Testing a Method for Evaluation of the Viability of Biofilm-Forming Bacteria after Exposure to Disinfectants.

A method for determining the viability of opportunistic pathogenic bacteria at the stage of biofilm formation after exposure to disinfectants with different active components was tested. The method is based on oxidation of tetrazolium salts by metabolically active cells with the formation of colored formazan derivatives and their quantitative spectrophotometry. The cell viability in the biofilm decreased after exposure to quaternary ammonium compounds and chlorine-containing disinfectants, but their effect was reversible. Dissemination of cells that had retained viability from the biofilm occurred after 24 h. The algorithm of testing, necessary controls, counting, and data interpretation are specified. The method can be recommended for use in laboratory diagnostics and clinical practice.

Effects of аntimicrobials on Pseudomonas aeruginosa biofilm formation.

Pseudomonas aeruginosa is one of the most problematic pathogens in medical institutions, which may be due to the ability of this microorganism to exist in a biofilm, which increases its resistance to antimicrobials, as well as its prevalence and survival ability in the external environment. This work aimed to evaluate the antimicrobial susceptibility of P. aeruginosa strains in planktonic and biofilm forms. We studied 20 strains of P. aeruginosa collected during 2018-2021 by specialists from the Laboratory of Microbiome and Microecology of the Scientific Centre for Family Health and Human Reproduction Problems. The identification of strains was carried out using test systems for differentiating gram-negative non-fermenting bacteria (NEFERMtest 24 Erba Lachema s.r.o., Czech Republic), and confirmed by mass spectrometric analysis and 16S rRNA gene sequencing. Antimicrobial activity was assessed by the degree of inhibition of cell growth in planktonic and biofilm forms (on a flat-bottomed 96-well plastic immunological plate). All clinical isolates of P. aeruginosa were biofilm formers, 47.6 % of the isolates were weak biofilm formers, and 52.4 % of the isolates were moderate biofilm formers. Planktonic cells and the forming biofilm of the tested P. aeruginosa strains were carbapenems-resistant. Biofilm formation was suppressed in more than 90 % of cases by the agents of the cephalosporin and aminoglycoside groups. Antimicrobial susceptibility of P. aeruginosa strains in the formed biofilm was significantly lower (p < 0.05). Carbapenems and cephalosporins did not affect the mature biofilms of the tested P. aeruginosa strains in more than 60 % of cases. Only non-beta-lactam antibiotics (ciprofloxacin and amikacin) suppressed the growth of planktonic cells and destroyed the mature biofilm. The revealed differences in the effect of the tested antimicrobials on the P. aeruginosa strains biofilms correlate with resistance to a number of antibiotics. To prevent biofilm formation in the hospital strains of P. aeruginosa, the use of ceftazidime may be recommended, and antimicrobials such as ciprofloxacin and amikacin may be used to affect mature biofilms of P. aeruginosa.

Effect of Disinfectants with Different Active Ingredients on Biofilm Formation in Pseudomonas aeruginosa.

We compared the effects of disinfectants on biofilms of 10 Pseudomonas aeruginosa strains isolated from different loci of patients with purulent-septic infections. Identification was carried out by standard bacteriological methods. To substantiate the prospects of using in hospitals and to assess the effect of disinfectants on P. aeruginosa biofilms, the following disinfectants were used in various concentrations: Sekusept Aktiv, A-DEZ, and Monitor Oxy. All clinical strains of P. aeruginosa showed the ability to form biofilms. Both oxygen-containing and quaternary ammonium compounds effectively inhibited the formation of biofilms. In more than 50% cases, disinfectants with different active ingredients did not destroy pre-formed of P. aeruginosa biofilms.

Simulation of bacterial biofilms and estimation of the sensitivity of healthcare-associated infection pathogens to bactericide Sekusept active.

