[EFFECT OF ANTIBACTERIAL PREPARATIONS ON VIBRIO CHOLERAE EL TOR BIOFILMS].

AIM: Study the effect of antibacterial preparations on biofilms of Vibrio cholerae El Tor. MATERIALS AND METHODS: Sensitivity of V cholerae El Tor (6 strains) to various concentrations of antibacterial preparations (doxycycline,.tetracycline, levomycetin, rifampicin, gentamycin, ceftazidime) was determined (MD 4.2.2495-09). Transmission electron microscopy was used for visualization of the effect of preparations on biofilms. RESULTS: The values of minimal inhibiting concentrations of antibacterial preparations against biofilms have increased by 5 - 100 times compared with plankton cultures. Certain smoothing of strands between the bacterial cell and substrate, alteration of vibrios' form, reduction of electron density of the matrix with an increase of its transparency were observed during electron-microscopy of the effect of antibacterial preparations on the biofilm. CONCLUSION: Study of the effect of antibacterial preparations on biofilms could increase effectiveness of rational antibiotics therapy of infec- tions by selection of preparations that disrupt functioning of microbial communities.

[The analysis of biofilms Vibrio choleraе using technique of transmissive electronic microscopy.]

The cholera continues to be one of the priority problems of world health care that determines necessity of its permanent monitoring and also further investigation of this infection in microbiological and ecological aspects. The phenomenon of biofilm formation plays a significant role among mechanisms permitting comma bacillus year by year to retain its ecological niche and to spread in various basins over a period of decades. The application of techniques of electronic microscopy played a decisive role in studying the process of biofilm formation. The transmission electronic microscopy has a number of significant advantages, but studying of biofilms using the given technique has certain limitations and peculiarities affecting derivable results. The purpose of study is modeling of biofilms of comma bacillus using a new original technique and analyzing them implementing transmission electronic microscopy. An original substrate was developed to cultivate biofilms of comma bacillus adjusted to convenient sampling preparation for transmission electronic microscopy. The application of the given technique manifold simplifies the process sampling preparation and permits to retain at most the native structure of biofilms. By means of combining alternatives of contrasting and comparing of obtained results the role of various components in the process of biofilm formation is determined. Also, the structure of matrix of biofilms and morphologic phasic picture of process of biofilm formation of comma bacillus is demonstrated. The electronic microscopic indications of biofilm forms of comma bacillus are marked out. The presented data supports optimization of studying biofilms using transmission electronic microscopy and better understanding phenomenon of biofilm formation.

[TYPING OF VIBRIO CHOLERAE NON O1/NON O139 STRAINS, ISOLATED IN ROSTOV REGION IN 2014].

AIM: Comparative study of antibiotics resistance and VNTR-typing of Vibrio cholerae non O1/ non O139 strains, isolated on the territory of Rostov region in 2014. MATERIALS AND METHODS: Antibioticogramms of strains were determined by serial dilution method in dense nutrient medium according to MG 4.2.2495-09 (2009). Pheno-, sero- and VNTR-typing was carried out by conventional-methods. RESULTS: The studied strains belonged to V. cholerae species, did not agglutinate with O1 and O139 sera, were atoxigenic hemolysis-positive, did not contain genes of cholera toxin and toxin-coregulating pili of adhesion, contained genes of hemagglutinin/protease, protease PrtV, collagenase, cytotonic factor Cef, outer membrane protein-OmpW, tol- and -vps-clusters, regulatory genes toxR and hapR. Antibioticogramms of the strains have shown the presence of cultures, resistant to ampicillin, ceftazidime-furazolidone, trimethoprim/sulfamethoxazole with intermediate resistance to streptomycin, kanamycin, gentamycin, amikacin, netilmicin, Approximately 20% of isolates had multiple drug resistance. Data of VNTR- and genotyping confirmed a possibility of water transmission route of the infection. CONCLUSION: Execution of monitoring of cultures from environmental samples is necessary for timely detection of genetic characteristics, antibiotics resistance.

[The modeling of biofilms of comma bacillus on solid surfaces (glass and plastic) and their visualization in light and luminescent microscopes].

Nowadays, necessity is shown up concerning modeling of certain ecological processes actually emerging in various biotops and hence step-by-step study of development of biofilms on solid surfaces (glass and plastic). To implement such kind of studies the light and luminescent microscopy are the most available and rather informative tools. The study used strains V.cholerae O1 and O139 serogroups with different genetic characteristics. The biofilms were developed on cover glass and/or plastic plates which were placed in bottles with tap autoclave water in vertical position in special device. The lifetime visualization of stages of development of biofilm was implemented using luminescent microscopy by coloring biofilm with solution of acridine yellow. The biofilms colored with Congo read and fuchsine were analyzed using light microscopy. The light and luminescent microscopy permit investigating biofilms in dynamics at various stages of development, to evaluate condition of its physiological functioning in quantitative and qualitative dimensions and to trace synthesis of exopolysaccharides in different serogroups of vibrios that has significance in prognosis of velocity of occurrence of biofilms on abiotic objects.

