Vol 88, No 1 (2011)

Aim . Comparative analysis of species diversity of sample of coagulase-negative staphylococci (CNS) isolated in hospitals of different specializations. Materials and methods . For identification of 102 CNS strains, biochemical systems manufactured by NPO «Diagnostic Systems», VITEK® 2 Compact, and BBL™ Crystal™ as well as sequencing of fragments of tuf and gap genes were used. Results . Greater differentiating capability of genotyping compared with phenotyping methods for species identification of staphylococci was demonstrated. Six CNS species were identified in the sample: S.epidermidis, S.haemolyticus, S.hominis, S.warneri, S.capitis, and S.pasteuri . The largest species diversity was noted for strains from maternity hospitals in Nizhny Novgorod and Kulakov Scientific Center for Obstetrics, Gynecology and Perinatology. Strains isolated from blood of patients in Bakulev Center for Cardiovascular Surgery were represented mostly by S.epidermidis and S.haemolyticus . Differences in species diversity of CNS — causative agents of neonatal conjunctivitis and omphalitis — were observed. Conclusion . Two species of CNS: S.epidermidis and S.haemolyticus pose special threat as nosocomial pathogens both in hospitals for adults and obstetrical facilities. Additionally, in neonatal units it is necessary to control such species as S.warneri, S.capitis, S.pasteuri .

Aim. Detection of bacteriocins and phages in pathogenic bacteria of Burkholderia genus and study of their specificity range. Materials and methods. Sixty strains of B. pseudomallei, 11 strains of B. mallei , 18 strains of B.cepacia, 5 strains of B.thailandensis, and 3 strains of B.gladioli were used in the study. The agar-overlay method was used to determine bacteriocin activity. For the accumulation of bacteriocins, strains-producers were grown on nutrient broth, inactivated by chloroform and an aqueous phase was spread on the culture surface of indicator strains cultivated on semisolid agar. Phages were isolated with Gratia agar method. Microscopy of phage particles was performed using the electron microscope JEM-100 SX by instrumental magnification 50,000—60,000. Results . It was shown that all studied clinical and collection strains of pathogenic Burkholderia — B.pseudomallei , B.cepacia , B.thailandensis , B.gladioli (total: 97 strains) produced bacteriocins and bacteriophages. The range of their inhibiting activity includes both strains of the same species and heterologous Burkholderia , including B.mallei , which does not have neither bacteriocins nor phages. For the first time presence of bacteriocins in B.pseudomallei strains were detected. Phage B.cepacia B623 effectively lysing B.mallei and not reproducing on B.pseudomallei cultures was identified which is suitable for differentiation of these two species. High sensitivity to the phages of heterologous Burkholderia has been established for B.thailandensis . Set of strains of the latter species allows to detect phagoproduction in virtually all lysogenic cultures of studied Burkholderia spe- cies. Conclusion . Pathogenic Burkholderia being inhabitants of the environment ( B.pseudomallei , B.cepacia , B.thailandensis , B.gladioli ) possess an- tagonistic factors that were lost in the process of evolution in strictly pathogenic B.mallei species.

Aim . Assessment of genetic diversity of influenza virus A/H1N1(sw2009) variants circulated in Russia, study of virus’ pathogenicity in humans and potential resistance to antiviral drugs. Materials and methods. Sequencing of PCR-fragments of genome of influenza viruses isolated from clinical and autopsy samples of 436 patients. Four full genome sequences of influenza viruses A/H1N1(sw2009) were obtained. Phylogenetic analysis was performed. Results. High degree of homology (98.9—100%) was found among influenza A/H1N1(sw2009) viruses in HA and NA genes as well as in their aminoacid sequences (1.3 and 1.4% respectively). Differences in other proteins did not exceed 1.1%. Diversity was found in position 222 of receptor-binding locus of HA and single amino acid polymorphism — in several internal proteins. Known mutations determining resistance to Tamiflu and Arbidol were not detected. All viruses were resistant to remantadine. Molecular markers of high pathogenicity were not found. Conclusion. High homology of influenza viruses determines low level of antigenic differences although in populations of viruses there are variants with different levels of adaptation to human organism and different affinity to receptors of upper and lower respiratory tract that can determine their different transmissibility.