The effect of bactericide Sekusept active (B SA), a peracetic acid-based preparation, on microbial strains, isolated from patients with severe infectious diseases who were treated in a regional children's multi-specialty hospital, was studied. Based on the biochemical identification, the strains were classified as gram-negative non-fermenting bacteria (22 strains), Enterobacteriaceae family (18 strains), and bacilli - 3 strains. The biocidal activity of B SA was evaluated by the degree of inhibition of the growth of bacterial cells, existing in the planktonic form and in the form of biofilm (on a flat-bottomed plastic immunological tablet). It was shown that all the studied strains had the ability to biofilm formation, most of them (67,4%) formed moderately pronounced biofilms, and non-fermenting bacteria had a particularly pronounced coefficient of biofilm formation. The selected concentrations of B CA inhibited the growth of planktonic cells, and the ability of bactericide to prevent the formation of biofilms depended on the concentration (the most effective concentrations were 0,8 and 3,0%). Sensitivity of the strains existed in the aged biofilm to the bactericide was significantly lower, especially resistant to this effect were biofilms formed by non-fermenting bacteria and representatives of fam. Enterobacteriaceae. Our results confirm the importance of testing the effectiveness of biocides not only in accordance with standard methods developed for microorganisms in planktonic form, but also for biofilms.

[Assessment of external risk factors of hepatocellular cancer development and markers of genetic predisposition to its development in the ethnic group of yakut - men].

AIM: To establish the main external and genetically determined risk factors for the development of hepatocellular cancer in the ethnic group of male Yakuts living in the Republic of Sakha (Yakutia) [RS (Y)] in the epidemiologically unfavorable conditions of the incidence of viral hepatitis. MATERIALS AND METHODS: A total of 97 male Yakuts were examined, including 44 people diagnosed with hepatocellular cancer and 53 people diagnosed with chronic viral hepatitis. HCC risk factors were identified by analyzing medical records and questioning patients. In the experimental and control groups, genetic studies of single nucleotide polymorphisms of genes mapped on the X-chromosome and involved in the activation of antiviral immunity along the TLR7 signaling pathway were performed. RESULTS AND DISCUSSION: In 100% of patients with hepatocellular cancer, infection with hepatitis B, C, D viruses or co - infection with these agents was detected. Every fourth patient with HCC in the RS (Y) was infected with hepatitis D. The course of hepatocellular cancer associated with HDV was characterized by rapid progression of liver cirrhosis, development of portal hypertension, bleeding from varicose veins of the stomach and esophagus (36.4%) and edematous ascitic syndrome (63.6%). In addition to viral agents, additional risk factors for liver cancer were identified, such as alcohol abuse, overweight, diabetes mellitus, and smoking. Among the studied variation sites of genes localized on the X-chromosome and encoding the reaction of innate antiviral immunity, no genetic marker was found with a sufficient degree of confidence determining the likelihood of hepatocellular cancer developing. CONCLUSIONS: The high incidence of hepatocellular carcinoma of the male population in the RS (Y) is due to the widespread prevalence of parenteral viral hepatitis, especially viral hepatitis D. Due to the introduction of mass vaccination of the population against hepatitis B in the Russian Federation in the foreseeable future in the RS (Y) we should see a decrease in the proportion of hepatocellular cancer associated with hepatitis B and D viruses, and therefore the focus should be on the treatment and prevention of hepatitis C virus and non - infectious risk factors.

Prognostic Model for Progradient Tuberculosis Course in HIV-Infected Children.

The aim of the study was to define independent predictors and create a prognostic model for the progradient course of tuberculosis in HIV-infected children. MATERIALS AND METHODS: The prospective observational study of tuberculosis cases of HIV-infected children under 15 years of age has been carried out over 2000-2014 using a continuous sampling method (n=65). Two observation groups were formed: the main group comprising children with a progradient (progressive) type of tuberculosis (n=37) and the comparison group of children with a regradient (regressive) type of the disease (n=28). The logistic regression method was used to create a prognostic model. The quality of model approximation was assessed using maximum likelihood function. Indicators of model goodness of fit are the coefficient of concordance (a permissible level of frequency disagreement is less than 20%) and Hosmer-Lemeshow test. RESULTS: As a result of a paired comparative analysis based on 17 medico-biological, epidemiological and social signs, 11 statistically different parameters have been distinguished. On their basis, a functional prognostic model has been created including six independent predictors: flaws in children observation in the general medical network (b=23.962), absence of Bacillus Calmette-Guérin vaccination (b=20.404), fatal course of tuberculosis in the human source of infection (b=2.762), tuberculosis identification in children under 3 years of age (b=2.620), absence or low adherence to therapy of the latent tuberculosis infection before tuberculosis detection (b=1.859), marked or severe immunodeficiency (b=1.693). The degrees of the risk factors for the progradient tuberculosis course have been established at the following probability values (decile): at 0.3-0.4 the risk is minimal; at 0.5-1.0 the risk is high; at values of 0-0.2 there is no risk of the disease. Programs for quantitative and qualitative assessment of the risk of progradient tuberculosis course in children with HIV infection have been designed to facilitate the model use. CONCLUSION: The presented prognostic model is based on the analysis of the obligatory data in the diagnostic search making its use convenient at any stage of rendering medical aid to HIV-infected children.

