Vol 91, No 4 (2014)

Aim. Study the prevalence and mechanisms of resistance in circulating C. diphtheriae strains. Materials and methods. 664 C. diphtheriae strains isolated in 1987 - 2013 in various regions of Russia and sent to the reference center of Gabrichevsky Moscow Research Institute of Epidemiology and Microbiology were the object of the study. Antibiotic sensitivity of the strains was studied by disk-diffusion and E-test methods using 10 antimicrobial preparations. Nucleotide sequence analysis was carried out by using BLAST program and EMBL/GenBank database. Results. Most of the studied strains turned out to be sensitive to all the antibacterial preparations used. 1.2% of C. diphtheriae strains turned out to be resistant to penicillin and 6.0% had intermediate level of resistance. 0.4 - 0.6% of the strains had intermediate level of resistance to macrolides, and 4.0 - 4.4% were resistant. 2.0% of the strains had multiple resistance. Erm(X)-specific PCR carried out in this study showed that all the C. diphtheriae strains resistant to macrolide antibiotics carry erm(X) gene. Conclusion. The results of the study indicate a fairly high level of prevalence for C. diphtheriae strains resistant to antibiotics.

Aim. Study specific activity and safety of vaccine preparations based on circulating B. рertussis strains with currently predominating allele variants of pertussis toxin (ptxA1) and pertactin (prn2) genes. Materials and methods. B. pertussis strains isolated from pertussis patients in Moscow in 2001-2010 were grown in dense and liquid media. The content of separate antigens in B. pertussis strains was determined by EIA. Immunogenicity and safety of the preparations was determined in F 1(СВАxС 57Bl6) line mice. Results. All the studied circulating B. pertussis strains expressed pertussis toxin (PT), filamentous hemagglutinin (FHA) and agglutinogens corresponding to the serovar. Whole-cell and acellular pertussis vaccines were prepared based on the circulating strains, and a highly productive recently isolated toxigenic B. pertussis strain that could be used for production of pertussis vaccines was selected as a result of studies of immunogenic, toxic and sensibilizing properties. Conclusion. Vaccine preparations based on a B. pertussis strain adapted to growth in liquid media with pertussis toxin and pertactin ptxA1 - prn2 gene allele variation characteristic for contemporary population are specifically active and safe.

Aim. Determination of frequency of occurrence and clinical significance of interleukin-28B (IL28B) and RNAse L gene polymorphism in patients with chronic hepatitis C (CHC). Materials and methods. 104 hospital patients with CHC (65% male; 63% with genotype 1 hepatitis C virus - HCV) were examined. 70 patients received therapy with interferon (IFN) and ribavirin (RBV). Single nucleotide polymorphism (SNP) of IL28B gene 39743165T>G (rs8099917), SNP 39738787C>T (rs12979860) and RNAse L gene (1385G>A) were determined by polymerase chain reaction. Results. The frequency of detection of «favorable» SNP allele variants of IL28B gene in patients with CHC was lower than in population of the European region. In patients with genotype 1 HCV, mutant alleles in SNP 39743165T>G (р=0.045) and 39738787C>T (р=0.005) occurred more frequently than in patients with other virus genotypes. Higher values of alanine aminotransferase in patients with genotype CC 39738787C>T were detected. Frequencies ofSNP variants of IL28B and RNAse L gene did not differ depending on the speed of disease progression (p>0.5). Response to IFN/RBV therapy was higher in «favorable» ТТ (SNP 39743165T>G) and СС (SNP 39738787C>T) variants. Conclusion. Examination for IL28B gene SNP 39738787C>T is recommended before the start of IFN/RBV therapy in all the patients with genotype 1 HCV as a prognostic factor on the therapy response. RNAse L gene SNP 1385G>A does not have a clear clinical significance in CHC.

Aim. Evaluate resolution and diagnostic significance of real-time multiplex PCR (MP RT-PCR) as a platform for group A rotavirus G/P genotyping test-systems. Materials and methods. Primer and DNA probe construction for an experimental test-system based on MP RT-PCR was carried out by using specialized PC programs and sequence databases GenBank NCBI, EMBL Nucleotide Sequence Database etc. The experimental genotyping test-system was tested using 116 clinical samples with confirmed rotavirus infection and 14 biosamples negative for group A rotavirus RNA. Selective sequencing of VP7, VP6, VP4 gene marker loci was carried out as a reference method for verifying determination of rotavirus genotype. Results. Specific interaction between primers and DNA probes with genotype-specific loci of retrovirus genome segments and a lack of false-negative signals, complete match of genotyping results obtained by MR RT-PCR and sequencing methods were established. Conclusion. The resolution of MP RT-PCR methods allows designing test-systems that can confidently identify rotavirus genotypes with effectiveness of 90% and above.

