RU2717026C1 - Strain of bacteriophage pseudomonas aeruginosa n 325 (500319), intended for preparation of mono- and polyvalent treatment-and-prophylactic preparations of bacteriophages - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a strain of bacteriophage Pseudomonas aeruginosa used in production of medical biological antibacterial drugs – bacteriophage preparations, intended for preventing and treating pyoinflammatory and enteral diseases caused by Pseudomonas aeruginosa bacteria. Strain has lytic activity against Pseudomonas aeruginosa. Strain is deposited in the State Collection of Pathogenic Microorganisms (GKPM) of the Federal State Budgetary Institution "Scientific Centre for Expert Evaluation of Medicinal Products" of the Ministry of Health of the Russian Federation (FSBI "SCEEMP" of the Russian Ministry of Health) under registration number No. 325 (500319).

EFFECT: invention has a wide spectrum of antibacterial activity in relation to epidemiologically significant hospital and isolated from patients with various pathologies of clinical strains of pathogenic bacteria Pseudomonas aeruginosa, 100 % resistant to one or more antibiotics, obtained with maximum expansion of geography of isolation regions.

1 cl, 1 dwg, 5 ex

Description

The invention relates to medical virology and microbiology, in particular to a strain of the bacteriophage Pseudomonas aeruginosa used in the manufacture of medical biological antibacterial drugs - preparations of bacteriophages intended for the prevention and treatment of purulent-inflammatory and enteric diseases caused by bacteria Pseudomonas aeruginosa.

Pseudomonas aeruginosa bacteria (lat. Pseudomonas aeruginosa) (hereinafter referred to as P. aeruginosa) are characterized by high both natural and acquired resistance (50-100%) to most antibiotics and have the ability to infect all organs and tissues of the patient’s macroorganism [1, 2, 3 ]. Bacteria P. aeruginosa are one of the main causative agents of nosocomial pneumonia, infections of the respiratory tract, lungs and pleura and cause severe diseases in the form of cystic fibrosis - from 20% to 61% of all nosocomial infections [2, 4]; from 11% to 30% suppuration of burn wounds [4]; more than 30% of all purulent-inflammatory gynecological and urological infectious diseases; they are the cause of 20-25%) purulent surgical infections and generalized septic diseases - sepsis, with a mortality rate of 18% -61% [4, 5].

Today, mono- and polyvalent therapeutic and prophylactic preparations of bacteriophages containing bacteriophage strains of P. aeruginosa are known: “Pseudomonas aeruginosa bacteriophage”, “Purified polyvalent pyobacteriophage”, “Intesti-bacteriophage”, which, along with antibiotics, are used to treat and prevention of pseudomonas infections.

In accordance with the Russian requirements of the General Pharmacopoeia article “Bacteriophages. OFS.1.7.1.0002.15 [6] (“State Pharmacopoeia of the Russian Federation. XIII edition. Volume II”), enforced by Order of the Ministry of Health of Russia dated 10.29.2015 No. 771 from 01.01.2016, and international requirements established by the Decision of the Eurasian Council Economic Commission dated 03.11.2016 No. 89 “On the Approval of the Rules for Research on Biological Medicines of the Eurasian Economic Union” [7], Directive No. 2001/83 / EU of the European Parliament and the Council of the European Union “On the Community Code of Medicines for Human Use” ( was adopted in Brussels on November 6, 2001) [8] to guarantee clinical efficacy, a wide range of antibacterial activity and genetic safety of their therapeutic use, the therapeutic and prophylactic preparations of bacteriophages of P. aeruginosa should include bacteriophages biologically, morphologically and genetically characterized with a wide spectrum antibacterial activity belonging to the genetic lytic groups that do not contain genes for drug resistance, lysogenicity and toxigenicity, with the ability to Tew transmit drug resistance and toxigenicity to other bacteria in the patient's body.

A disadvantage of the known therapeutic and prophylactic preparations of P. aeruginosa bacteriophages is the absence of the above characteristics of the biological properties of bacteriophages in the composition of the preparations due to the passage of phage isolated from soil samples, wastewater, and wounds on bacterial strains of P. aeruginosa.

The criterion for the activity of the selected "passage" phages is the degree and stability of their lytic activity in the Appelman test in relation to production strains of P. aeruginosa bacteria.