Aim. To study antiviral activity of extracts obtained from basidial fungi against influenza viruses of different subtypes. Materials and methods. Antiviral activity of extracts obtained from basidial fungi against influenza virus A/chicken/Kurgan/05/2005 (H5N1) was determined in in vitro experiments. Changes in infectiousness of pandemic influenza virus A/Moscow/226/2009 (H1N1)v caused by extracts of basidial fungi was studied in experiments in vitro and in vivo . Results. Seventy water extracts of basidial fungi were studied, of which 10 were able to inhibit infectiousness of influenza virus strain A/chicken/Kurgan/05/2005 (H5N1) in MDCK cell culture. Also, several studied extracts decreased infectiousness of pandemic influenza virus strain A/ Moscow/226/2009 (H1N1)v in MDCK cells and inhibit its reproduction in lungs of infected mice. Conclusion. High antiviral activity of extracts obtained from basidial fungi against influenza viruses opens perspectives for development of drugs with preventive and treatment effects.

Aim. To experimentally assess protective effect of Immunovac-VP-4 vaccine against avian influenza virus H5N2. Materials and methods. Immunization of mice with polycomponent vaccine Immunovac-VP-4 was performed using oral or mucosal route of administration (intranasally, orally, and with com- bined nasal-oral method). Immunized mice were inoculated intranasally by influenza virus H5N2 adapted for mice. Survival of mice in experimental and control (intact) groups was assessed daily during 14 days. Survival and death rates of mice were determined. Levels of cytokines in sera of mice from both groups were measured by enzyme immunoassay. Results. Half of experimental animals survived after triple subcutaneous administration of vaccine in dose 20 mcg and subsequent intranasal challenge with avian influenza virus H5N2. Single subcutaneous immunization with dose 400 mcg resulted in survival of 80±12.6% of mice after challenge. Triple intranasal and combined intranasal-oral immunization as well as after triple subcutaneous immunization resulted in survival of half of challenged mice. In control group challenge was lethal for 90—100% of mice. Same methods of immunization lead to increase of IL-6, IL-12, IL-15, and IFN-γ levels. Conclusion. Data about significant protective effect after immunization with Immunovac-VP-4 against avian influenza virus H5N2 were obtained. Immunovac-VP-4 administered by mentioned routes activated nasal-associated lymphoid tissue providing first line defense at entry site of influenza infection, which demonstrates need to further study of this vaccine during development of strategy for non-specific prophylaxis of influenza infection.

Aim. To obtain monoclonal antibodies (MCAs) to glycoprotein E 1 of rubella virus, to assess their immunochemical characteristics and ability to use fluorescent MCA for rapid identification of rubella virus. Materials and methods. Rubella virus strain C-74 (Moscow), vaccine strains «Orlov» (Saint-Petersburg), Wistar RA 27/3 (USA) as well as strain Judith (Germany) were used. Viral antigens were obtained using diploid cells L-68 and cell lines VNK-21-F and Vero E6. MCAs were produced by conventional method and their isotype was determined: Immunoblotting, immunoenzume assay (IEA), hemagglutination inhibition assay (HIA) and immunofluorescence assay (IFA) were performed. Results. Five monoclonal antibodies — Kh-252.1, Kh-347.2, Kh-183.3, Kh-214.4, Kh-187.5 — to antigens of rubella virus strain C-74 were obtained. Isotypes of these antibodies were determined and their reactivity with native and denaturated antigens of other strains («Orlov», Wistar RA 27/3, Judith) was characterized. IEA showed that all MCAs interacted with rubella virus glycoprotein E1 at high titers ranging from 1/1600 to 1/200,000. Immunoblotting demonstrated that 4 MCAs (Kh-252.1, Kh-347.2, Kh-183.3, Kh-214.4) had aforementioned feature. MCAs inhibited hemagglutinating activity of Judith strain in titer from 1/16 to 1/1024 in HIA. FITC conjugate of MCA Kh-347.2 (most sensitive variant) allowed to detect rubella virus in infected Vero E6 cells after 24 hours since infection, whereas FITC conjugates of 3 MCAs (Kh-183.3, Kh-214.4, Kh- 187.5) — after 72 hours since infection. Conclusion. Use of FITC conjugates of MCAs is a perspective tool for identification of rubella virus glycoprotein E1 in infected cell cultures and nasopharyngeal swabs.

Aim. To detect virulence genes in clinical isolates of Escherichia coli O1 using polymerase chain reaction (PCR). Materials and methods. One hundred and twenty strains of E.coli O1 strains isolated from faeces of patients with acute diarrhea (n=45) and healthy persons (n=75) were studied. PCR with primers for rfb and fliC genes, which control synthesis of O- and H-antigens respectively, was used. Fourteen virulence genes (pap, aaf, sfa, afa, eaeA, bfpA, ial, hly, cnf, stx1, stx2, lt, st, and aer) were detected by PCR primers. K1-antigen was determined by Pastorex Meningo B/E.coli O1 kit (Bio-Rad). Results. rfb gene controlling O-antigen synthesis in serogroup O1 as well as fliC gene controlling synthesis of H7 and K1 antigens were detected in all strains. Thus all E.coli strains had antigenic structure O1:K1:H-:F7. Virulence genes aaf1, sfa, afa, eaeA, bfpA, ial, hly, cnf, stx1, stx2, lt, and st were not detected. All strains owned pap and aer genes regardless of the presence of acute diarrhea symptoms. Conclusion. It was shown that E.coli O1:K1:H-:F7 strains do not have virulence genes which are characteristic for diarrhea-causing Escherichia. In accordance with the presence of pap and aer genes they could be attributed to uropathogenic Escherichia (UPEC) or avianpathogenic Escherichia (APEC). It is necessary to detect virulence factors in order to determine E.coli as a cause of intestinal infection.

