Alpha Diversity Indices as Indicators of the Variability of Gut Microbiota in Obese Adolescents of Different Ethnicities.

We compared alpha diversity indices of the intestinal microbiota in adolescents with obesity and normal body weight, taking into account their ethnicity. Intestinal biocenosis was studied by metasequencing of amplicon libraries of V3-V4 fragments of the 16S rRNA gene. The alpha diversity of the microbiota was assessed using classical and alternative indices. Statistically significant differences in intestinal microbiota were observed between Russians with obesity and Buryats with normal body weight, as well as between Russians with obesity and Buryats with obesity when assessing the Shannon-Weaver, Chao1 indices, Faith phylogenetic diversity index, ACE, Fisher, Gini coefficient, Margalef, and Menkhinik indices. It was shown that alpha diversity indices can be used to assess significance of differences and variability of the intestinal microbiota in multifactorial diseases such as obesity in adolescents; however, the scope of application of the criteria should be considered.

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.

Differences in Gut Microbiota Composition and Predicted Metabolic Functions: a Pilot Study of Adolescents with Normal Weight and Obesity.

Understanding the principles underlying the stability and sustainability of the gut microbiome of adolescents with normal weight and obesity will make it possible to implement a personalized approach to the correction of metabolic disorders. The article presents the results of a pilot study of the diversity and metabolic potential of the gut microbiome in adolescents with normal body weight and obesity. Biological material was studied using metagenomic sequencing of the V3-V4 variable regions of the 16S rRNA gene. In all adolescents with normal weight, similar degree of the phylogenetic relationship between the bacterial taxa of the community was demonstrated. In contrast, obese adolescents were characterized by the presence of phylogenetically distinct taxa in the gut microbiota. However, even with differences in taxonomic composition, the gut microbial community can compensate for the absence of certain taxonomic groups by implementing the necessary metabolic functions using other phylogenetically close taxa.

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.

Diversity and Metabolic Potential of the Gut Microbiome in Adolescents with Functional Bowel Disorder.

The article presents the results of a pilot study of the diversity and metabolic potential of the gut microbiome in adolescents with various forms of functional bowel disorder. The participants were the patients of the Clinic of Research Center for Family Health and Human Reproduction Problems. Biological material was studied using metagenomic sequencing of V3-V4 variable regions of the 16S rRNA gene. We showed that the composition of the minor component of the intestinal microbiome in adolescents with functional bowel disorder differs from that in the healthy subjects (control). Different types of transit disturbances in functional bowel disorder also differ from each other. According to the metabolic potential, adolescents can be divided into two groups irrespective of the pathophysiological manifestations: for one group, a more intensive metabolism in amino acid and lipid biosynthesis pathways was predicted than for the other.

The relationship between the composition of the microbiota and functional intestinal disorders in obese adolescents.

Functional gastrointestinal disorders (FGIDs) in children and adolescents are one of the most common pathologies of the digestive tract. The aim of the study was to study the structure of the colon microbiota in obese adolescents and to establish its relationship with the clinical manifestations of the disease FGIDs. The study included 20 adolescents aged 11-17 years. The main group included 13 adolescents with a clinically confirmed diagnosis of obesity (the standard deviation of the body mass index (SDS BMI) is more than 2,0) in combination with FGIDs. The control group consisted of 7 adolescents with normal body weight and no abdominal complaints (SDS BMI=0,66). Clinical symptoms from the bowel re diagnosed on the basis of the Rome IV. The consistency of feces was evaluated using the Bristol Stool Form Scale. The study of biological material was carried out by standard bacteriological methods. It is shown that in adolescents with obesity and FGIDs, there is a decrease in the concentration of representatives of bifidoflora, an increase in the microbial density of Escherichia coli with altered properties and an excessive growth of potentially pathogenic enterobacteria. It is established that the presence of conditionally pathogenic bacteria is sem. Enterobacteriaceae is associated with abdominal pain during defecation. In obese adolescents in the group with unformed stools, Clostridium spp was recorded as part of the intestinal microbiota.

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.

Еvaluation of the sensitivity of bacteriophage preparations to Klebsiella pneumoniae strains isolated from the colon microbiota in children with functional gastrointestinal disorders.