[Sensitivity to antibacterial preparations of Vibrio cholerae El Tor strains isolated from the environmental objects in Russia in 2005 - 2012].

AIM: Analysis of antibiotic resistance profiles of Vibrio cholerae O1 strains isolated from the environmental objects in the territory of Russia in 2005 - 2012. MATERIALS AND METHODS: Antibiotocograms of 52 strains of V. cholerae were determined by serial dilution method in dense nutrient medium. Interpretation of the results was carried out in accordance with guidelines MI 4.2.2495-09 (2009). RESULTS: All the cultures turned out to be sensitive to tetracyclines, ciprofloxacin, cephalosporins: Isolates from Stavropol region were resistant to furazolidone (33.3%), trimethoprim/sulfamethoxazole (100%). Strains resistant to ampicillin, streptomycin, rifampicin (7%), furazolidone (43%), trimethoprim/sulfamethoxazole (100%) were isolated in Primorsky region. In Irkutsk region and Kalmykia--to furazolidone and trimethoprim/sulfamethoxazole (11 - 89%), ampicillin (8.3 - 11%). Analysis of antibioticograms gives evidence on the occurrence in the studied strains of 1 to 5 r-determinants of antibiotic resistance in various combinations. CONCLUSION: The data obtained give evidence on preservation of the tendency to expand the specter of antibiotic resistance in V. cholerae O1 isolated from environmental objects that necessitates a more rational and effective use of antibacterial preparations, determination of antiobioticogram for every isolated culture, strict bacteriologic control during the course of etiotropic therapy for the prevention of increase of number of resistant strains.

[Antibiotic Susceptibility of Vibrio cholerae non O1/non O139 Serogroups Isolated from Environment in the Rostov Region].

Analysis of the antibioticograms of 22 strains of Vibrio cholerae non O1/non O139 serogroups (ctxA- tepA-) isolated from the environment in the Rostov Region in 2011 showed that all the cultures were susceptible to ciprofloxacin, aminoglycosides, ceftriaxone, trimetoprime/sulfamethoxazole and resistant to levomycetin and furazolidone. 32%, 18% and 9% of the isolates were resistant to tetracycline, rifampicin and nalidixic acid respectively. No strains of V. cholerae susceptible to all the tested antimicrobials were detected. 37% of the V. cholerae isolates was resistant to two antibacterials and the others showed multiple resistance and contained 3-6 r-determinants of antibiotic resistance. Since the antibiotic resistance genes in Vibrio cholerae non O1/non O139 serogroups are often located on mobile genetic elements (plasmids, interferons, SXT elements), many strains of such organisms, the same as the natural environment, could serve as reservoirs of antibiotic resistance. The presence of antibiotic resistance r-determinants in the investigated strains in various combinations, the antibiotic resistance variability in the isolates collected on the same territory within a relatively short period of time require monitoring of antibiotic susceptibility in them and the use of the antibiotic for the etiotropic therapy only in strict accordance with the antibioticogram of the culture isolated from the concrete patient.

[Design of a polymer drug for serological diagnosis of hepatitis C].

A new immunobiological polymer drug has been designed for the serological identification of hepatitis C. The drug is able to reveal specific antibodies in the sera of patients with hepatitis C, meets the current requirements of diagnostic test systems, and shows a high sensitivity and specificity. It is based on polyacroleinic microspheres; the concentrated cell culture biomass of hepatitis C virus (HCV), which contains an adequate set of viral antigens, is used as sensitin. A new diagnosticum is proposed to be used during primary (screening) laboratory studies based on the serological detection of total antibodies to HCV antigens in the volume agglomeration test. The latter is both one of the alternative methods during serological studies and an additional procedure when a set of diagnostic techniques is used.

[New nutrient medium for the cultivation and isolation of the plague microbe ChDS-37 as an element of the mobilization reserve of specialized antiepidemic teams of the Russian Inspectorate for the Protection of Consumer Rights and Human Welfare].

A new nutrient medium has been designed to culture and isolate the plague microbe ChDS-37 on the basis of the pancreatic digest of baker's yeast. The results of laboratory tests of the designed medium, by using 10 plague microbe strains and those of approval during the tactical and special training of a specialized antiepidemic team (SAET), suggest that the medium has some advantage over reference media and creates prerequisites for being incorporated into the mobilization reserve of a SAET.

[Antibioticograms of Vibrio cholerae non-01/non-0139 strains isolated from humans within 1968-2009].

