in the presence of lysis. At that the diagnostic phage preparation is prepared by mixing phages FC-44 and FC-46, prepared based on the strain Vibrio parahaemolyticus KM-97, in the ratio of 1:1 in the titre n·108 PFU/ml.

EFFECT: present invention enables to accelerate the identification of strains and can be used in the laboratory diagnostics of acute intestinal infections caused by this microorganism.

2 cl, 1 tbl, 3 ex

Description

The present invention relates to medical microbiology, in particular, laboratory diagnosis of acute intestinal diseases caused by Vibrio parahaemolyticus.

Currently, in the laboratory diagnosis of most bacterial intestinal infections, specific bacteriophages are used to identify isolated microbial strains with a high degree of certainty.

Due to the increasing incidence of food poisoning caused by Vibrio parahaemolyticus, the importance of phagodiagnostics of halophilic microorganisms is enhanced and relevant in the search for new, simple and effective methods for their detection, since it allows one to identify the specific properties of a certain type of vibrios when interacting with phages, to differentiate from closely related microorganisms childbirth, families, and the absence of a specific phage preparation for the diagnosis of Vibrio parahaemolyticus reduces the effectiveness of the counter idemicheskih and preventive measures. Known methods for diagnosing diseases caused by parahemolytic and other vibrioes pathogenic for humans (see Guidelines MUK 4.2.1793-03, Moscow, 2004, p. 23), which consist in serological typing of parahemolytic vibrios, which is carried out according to the scheme. A set of agglutinating O and K serums for serological typing on glass is produced by the Japanese company Toschiba Kazaki Co. Ltd.

The disadvantage of this method is that serological typing is a time-consuming process, as each serotyped strain requires an individual agglutinating serum, and this circumstance significantly increases the time of one laboratory analysis and, in addition, agglutinating serums (Japan) are not available and expensive.

For the prototype, a method for the phagodiagnostics of parahemolytic vibrios of serotype O5: K15 (see AS of the USSR No. 1671690, class C12 No. 7/00, August 23, 91) was selected, which consists in the use of the phage strain V. parahaemolytici F. 139, which is propagated using the Grazia method on alkaline media containing 3% NaCl and an indicator culture added to agar.

However, using phage F-139 to identify V. parahaemolyticus of other O and K serovars is not possible due to the limited lytic properties of the phage within the serogroup O5: K15 strains of V. parahaemolyticus.

The technical task of the invention was to develop an effective method that allows to accelerate the identification of strains of V. parahaemolyticus of various serovars, expanding its spectrum of action.

This object is achieved in that in the method for detecting microorganisms of the species Vibrio parahaemolyticus, including the use of an indicator strain followed by lysing the test culture with phage, Vibrio parahaemolyticus KM-97 is used as an indicator strain, from which 1.0 ml of FC- phage is prepared before use 44 and 1.0 ml of phage FC-46 in a ratio of 1: 1, in a titer of n · 10 ⁸ PFU / ml, then the test strain is inoculated onto the culture lawn and a drop of the obtained diagnostic phage preparation is applied to the center of the plate, the crops are kept at at a temperature of 37 ° C for 20-24 hours and the presence of phage lysis of the studied strain confirms its belonging to microorganisms of the species V. parahaemolyticus.

In this case, the lawn of the culture of the studied strain is prepared by pre-growing the latter in an incubator for 4 hours at 37 ° C after plating in Marten broth with 3% NaCl, then 0.3 ml of the grown culture is added to 4.5 ml of molten and cooled to 45 ° C 0.7% Marten agar with 3% NaCl and the second layer is poured onto a plate of Marten agar with 3% NaCl in Petri dishes, plated, dried, followed by application of the phage preparation.

The method is as follows.

To carry out the method use: indicator strain R-14706, deposited in the Clinical Hospital "Microbe", Saratov, under the number KM-97, phage 1154 Vibrio parahaemolyticus, phage 3256 Vibrio parahaemolyticus.