[The detection of highly-transmissible genotypes of agent in clinical samples for prognosis of unfavorable course of tuberculosis.]

The bio-information search was carried out and the design of primers and TaqMan probes was developed to detect DNA of agent of tuberculosis subtypes CC1 and CC2-W148 of Beijing genotype and also Ural genotype in various clinical material (phlegm, spinal fluid, pleural fluid, etc.) using real-time polymerase chain reaction technique. The 180 clinical samples from 143 patients with tuberculosis of lungs were used to carry out an approval of sensitivity and specificity of the developed tests concerning studies at the genetic analyzer GeneXpert. The sensitivity of tests on CC1, CC2-W148 и Ural relating to polymerase chain reaction of analyzer Gene Expert made up to 91%, 106% and 81% correspondingly. In all cases, the specificity made up to 100%. In parallel studies the samples with DNA of СС2-W148 genotype were more often positive on mutation of resistance to Rifampicin-Rif (+) according the results of GeneXpert (χ² = 27,1; p < 0,01) related to other genotypes. At the same time, detection of СС2-W148 strain in patient was more often accompanied by discrepancy of results: GeneXpert - Rif (+) and resistance to Rifampicin in bacteriological study under ultimate validation of multiple medicinal resistance of tuberculosis (χ² = 5,1; p < 0,05). The analysis was applied to negative effect of combination of allele-336G of CD209 gene of patient with genotype of tuberculosis mycobacterium Beijing detected previously (Ogarkov et al., 2012). The significant prevalence was observed related to widespread medicinal resistance (χ² =4,3; p < 0,05) in patients with allele-336G of CD209 gene in combination with CC2-W148 clone in comparison with other combinations in patients. The obtained results testify a possibility of application of genetic typing of tuberculosis agent and patient for early diagnosis of development of various complications of tuberculosis at the stages of primary examination of primarily detected patients with tuberculosis.

Population Polymorphism of IFNL3 and IFNL4 Genes of Type 3 Interferon Associated with Spontaneous Clearance of Hepatitis C Virus in Representatives of Caucasian and Mongoloid Races.

The frequency of single nucleotide polymorphisms of IFNL3 gene (rsl12979860 and rs8099917) and dinucleotide polymorphism of IFNL4 gene (ss469415590) were estimated in healthy inhabitants of Mongolia and Irkutsk regions taking into account their races. Population and genetic studies were performed in 1520 conventionally healthy volunteers (blood donors), representatives of Caucasian and Mongoloid races. Significant race-related differences in the incidence of IFNL3 and IFNL4 gene polymorphisms associated with spontaneous clearance of hepatitis C virus were found in healthy volunteers.

[EPIDEMIOLOGIC MANIFESTATIONS OF VIRAL HEPATITIS C IN REPRESENTATIVES OF CAUCASIAN AND MONGOLOID RACES].