Passages of bacteriophages are carried out constantly to maintain the necessary activity and the possibility of their further use as a working seed when receiving uterine phages and receiving bacteriophage preparations themselves.

Separate strains of bacteriophages of P. aeruginosa are not isolated and are not studied, and the constant passage of bacteriophages on bacterial cells often enough, due to uncontrolled recombination of bacteriophages when they multiply together, leads to the “degeneration” of passages and their loss of lytic activity.

The indicated method of creating an antibacterial basis for bacteriophage preparations in practice leads to the inclusion of “passive phages” and to the preparations of moderate phages and lytic phages containing genes for drug resistance, lysogenicity and toxigenicity, which have the ability to transmit drug resistance and toxigenicity to other bacteria in the patient’s body .

The problem solved by the invention is to obtain a strain of lytic bacteriophage P. aeruginosa that does not contain genes for drug resistance, lysogenicity and toxigenicity, which has a wide spectrum of antibacterial activity against epidemiologically significant clinical and hospital strains of bacteria P. aeruginosa isolated from patients.

The technical result of the invention is the selection of a highly active strain of the bacteriophage P. aeruginosa, intended for the preparation of mono- and polyvalent therapeutic and prophylactic preparations of bacteriophages P. aeruginosa, belonging to the genetic lytic group (genus), which does not contain genes for drug resistance, lysogenicity and toxigenicity, which has a wide spectrum of antibacterial activity against epidemiologically significant clinical and hospital strains of bacteria P. aeruginosa isolated from patients and meets Russian and international requirements for bacteriophages as part of therapeutic and prophylactic drugs.

The isolated strain of bacteriophage P. aeruginosa was deposited in the State collection of pathogenic microorganisms (GKPM) of the Federal State Budgetary Institution Scientific Center for Expertise of Medical Drugs of the Ministry of Health of the Russian Federation (FSBI "NTsESMP" of the Ministry of Health of Russia) under registration number 325 (500319) and has a wide spectrum of antibacterial activity against drug-resistant clinical and hospital strains of bacteria P. aeruginosa, related by virion morphology and gene the chemical properties of lytic bacteriophages of the order Caudovirales, family Podoviridae, genus Litllikevirus, which do not contain genes for lysogenesis, toxigenicity, and drug resistance.

The scope of the bacteriophage strain No. 325 (500319) is the production of therapeutic and prophylactic preparations of bacteriophages intended for the treatment of purulent-septic and enteric diseases caused by P. aeruginosa bacteria: “Polyvalent purified pyobacteriophage”, “Intesti-bacteriophage”, “Pseudomonas aeruginosa bacteriophage” ".

Patent RU 2455355, 2011 [9] is known, in which it is proposed to use a bacteriophage strain P. aeruginosa ph57 isolated from animal feces of vivarium as the basis of a veterinary antiseptic for the treatment of purulent-septic diseases of animals caused by P. aeruginosa bacteria.

The disadvantages of the invention are:

- lack of genetic characteristics of the proposed bacteriophage rp57, only the type of its nucleic acid is determined - DNA;

- its membership in the moderate or lytic genetic genera of bacteriophages has not been determined;

- the presence or absence of genes for drug resistance, toxigenicity and lysogenicity has not been established, which does not guarantee the genetic safety of its clinical use even in veterinary medicine and contradicts Russian and international requirements [7,8,9].

- there is no data on the breadth of the spectrum of antibacterial activity of the proposed bacteriophage strain in relation to epidemiologically significant hospital and clinical - isolated from patients with different pathologies of P.aeruginosa bacterial strains, since the method for determining the activity is not described, and the spectrum of antibacterial activity of the phage strain is 100%, determined on 78 strains claimed in the patent are not confirmed by the data of examples, which show that the assessment of the activity spectrum was carried out by the authors not on 78, but only on 10 strains of P. aerugi bacteria nosa.

Known patent WO 2015059298, 2015 [10], offering 13 strains of bacteriophages P.aeruginosa, for use as active agents of pharmaceutical or veterinary drugs in various compositions in an amount of from 2 to 13 bacteriophages.

