A. P. Godovalov

T. I. Karpunina

Russian Journal of Infection and Immunity

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6032018, vol. 8, no. 4 9. Antibiotic resistance of microorganisms9.6 doi: 10.15789/2220-7619-2018-4-9.6PERINATAL LISTERIOSIS: THE MOUSE MODELS.A. Ermolaeva, K.A. Sobyanin, E.V. Sysolyatina, Ya.M. ChalenkoGamaleya State Research Center of Epidemiology and Microbiology, Moscow, RussiaThe Gram-positive bacterium Listeria monocytogenes is typical sapronontic pathogen. L. monocytogenes caus-es listeriosis, a severe disease with multiple manifesta-tions including stillbirths and meningitis of newborns, in humans and a wide range of domestic and wild ani-mals. The invasion factor of the internalin family InlB is involved in crossing the maternal-fetal barrier (Disson et al., 2008). Previously, we compared human and wild animal L. monocytogenes strains and described several naturally occurring InlB variants. We demonstrated that InlB variants differed in the ability to support intragastric infection in mice (Sobyanin et al., 2017). The aim of this work was to compare effects of InlB variants on perinatal infection. The mouse model was used. The InlB variants differing in 10 amino acid substitutions were expressed under the same promoter in the L. monocytogenes strain EGDeΔinlB. Work with animals was performed with ap-proval of local bioethical committee. Mice were intragas-trically infected on the 14th day of pregnancy, euthanized 1 and 3 dpi, bacterial loads were determined by plating. One of two InlB variants provided infection of both pla-centas and fetuses while another did not. Bacteria car-rying InlB variant 14 but not the variant 9 were revealed in placentas 24 and 72 hpi. 65% of placentas and only 20% of fetuses were infected. Fetus infections was correlated with placenta infection. Infection was unequal for differ-ent fetuses in the same animal with bacterial loads ranges from individual bacteria to 105 CFU per fetus. Obtained results suggested that some but not all InlB variants might promote perinatal infection upon intragastric in-fection and that the infection of each placenta happens individually.9.7 doi: 10.15789/2220-7619-2018-4-9.7PHENOTYPES AND GENOTYPES OF CLASSICAL AND HYPERVIRULENT KLEBSIELLA PNEUMONIAE CLINICAL STRAINS ISOLATED IN MOSCOW IN 2013–2018N.K. Fursova, E.I. Astashkin, A.I. Lev, T.S. Novikova, G.N. Fedyukina, N.V. VolozhantsevState Research Center for Applied Microbiology and Biotechnology, Obolensk, Moscow Region, RussiaKlebsiella pneumoniae is causative agent of commu-nity-acquired and healthcare-associated infections in-cluding pneumonia, bloodstream infection, surgical site infections, liver abscess and meningitis. Multidrug re-sistant (MDR) Klebsiella belonged to classical branch of K. pneumoniae (cKp) have been included recently into the ESKAPE group of pathogens. On the other hand, in the last two decades hypervirulent Klebsiella phyloge-netic branch (hvKp) emerged and spread around the world. In this study, we aimed to investigate phenotypes and ge-notypes of virulence and antibacterial resistance for 500 K. pneumoniae clinical strains collected in 2013–2018 from the patients of several Moscow hospitals.Virulence factors and antibiotic resistance profiles between classical and hypervirulent K. pneumoniae iso-lates were compared. It was shown that hvKp strains were attributed to international sequence types ST23, ST86, ST65, and to novel clones ST2174 and ST2280, while strains of cKp — to international clones ST218, ST395, ST11, ST39, ST48, ST147, ST833, ST20, ST13, and ST3346. All hvKp strains had hypermucoviscosi-ty phenotype, capsule types of K1, K2 and K57; carried 5–7 pathogenic genes (regulator of mucoid phenotype gene rmpA, aerobactin gene aer, iron uptake system gene kfu, allantoin metabo lism gene allR, lipopolysaccharide synthesis genes uge2 and wabG, and fimbrial gene fimH ). On the contrary, cKP strains had no hypermucoviscosity phenotype, identified capsule types were K57, K62, K47, K14, K27, K28, K60, and K420. Such strains carried four or less pathogenic genes (they did not have rmpA, aer, and allR genes).Major cKP strains in this study expressed the MDR phenotype, resistance to three or more classes of anti-bacterials; while more than 50% of them were resistant to six or more classes (beta-lactams, aminoglycosides, fluoroquinolones, sulfonamides, nitrofurans, phenicols). Molecular mechanisms of MDR include beta-lactamase genes (blaTEM, blaSHV, blaCTX-M, blaOXA, and blaNDM), class 1 integrons carrying gene cassettes (dfr, aac, aad,, aph, etc.), and