Analysis of the antibioticograms of the Vibrio cholerae non-01/non-0139 strains showed that in the cultures isolated in the Rostov Region in 1968--1975 there were present markers of resistance to ampicillin (7%), kanamycin (15.8%), rifampicin (3.5%) and trimetoprim/sulfamethoxazole (14%). Among the strains isolated in the Ukraine in 1975 14% was resistant to ampicillin. More than a half of the strains isolated in Uzbekistan in 1990 and 2000-2001, in the Arkhangelsk Region in 1999-2000 and in the Kalmykia in 1999-2005 was antibiotic resistant. In the above regions the strains were resistant to ampicillin (12.5-44.4%), kanamycin (11-55%), rifampicin (1.9-12.5%) and trimetoprim/sulfamethoxazole (25-62.5%). Among the cultures isolated in Uzbekistan in 1990 and 2000-2001 25 and 7.8% were resistant to furazolidone and 31.25% was resistant to streptomycin (1990). All the cultures isolated in the Rostov Region in 2005-2009 were resistant to ampicillin, 50% was resistant to ceftazidim, 57% was resistant to streptomycin and furazolidone, 7.2% was resistant to kanamycin and 14% was resistant to trimetoprim/sulfamethoxazole. The studies revealed an increase of the extent of the V.cholerae non-01/non-0139 resistance spectrum within 1968-2009, simultaneous presence of up to 5 diverse resistance markers and a variety of their combinations, that requires the use of antibacterials for the treatment of the diseases due to the vibrios in strict compliance with the pathogen antibioticogram and their early replace by more efficient drugs.

[In vitro induction of transmissive resistance to tetracycline, chloramphenicol and ampicillin chloramphenicol in Vibrio cholera non-O1/non-O139 serogroups isolated within 1990-2005].

Inducible character of resistance to tetracycline, chloramphenicol and ampicillin was investigated in 20 strains of Vibrio cholera non-O1/non-O139 serogroups isolated from inhabitants of Uzbekistan in 1990 (10 strains, ctx+) and in 2001 (5 strains, ctx-) and from inhabitants of Kalmykiya within 2003-2005 (5 strains, ctx-). Eight of the 20 isolates showed not only capacity for induction of the antibiotic resistance, but also its possible self transfer to Escherichia coli and reverse crosses in El Tor V. cholerae P-5879. It was shown that the effect of the antibacterial on the isolates phenotypic susceptibility could increase the resistance markers expression, when the genomes contained sites responsible for their expression, that required constant bacteriological control of the treatment efficacy and the use of the isolates antibioticograms for early replace of the inefficient drug by the efficient one. The prevalence of V. cholerae O1 and non-O1/non-O13 serogroups with multiple resistance to the antibacterial and the genetic potency for the antibiotic resistance development in the pathogen made difficult the choice of efficient drugs for prophylaxis and treatment of diseases caused by V. cholerae.

[Lack of levofloxacin and moxyfloxacin efficacy in experimental plague of albino mice infected with nalidixic acid resistant pathogen (Nal(r))].

The efficacy of levofloxacin and moxyfloxacin vs. the previously tested fluoroquinolones was studied on albino mice with experimental plague due to the Nal(r) mutants of Yersinia pestis 231 and 231 FI-. The plague microbe mutants resistant to nalidixic acid (Nal(r)) generated at a frequency of 10(-10)-10(-9). The resistance to nalidixic acid was not accompanied by the strains loss of the virulence. The Nal(r) mutants were cross resistant to fluoroquinolones (ciprofloxacin, moxyfloxacin). The LD50 for the nontreated animals did not differ from that for the mice treated with nalidixic acid and the fluoroquinolones (when the animals were infected with Nal(r) mutants). The results showed that the criteria of the plague microbe susceptibility/resistance to fluoroquinolones should be revised.

[Novel diagnostic kit on the basis of stained polymeric carriers for epidemiologic surveillance in natural foci of Crimean-Congo hemorrhagic fever].

AIM: To study diagnostic value of developed antigenic polymeric diagnostic kit for epidemiologic surveillance in natural foci of Crimean-Congo hemorrhagic fever (CCHF) in South federal district. MATERIALS AND METHODS: Novel antigenic diagnostic kit on the basis of polymeric microspheres for reaction of volume agglomeration was developed. The kit is designed for detection of virus-specific antibodies in human serum and in serum of agricultural animals. RESULTS: Laboratory and field trials of the kit showed its high diagnostic potency, it was included in methodical recommendations "Organization and accomplishment of measures against Crimean-Congo hemorrhagic fever on the territory of its natural foci in Russia". Use of antigenic polymeric kit for epidemiological surveillance allows for more complete and systemic understanding of CCHF epidemic process. CONCLUSION: At present, the diagnostic kit is successfully used, alongside with ELISA and PCR, on different levels of epidemiologic surveillance for CCHF in Rostov region.

