Vol 90, No 5 (2013)

Aim. Characteristic of morpho-functional reaction of Listeria monocytogenes bacterial cells to the effect of cationic peptide nisin. Materials and methods. Culture of L. monocytogenes 88-BK bacterial cells was grown in the presence of nisin (experiment) and without it (control). The samples obtained were studied by atomic-force microscopy method in contact mode by using scanning probe of SMM-2000 microscope. Study of elastic properties of bacterial cells was carried out by using AFM in force spectroscopy mode. Results. Microorganisms grown in the presence of sub-inhibitory concentrations of antibiotic were characterized by changes in cell morphology and mechanical properties that were recorded by using AFM as changes in size parameters of bacteria, visualized local lesions of surface with the increase of its roughness as well as decrease of mechanical durability of the cell wall. Conclusion. Use of atomic-force microscopy allows to detail mechanisms of biological activity of cationic peptide nisin on gram-positive bacteria, evaluate specter of changes of cell morphological and mechanical parameters that took place and were analyzed in the conditions close to native.

Aim. Molecular-genetic characteristic of M. tuberculosis strains isolated from operation material of patients with tuberculous spondylitis. Materials and methods. 107 strains of M. tuberculosis isolated in 2007-2011 from patients with spine tuberculosis were studied by methods of spoligotyping and MIRU-VNTR by 12 and 24 loci. Results. Strains of genetic family Beijing dominated (n=80), 78% ofthose had multiple drug resistance (MDR). Strains ofgenetic families T, H3 (Ural), LAM, Manu, H4 and S were also detected. Differentiating of80 strains of Beijing genotype by MIRU-VNTR method by 24 loci revealed 24 variants (HGI=0.83) including 7 clusters, the largest of those (100-32) included 23 strains (87% MDR). Conclusion. The leading role of Beijing genotype M. tuberculosis strains in development of tuberculous spondylitis with multiple drug resistance of the causative agent is shown.

Aim. Detection of circulation ofWest Nile virus (WNV) on the territory of Saratov Region and prerequisites for formation of natural focus of West Nile fever (WNF), determination of the role ofWNV in infectious pathology on the territory of the region. Materials and methods. Suspensions of organs of small mammals, birds, blood-sucking arthropods for the presence of WNV markers (antigens and/or RNA) were studied. Clinical material from patients with symptoms not excluding WNF was studied. Donor blood sera samples were analyzed with the aim of detection of immune layer against WNV in population of Saratov Region. Results. In 2010 WNV antigens were detected by EIA in 12 samples (7.1%) of mammal organ suspensions. In 2012 by using RT-PCR and EIA, markers of WNV were detected in 6 samples of bird brain suspensions (6.3%) and 1 sample of mammal organ suspension. Immune layer of population against WNV was 4% in 2011, 2.8% in 2012. In 2012 in 11 of 27 examined patients IgM against WNV in diagnostic titers and/or serconversion of IgG in paired sera were detected. In addition in 5 individuals virus RNA was detected in blood. Based on clinical, laboratory data and epidemiologic anamnesis 11 patients were diagnosed with WNF. Conclusion. The results obtained give evidence on the circulation of WNV on the territory of Saratov Region in 2010 - 2012. With the development of complications ofWNF epidemiologic situation in 2012 an expansion ofWNV areal onto the territory of the region took place and the process of formation of WNF natural foci is ongoing.

For the period from 2001 to 2011 zoological and epizootological studies in more than 100 points ofNorthwestern Caucasus including territories ofKrasnodar Region and Republic ofAdygea were carried out. 8723 specimens of small mammals represented by 20 rodent species and 7 insectivorous species were captured and examined. Organs and blood from 5057 specimens of creatures were studied for hantavirus infection. The presence of natural foci of circulation of 2 species of hantavirus - Dobrava/Belgrade and Tula - was established. Sochi viruses and presumably Kurkino with main natural hosts - Caucasian wood and field mice belong to the first species. Tula and Adler viruses with the main host - Microtus genus vole belong to the second species. Quantitative characteristics of infection of small mammals of various species during different seasons and years on the examined territories were obtained, that allowed to create a map of allocation of foci of hantavirus circulation that differ by structure.

Aim. Study intra-species immunogenic activity ofantigenic protein-polysaccharide components of S. pneumoniae. Materials and methods. Antigenic components of serotype 3, 6А, 6В, 14, 10А, 18А, 19А, 19F, 23F and unencapsulated S. pneumoniae strains were obtained by water extraction method. Synthetic hexasaccharide - corresponding to the structure of S. pneumoniae serotype 14 capsule polysaccharide repeated unit chain fragment was used as a reference preparation. Molecular mass of antigenic components was determined in SDS-electrophoresis. Antibody titers in blood sera of immunized mice were evaluated by solid-phase EIA method. Protective activity of preparations was studied in mice after 2 immunizations with consequent infection by virulent S. pneumoniae serotype 3 and 6B strains. Results. Preparations from serotype 6А, 6В, 14, 19А, 19F, 23F strains in reaction with anti-microbial sera were characterized by cross serologic activity (IgG titers of 1200 - 12 800). The lowest serologic activity was detected in S. pneumoniae serotype 3 and unencapsulated strain preparations. Conjugate of synthetic hexasaccharide and bovine serum albumin interacted only with homologous antimicrobial sera up to titers of 600+89.4 and did not react with sera against serotypes 19A and 19F. Cross serologic activity of preparations is probably determined by the presence of protein fractions that were detected in SDS-electrophoresis. This is confirmed by high intra-species cross protective activity of preparations from serotype 6B and 10A strains that protect 90 - 100% of mice from infection by heterologous S. pneumoniae strains. Conclusion. Use of strains with cross antigenic and protective activity for production of immunogenic protein-containing fractions with the aim ofenchanting and broadening specter ofprotective activity of vaccine preparations that are constructed based on capsule polysaccharides of S. pneumoniae is appropriate.

