RU2455355C1 - Pseudomonas aeruginosa BACTERIOPHAGE STRAIN USED AS BASE FOR PREPARING ASEPTIC FOR P AERUGINOSA - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is presented is a very virulent Pseudomonas aeruginosa ph57 bacteriophage strain recovered from vivarium excrement and depositied in the Collection of Microorganisms of Federal State Research Institution State Science Centre Vector, registration No. V-357.

EFFECT: strain shows a wide spectrum of Pseudomonas aeruginosa lytic activity and may be used as a base for preparing an antiseptic agent for P aeruginosa.

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Description

The invention relates to the field of medical microbiology, in particular to antiseptic agents based on bacteriophages.

To date, a known bacteriophage strain Pseudomonas aeruginosa (P. aeruginosa), used to treat inflammatory diseases (Chinishvili T.G. Bacteriophages: theoretical and practical issues. The main directions of scientific research on the problem of bacteriophage Tbilisi Scientific Research Institute of the Ministry of Health of the USSR. - M .: 1983, p. 1-26). The bacteriophage described was isolated from wastewater samples.

The disadvantage of such bacteriophages is the limited spectrum of lytic action, in addition, in the practical use of such bacterial viruses for therapeutic purposes, they can be inactivated by various factors of purulent exudate (HWAckermann, MS Dubow. 1987. Viruses of Prokariotes. Vol. 1. General properties of bacteriophages. CRC Press, Boca Raton, FL. P. 152).

Known pilospecific bacteriophage Pseudomonas aeruginosa Fi 03 (patent RU No. 2112800, publ. 10.06.1998). Pili (fimbriae) are one of the pathogenicity factors of Pseudomonas aeruginosa. Phage Phi 03 shows lytic activity against 55% test strains of P. aeruginosa. An advantage and at the same time a disadvantage of phage is the need for pili in a pathogenic bacterium. If there is no drink in the P. aeruginosa strain, then the bacteriophage is not able to lyse the bacterium.

The closest to the claimed strain, the prototype, is the bacteriophage strain Pseudomonas aeruginosa SSC PM N 02 (Phi 02) used to treat inflammatory diseases (patent RU 2113476, publ. 06/20/1998). The strain has a wide spectrum of lytic action in relation to test strains of P. aeruginosa and samples of purulent separable wounds from various patients collected from the clinical isolates collected in the Moscow region. A comparative check of this strain for bacterial isolates isolated from veterinary clinics in the Omsk and Novosibirsk regions showed that bacteriophage lyse only 50% of the tested P.aeruginosa strains.

An object of the invention is the creation of a highly virulent bacteriophage ph 57 with a wide range of lytic activity against Pseudomonas aeruginosa to combat purulent-septic infections.

The strain of the bacteriophage Pseudomonas aeruginosa ph 57 isolated from animal feces of vivarium, i.e. the bacterial virus is adapted to function in this ecological niche.

The strain of bacteriophage ph 57 was deposited in the collection of the Research Institute “Collection of microorganism cultures” of the State Scientific Center for Virology and Biotechnology “Vector” (Research Institute of KKM SSC VB “Vector”, Novosibirsk) under registration number V-357.

The inventive strain can be used as the basis for the preparation of an antiseptic agent against Pseudomonas aeruginosa

The bacteriophage is characterized by the following properties.

Morphological features:

The head is hexagonal in size 66-72 nm, the diameter of the tail process is 16-18 nm. On the lawn of bacterial cultures of P. aeruginosa, the phage forms transparent lysis spots with a diameter of negative colonies of 1.5-2.0 mm. The center is transparent, the edge is uneven, there is no secondary growth.

Serological characteristics.

The phage is not inactivated by antisera obtained for other bacterial viruses of P. aeruginosa from the collection of the Research Institute of KKM SSC WB Vector (11 different forms) The inactivation constant of the phage by homologous antiserum (K) is 85 min ^-1 .

The type of nucleic acid of a phage strain is DNA.

Physico-chemical properties of phage ph 57.