AIM: Present comparative epidemiologic characteristics of viral hepatitis C in Mongolia and Irkutsk Region taking into account racial origin of the studied populations. MATERIALS AND METHODS: The studies were carried out in 2009-014 on the territory of Irkutsk Region in Mongolia. Prevalence of viral hepatitis based on serological monitoring, virus RNA detection, risk factors, change in structure of circulating genotypes, hepatocellular carcinoma morbidity were studied. RESULTS: Epidemiologic manifestations of viral hepatitis C in Mongolia, in contrast to Irkutsk Region, are characterized by, a wider prevalence of the disease, predominance of the fraction of seropositive individuals in age category of above 50 years and predominance of genotype 1 virus in circulation. In recent years an evolution of diversity of circulating' irus genotypes, ook place towards a reduction of the fraction of genotype in Mongolia and Russia due tor ni ncrease of the fraction of genotype-3. Expressed,differences in average-annual values of hepatocellular carcinoma morbidity were detected, that were more than 10 times higher among Mongoloids compared with Caucasians. CONCLUSION: Pronounced differences were detected in manifestations of epidemic process of viral hepatitis C in Mongolia and Asian part of Russia, represented by Eastern Siberia, that are associated with ethnic, social and, cultural living conditions of the indigenous population.

Undertreated HIV and drug-resistant tuberculosis at a referral hospital in Irkutsk, Siberia.

SETTING: A referral hospital for tuberculosis (TB) in Irkutsk, the Russian Federation. OBJECTIVE: To describe disease characteristics, treatment and hospital outcomes of TB-HIV (human immunodeficiency virus). DESIGN: Observational cohort of HIV-infected patients admitted for anti-tuberculosis treatment over 6 months. RESULTS: A total of 98 patients were enrolled with a median CD4 count of 147 cells/mm(3) and viral load of 205 943 copies/ml. Among patients with drug susceptibility testing (DST) results, 29 (64%) were multidrug-resistant (MDR), including 12 without previous anti-tuberculosis treatment. Nineteen patients were on antiretroviral therapy (ART) at admission, and 10 (13% ART-naïve) were started during hospitalization. Barriers to timely ART initiation included death, in-patient treatment interruption, and patient refusal. Of 96 evaluable patients, 21 (22%) died, 14 (15%) interrupted treatment, and 10 (10%) showed no microbiological or radiographic improvement. Patients with a cavitary chest X-ray (aOR 7.4, 95%CI 2.3-23.7, P = 0.001) or central nervous system disease (aOR 6.5, 95%CI 1.2-36.1, P = 0.03) were more likely to have one of these poor outcomes. CONCLUSION: High rates of MDR-TB, treatment interruption and death were found in an HIV-infected population hospitalized in Irkutsk. There are opportunities for integration of HIV and TB services to overcome barriers to timely ART initiation, increase the use of anti-tuberculosis regimens informed by second-line DST, and strengthen out-patient diagnosis and treatment networks.

GENETIC DIVERSITY OF THE MYCOBACTERIUM TUBERCULOSIS ISOLATES IN THE REPUBLIC SAKHA (YAKUTIA), RUSSIA.

The population structure of the M. tuberculosis in Yakutia was estimated by the MIRU-VNTR method of 24 loci genotyping. 199 strains from 199 patients with pulmonary tuberculosis were tested. The greatest number of the strains (34.2%, 68/199) belonged to the genotype Beijing. The significant predominance (X² = 15.5; p < 0.001) of the multidrug and extensively drug-resistance (MDR/XDR) among the isolates of Beijing genotype was revealed in subtype CC2/ W148 - 9.5% (19/199). Strains of the genotype S (15.6%, 31/199) were the second most common genotype after Beijing. The majority of S-strains had an identical profile 233325153325141344222372. S genotype strains also significantly more frequently carried the MDR/ XDR (X² = 59.8;p<0.001) among non-Beijing isolates. The genotype strain Ural ranks the third in the prevalence - 10.0% (20/199). The strains belonging to the family LAM (8.5%, 17/199) had considerable genetic heterogeneity. A great genetic diversity was also found in minor genotypes T and Haarlem. A phylogenetic reconstruction of the epidemic spread of the S-genotype and subtype CC2/W148 of the Beijing genotype in Yakutia was performed with estimation of the probable time of origin in the scale proposed by Merker M. et al. (2015). It was shown that the strains of the subtype CC2/W148 had been formed from four distinct phylogenetic sublines in recent historical period (XX century). It was estimated that phylogenetic relationships accounted for 30 MIRU-VNTR profiles of S-strains from Yakutia and 31 reference S-profiles from Europe and Canada. The profiles of the S-genotype from Yakutia form a phylogenetically compact group, indicating that all evolutionary history of these strains happened in the Sakha Republic. The time of the ancestral S-genotype spreading in Yakutia was estimated to be in the range from 300 to 600 years.

