RU2112800C1 - Strain of pili-specific bacteriophage of pseudomonas aeruginosa "гнцпм" n 03 used for preparing medicinal preparation against pyocyanic rod - Google Patents

Abstract

FIELD: medicinal microbiology, phages. SUBSTANCE: new virulent pili-specific phage "ГНЦПМ" N 03 lyses broad spectrum of pyocyanic rod strains. Pili (fimbria) are one of factors of pathogenicity in Pseudomonas aeruginosa. Phage ⌀ 03 shows lytic activity with respect to 55% test-strains of P. aeruginosa. In this connection inclusion of bacterial virus ⌀ 03 to composition of multipartial medicinal preparation consisting of pyocyanic rod phages of different receptor specificity is considered to be expediently. EFFECT: new strain of phage indicated above.

Description

 The invention relates to medical microbiology, in particular to therapeutic bacteriophages.

 To date, the phage Pseudomonas aeruginosa is known to be used in the treatment of purulent-inflammatory diseases [1]. The bacteriophage described was isolated from wastewater samples.

 However, it is important to note that in the study of such phages for therapeutic purposes, their inactivation by various factors of purulent exudate is possible [2].

 The purpose of the invention is a pilospecific virulent phage ⌀ 03 used to prepare a multivalent therapeutic preparation of Pseudomonas aeruginosa phages.

 The bacteriophage ⌀ 03 Pseudomonas aeruginosa was isolated from a sample of a purulent discharge wound, i.e. the bacterial virus is adapted to function in this ecological niche.

 Saw-specific phages of Pseudomonas aeruginosa have not previously been used as therapeutic antimicrobial agents.

 Bacteriophage ⌀ 03 is deposited in the collection of phages of the GNTsPM under number 03.

 The bacteriophage is characterized by the following properties.

 Morphological signs.

 Phage ⌀ 03 has a slightly elongated head (95 • 60 nm) and a non-contracting caudal process (165 • 12 nm). The distal end of the caudal process resembles a truncated cone, from the top of which 3 terminal fibrils 10–12 nm long originate. Terminal fibrils have thickenings at the distal ends. At a distance of 15–17 nm from the apex of the process cone, subterminal adsorption organelles are visible — formations 15–18 nm long and 6–7 nm wide. Using subterminal organelles, phages are adsorbed on the saw blades of P. aeruginosa. Terminal fibrils are necessary for the adsorption of ⌀ 03 on the outer membrane of the microorganism.

 Patterns of interaction of the phage with the host cell.

 Phage ⌀ 03 forms on the lawns of sensitive bacterial cultures transparent lysis spots with a diameter of 0.5 - 1.5 mm.

The frequency of formation of phage-resistant forms of P. aeruginosa per cell is 10 ^-6 .

 Physiological signs.

The adsorption kinetics of ⌀ 03 on the host cell was characterized in the traditional way. During incubation of the phage-cell system (cell titer 10 ⁸ cells / ml and a multiplicity of infection 1:10) for 15 min at 37 ^o C 72 + 5% of particles ⌀ 03 are adsorbed on microorganisms.

 When reproducing phage in a cell, the latency period is 50-60 minutes, and the yield is 30-40 particles per microorganism.

 Serological properties of the phage.

Phage ⌀ 03 is not inactivated by antisera obtained against other bacterial viruses of P. aeruginosa from the GNCPM collection (20 different forms). The inactivation constant ⌀ 03 by homologous antiserum (K) is 42 min ^-1 .

 Physico-chemical properties of the phage.

The lytic activity of the bacterial virus does not change when the suspension is heated for 60 minutes at a temperature of 55 ^o C. The permissible pH in suspensions at which the phage is active is 4.5 - 11.

 A single freeze - thawing of the sample does not lead to a decrease in the titer of active particles ⌀ 03. The phage is resistant to chloroform (in a ratio of 1:20).

 The lytic spectrum of the bacterial virus. 03.

 To assess the range of phage host strains, a collection of 200 aeroginosa P. strains was used. The collection includes 100 clinical isolates of P. aeruginosa and 100 strains presented by the Museum of Bacterial Cultures of the State Scientific Center for Microbiological Medicine. Phage ⌀ 03 is able to lyse 110 strains of Pseudomonas aeruginosa. In separate experiments, it was shown that most phage-resistant forms of P. aeruginosa lack drank.

 Example 1. Adsorption of phage ⌀ 03 on the host cell. The evidence of the primary interaction of phages with P. aeruginosa saws is the data of electron microscopic studies of the ⌀ 03 - cell system. Samples of this system at different points in time were fixed with 1% glutaraldehyde prepared in 0.1 M phosphate buffer (pH 7.2) and analyzed under an electron microscope. It was shown that, after initial adsorption on polar saws, частицы 03 particles are translated by fimbriae to the surface of P. aeruginosa and are fixed on the outer membrane by terminal fibrils.

Example 2. Obtaining phage ⌀ 03 in a dense nutrient medium. Growing ⌀ 03 by the two-layer method provides for the initial production of a phagolysate in a layer of semi-liquid (0.7%) meat peptone agar (MPA). After low-speed centrifugation of the system (5 thousand g, 10 min), the phage titer in the supernatant is usually 10 ⁹ - 10 ¹⁰ hours / ml.

Example 3. Obtaining phage-resistant cells of P. aeruginosa and checking for lysogenicity. A suspension of susceptible P. aeruginosa cells (10 ⁸ cells / ml) with phage (final titer of ⁹ hours / ml) was incubated in meat-peptone broth (MPB) at 37 ^° C for 30 minutes and 0.1 ml of the sample was distributed in a Petri dish the surface of 1.5% MPA. The plates were incubated for 20 hours in an incubator (37 ^° C). The grown phage-resistant colonies are cloned by triple reseeding on MPA. Clone 50 to 100 colonies, randomly selected and then the resulting strains are analyzed for the presence of moderate phages.

The cells of each clone are resuspended in BCH. The initial concentration of microorganisms is 10 ⁷ cells / ml. Suspensions are incubated in a thermostat at 37 ^o C for three hours. Then, the mitomycin C inducer is introduced into the systems (final concentration 1 μg / ml) and the samples are incubated for another 3 hours. At the next stage, the systems are centrifuged (5 thousand g, 10 min). Fractions of the supernatant are titrated using the agar layer method on ⌀ 03 sensitive (indicator) cultures.

 Checking the ⌀ 03 - phage-resistant forms (100 colonies) of P. aeruginosa for the presence of moderate phages showed that ⌀ 03 does not lyse the Pseudomonas aeruginosa cells. Therefore, ⌀ 03 is a virulent phage.

Sources of information
1. The main directions of scientific research on the problem of bacteriophagy of the Tbilisi NIIIVS Ministry of Health of the USSR - Chinishvili T.G. Bacteriophages: theoretical and practical issues. - M., 1983, p. 1-26.

 2. H.W. Ackermann, M.S. Dubow. 1987. Viruses of Prokariotes. vol. 1. General properties of bacteriophages. CRC Press, Boca Raton, FL. p. 152.

Claims (1)

 The strain pylonspecific bacteriophage Pseudomonas aeruginosa GNCPM N 03 used to prepare a therapeutic drug against Pseudomonas aeruginosa.