The lytic activity of ph 57 bacteriophage is not inactivated by heating the suspension for 30 min at a temperature of 55 ° C (Table 1). The permissible pH values at which the phage is active are 4.5–9.5 (Table 2). Threefold freezing - thawing of the sample does not lead to a decrease in the titer of active particles of phage ph 57 (Table 3). The phage is resistant to chloroform in the ratio (1:20) (Table 4). Exposure to ultraviolet (Table 5) and with hydrogen peroxide (Table 6) leads to a decrease in the lytic activity of the bacteriophage. Within 15 minutes, 1% chloramine inactivates the bacteriophage.

The lytic spectrum of the bacterial virus.

To assess the range of phage host strains, a P. aeruginosa collection of 78 strains was used. The phage has the ability to lyse 100% of the tested cultures of Pseudomonas aeruginosa (Table 7).

Specificity range: lyses P. aeruginosa, does not lyse dysenteric sticks (Shigella Sonnet, Flexner), freshly isolated and museum strains of E. coli and other representatives of normal microflora.

Optimal breeding conditions.

Cultivation conditions. The phage strain propagates on a sensitive culture of Pseudomonas aeruginosa (culture 663, Table 7) on ordinary nutrient media (meat-peptone broth, simple nutrient agar) at a temperature of 37 ° C for 16-18 hours of incubation.

Storage conditions: ph 57 bacteriophage retains lytic activity when stored at a temperature of + 4 ° С - + 6 ° С in peptone water for 1 year.

Frequency of reseeding 1 year.

Reproduction method: when reproducing phage in a host cell, the latent period is 45-50 minutes, yield - up to 100 particles per cell.

Example 1. Growing bacteriophage ph 57

The cultivation of phage ph 57 in meat-peptone broth is carried out using a thermostatically controlled rocking chair. Cultivation mode: temperature 37 ° С, flask rotation frequency - 200 rpm. Initially, 5 ml of Pseudomonas aeruginosa overnight culture was introduced into 250 ml flasks with medium and cell biomass was grown for 4-6 hours. At the next stage, phage are introduced into the cell suspensions (multiplicity of infection 1:10), after which the system is continued to incubate in the same mode for 12 hours. Typically, the concentration of phage ph 57 in the resulting lysates is (1-5) × 10 ¹⁰ ppm / ml.

Example 2. Titration of phage ph57 by the method of agar layers

Initially, dilutions of the analyzed phage suspension in physiological saline are prepared. A sample (100 μl) of the appropriate dilution is mixed with 100 μl of Pseudomonas aeruginosa overnight culture. 10 ml of 0.7% agar melted to 42 ° C are added to the resulting suspension, and after rapid stirring, the contents of the tube are poured onto the surface of a 1.5% meat-peptone agar in a Petri dish. After the top layer has hardened, the plate is turned over and incubated in a thermostat at 37 ° C for 16-18 hours. Ph 57 phage lysis spots on the bacterial culture lawn are counted to determine the concentration (titer) of the bacterial virus in the analyzed system.

The results of testing 10 strains of Pseudomonas aeruginosa for the lytic activity of phage ph 57 are presented in table 7.

Example 3. The use of bacteriophage ph57 as an aseptic agent against Pseudomonas aeruginosa

Sown strains from patients with a diagnosis of diabetic foot. The strains belonged to the pathogenic species Pseudomonas aeruginosa and showed multiple resistance to antibiotics recommended for the treatment of Pseudomonas aeruginosa. Model animals infected with this strain were daily cleansed for 5 days with a solution containing the inventive ph57 bacteriophage with a titer of 2.4 × 10 ⁷ PFU / ml. After 5 days, there was a complete cleansing of the wounds from purulent-necrotic contents.

Example 4. Two patients were diagnosed with pancreatic necrosis, in which, after a series of sanitation of the abdominal cavity and taking crops, Pseudomonas aeruginosa began to be sown. The abdominal cavity was washed twice with a solution containing ph57 bacteriophage with a titer of 1.4 × 10 ⁷ PFU / ml. On subsequent relaparotomies, it was found that these patients had an adhesive fibrin-free process in the abdominal cavity, and subsequent microflora cultures were sensitive to antibiotic therapy.