The disadvantages of the invention are:

- the lack of convincing data confirming (90%) the wide spectrum of antibacterial activity of the proposed 13 strains of bacteriophages Pseudomonas aeruginosa and their combinations (compositions), since epidemiologically significant hospital and clinical strains isolated from patients with various pathologies were not used to study their spectrum and degree of antibacterial activity P. aeruginosa bacteria, and only 20 collection reference laboratory strains of P. aeruginosa bacteria from the international LMG collection (BCCM LMG / bacteria Collection) were used;

- lack of information about the lytic nature of the proposed 13 phages - the presence or absence of drug resistance, toxigenicity and lysogenicity genes was not established, which does not guarantee the genetic safety of their clinical use and is contrary to Russian and international requirements [6, 7, 8].

The patent EP2248890, 2010 [11] is known, which proposes the use of 6 bacteriophage strains of P. aeruginosa of the Myoviridae family of lytic genera CANCER and CHA to create phage compositions intended for the prevention or treatment of cystic fibrosis - pulmonary infections in mammals and humans caused by bacteria P .aeruginosa strains of CANCER and CHA. The disadvantages of the invention are:

- lack of information on the presence of drug resistance, toxigenicity and lysogenicity genes in patented strains of bacteriophages, a fairly narrow spectrum (only 30-40%) of the antibacterial activity of the proposed bacteriophages and their combinations, and activity only against bacterial strains of P. aeruginosa PAA and CHA, causing cystic fibrosis ;

- the lack of data on the breadth of the spectrum of antibacterial activity in relation to epidemiologically significant hospital and clinical strains of bacteria P.aeruginosa isolated from patients with various pathologies, as well as the small number of tested P.aeruginosa bacteria strains when determining the breadth of the spectrum of antibacterial activity (total 40 strains of bacteria P. aeruginosa CANCER and CHA) and the lack of data on the spectrum of antibacterial activity of the proposed bacteriophages in relation to representatives of other genetic species of bacteria P. aeruginosa.

Known patent PL 007472, 2006 [12], in which 20 strains of bacteriophages of P. aeruginosa are proposed that are active against drug-resistant bacterial strains of homologous species and are intended for the treatment of cystic fibrosis.

The disadvantages of the invention are:

- insufficient genetic characteristics of the genome of the patented phages — only family membership has been determined, genetic status has not been established, membership in lytic or moderate phages has not been characterized;

- the lack of information about the presence of drug resistance, toxigenicity and lysogenicity genes in patented bacteriophage strains and a fairly narrow spectrum of antibacterial activity of most strains against bacteria of a homologous species - only 2 phage strains of P. aeruginosa were active against 43% and 64% P. aeruginosa.

Known patent RU 2186574, 2001 [13], which proposed the preparation of bacteriophages Combiphage, intended for the treatment of infections caused by bacteria P. aeruginosa, which is a suspension in peptone water of a mixture of 4 strains of bacteriophages P. aeruginosa (No. 05, 03, 06 and 07), optionally containing components of a nutrient medium and auxiliary additives.

The disadvantages of the invention are:

- the lack of genetic characteristics of the proposed strains of bacteriophages of P. aeruginosa (No. 05, 03, 06 and 07), determining their affiliation with moderate or lytic genetic species of bacteriophages, belonging to the family, genus, group;

- the lack of information about the presence of drug resistance, toxigenicity and lysogenicity genes in patented bacteriophage strains, which leads to the lack of guarantees of the genetic safety of the clinical use of Combiphage.

Known patent RU 2112800, 1996 [15], in which it is proposed to use a saw-specific bacteriophage of P. aeruginosa - phage No. 03, which has antibacterial activity against saw-bearing clinical bacterial strains of P. aeruginosa, for inclusion in the composition of a medicinal product for the treatment of Pseudomonas infections . Meanwhile, they drank (fimbriae) bacteria P. aeruginosa are one, but not the only one of the pathogenicity factors of clinical bacteria strains of P. aeruginosa.

Moreover, the authors showed that in most phage-resistant forms of P. aeruginosa they did not drink, the proposed phage No. 03 was inactive against P. aeruginosa bacteria that did not contain pili, which, according to the authors, refers to the majority of phage-resistant forms of P. aeruginosa.