Aim. Study sensitivity of laboratory animals to a causative agent of Argentine hemorrhagic fever. Materials and methods. Junin virus strain XJ P37 was obtained from the State Collection of Causative Agents of Viral Hemorrhagic Fevers of the Pathogenicity Group I ofScientific Research Center of the 33 rd Central Scientific Research Test Institute (SRC of the33 rd CSRTI). Junin virus strain XJ P37 culture with biological activity of 5.2 lg PFU x ml was used in the experiments. Mice (2 - 4 and 7 - 14 days old), guinea pigs (250 - 300 g), 1.8 - 2.5 kg shinshilla breed rabbits, 2.0 - 3.0 kg javanese macaque monkeys were obtained from vivarium of the SRC of the 33 rd CSRTI. Vero (B) and GMK-AH-1 (D) cell cultures were obtained from cell culture collection of the SRC of the 33 rd CSRTI. Biological activity calculation of Junin virus was carried out by Kerber in I.P. Amsharin modification. Results. Lethality in animals was from 12.5 to 50% after intranasal and intraperitoneal infection of guinea pigs, intramuscular, intraperitoneal and subcutaneous infection of rabbits, intracerebral and intranasal infection of mice at the doses from 0.4 to 1.0 x 10 5 PFU. Death of infected monkeys after intramuscular administration of the virus at 1.0 x 10 4 PFU dose was not observed. In 100% of surviving animals formation of virus-neutralizing antibodies was registered. Conclusion. Evaluation of sensitivity of laboratory animals to Junin virus has shown that intracerebrally infected mice may be used to maintain causative agent culture, infected guinea pigs - to prepare virus-containing cultures and modelling infection exacerbation in humans. Intramuscularly infected rabbits may be used to obtain hyper-immune sera.

Aim. Study the sensitivity of nosocomial purulent-septic infection (PSI) causative agents to disinfectants (DA) and antibiotics (AB). Materials and methods. Sensitivity to DA and AB of209 PSI causative agent strains isolated from patients and the environment of 2 obstetric and 3 surgical hospitals was studied in 2009-2011. Sensitivity to DA of 94 strains and to AB of 189 strains of Рseudomonas аeruginosa isolated from patients with signs of PSI of reanimation and intensive therapy and surgical departments of a multi-field hospital was studied in 2012. Sensitivity to DA was determined on test-surfaces and in solution according to guidelines by V.V. Shkarin et al., 2010; sensitivity to AB - by disc-diffusion method. Results. Among PSI causative agents resistant to DA the portion of poly-antibiotic resistant strains is higher than among microorganisms sensitive to DA, and among antibiotic resistant bacteria the number of strains resistant to DA is higher than among sensitive to antibiotics. The increase of resistance to DA and AB of P. aeruginosa strains is observed in parallel to the increase of volume of the antibacterial preparations used. Conclusion. The results obtained give evidence of the possibility of formation of combined (associated) resistance to DA and AB by nosocomial PSI causative agents against the background of increase of their consumption.

Aim. Analysis of frequency and reasons of complications after BCG vaccination in 2008 - 2012 in Krasnodar Region for improvement of prevention measures. Materials and methods. Data on frequency and structure of post-vaccination complications during vaccination against tuberculosis of Centre of Hygiene and Epidemiology in Krasnodar Region and Regional Tuberculosis Hospital in 2008 - 2012 were used. Reasons of the complications were analyzed. Results. During the observation period 16 complications related to immunization with tuberculosis vaccine were noted with frequency index of 5.1 - 40.4 per 100 000 immunized newborns and a tendency of decrease from 2008 to 2012. In the structure of complications cold abscesses (37.5%), lymphadenitis (31.25%) and ostitis (31.25%) occurred at a similar frequency. Conclusion. Predominance of cold abscesses in the structure of post-vaccination complications indicates violations in vaccination technologies in maternity hospitals and children’s clinics in Krasnodar city, and late periods of post-vaccination ostitis diagnostics - a decrease of epidemiologic awareness of general medical network physicians regarding BCG-vaccination complications, that requires enhancement of immunization control and requalification of personnel carrying it out.

Aim. Study the effect of cycloferon in experimental and clinical conditions on persistence properties of aurococci as well as features of their morpho-functional reaction by atomic force microscopy. Materials and methods. The study was carried out in 12 Staphylococcus aureus clones isolated from mucous membrane of nose anterior part of a resident carrier. The effect of cycloferon in vivo was evaluated in 26 resident staphylococci carriers under the control of anti-carnosine activity of staphylococci. Anti-carnosine activity was determined by O.V. Bukharin et al. (1999), biofilm formation - by G.A. O’Toole et al. (2000). Staphylococci treated with cycloferon were studied by atomic force microscopy in contact mode using scanning probe SMM-2000 microscope. Results. The decrease of persistence properties of staphylococci under the effect of cycloferon in vitro and in vivo may be examined as one of the mechanisms of biological activity of the preparation. A significant increase of S. aureus surface roughness and changes in their morphology under the effect of cycloferon allow stating the disorder of barrier functions in the aurococci cell wall. Conclusion. The data obtained expand the understanding of cycloferon biological activity mechanisms.

Analysis of summarized data obtained by us on ultrastructure of microbial biofilms of opportunistic bacteria is presented. A complex ultrathin organization of lactobacilli, enterobacteria, staphylococci and enterococci biofilms discovered during electron microscopy is described. The presence of surface film and polysaccharide matrix that determine increased resistance of intrabiofilm bacteria against the effect of protective immune factors of the organism and etiotropic preparations is demonstrated in all the studied comminutes. A varied response of bacterial cells contained in the biofilm during antagonistic effect of symbiont probiotic bacteria was discovered.

Literature data on molecular mechanisms of resistance to β-lactam antibiotics have been summarized. The following mechanisms are examined: appearance of penicillin-binding proteins (PBP) with lower affinity to β-lactam antibiotics; production by microorganisms of enzymes β-lactamases) that hydrolyze β-lactam ring; disruption of microbial cell outer membrane permeability and active secretion of antibiotics from the microbial cell (efflux-effect). Characterization of extended spectrum β-lactamases (ESBL) as well as data on structure of SCCmec element of MRSA and bacterial efflux system (RND) is presented.