In all cases of the use of an antiseptic based on the claimed ph57 phage, a pronounced positive clinical effect was noted. In patients with urinary tract infections and pneumonia, the general condition quickly improved, the temperature decreased, and inflammation in the affected organs subsided. In patients with purulent wounds, necrotic tissues were cleansed, granulations appeared, and healing was accelerated.

In addition to assessing the clinical effect in individual patients, an assessment was made of the possibility of achieving an anti-epidemic effect in the patient population of the purulent department of the trauma hospital. After the use of the antiseptic agent pseudomonas phage ph57 in the hospital, there was a sharp decrease in the frequency of nosocomial infections with purulent-septic infections caused by P. aeruginosa from 40.8 to 8.9%.

The inventive strain of bacteriophage ph57, having a wide spectrum of lytic activity against Pseudomonas aeruginosa, can be used as the basis for the preparation of an antiseptic agent for the treatment of purulent-septic infections.

Table 1 No. p / p Incubation time, min Titer, PFU / ml one 0 (ref) 1.0 × 10 ⁸ 2 5 1.2 × 10 ⁸ 3 10 1.0 × l0 ⁸ four twenty 1.1 × l0 ⁸ 5 thirty 1.7 × 10 ⁸ 6 60 2.9 × 10 ⁷ 7 90 2.9 × 10 ⁷

table 2 Time Titer, PFU / ml pH 1 pH 3 PH 4.5 pH 8 pH 9.5 pH 11 1 hour 0 0 1.2 × 10 ⁸ 1.6 × 10 ⁸ 2.0 × 10 ⁶ 0 48 hour - - 5.6 × 10 ⁶ 1.15 × 10 ⁸ 1.7 × 10 ⁶ -

Table 3 No. p / p Number of Freezes Titer, PFU / ml one 0 (ref) 2.0 × 10 ⁷ 2 one 7.4 × 10 ⁷ 3 2 8.7 × 10 ⁷ four 3 6.6 × 10 ⁷ 5 four 2.4 × 10 ⁶ 6 5 1.4 × 10 ⁶ 7 6 8.5 × 10 ⁴ 8 7 3.4 × 10 ⁴ 9 8 8.6 × 10 ³ 10 9 6.0 × 10 ³

Table 4 No. p / p Incubation time, min Titer, PFU / ml one 0 (ref) 2.0 × 10 ⁷ 2 fifteen 2.7 × 10 ⁷ 3 thirty 1.6 × 10 ⁷ four 60 1.87 × 10 ⁷ 5 Day 1.4 × 10 ⁷

Table 5 No. p / p Incubation time, min Titer, PFU / ml one 0 (ref) 2.0 × 10 ⁷ 2 5 4.0 × 10 ⁶ 3 10 1.6 × 10 ⁵ four fifteen 7.0 × 10 ⁴ 5 35 4.6 × 10 ³ 6 60 0

Table 6 No. p / p Incubation time, min Titer, PFU / ml one 0 (ref) 2.0 × 10 ⁷ 2 fifteen 2.0 × 10 ⁴ 3 thirty 0 four 60 0

Table 7 No. p / p Strain P. aeruginosa (clinical isolates) Lytic activity ph 57, PFU / ml one 671 5.4 × 10 ⁷ 2 662 2.0 × 10 ⁸ 3 663 2.6 × 10 ⁸ four 664 4.8 × 10 ⁵ 5 665 2.6 × 10 ⁸ 6 666 1.54 × 10 ⁸ 7 667 4.1 × 10 ⁵ 8 668 5.2 × 10 ⁵ 9 669 8.0 × 10 ⁴ 10 670 1.8 × 10 ⁸

Claims (1)

The strain of bacteriophage Pseudomonas aeruginosa, deposited in the collection of the Research Institute of KKM SSC VB "Vector" under registration number V-357, with lytic activity against bacteria Pseudomonas aeruginosa, used as the basis for the preparation of antiseptic against Pseudomonas aeruginosa.