[Shaping approaches to standardization of oropharynx mucous membrane assessment of immunological parameters]. / Formirovanie podkhodov k standartizatsii issledovaniya immunologicheskikh parametrov slizistoi obolochki rotoglotki.

Basis of acute pharyngitis pathogenesis is an inflammatory process at the entrance gate of the infection. Therefore, local immunity study proves to be the most informative. Difficulty in making that type of assessment is lack of generally accepted reference values and biological sampling techniques. OBJECTIVE: Validation of biological sampling techniques to study the parameters of local mucosal immunity in oropharynx acute inflammatory diseases. MATERIAL AND METHODS: 30 people with acute catarrhal pharyngitis with intoxication syndrome were examined. The sampling was carried out in 7 different ways. 1. Collect saliva samples using the passive drool collection method. 2. Collect saliva, using cotton swabs placed into the mouth for 3 minutes. 3. Cotton swabs wrapped around a metal probe was placed on palatine tonsils and lateral walls of the oropharynx. 4. Instead of a cotton swab, a disc of laboratory filter paper with a diameter of 0.7 cm was used. 5. Scrape by the mucous membrane of the palatine tonsils and lateral walls of the oropharynx were made with a cytobrush. 6. Using a cytobrush, scrapings were made from the mucous membrane of only the posterior pharyngeal wall, excluding the region of the palatine tonsils. 7. Using a cytobrush to make scrapings from the only palatine tonsils mucous membrane. RT-PCR was used to determine IL-1ß mRNA. RESULTS: Minimal IL-1ß mRNA values were detected in saliva collected by passive flow (0.095 [0; 3.45] units) and on a cotton swab (0.21 [0.1; 3.82] units). IL-1ß mRNA in the material collected by methods No. 3 and No. 4 on a cotton swab and a paper disk did not differ significantly from each other. Its level was higher than in saliva and lower than in scrapings. The maximum result was revealed with method No. 5 when simultaneously taking scrapings from the palatine tonsils and posterior pharyngeal wall mucous membrane (4.76 [0.92; 8.13] units). The expression of IL-1ß mRNA in the material obtained by methods No. 6 and No. 7 did not differ significantly from each other. CONCLUSION: Separated scrapings collecting from the palatine tonsils or posterior pharyngeal wall mucous membrane will allow assessing the inflammatory response autonomously in the lymphoid tissue and separately on the mucosa of the posterior pharyngeal wall. The mucosal scraping technique was the most effective for assessing cytokines in the oropharyngeal mucosal membrane.

Molecular Epidemiology of Hypervirulent K. pneumoniae and Problems of Health-Care Associated Infections.

The review describes virulence factors of hypervirulent K. pneumoniae (hvKp) including genes determining its virulence and discusses their role in the development of health-care associated infections. The contribution of individual virulence factors and their combination to the development of the hypervirulence and the prospects of using these factors as biomarkers and therapeutic targets are described. Virulence factors of hvKp and "classical" K. pneumoniae strains (cKp) with no hypervirulence genes were compared. The mechanisms of biofilm formation by hvKp and high incidence of its antibiotic resistance are of particular importance for in health care institutions. Therefore, the development of methods for hvKp identification allowing early prevention of severe hvKp infection and novel approaches to abrogate its spreading are new challenges for epidemiology, infection diseases, and critical care medicine. New technologies including bacteriological and molecular studies make it possible to develop innovative strategies to diagnose and treat infection caused by hvKp. These include monitoring of both genetic biomarkers of hvKp and resistance plasmid that carry of virulence genes and antibiotic resistance genes, creation of immunological agents for the prevention and therapy of hvKp (vaccines, monoclonal antibodies) as well as personalized hvKp-specific phage therapies and pharmaceuticals enhancing the effect of antibiotics. A variety of approaches can reliably prepare our medicine for a new challenge: spreading of life-threatening health-care associated infections caused by antibiotic-resistant hvKp strains.

The Effect of TLR Agonists and Myokines on Secretory Activity of Adipogenically Differentiated MSC Cultures.

We studied the effect of bacterial pathogen-associated molecular patterns and myokines on the secretion of adipokines by mesenchymal stem cells (MSC) and products of their adipogenic differentiation. The secretion of adiponectin, adipsin, leptin, and insulin by adipogenically differentiated cell cultures was quantitatively determined using multiplex ELISA. MSC obtained from the stromal vascular fraction of human subcutaneous adipose tissue were shown to secrete a known adipokine adipsin. The ability of white adipocytes to secrete significant amounts of insulin (in vitro) has been shown for the first time. Control cultures of white adipocytes secreted much higher levels of adiponectin, leptin, and insulin when compared to other adipocytes cultures. On the other hand, beige and brown adipocyte cultures secreted more adipsin than white adipocyte cultures. The influence of myokine ß-aminoisobutyric acid on the secretion of adipsin in MSC, white, beige, and brown adipocytes was also studied.