Phage 1154 Vibrio parahaemolyticus is deposited and stored under the number FK-44 (1154) in the collection of the laboratory of bacteriophages FKUZ Growing NIPS. In electron microscopic studies, the phage is characterized by a III morphogroup according to the classification of A.S. Tikhonenko. By serological properties, phage FC-44 belongs to the III serotype. The range of lytic activity extends to 80.4% of Vibrio parahaemolyticus strains of various O-serovars.

Phage 3256 Vibrio parahaemolyticus is deposited under the number FK-46 (3256) and is stored in the collection of the laboratory of bacteriophages FKUZ Growing NIPS. An electron microscopic study found that the phage belongs to the IV morphological group according to the classification of A.S. Tikhonenko. The results of the study of serological properties showed its belonging to the IV serotype of phages of parahemolytic vibrios. An important feature of the phage is the specificity of the logical action against 60.1% of V. parahaemolyticus strains of various O-serovars.

To propagate the bacteriophages FC-44 (1154) and FC-46 (3256), the indicator strain Vibrio parahaemolyticus KM-97 (P-14706) is used. Phages FK-44 and FK-46 are stored in ampoules, the shelf life is 1 year. The titer of phages is n · 10 ⁸ -10 ⁹ PFU / ml.

The phage preparation (DIF) is obtained before its direct use for high preservation of active phage particles. A bivalent mixture of phages FC-44 and FC-46 is prepared in a ratio of 1: 1 in a titer of n · 10 ⁸ PFU / ml, 1.0 ml of phage FC-44 and 1.0 ml of phage FC-46 are taken, which are connected in a test tube and DIF is ready for use.

The next step is to prepare the lawn of the culture of the studied strain, which can be isolated from the patient, from food products, water samples, fish, hydrobionts. According to the Grazia method, the lawn is bilayer. The bottom layer represents an agar medium pH 7.6 with the addition of 3% NaCl. The top layer is obtained from 4.0 ml of the test sample, which is grown in Marten broth with 3% NaCl at 37 ° C for four hours, then 0.3 ml of the grown culture is added to 4.5 ml of molten and cooled to 45 ° C 0 , 7% Marten agar with 3% NaCl and poured in a second layer onto agar plates in Petri dishes. After solidification, the agar is dried. Then, the DIF preparation is applied drop in the form of a track to the lawn of the studied culture and incubated at 37 ° C for 20-24 hours.

Analysis of the results, namely the identification of Vibrio parahaemolyticus, is carried out by the presence of a phagolysis zone in the grown culture. A transparent lysis zone confirms the presence of V. parahaemolyticus, and its absence precludes belonging to this type of microbe.

Example 1, confirming the specificity of the action of the DPh phage against vibrios and other closely related organisms.

When creating the drug DIF, 9 different types of phages were selected taking into account their biological characteristics (phages 109, 123, 154, 616, 124, 175, 227, 1154, 3256). The range of action of these phages (by lysis of strains) ranged from 7.0 to 34.7%. Then 3 variants of mixtures are combined: 1) 1154 + 3256; 2) 1154 + 227; 3) 3256 + 154. Test these mixtures on 184 strains of parahemolytic vibrios of various serotypes (see table 1). The first mixture lysed 92.5% of the studied strains, the second - 72.09%; the third - 41.39%. The table shows that the first version of the mixture, the preparation of the DIF phage, has a high specific effect on vibrios and other closely related organisms. The phage titer was n · 10 ⁸ PFU / ml.

Phage DIF lyses 92.5% of Vibrio parahaemolyticus strains and does not exert a lytic effect against bacteria of the families Vibrionaceae, Pseudomonadaceae, Enterobacteriaceae.

Example 2. The use of the preparation of phage DIF to identify V. parahaemolyticus.