Aim. To assess level of pertussin toxin (PT) production by vaccine strains of Bordetella pertussis and strains isolated from patients with whooping cough. Materials and methods. Concentration of PT in supernatants of microbial cultures of 3 vaccine strains and 25 strains of B.pertussis isolated from patients with pertussis in 2001—2005 was measured with enzyme immunoassay using gamma-globulin fractions of rabbit antiserum to PT as immunosorbent or included in peroxidase conjugates. Results. Level of PT production by strains isolated from infected persons varied from 3±0.5 to 64.8±12.2 ng/MFU/ml: in 9 strains — from 3±0.5 to 9.4±2.1 ng/MFU/ml, in 7 — 10.5±1.8 to 18.4±2.6 ng/MFU/ml, and in 9 — 23.6±4.5 to 64.8±12.2 ng/MFU/ml. Conclusion. B.pertussis strains isolated from patients were heterogeneous on level of PT production. Difference in expression of PT between strains were as high as 20-fold. Conditionally low, moderate and high levels of PT production had 9 (36%), 7 (28%), and 9 (36%) of 25 studied strains. Three vaccine strains had levels of toxin production similar to recently isolated strains with moderate level of its production.

Aim. To study efficacy of anaferon pediatric in mice infected by pandemic influenza virus A(H1N1/09)v. Materials and methods. Influenza virus strain A/California/07/2009 (H1N1)v was used. Three groups of BALB/c mice intranasally inoculated with influenza virus were studied. First group received solution of Anaferon pediatric during 5 days before and 8 days after inoculation, 2 nd group received Tamiflu during 5 days after inoculation. Distilled water was administered orally to mice from control group. Results. It was shown that Anaferon pediatric used as preventive and treat- ment agent in mice intranasally inoculated with 100% infectious dose of influenza virus strain A/California/07/2009 (H1N1)v had antiviral effect, which expressed in 10-fold decreased reproduction of influenza virus in lungs of infected mice compared to control group measured 4, 6, and 8 days after inoculation. Conclusion. Use of anaferon pediatric before and after inoculation with influenza virus A(H1N1/09)v was not less effective than use of Tamiflu after inoculation.

Aim. To study efficacy of complex therapy of urogenital infections caused by Chlamydia and Mycoplasma using immunomodulator Superlimph. Materials and methods. Fifty males and thirty six females ages 22—47 years old with chronic urogenital infections – cervicitis and vulvovaginitis (females), prostatitis (males) — were studied. PCR and bacteriologic methods were used for diagnostics of mixed infection and microbiota efficiency. Patients were divided on 3 groups according to treatment protocol. Twenty patients (group 1) — standard therapy (josamycin), 48 patients received immunomodulator(suppositorium) before treatment with josamycin (group 2), 18 patients were simultaneusly treated with josamycin and immunomodulator (group 3). Results. Combinations of Chlamydia trachomatis, Ureaplasma urealyticum, Gardnerella vaginalis and Mycoplasma genitalium were identified in 30—35% of cases before treatment. After treatment with josamycin (group 1) or simultaneous therapy with josamycin and immunomodulator (group 3) considerable suppression of growth and elimination of both main pathogens and members of microbiota. Use of immunomodulator (group 2) in some cases resulted elimination of main pathogens and associated opportunistic microflora. Conclusion. Microbilogical monitoring in 97% cases demonstrated therapeutic effect of immunomodulator Superlymph on urogenital infections associated with Chlamydia and Mycoplasma and disbiotic microbiota of urogenital tract.

Staphylococci are able to cause chronic (persistent) infections, which develop in native tissues as well as on invasive materials artificially introduced into an organism. Such infections are associated with formation of biofilms. The review determines the definition of biofilms, describes factors, which contribute to their formation, characterizes adaptive stability of staphylococcal biofilms, which provides their long-term persistence in host organism. Genetic organization and regulation of polysaccharide and protein biofilms of staphylococci are described. Strategy for prevention and treatment of staphylococcal biofilm diseases is discussed.