efflux pumps (oqxAB, mph, cml, etc.). Among hvKp strains two groups were described: (1) most of them were mainly susceptible to antibacterials carrying few resistance genes, (2) some strains accepted MDR plasmids carrying resistance genes, and expressed MDR phenotype.This work was funded by the Russian Science Foundation (grant no. 15-15-00058-P).9.8 doi: 10.15789/2220-7619-2018-4-9.8RELATIONSHIP BETWEEN MICROORGANISMS IN THE VAGINAL BIOTOPE OF SUBFERTIL WOMENA.P. Godovalov, T.I. KarpuninaAcad. E.A. Wagner Perm State Medical University, Perm, RussiaThe aim of investigation was to evaluate the features of microecology of the lower genital tract of women with infertility.A retrospective analysis of microbiological data of the vaginal discharge of 345 subfertil women was carried out. To assess the share of different types of microorganisms in the structure of the microbiota the coefficient of species constancy was used. To quantify the interaction between members of the microbiocenosis, the Jacquard similarity coefficient was used.The nature of the relationship between the main mem-bers of the microbial community in the vaginal biotope of women with infertility should be considered antago-nistic. The phenomenon of mutualism was characteristic only between Lactobacillus spp. and Pepto streptococcus spp. Typical E. coli have a significant ecological commu-nity with these bacteria, the relationships of their can be characterized as synergistic. Similar ecological synergism was revealed for S. epidermidis and lactobacilli. It was shown that in subfertil women E. coli acquires the func-tions of stabilizing strain and its activity often associated with both a change in the species composition of lactoba-cilli and their functional characteristics. In a similar situa-tion despite the prevalence of microbial antagonism in the vaginal microbiota, Lactobacillus spp. “admit” the exis-tence of E. coli, Enterobacter spp., S. aureus, S. epidermidis, S. haemolyticus, S. agalactiae, Enterococcus spp. and C. al-bicans. Under such conditions, the negative influence of commensal microorganisms on lactobacilli is enhanced and its have manifestation by a marked decrease in their numbers and functional activity, as well as a decrease in the sensitivity of the associates to the biocidal factors of lactobacilli when coexisting.604Russian Journal of Infection and ImmunityAbstracts of International conferenceThus, the microecological approach to assessing the state of the microbiota provides the necessary informa-tion on the relationship between individual microorgan-isms in its composition and can be a valuable tool in deci-phering the mechanisms for reducing fertility associated with nonspecific infections.This work was supported by RFBR grants No. 16-44-590429, No. 17-44-590404 and Administration of Perm Region.9.9 doi: 10.15789/2220-7619-2018-4-9.9IMPROVEMENT OF BACTERIAL BIOFILM’S INVESTIGATIONA.P. Godovalov, L.D. Ryabova, T.I. KarpuninaAcad. E.A. Wagner Perm State Medical University, Perm, RussiaExisting approaches to the investigation of biofilms haven’t a unified methodology. Researchers use different solvents, allow deviations from the original technique. Each of them distorts the results and doesn’t allow them to be compared.The aim of investigation was to evaluate the possibility of using alcohol and acetic acid for dissolution of crystal violet, as well as additional dye Lugol’s solution for the co-loring biofilms.The studies were carried out on S. aureus, S. epider-midis, E. coli. To form biofilms, the strains were cultured in flat-bottomed plates for 48 h. Biofilms were stained with a 0.1% solution of crystal violet (CV). In part of the studies after the coloration of biofilm with CV, Lugol’s solution (LS) was used for 2 min. Extraction of dyes was performed with 70 and 95% alcohol and 33% acetic acid. Results were taken into account by measuring the optical density of solutions at a wavelength of 590 nm. Statistical analysis of the data was carried out using the paired version of the Student t-test. The threshold value was taken as p < 0.05. The data are given as the arithmetic mean (M) and its error (m).It was shown that the use of 70% alcohol qualitatively elutes CV from biofilms than 95% one. This may be due to the fact that 95% alcohol narrows the pores of the cell wall and the dye is less efficiently released into the solu-tion. The acetic acid more efficiently elutes the CV from biofilms formed by grampositive microorganisms. The use of LS after staining by CV showed the best results, but only if the