The disadvantages of the invention are:

- the lack of mandatory complete genetic characteristics of the proposed bacteriophage P. aeruginosa No. 03;

It was established that in tests with Mitomycin-C, phage P. aeruginosa No. 03 does not lyse the bacteria cells of P. aeruginosa, based on which it was concluded that this is a virulent (lytic) phage;

- the phage of P. aeruginosa No. 03 does not determine the specific genetic order, family, and genus; there is no evidence of the presence or absence of drug resistance, toxigenicity, and lysogenicity in the phage, which does not guarantee the genetic safety of its clinical use and is contrary to Russian and international requirements [ 6, 7, 8].

Known patent RU 2113476, 1996 [15], in which a bacteriophage strain P. aeruginosa No. 02 is proposed for inclusion in the composition of a multivalent therapeutic drug intended for the treatment of infectious diseases caused by bacteria P. aeruginosa.

The disadvantages of the invention are:

- the lack of mandatory complete genetic characteristics of the proposed strain of bacteriophage P. aeruginosa No. 02, only the type of nucleic acid is indicated - DNA;

- the lack of information about the lytic nature of the phage strain No. 02, its membership in moderate or lytic bacteriophages, its belonging to the order, family, and genus has not been determined; the presence or absence of drug resistance, toxigenicity, and lysogenicity genes in the phage strain P. aeruginosa No. 02 has not been established, which does not guarantee the genetic safety of the clinical use of the strain and is contrary to Russian and international requirements [6, 7, 8].

Known patent No. 2366437, 2007 [16], in which, to ensure stable virulence of bacteriophages in the composition and their adaptation to circulating pathogens of infections by obtaining bacteriophages by successive repeated passages of phage preparations of bacteriophages through test strains and freshly isolated isolates of non-isogenic bacteria with the addition of into the culture medium of an inducing agent, for example, mitomycin C.

The disadvantages of the invention are:

- the use of "passive" phages - mixtures of phages of P. aeruginosa, rather than individual strains of bacteriophages of P. aeruginosa with the studied biological and genetic properties, to obtain a multivalent bacteriophage preparation;

- the absence of guarantees to obtain stable induced virulence in moderate bacteriophages when using the mutagen Mitomycin C and guarantees that the drug will not contain moderate phages. The patent does not contain information about the presence in the "passive" phages treated with mitomycin C of lytic phages that do not have undesirable genes for drug resistance, toxigenicity, and lysogenicity;

- lack of complete information about the biological properties of bacteriophages in the composition of the drug - the morphology of phage virions, their belonging to the order, family, genus, there is no information on the nucleotide sequence of DNA phages in the composition of the drug with convincing evidence of the absence of undesirable genes for drug resistance, toxigenicity, lysogenicity, which contradicts Russian and international requirements and does not ensure the genetic safety of the therapeutic use of the bacteriophage preparation.

The analysis of the inventions indicates that none of the analogues can be selected as a prototype. However, most analogues do not contain information proving a wide range of antibacterial activity of patented bacteriophages in relation to epidemiologically significant hospital and clinical strains of bacteria P. aeruginosa isolated from patients.

The above analogues do not contain the full information on the genetic properties of the patented bacteriophages provided for by Russian and international requirements; belonging to moderate or lytic phage groups; belonging to a specific genetic order, family, genus; the presence or absence of drug resistance, lysogenicity and toxigenicity genes in patented phages, and therefore their presence in bacteriophage therapeutic and prophylactic preparations does not ensure the effectiveness and genetic safety of the therapeutic use of drugs.

Unlike most analogues of the bacteriophage strain P. aeruginosa No. 325 (500319), proposed for inclusion in the composition of mono- and polyvalent therapeutic and prophylactic preparations of bacteriophages, the essential distinguishing features are:

1. the presence of characteristics of biological properties - the lytic nature of the interaction with bacterial cells, a high degree (90%) and adsorption rate (10 min), the duration of the latent period (30 min), yield (10-12 phage virions / bact. Cells), morphology negative colonies;

2. the presence of morphological characteristics of bacteriophage virions of the phage strain No. 325 (500319) obtained by electron microscopy;

3. The presence of characteristics of the genetic properties of the DNA of phage strain No. 325 (500319) obtained by high-throughput genome-wide sequencing of its DNA followed by bioinformation analysis, in which the phage strain belongs to lytic bacteriophages of the order Caudovirales, the Podoviridae family, the genus Litllikevirus and the absence of the phage strain No. 325 (500319) of the genes for lysogenesis, toxigenicity and drug resistance;

4. the availability of data obtained by the Appelman method [6] indicated in the State Pharmacopoeia of the Russian Federation on the wide range of antibacterial activity of phage strain No. 325 (500319) (53.94%) in relation to 917 epidemiologically significant hospital pathogens and pathogenic bacteria P isolated from patients .aeruginosa in 100% resistant to one or more antibiotics obtained from sanitary-epidemiological services and hospitals with the maximum expansion of the geography of the regions of separation - from 30 geographically remote regions of Russia and the CIS countries.