The level of sensitivity of Klebsiella pneumoniae strains isolated from children of the first half of life with functional gastrointestinal disorders (FGID) to commercial bacteriophage preparations was assessed. The material was the feces of 67 children of the first half of life with FGID who are breastfed. Culture of K. pneumoniae isolated from faeces of children, amounted to two comparison groups, depending on the age of the patients. The first group included 43 K. pneumoniae strains isolated from the colon of children aged from birth to three months, in the second group - 24 strains, from children aged three to six months. The composition of the colon microbiota was studied using standard methods, and the results were evaluated in accordance with Industry Standard 91500.11.0004-2003. Identification of K. pneumoniae was performed by bacteriological methods. Determination of the level of lytic activity of K. pneumoniae, Klebsiella polyvalent and sextaphage bacteriophages to K. pneumoniae strains was conducted by the drip method (spot-test) according to clinical recommendations. It is shown that the formation of FGID symptoms in children correlates with age - the frequency of regurgitation decreased in children up to six months (from 23.3% to 4.2%) against the background of the formation of a symptom complex associated with defecation disorder. Bacteriological analysis showed that in General, phages show a low level of lytic activity, sensitivity to them Klebsiella also correlated with age and was higher in children of three to six months. Thus, in the first comparison group, the absence of Klebsiella lysis in relation to the Klebsiella pneumoniae bacteriophage was registered three times more often (30.2%, p<0.05) and twice less often (30.2%, p<0.05), the low level of lysis of K. pneumoniae strains to the Klebsiella polyvalent phage compared to the second group. The sensitivity of K. pneumoniae strains to sextaphage was comparable in comparison groups and varied from 2 to 10%. Thus, among the studied K. pneumoniae observed a low level of sensitivity to specific drugs - bacteriophage Klebsiella pneumoniae, Klebsiella polyvalent and sextaphage. This fact reflects the insufficient activity of phages and predicts low effectiveness of empirical phage therapy without elimination from the intestinal biocenosis of K. pneumoniae in children with FGID who are breastfed.

Metasequencing of V3-V4 Variable Regions of 16S rRNA Gene in Opportunistic Microbiota and Gut Biocenosis in Obese Adolescents.

Opportunistic microorganisms in the gut biocenosis were studied in adolescents with normal body weight and obesity (patients consulted at the Clinical Department of Research Center of Family Health and Human Reproduction Problems). The biological material was studied by standard bacteriological methods, representatives of Enterobacteriaceae family were also characterized using metagenomic sequencing of V3-V4 variable regions of 16S gene rRNA. Gut microbiota of obese adolescents was unbalanced and was characterized by low levels of bifido- and lactoflora representatives, a spectrum of E. coli associations, and high prevalence of opportunistic microorganisms and their associations. Representatives of Enterobacteriaceae family were most often found in the gut microbiota of obese adolescents.

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.

Composition and Structure of Gut Microbiome in Adolescents with Obesity and Different Breastfeeding Duration.

Gut microbiome of adolescents with obesity and different duration of breastfeeding was analyzed by metagenomic analysis of V3-V4 variable domains of the 16S rRNA gene. In subgroup with breastfeeding duration <3 months, intrapopulation structure of gut microbiome by alpha diversity indices was similar in adolescents with obesity and normal body weight. The decrease in phylotype abundance in the structure of communities was associated only with obesity, while dysbiotic state persisted in both lean and overweight adolescents, which confirmed the effect of breastfeeding duration on stability of gut microbiome.

[The structure of opportunistic microbiota of nasopharynx and vaginal tract in women with reproduction disorders and chronic endometritis.]

The study was carried out to investigate microbiocenosis of nasopharynx and vaginal tract in women with reproductive disorders and chronic endometritis. The females were separated on two groups. The first group included females with morphologically verified chronic endometritis (n=32). The second group included females without chronic endometritis (n=32). The chronic tonsillitis was established in 21 out of 65 females from first group and in comparison group only in 9 females. In the analyzed biotypes resemblance of main infection agents (staphylococcus,enterococсus, colibacillus) was established. In nasopharynx biotope normocenosis was established in first and second groups in 33.3% and 45% of cases correspondingly. The first degree of dysbiosis was determined in both groups with equal rate (55,5%), second degree of dysbiosis was established only in women with chronic endometritis and chronic tonsillitis (11.1%).

A comparative study on microbiota from the intestine of Prussian carp (Carassius gibelio) and their aquatic environmental compartments, using different molecular methods.

AIMS: The aim of this study was to evaluate, via various molecular methods, the possible correlations between microbial community structure of Prussian carp and the environmental compartments of their habitat. METHODS AND RESULTS: Microbial communities in the intestine and environmental compartments were studied using PCR-screening, cloning and next-generation high-throughput sequencing of the 16S ribosomal RNA genes. The 16S rDNA metagenomic sequencing showed higher bacterial diversity in comparison with clone libraries, while group-specific PCR showed positive detection of nine bacteria phyla. Proteobacteria, Bacteroidetes, Firmicutes, Cyanobacteria and Actinobacteria were most abundant both in the intestine and habitat environments. The comparative analyses reveal that the bacterial community in the Prussian carp intestine is most similar to that identified from the chironomid. CONCLUSIONS: This study demonstrated some differences between molecular methods and showed advantages and limitations associated with them. These differences have the potential to reduce bias in results obtained from analysis of the community structure. The advantages of each molecular technique can be used for a better understanding of microbial diversity. The microbiota of Prussian carp intestine is most similar to those from the chironomids. SIGNIFICANCE AND IMPACT OF THE STUDY: We investigated the diversity of the intestinal microbiota in an economically important aquaculture species, the Prussian carp (Carassius gibelio). The results provide significant information to discuss possible functions of these bacteria for further understanding of Prussian carp health.