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.

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 THE INTESTINAL MICROBIOTA AND THE FREQUENCY OF DETECTION OF PATHOGENICITY GENES (STXI, STX2, BFP) IN ESCHERICHIA COLI WITH NORMAL ENZYMATIC ACTIVITY ISOLATED FROM CHILDREN DURING THE FIRST YEAR OF LIFE.

E. coli is an intestinal commensal of vertebrates. The ability to produce Shiga toxins (Stx) is the major virulence feature of Shiga toxin-producing E. coli (STEC). These potent cytotoxins block the protein synthesis by inactivating ribosomes. Their action on the target cells is responsible for the most severe forms of STEC-induced disease, such as hemorrhagic colitis and the life-threatening hemolytic uremic syndrome (HUS). Enteropathogenic E. coli (EPEC) also continues to be an important cause of infantile diarrhea in developing countries. Bundle-forming pili (bfp) are essential for the full virulence of enteropathogenic E. coli (EPEC). Exchange of genetic material between different types of bacteria, as well as with other members of the family Enterobacteriaceae in the intestinal ecosystem, leads to the appearance of normal variants of E. coli with phenetic features of pathogenicity that can serve as a theoretical basis for attributing these strains to pathobionts. PCR was used to examine 68 strains of E. coli (E. coli with normal enzymatic activity) for the presence of genes encoding the synthesis of Shiga toxins (stxl and stx2) and genes encoding the bundle-forming pilus (bfp). They were isolated from children with functional disorders of the gastrointestinal tract. The presence of the desired amplicon specific for stxl and bfp genes has resulted in formation of E. coli strains with more intense pathogenicity.

[The complex evaluation of condition of intestinal and nasopharyngeal microbiocenosis in alumni of children's home].

The examination of nasopharyngeal and intestinal microbiocenoses was implemented in orphan children aged 1-3 years residing in children;s home of Cheremkhovo of the Irkutskaia oblast. All children had compromised anamnesis: prematurity, hypotrophy, chicken pox, frequent acute respiratory viral infections, intestinal infections, atopic dermatitis. The study was carried out to comprehensively evaluate conditions of intestinal and nasopharyngeal biotops in children residing in closed children's institution - children's home. The microbiological analysis of qualitative and quantitative composition of content of intestine and nasopharynx was implemented according standard techniques. The analysis established in 81.2±6.90% of examined children deficiency of Bifidobacterium flora, decreasing of level of population density of bifidobacteria up to 6.9±1.53% lg KOE/g, in 31.2±8.1% - deficiency of normal colibacillus, in 78.1±7.3% - increased level of opportunistic flora. The analysis also established high rate of isolation of Escherichia coli with decreased enzyme activity and in 28.1±7.9% - Escherichia coli with hemolytic activity. The enterococci were permanent participants of nasopharyngeal and intestinal biotop (58.6±8.7%). From opportunistic flora, in nasopharynx were registered pathogenic streptococci - S.pyogenes, S.pneumonia and also pathogenic fungi Candida andpoly-resistant strains S. aureus. The study results demonstrated characteristics of microbial ecology of intestinal and nasopharyngeal biotops of orphan children in conditions of children institution of closed type where the circulation of pathogenic and opportunistic microorganisms occurs intensively, including strains with high medicinal resistance. All this determines necessity to attribute alumni of children's homes to risk group of infectious pathology and requires constant micro-ecological monitoring for timely correction of microbiocenoses.

[The structure of intestinal dysbioses in children of preschool age during long-term period of monitoring].

The study was organized to examine long-term (1990-2011) structure of intestinal dysbioses in children of preschool age residing in Irkutsk. The significant decrease of expression of micro-ecological shifts (IV and III degrees) to the end of period of monitoring and almost total lacking of cases of eubiosis and statistically reliable (p ≤ 0.05) increasing of rate of dysbioses of I and II degrees were established. The given circumstance can be related to ongoing on the territory ecological pressure on organism of negative factors of environment including factors of anthropogenic character.

[The detection of occurrence rate of genes coding capability to form pili binding in auto-strains of Escherichia coli].

E. coli is a commensal of intestine of the vertebrata. The exchange of genetic material of different types of bacteria between themselves and with other representatives of family of Enterobacteriaceae in intestinal ecosystem results in development of types of normal colibacillus with genetic characteristics of pathogenicity that can serve as a theoretical substantiation to attribute such strains to pathobionts. The entero-pathogenic colibacillus continues be an important cause of diarrhea in children in developing countries. The gene responsible for formation of pili binding is a necessary condition for virulence of entero-pathogenic colibacillus. The polymerase chain reaction was applied to examine 316 strains of different types of E. coli (normal, with weak enzyme activity and hemolytic activity) isolated from healthy children and children with functional disorders of gastro-intestinal tract for presence of genes coding capability to form pill binding. The presence of this gene in different biochemical types of E. coli permits to establish the fact of formation of reservoir of pathogenicity in indigent microbiota of intestinal biocenosis.

[The detection of strains of Esherichia coll producing Shiga toxin in populations of normal intestinal microbiota in children with functional disorders of gastrointestinal tract].

In intestinal ecosystem, interchange of genetic material between different types of bacteria and other representatives of family Enterobacteriaceae results in development of types of normal colibacillus with genetic characteristics of pathogenicity. This occurrence can be considered as a theoretical substantiation for labeling such strains as pathobionts. The polymerase chain reaction was implemented to analyze 96 strains of different types of Escherichia coli (with normal and weak zymogenic activity and hemolytic activity) isolated from children with functional disorders of gastrointestinal tract. The purpose was to detect presence of gens coding capacity of toxin production (six1, stx2). In intestinal biotope of children, circulation of strains of Escherichia coli producing shiga toxin having no relation to pathogenic group being representatives of normal indigenous microbiota. The presence of gens stx1 and stx2 in various biochemical types of Escherichia coli permits establishing fact of forming of reservoir of potential pathogenicity in non-pathogenic forms of Escherichia coli. The presence of gen (verotoxin 1) in genome of various types of Escherichia coli isolated from one single biotope testifies possible horizontal transmission of factors of pathogenicity in intestinal biotope.

[The specific features of microbiocenoses in children living under conditions of anthropogenic pressing].

The specific features of enteric and nasopharyngeal microbiocenoses and the species composition of bifidobacteria have been studied in children living in the industrial towns of the Irkutsk Region under the existing anthropogenic load. Ambient air pollution is characterized and a presumptive human health risk assessed.