The proposed combination of essential features of the invention is new, and the effect of significant features distinctive from analogues on the achievement of a technical result does not follow from the prior art.

For the preparation of multivalent therapeutic and prophylactic preparations of bacteriophages, a bacteriophage strain P. aeruginosa is proposed, deposited in the State collection of pathogenic microorganisms (GKPM) of the Federal State Budgetary Institution Scientific Center for Expert Advice of Medical Applications of the Ministry of Health of the Russian Federation (FSBI "NTsESMP" of the Ministry of Health of Russia) under registration No. 325 (500319), comprehensively biologically, morphologically and genetically studied, related to lytic bacterium ofagam of order Caudovirales, family Podoviridae, genus Litllikevirus, which does not contain genes for lysogenesis, toxigenicity and drug resistance, has a wide spectrum of antibacterial activity (53.94%) in relation to 917 epidemiologically significant hospital and isolated from patients with various pathologies of clinical strains of pathogenic bacteria P. aeruginosa in 100% resistant to one or more antibiotics obtained with the maximum expansion of the geography of the regions of separation from sanitary-epidemiological services and hospitals 30 geographically remote Russia's regions and CIS countries.

The bacteriophage strain P. aeruginosa No. 325 (500319) ensures the presence of biologically, morphologically and genetically studied lytic bacteriophage in the treatment and prophylactic preparations of bacteriophages, and also provides a wide range and stability of the antibacterial activity of the prophylactic drug based on it in relation to drug-resistant clinical and hospital bacterial strains of P. aeruginosa, safety, efficacy, and genetic safety of its therapeutic use.

The bacteriophage strain P. aeruginosa No. 325 (500319) is characterized by the following properties.

1. The strain of the bacteriophage P. aeruginosa was deposited in the State collection of pathogenic microorganisms (GKPM) of the Federal State Budget Institution “Scientific Center for Expertise of Medical Devices” of the Ministry of Health of the Russian Federation (FSBI “NTsESMP” of the Ministry of Health of Russia) under registration number No. 325 (500319).

The author's designation of the strain with which the bacteriophage strain P. aeruginosa was deposited is the bacteriophage P. aeruginosa PaUfa№P11.

The date of deposit with the State Public Institution of Healthcare Federal State Institution “National Center for Healthcare and Social Development” of the Ministry of Health of the Russian Federation is 01.11.2018.

Location GKPM FSBI "NTsESMP" Ministry of Health of Russia: 127051, Moscow, Petrovsky Boulevard, d. 8, p. 2.

The bacteriophage strain P. aeruginosa No. 325 (500319) was found in 2017 in vivo in the wastewater of the city of Kaliningrad, studied and characterized by the depositor at the branch of NPO Microgen in Ufa Immunopreparat.

The test strain of the bacterium P. aeruginosa, which is used to propagate the strain of the bacteriophage P. aeruginosa No. 325 (500319), is isolated from clinical material and deposited in the State collection of pathogenic microorganisms (GKPM) of the Federal State Budgetary Institution “Scientific Center for Expertise of Medicinal Products” of the Ministry Healthcare of the Russian Federation (FSBI "NTsESMP" of the Ministry of Health of Russia) under the number No. 321 (500315).

The test strain of the bacterium P. aeruginosa No. 321 (500315) was isolated from the patient from clinical material in 2009, is typical for its cultural, morphological, tinctorial and biochemical properties, studied and characterized by the depositor in the branch of NPO Microgen JSC in Ufa "Immunotherapy."

The author's designation of the test strain received for deposit is the bacterial strain P. aeruginosa No. 376.

The date of deposit with the State Public Institution of Healthcare Federal State Institution “National Center for Healthcare and Social Development” of the Ministry of Health of the Russian Federation is 01.11.2018.