From the feces of the patient (48 years old) with signs of foodborne toxicosis, a culture suspicious of morphological signs on V. parahaemolyticus is isolated. The phagodiagnostics of this culture is carried out as follows: the initially studied culture in the amount of 4.0 ml is grown in Marten broth with 3% NaCl for 4 hours at 37 ° C. After that, 0.3 ml of the grown culture is mixed with 4.5 ml of 0.7% marten agar molten and cooled to 45 ° C with 3% NaCl and the second layer is poured onto a plate of frozen 1.5% marten agar (pH 7.6) with 3% NaCl in Petri dishes. After solidification, the agar is dried and a drop of DIF phage is pipetted into the center of the plate. Crops are left at 37 ° C for 20-24 hours and a clear zone is detected at the site of application of the DIF phage. As a result, the test culture is classified as V. parahaemolyticus.

Example 3. The culture isolated from samples of sea water (a sample from the Black Sea water for monitoring vibrioflora, August 2012), suspicious of V. parahaemolyticus, was studied according to the method described in example 1. After lysing the test culture with the DIF phage for 24 hours diagnosed the presence of halophilic vibrios of V. parahaemolyticus in the samples.

Using the proposed method makes it possible to effectively use it in laboratory diagnostics, since the expressed sensitivity to the DPh phage in Vibrio parahaemolyticus, the growing conditions in the presence of NaCl in the medium allows it to be used as an additional test for differentiation of foodborne toxicoinfection pathogens, gastroenteritis, which can be used in laboratory diagnostics in order to:

- to use during the epidanalysis of acute intestinal diseases with foodborne toxicoinfections;

- used in laboratory diagnosis of strains of V. parahaemolyticus isolated from patients and from the environment (from sea water, aquatic organisms, etc.);

- to carry out typing with the phage of the differentiated strains of V. parahaemolyticus of various K and O-serovars;

- differentiate phagosensitive strains of V. parahaemolyticus from phage-resistant bacteria;

- the cost-effectiveness and ease of analysis, accelerated results, the availability of any laboratory make it possible to recommend the WPPT for widespread use, especially in mass studies and emergency epidemiological circumstances. Moreover, the effectiveness of diagnostic measures can be significantly increased through the use of diagnostic phage.

Table Family Kind View Number of strains Of these, are sensitive to Dif (%) Vibrionaceae Vibrio V. parahaemolyticus 184 92.5 V. cholerae cholerae 56 0 V. cholerae eltor 252 0 V. cholerae non O1 / non O139 57 0 V. cholerae O139 36 0 V. metschnikovii 7 0 V. mimicus 5 0 V. fluvialis one 0 V. vulnificus four 0 V. furnissii 5 0 V. diazotrophicus 2 0 V. splendidus 2 0 V. cambellii 2 0 V. pelagius one 0 V. orientalis one 0 Aeromonas 13 0 Pseudomonadaceae Pseudomonas 19 0 Enterobacteriaceae Escherihia 5 0 Schigella 5 0 Salmonella 12 0

Claims (2)

1. A method for detecting a microorganism of the species Vibrio parahaemolyticus, comprising using an indicator strain followed by lysing the test culture with a phage, characterized in that Vibrio parahaemolyticus KM-97 is used as an indicator strain, from which a diagnostic phage is prepared by mixing 1.0 ml of phage FC -44 and 1.0 ml of phage FC-46 in a ratio of 1: 1, in a titer of n · 10 ⁸ PFU / ml, then the test strain is inoculated onto the culture lawn and a drop of the obtained diagnostic phage preparation is applied to the center of the plate, the crops are kept at at a temperature of 37 ° C for 20-24 hours and the presence of phage lysis of the studied strain confirms its belonging to microorganisms of the species V. parahaemolyticus. 2. The method according to claim 1, characterized in that the lawn of the culture of the studied strain is prepared by pre-growing the latter in an incubator for 4 hours at 37 ° C after sowing in Martin broth with 3% NaCl, then 0.3 ml of the grown culture add in 4.5 ml of molten and cooled to 45 ° C 0.7% Marten agar with 3% NaCl and pour it a second layer on a Marten agar plate with 3% NaCl in Petri dishes, sowing is dried, followed by application of the phage preparation.