extraction was carried out with a solution of acetic acid prepared with alcohol. This approach seems most optimal. LS fixes the dye in the cell and dissolution with a mixture of alcohol and acetic acid makes it more possible.It was shown that extraction of CV is best performed with 70% alcohol. When fixing CV by LS, it is more appro-priate to use a solution of acetic acid in alcohol.This work was supported by RFBR grants No. 16-44-590429, No. 17-44-590404 and Administration of Perm Region.9.10 doi: 10.15789/2220-7619-2018-4-9.10EFFECT OF METAL OXIDE NANOPARTICLES ON THE EXCHANGE OF GENETIC MATERIAL BETWEEN BACTERIAP.A. Kalikina, E.I. KoshelITMO University, St. Petersburg, RussiaThe development of resistance to antibiotics in bac-teria is one of the most serious threats to global pub-lic health. The spread of resistance genes to antibio-tics is caused by the increase and misuse of antibiotics in medicine and in animal feed. Nanomaterialscan in-crease the efficiency of horizontal transmission of mul-tiresistance genes localized in plasmids between bac-teria. Some studies have indicated that nanomaterials can cause damage to bacterial membranes, possibly by forming reactive oxygen species and can deliver DNA or RNA molecules to animal or plant cells. It was shown earlier the increase in efficiency of horizontal transmis-sion of multidrug-resistant genes localized in plasmids by alumina nanoparticles (NPs) [1]. In this study, we observed the effect of different metal oxide NPs on the horizontal transfer of antibiotic-resistance genes be-tween different Escherichia coli strains.For the analysis of plasmid horizontal junctions, NPs of metal oxides Ta2O5, HfO2, Fe3O4, ZrO2, TiO2, Al2O3 were used at a concentration of 5 mmol/L. The studies were conducted between E. coli strains containing different plasmids with different resistant cassettes. The transcon-jugants growth after incubation with metal oxides NPs was fixed on medium with appropriate antibiotics. The effect of metal oxide NPs was compared with control samples without addition of NPs.In this study, we showed that the addition of NPs metal oxides can enhance the efficiency of horizontal gene trans-fer between different bacteria.This work was supported by the Ministry of Education and Science of the Russian Federation (Project 4.8955.2017/8.9).9.11 doi: 10.15789/2220-7619-2018-4-9.11SONOCHEMICAL NANOSTRUCTURING OF ANTIBIOTICS IS A NEW APPROACH TO INCREASING THEIR EFFECTIVENESS AGAINST RESISTANT STRAINSI.S. Kassirov, S.A. Ulasevich, E.V. Skorb, E.I. KoshelITMO University, St. Petersburg, RussiaOne of the most urgent problems of modern medi-cine is bacterial resistance to antibiotics. Development of new treatment approaches is a laborious and expensive process. One of the strategies for developing new anti-microbial agents is a modification of existing antibiotics. Sonochemical nanostructuring of antibiotics can become a cheap alternative to modern complex methods. In this regard, the aim of this research was to analyse the antimi-crobial activity of sonochemically-modified tetracycline against sensitive and resistant strains.Escherichia coli Nova Blue TcR (with antibiotic resist-ance) and E. coli 292–116 (without drug resistance) were used in this study. Tetracycline, a broad-spectrum antibio-tic, was modified using industrial sonicator UIP1000hdT (Hielscher, Germany). The effectiveness of antibacterial properties was estimated using the disc-diffusion method and spectrophotometry analysis of liquid cultures. The re-sults were confirmed by flow cytometry after staining with propidium iodide and Syto-9 dyes. The antimicrobial ac-tion of the modified antibiotic solution during long-term storage has also been studied.The ultrasound processing time determines the change in antimicrobial properties against both sensitive and re-sistant cells. As a result of sonochemical treatment, the ef-fectiveness of antibacterial properties increases up to 25% against the resistant strain and up to 100% against the sen-sitive strain. The long-term storage at +4°C does not re-duce the antimicrobial properties.The obtained data shows that sonochemical modifica-tion of antibiotics can be a new promising and cheap ap-proach to the development of new drugs effective for anti-biotic therapy against drug resistance strains.

RELATIONSHIP BETWEEN MICROORGANISMS IN THE VAGINAL BIOTOPE OF SUBFERTIL WOMEN

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RELATIONSHIP BETWEEN MICROORGANISMS IN THE VAGINAL BIOTOPE OF SUBFERTIL WOMEN

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