Location GKPM FSBI "NTsESMP" Ministry of Health of Russia: 127051, Moscow, Petrovsky Boulevard, d. 8, p. 2.

2. The method of isolation of the strain of bacteriophage No. 325 (500319).

The phage strain was isolated by the method of cloning by injection from negative colonies obtained by sieving a sample of wastewater and a test strain of the bacterium P. aeruginosa No. 321 (500315) using the two-layer Grazia method on 0.7% –1.5% agar medium [17].

3. The conditions for the propagation of the strain of bacteriophage No. 325 (500319).

To propagate the bacteriophage strain P. aeruginosa No. 325 (500319), an 18-24 hour broth culture of the test strain of the bacterium P. aeruginosa No. 321 (500315), liquid (broths) and agar (0.7%, 1.5%) nutrients are used media based on pancreatic meat or casein hydrolysates or meat-peptone broth, pH 7.0-7.7.

The bacterial strain P. aeruginosa No. 321 (500315) and the bacteriophage strain P. aeruginosa No. 325 (500319) are simultaneously introduced into the sterile broth in the ratio (phage / bacterium) 1/10, incubation is carried out at a temperature of 37 ° C for 18-24 hours in conditions of periodic static cultivation. The end of the cultivation process of the phage strain is indicated by visual clarification of the culture fluid (phagolysate).

The bacteriophage strain No. 325 (500319) is not sensitive to chloroform, therefore, the phagolysate containing the phage strain is preserved by adding chloroform in a final concentration of 5-10% of the volume. The concentration of phages in the phagolysate after cultivation is determined by the Grazia method [17] and amounts to (0.5–5.0) × 10 ⁹ phage virions in 1 ml.

4. Storage of the bacteriophage strain No. 325 (500319).

The bacteriophage strain is resistant to chloroform, retains antibacterial lytic activity for 5 years when stored as a phagolysate in meat-peptone broth (MPB) containing 5-10% chloroform, at a temperature of + 2 ° C to + 8 ° C.

When storing the lyophilized dried bacteriophage strain P. aeruginosa No. 325 (500319) in a sucrose-gelatin medium at a temperature of + 2 ° C to + 8 ° C, the bacteriophage strain retains antibacterial lytic activity for 15 years.

5. Morphology of the negative colonies of the bacteriophage strain No. 325 (500319).

When the phage strain is plated with the Grazia two-layer method, the P. colugia No. 321 (500315) bacteria test strain negative bacteria form negative colonies with a diameter of (3.5 ± 0.5) mm and an incomplete lysis zone width of (1.2 ± 0.5) mm .

6. Morphology of phage virions of the bacteriophage strain No. 325 (500319).

According to electron microscopy, the bacteriophage strain No. 325 (500319) belongs to the Podoviridae family, the capsid diameter is 55 nm, the process length is 10-12 nm (Fig. 1).

7. The features of the phases of interaction of the bacteriophage strain with bacterial cells, studied in a single reproduction cycle [17] in modification [18] using the bacterial test strain P. aeruginosa No. 321 (500315) indicate that the bacteriophage strain No. 325 (500319) has a lytic a cycle of interaction with bacterial cells of a homologous type of bacteria — it multiplies intracellularly, lyses bacterial cells and is characterized by the following parameters of interaction with them:

- the degree of maximum adsorption on bacterial cells of Pseudomonas aeruginosa - 90%, the time of maximum adsorption - 10 min;

- the duration of the intracellular cycle of reproduction - 30 min;

- the output of phage from one infected bacterial cell (yield) is 10-12 phage. virions / bact. class

8. The spectrum of antibacterial activity of the bacteriophage strain P. aeruginosa No. 325 (500319).

The breadth of the spectrum of antibacterial activity of the bacteriophage strain P. aeruginosa No. 325 (500319), determined in the Appelman test, is 53.94% for 917 isolated from patients with various pathologies and hospital strains of P. aeruginosa epidemiologically significant, typical for their cultural, morphological, tinctorial and biochemical properties, 100% resistant to one or more antibiotics, obtained from hospitals and sanitary-epidemiological services in 30 cities of different territorially remote regions of Russia and the CIS countries.