Aim. Construct composition of polycomponent probiotic based on lactobacilli cultural fluid (CF) supernatant. Materials and methods. Species membership of lactobacilli strains was specified by analysis of nucleotide sequences of 16S RNA genes. For comparison of probiotic effect of CF, supernatant and ultrafiltrate of a consortium of lactobacilli strains, studies of specter of peptides, amino- and other organic acids contained in them as well as profile of bactericidal and fungicidal activity was carried out. Results. Technology of production of amicrobial probiotic agent based on CF of the consortium of lactobacilli strains was developed. During determination of concentration of metabolites of protein nature analogy of parameters was shown in CF supernatant and native lactobacilli cultures. In both the studied supernatants and CF the same set of organic acids was also reported to be present. During comparison of antimicrobial and anticandidosis activity of lactobacilli native culture and CF supernatant against Staphylococcus aureus 209, Enterococcus faecalis 1154 and Candida albicans 5 test-strains, an equivalent effect of both samples was shown. Conclusion. Polycomponent metabolite probiotic based on CF supernatant of Lactobacillus helveticus NKJC, Lactobacillus helveticus JCH and Lactobacillus casei КАА strains completely retains bactericidal and fungicidal activity of the native culture of the consortium of strains and at the same time lacks disadvantages of compositions containing live microorganisms.

Aim. Development of multiplex PCR for indication and differential analysis of CFU in a probiotic preparation containing 3 various species of bifidobacteria (Bifidobacterium bifidum, B. longum, B. adolescentis) and 2 species of lactobacilli (Lactobacillus acidophilus, L. plantarum). Materials and methods. Multiplex PCR with primers to 16S RNA genes of 5 species of bacteria was applied. Samples of bacterial colonies were removed from solid nutrient medium and transferred into test-tube for multiplex PCR execution. Bacterial lysates were used as target-matrix for amplification and determination of presence of the required amplicons. Results. Multiplex PCR for detection of CFU of each of the bifidobacteria and lactobacilli strains in samples of 5-component probiotic with accuracy up to species was developed. A reagent kit was fine-tuned and high reproducibility and specificity of the multiplex PCR method with the accuracy of 90-95% of cases was shown. Conclusion. The multiplex PCR method developed is recommended to be applied for CFU control of each bacterial component in a complex probiotic preparation.

Aim. Determine etiologic significance of clinical strains of E. coli О145:Н28 isolated from feces of a patient with hemolytic-uremic syndrome (HUS). Materials and methods. 20 E. coli strains isolated from feces of a patient with HUS that developed after an acute intestine infection were studied. Antigenic structure of strains was determined by sequencing of rfb and fliC genes; presence of virulence genes (pap, aaf, sfa, afa, eaeА, bfpA, ial, hly, cnf, stx1, stx2, LT, ST and aer) - in PCR; ESBL production - by double disk method, ESBL genes - in PCR. Results. The strains contained rfb gene coding О145, fliC gene coding H7. Genes coding synthesis of stx2-toxin and intimin (eaeA) were detected. The strains were resistant to P-lactams due to production of CTX-M class ESBL. Conclusion. A causative agent E. coli О145:Н28 was isolated from a patient with HUS that produces stx2-toxin and CTX-M class ESBL and has not been previously registered in Russian Federation.

Contemporary features of distribution of various subspecies and biovars of plague causative agent by landscape-geographical zones and mountain belts on the territory of Russia and other CIS countries are examined. The most widely spread in plain and mountain natural foci were noted to be Yersinia pestis main subspecies medieval biovar strains. Strains of Y. pestis non-main subspecies are spread in mountain landscapes of Altai, Caucasus, Tian Shan. Change of dominating species of rodents considered as the main carriers of plague was noted not to result in change of genetic and biochemical characteristics of Y. pestis strains. Perspectives of study of «micro-focality» of plague are emphasized for deciphering the mechanism of the enzootic.

Analysis of influenza morbidity in pregnant women shows high frequency of severe variants of infection course with fatal outcome. The highest number of influenza complications accounts for women in the case of their infection in the 3 rd trimester of pregnancy, as well as having complicated pre-morbidity background in the form of bronchial asthma, obesity and diabetes mellitus. Healthy women at the 3 rd trimester of pregnancy were shown to have the same risk of complicated course of respiratory infection as non-pregnant women with chronic concomitant pathology. Vaccination against influenza in pregnant women has a well demonstrated safety and effectiveness. Contemporary vaccines currently used allow with a high significance to decrease the risk of development of obstetric and pediatric pathology connected with previous influenza infection.