[Microbiological monitoring of opportunistic Enterobacteriaceae of the Lena river].

The aim is the study of the circulation, biological activity, persistent potential and genotypic characteristics of opportunistic Enterobacteriaceae of microbial community of the Lena River which is the one of the largest sources of water in Russia and Siberia. Gram-negative component of microbial community in the Lena River was mainly represented by Enterobacteriaceae (80%), including dominated Escherichia (dominant class) and Enterobacter and Klebsiella (subdominant class). In conditions of anthropogenic pollution there is a reorganization of microbial community of the Lena River in the direction of the increase in the proportion of opportunistic pathogens, their species diversity; there is a change of the biological activity of microorganisms, gains of share of strains characterized by antilysozyme activity, hemolytic activity, production DNase, phosphatase. The parameters of system "lysozyme-antilysozyme" are changing, the frequency of the occurrence of bacteria with antilysozyme activity is increasing. This shows the restructurization of the microbial community of water objects. Microorganisms of water objects in territories of anthropogenic pollution are characterized by multiple antibiotic resistance. The proportion of gram-negative opportunistic Enterobacteriaceae in microbial communities of water ecosystems determines the potential danger of water objects and the impact on the level of antibiotic resistance of bacteria. Opportunistic Enterobacteriaceae of microbial community of the Lena River near the city of Yakutsk are characterized by the presence of genetic determinants of pathogenicity (hlyA and sfaG). This is indicative of their potential epidemiological relevance. Microorganisms with high biological activity are markers of their epidemiological danger.

[An assessment of the immune status of the children population as a marker of technogenic pollution of the environment].

This article describes results of the immunological study of school-aged children residing in cities with different levels of the technogenic air pollution. Children from cities with the highest level of the technogenic pollution had a high number of immature neutrophils (band cells) and eosinophils. The children living in these ecologically unfavorable areas have presented a reduction of T-cell antigen receptor CD3, CD4, CD8, CD20, CD16, CD95. This indicates to that both T-cell and B-cell immunity is suppressed. The decline of the phagocytic function in neutrophils indicates to the suppression of the nonspecific host defense mechanisms also.

[Sanitary-Epidemiological Status of Siberian Population (Medico-Demographical and Epidemiological Characteristics)].

Review on the problem of sanitary-epidemiological welfare of the population in the Siberian Federal District (SFD) was conducted based on literature data and authors own research in the period of 2002−2014. Authors provided broad information on the health and demographic and epidemiological characteristics of SFD population. SFD in comparison with other regions of the Russian Federation overcomes one of the most adverse situations including mortality rates from external causes. SFD population's infectious and somatic morbidity rates were analyzed. Analysis demonstrated that the situation relating to priority epidemiologically and socially important infections (HIV-infection, parenteral viral hepatitis, tuberculosis etc.) on the territory of the SFD remains tense. Authors provided information on the increase in the level of the actual for Siberian regions natural-foci tick-borne infections. Detailed analysis for the environment anthropogenic pollution impact for the epidemic, infectious and vaccine induced processes. Authors suggest that anthropogenic (biological) environmental pollution is one of the most important factors influencing the epidemiological welfare of the Siberian population. A new strategic direction in epidemiological research associated with the problem of comorbid diseases is planned.

[Technogenic environmental pollution and the public health: analysis and prognosis].

Technogenic risk factors are very aggressive for a human health. Due to the progressive increase in environmental pollution the problem of the adverse impact of these factors on the health of both the human population as a whole, and individual groups every year is becoming increasingly important. At that the influence of anthropogenic pollution on the various manifestations of infectious pathology in the scientific literature is presented very modestly. In this paper there is presented a review of research devoted to the problem of the interrelationship of man-made pollution of the environment and public health.

[Identification of ubiquitous and endemic Mycobacterium tuberculosis genotypes in the Republic of Buryatia].