9. Genetic properties of the bacteriophage strain P. aeruginosa No. 325 (500319).

Genome characteristic of the bacteriophage strain P. aeruginosa No. 325 (500319) - genome size 72,190 bp, double-stranded DNA, 54.9% HC content.

According to the data of high throughput genome sequencing and subsequent bioinformation analysis, the phage strain No. 325 (500319) belongs to lytic bacteriophages of the order Caudovirales, family Podoviridae, genus Litllikevirus.

The genome of the bacteriophage strain lacks genes for lysogenesis, toxigenicity, and drug resistance.

The complete nucleotide sequence of the DNA of the bacteriophage strain P. aeruginosa No. 325 (500319) was determined by high throughput genome sequencing on the Illumina Miseq platform with de novo assembly.

Example 1. Conditions for the isolation, reproduction and storage of a bacteriophage strain P. aeruginosa No. 325 (500319).

The strain of bacteriophage No. 325 (500319) was isolated by cloning [17] by injection from a negative colony obtained by sieving by a two-layer method of Grazia on 0.7% -1.5% agar medium of a Kaliningrad wastewater sample and a test strain of the bacterium P. aeruginosa No. 321 (500315).

To propagate the phage strain, nutrient media are used - sterile meat-peptone broth or broths with growth bases in the form of pancreatic meat or casein hydrolysates.

To propagate the phage strain, the bacterial strain P. aeruginosa No. 321 (500315) (18-24 hours culture) is introduced into the sterile broth in the initial concentration of bacteria (4.0-5.0) × 10 ⁷ bacteria. cells / ml and simultaneously bring bacteriophage No. 325 (500319) in the ratio of phage / bacterium 1/10.

Incubation is carried out for 18-24 hours at a temperature of 37 ° C under conditions of periodic static cultivation.

The completion of the phage propagation process is indicated by visual enlightenment of the culture fluid (phagolysate).

The strain of phage No. 325 (500319) is not sensitive to chloroform, so the phagolysate is preserved by the addition of chloroform (5-10)%. The concentration of phages in the phagolysate after the end of cultivation is determined by the Grazia method and is (0.5-5.0) × 10 ⁹ phage virions in 1 ml.

The strain of phage No. 325 (500319) retains lytic activity when stored in the form of a phagolysate containing 5-10% chloroform, at a temperature of + 2 ° C to + 8 ° C in BCH for 5 years.

When storing the lyophilized dried bacteriophage strain P. aeruginosa No. 325 (500319) in a sucrose-gelatin medium at a temperature of + 2 ° C to + 8 ° C, the bacteriophage strain retains antibacterial lytic activity for 15 years.

Example 2. Characterization of the biological properties of the bacteriophage strain P. aeruginosa No. 325 (500319).

The morphology of the negative colonies of the bacteriophage strain No. 325 (500319).

The phage strain when seeding dilutions of the phage by the two-layer method of Grazia in 1.5% -0.7% agar medium [17] forms negative colonies with a diameter of (3.5 ± 0.5) mm, the width of the zone of incomplete lysis (1.2 ± 0, 5) mm.

The features of the interaction phases of the bacteriophage strain P. aeruginosa No. 325 (500319) with bacterial cells are studied in a single reproduction cycle [17] in modification [18] using the bacterial test strain P. aeruginosa 321 (500315).

The results indicate that the bacteriophage strain has a lytic cycle of interaction with bacterial cells of a homologous type of bacteria — it multiplies intracellularly and lyses bacterial cells and is characterized by the following parameters of interaction with them:

- the degree of maximum adsorption on bacterial cells of P. aeruginosa - 90%, the time of maximum adsorption - 10 min;

- the duration of the intracellular cycle of reproduction on bacterial cells is 30 minutes,

- the output of phage from one infected bacterial cell (yield) is 10-12 phage. virions / bact. class

Example 3. The morphology of the virions of the phage strain P. aeruginosa No. 325 (500319).

The morphology of the virions of the bacteriophage strain is studied by electron microscopy. Suspensions of bacteriophage virions for electron microscopy were purified from bacterial impurities by ultracentrifugation in a gradient of cesium chloride [19].

Taxonomic affiliation was studied using a JEM-1400 electron microscope (Jeol, Japan) equipped with a Veleta digital lateral input camera (SIS, Germany).

The size and morphology of the virion particles of the bacteriophage strain according to electron microscopy is the Podoviridae family, capsid size 55 nm, the process length is 10-12 nm (Fig. 1).