[Study of Legionella pneumophila strains isolated from environmental objects].

The paper presents the results of studying the biological properties of Legionella pneumophila strains isolated from environmental objects. Elective legionellosis medium (ELM) has been found to be suitable for the isolation of the causative agent from the starting material and to be as sensitive as CYE (Oxoid company) containing growth and selective additives. Polymerase chain reaction (PCR) with a home-produced commercial test system used to detect L. pneumophila DNA enables identification of the causative agent, including its species. Hyperimmune sera against L. pneumophila 1-7 serogroups used in slide-agglutination and agglutination, as well as a series of co-agglutinating diagnosticums for legionellosis 1-7 serogroups make it possible to identify even the serogroups of L. pneumophilla. Comparative analysis of the virulence of L. pneumophila cultures in vivo and in vitro allows recommendation that practical laboratories should employ a simple NaCl resistance test, which can be used as a guide virulence test.

[Serologic diagnostics of Legionella infection].

Technical approaches to construction of preparations for serologic diagnostics of Legionella infection were presented in the article; antigenic- and immunoglobulin-based diagnostic kits with known characteristics were developed. Immunogenic properties of protein and lypopolysaccharide antigens, which have diagnostic value, were studied; similarity of protein antigens from 7 serogroups of L. pneumophila was demonstrated. Soluble antigen with known composition was obtained and used for the development of antigen-based polymeric kit for diagnostics of Legionella infection. On the basis of hyperimmune sera, immunoglobulin-based polymeric diagnostic kit and array of coagglutinating diagnostic kits for the mentioned 7 serogroups were developed. Antigen-based polymeric diagnostic kit was recommended for licensure.

[The cytokine-inducing activity of the Yersinia pestis "murine" toxin]. / Tsitokinindutsiruiushchaia aktivnost' "myshinogo" toksina Yersinia pestis.

In this work the data obtained in the study of cytokinin-inducing activity of Y. pestis "mouse" toxin (MT) are presented. The study revealed that MT induced the synthesis of IL 1, IL 2 and TNF alpha. The most pronounced activity in MT was IL 2 inducing activity having dose-dependent character: the effect increased with the decrease of the dose of the preparation. The maximum level of the synthesis of IL 2 was observed when very low doses of the preparation (0.01 microgram/ml) were used, which was characteristic of superantigens. The presented data suggest that IL 2 plays an essential role in the mechanism of immunopathological reactions stipulated by MT.

[The effect of a modification to the lipopolysaccharide of Yersinia pestis on its neutrophilokine-inducing activity]. / Vliianie modifikatsii lipopolisakharida chumnogo mikroba na neitrofilokin-indutsiruiushchuiu aktivnost'.

The ability of the isolated and modified Yersinia pestis LPS to induce the synthesis of the neutrophilokines that regulate the macrophage functional activity was studied. It is established that the Y. pestis LPS detoxication, especially by the method of deacylation, does not lead to the decrease in biological, in particular neutrophilokine-inducing activity of these preparations, but actually even increases it. These results are in agreement with many reports showing the possibility of decreasing the LPS toxicity without reducing immunostimulatory activity of this important component of the outer membrane of gram-negative bacteria.

[The evaluation of the efficacy of immunization against plague by the modulation in the index of the macrophagal transformation of of the peripheral blood monocytes]. / Otsenka éffektivnosti immunizatsii protiv chumy po moduliatsii pokazatelia makrofagal'noi transformatsii monotsitov perifericheskoi krovi.

The subcutaneous and aerogenous immunization of experimental animals with live dried antiplague vaccine has been shown to modulate the macrophagal transformation index of peripheral blood monocytes (PBM) which correlates with the resistance of the animals to the causative agent of plague. This finding allows to recommend the use of PBM for the evaluation of the effectiveness of immunization with antiplague vaccine.

[A new method of assessing plague immunity in humans]. / Novyi metod otsenki protivochumnogo immuniteta u liudei.

Subcutaneous and aerogenic vaccination of humans with live dry vaccine may be assessed using a new test: index of macrophageal transformation of peripheral blood monocytes. In addition, this method will help choose the optimal dose of vaccine.

[The protective activity of the adhesion pili of Yersinia pestis]. / Izuchenie protektivnoi aktivnosti pilei adgezii Yersinia pestis.

Y. pestis adhesion pili (AP) in the native form and in the subunit form, used for immunization in one or two injections in a dose of 12-100 mu g on aluminum hydroxide, did not protect white mice and guinea pigs from experimental Y. pestis infection. The study revealed that AP produced a pronounced cytotoxic effect on macrophages and practically no influence on leukocytes. This result was confirmed in the study of luminol-dependent chemiluminescence and in the analysis of 5'-nucleotidase activity of phagocytes. These data make it possible to regard AP as Y. pestis antimacrophagal pathogenicity factor.