The MIRU-12 and MIRU-24 methods were used for genotyping and estimation of M. tuberculosis population structure in Buryatia. It was studied in 283 strains isolated from individuals from the regional TB dispensary and medical clinics of the penitentiary system of the republic. It was found that the prevalence of pandemic genotypes Beijing MIT17 and MIT16 and also subtype Beijing MIT642 (24.5% - 46/188) in the studied area were dominant. The isolates of the Beijing MIT642 were common among all groups of patients with pulmonary tuberculosis with the tendency to formation of MDR and chronic course of the disease.

[Heterogeneity of microbial communities of surface waters on indices of antibiotic resistance of bacteria].

Microorganisms isolated from reservoirs within urbanized areas, have acquired multiple antibiotic resistance, in this connection during the microbiological monitoring of water objects as an additional criterion of anthropogenic pollution it is worth to take into account indices of antibiotic resistance of bacteria. The study of heterogeneity of microbial communities of largest reservoirs in Eastern Siberia (Angara River and Lena) in terms of antibiotic resistance bacteria was performed from the positions of cluster structuredness. Opportunistic gram-negative bacteria and allochthonous representatives of microbiocenoses of the water bodies were established to be characterized by belonging to four clusters (antibiotic sensitive, mono-, moderately--and poly-antibiotic resistant). At the same time the proportion of microorganisms belonging to different clusters, vary significantly both in terms of different water bodies, and in separate parts of the same body of water, being largely dependent on anthropogenic load on water. Thus, the heterogeneity of the microbial populations permits to present comparative quantitative assessment of reservoirs, as well as their portions, in terms of antibiotic resistance of microorganisms. The increment in the proportion of resistant strains reflects the sanitary and epidemiological importance of this property, because provides evidence of the contamination of water supplies by household sewage.

[Genetic determinants of pathogenicity of opportunistic enterobacteria isolated from children with acute intestinal infections].

AIM: Detection of nucleotide sequences of genes controlling synthesis of pathogenicity factors in clinical strains of opportunistic enterobacteria isolated from children with acute intestinal infections (AII), as well as their association with resistance to antibiotics and the course of the infectious process. MATERIALS AND METHODS: 175 clinical strains obtained from children with AII undergoing treatment in Irkutsk state infectious diseases hospital (2007-2010) were studied. Primers to a number of genes detected in Escherichia coli pathogenicity islands, controlling type S and type 1 adhesion; formation of hemolysins; iron-regulatory protein synthesis; capsule formation were used in the study. PCR products analysis was performed by agar gel electrophoresis. RESULTS: Genetic determinants of pathogenicity were detected in bacteria genera Klebsiella, Citrobacter, Enterobacter, Proteus, Kluyvera, Morganella, Pantoea, Serratia. Fragments of hlyA and hlyB genes (hemolysin production) were detected more frequently; less frequently--sfaA, sfaG, fimA (adhesion), as well as irp-2 gene (synthesis of iron-regulatory protein). The largest set of genetic determinants of pathogenicity was noted in clinical strains of Klebsiella spp. Cultures with DNA fragments specific to genes of E. coli pathogenicity clusters were obtained predominately from children aged up to 3 years, had multiple antibiotic resistance and were isolated significantly more frequently in severe forms of AII when compared with strains in which these determinants were not detected. CONCLUSION: The studies performed showed that clinical strains of opportunistic bacteria isolated from patients with AII have a certain pathogenic potential, as evidenced by the presence of genetic pathogenicity markers in them.

[Evolution of the epidemic process under current conditions].

The on-going evolution of the epidemic process is under active influence of a new regulatory risk factor, environmental pollution of technogenic origin. The negative influence of ecological pressure on the manifestations of infectious diseases is apparent as destabilization of the epidemic processes, increased severity of infections, impaired epidemiological and immunological efficacy of vaccine prophylaxis in the child population. The most vulnerable component in the epidemic process is the minimal period of infectious morbidity. As shown in an epidemiological experiment, it is in this period that especially active preventive measures must be taken to ensure efficacious management of infection.

[Microbiological monitoring of water ecosystems].

The paper presents the results of a study of the microbial community of the Lena River. Active anthropogenic pollution induces structural changes in the microbiocenoses of aquatic objects and increases in the quantity of opportunistic gram-negative microorganisms and in that of antibiotic-resistant bacteria.