Example 4. The spectrum of antibacterial activity of the bacteriophage strain P. aeruginosa No. 325 (500319).

To determine the breadth of the spectrum of antibacterial activity of a bacteriophage strain, the Apelman test is used at a dilution of 10 ^-1 [6] and epidemiologically significant bacterial strains of P. aeruginosa (917 pcs.), Isolated from patients with various pathologies and hospital, typical for their cultural, tinctorial, and morphological and biochemical properties that are 100% resistant to one or more antibiotics, obtained from sanitary-epidemiological services and hospitals in 30 cities of different geographically remote regions of Russia and the CIS countries.

To assess the antibiotic sensitivity of 917 clinical and hospital bacterial strains of P. aeruginosa, use 1.5% MPA, the method of antibiotic discs, including a set of antibiotic discs No. 9 recommended for bacteria P. aeruginosa (Federal budget institution of science "St. Petersburg Scientific Pasteur Research Institute of Epidemiology and Microbiology ").

Testing the antibiotic sensitivity of clinical and hospital strains of P. aeruginosa bacteria shows that P. aeruginosa bacteria (917 strains) are 100% resistant to one or more antibiotics, including ceftazidime antibiotics (35.7%), gentamicin (41.0%) ), amikacin (43.5%), aminoglycosides (60-85%), fluoroquinolam (77.0%), cephalosporins (60.0% -76.0% -85.0%), ciprofloxacin (47.0 %) and ticarcillin (53.5%), carbopenem - meropinem, ipinem (70.0%).

100% of P. aeruginosa bacterial strains were resistant to penicillin and tetracycline antibiotics.

Parallel testing of the antibacterial activity of the bacteriophage strain P. aeruginosa No. 325 (500319) in the Appelman test at a dilution of 10 ^-1 on 917 clinical and hospital strains of P. aeruginosa bacteria that are 100% resistant to one or more antibiotics indicates a wide spectrum and stability of antibacterial activity strain of bacteriophage P. aeruginosa No. 325 (500319) - the activity of 53.94% against bacteria P. aeruginosa.

Example 5. The study of the genetic properties of the bacteriophage strain P. aeruginosa No. 325 (500319).

To study the genetic properties of the bacteriophage strain P. aeruginosa, the method of high-throughput genome sequencing followed by bioinformation analysis was used.

To analyze the genomes of bacteriophage strains of P. aeruginosa, the DNA of bacteriophage strains was isolated by phenol-chloroform extraction with preliminary enzymatic treatment with RNase A, DNase I, and Proteinase K (Thermo Fisher Scientific, USA) [20].

DNA library sequencing was performed on the Illumina Miseq platform.

The de novo nucleotide sequence assembly was performed using the ABYSS package.

The search for open translation frames (ORT), taxonomic and functional identification of these ORTs was carried out using the MG-RAST software using DNA sequences found in the GenBank databases and others.

The characteristic of the genome of the bacteriophage strain P. aeruginosa is the genome size 72190 bp, double-stranded DNA, the content of HC is 54.9%.

According to high-performance full-genome sequencing and subsequent bioinformation analysis, it was found that the bacteriophage strain No. 325 (500319) belongs to the lytic bacteriophages of the order Caudovirales, family Podoviridae, genus Litllikevirus. It has been established that the genome of a bacteriophage strain lacks genes for lysogenesis, toxigenicity, and drug resistance.

The complete nucleotide sequence of DNA of the bacteriophage strain P.aeruginosa No. 325 (500319) was determined by high-throughput genome sequencing on the Illumina Miseq platform with de novo assembly.

Sources of information

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Claims (1)

The strain of the bacteriophage Pseudomonas aeruginosa, deposited in the State collection of pathogenic microorganisms (GKPM) of the Federal state budget institution "Scientific center for the examination of medical devices" of the Ministry of Health of the Russian Federation (FSBI "NTsESMP" of the Ministry of Health of Russia) under registration number N325 (500319), which has antibacterial activity in against drug-resistant clinical and hospital strains of bacteria P. aeruginosa, intended for the preparation of mono- and polyvalent preparations Ratov bacteriophages used for the treatment and prevention of infectious diseases caused by bacteria Pseudomonas aeruginosa

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