The Effect of LPS and Flagellin on the Process of Lipolysis in Mesenchymal Stromal Cells during Adipogenic Differentiation.

We analyzed the effects of bacterial pathogen-associated molecular patterns (LPS and flagellin) and adrenergic agonist isoproterenol on the content of total and phosphorylated (Ser552) hormone-sensitive lipase in mesenchymal stromal cells and cell products of their adipogenic differentiation. The expression of hormone-sensitive lipase and an increase in the content of its activated phosphorylated form were demonstrated by Western blotting in cells of all three lines of adipogenic differentiation. Under the influence of flagellin, the content of total and phosphorylated forms of hormone-sensitive lipase increased in brown adipocytes, while LPS induced a decrease in the content of total hormone-sensitive lipase in white adipocytes. We hypothesize that bacterial pathogen-associated molecular patterns can activate lipolysis under pathological conditions associated with slow remodeling of the adipose tissue.

[The study of tryptophan metabolite concentrations in blood serum and fecal extracts from obese children]. / Izuchenie soderzhaniia metabolitov obmena triptofana v syvorotke krovi i kalovykh ékstraktakh u detei s ozhireniem.

We found that changes in the concentrations of tryptophan metabolites in the blood serum and in the intestinal contents are one of the mechanisms for the formation of metabolic coupling in the system "macroorganism-intestinal microbiota", which undergoes significant changes in the development of obesity. Although blood kynurenine remained basically unchanged in obese children we found an increase in some of its serum metabolites: anthranilic, kynurenic and xanthurenic acids. It is noteworthy that in the analysis of fecal matter in obese children, revealed a 2-fold increase in the level of kynurenine while the concentration of kynurenine pathway metabolites corresponded to the level of the group of healthy children. This may indicate the metabolic activation of the microbiota associated with the intestinal mucosa. This is also supported by the absence of statistically significant differences in the concentration of indole in healthy children and in obese children in fecal analyses, and a significant increase in the concentration of indole-3-lactate and indole-3-acetate in the blood serum of obese children.

[Generation of Antibiotic Tolerant Bacterial Persisters in Immunocompromized Patients with Hematologic and Malignant Diseases: A New Problem of Health-Care Associated Infections].

Background: Antibiotic tolerance (AT) represents one of the causes of the phenomenon of antibiotic resistance that allows escape of non-replicating metabolically inert microorganisms (persisters) from any antibiotics attack because molecular targets of antibiotics are lacking thereby creating the potential for chronic infections. Aims: Determine the heterogeneity of the strains of opportunistic pathogens E. coli and P. aeruginosa isolates from children with hematologic malignancies containing bacterial persisters that cause the AT phenomenon. Methods: Children with hematological malignancies were divided into 2 groups according to the intensity of antibiotic treatment of infectious complications. Ciprofloxacin-induced persisters were quantitatively determined in the biological materials obtained from sick children. Results: Within the clinical isolates of E. coli and P. aeruginosa, about a third of the strains belong to high-persisting. The numbers of persistent forms of bacteria did not correlate with a minimal inhibitory concentration values ciprofloxacin (r=0.148, n=25, p>0.05). Interestingly, higher level of formation of persistent E. coli and P. aeruginosa, is associated with higher frequencies of infection attacks, massive antibiotic use and unfavorable course of the disease in children. Conclusions: Therefore, detecting the persistent forms of bacterial pathogens including those associated with the health-care associated infection, specifically, in immunocompromised patients, should be included into the contemporary algorithms of microbiological observation and monitoring of patients and intrahospital environment.

[Applicability of MALDI Mass Spectrometry for Diagnostics of Phase Variants in Bacterial Populations].

Efficiency of MALDI mass spectrometry for differentiation between phenotypic phase variants (in colony morphology and virulence/avirulence) was investigated.for saprotrophic and opportunistically pathogenic bacteria of five genera (Acinetobacter, Arthrobacter, Rhodococcus, Corynebacterium, and Escherichia). Analysis of MALDI spectra (on the SA and HCCA matrices) included: (1) determination of similarity of the protein spectra as a percentage of the common protein peaks to the total amount of proteins, which reflects the phylogenetic relationships of the objects and has been recommended for identification of closely related species; (2) comparison of intensities of the common peaks; and (3) the presence of specific peaks as determinative characteristics of the variants. Under the standard analytical conditions the similarity between the MALDI profiles was shown to increase in the row: genus-species-strain-variant. Assessment of intensities of the common peaks was most applicable for differentiation between phase variants, especially in the case of high similarity of their profiles. Phase variants (A. oxydans strain K14) with similar colony morphotypes (S, R, M, and S(m)) grown on different media (LB agar, TSA, and TGYg) exhibited differences in their protein profiles reflecting the differences in their physiological characteristics. This finding is in agreement with our previous results on screening of the R. opacus with similar colony morphology and different substrate specificity in decomposition of chlorinated phenols. Analysis of MALDI spectra is probably the only efficient method for detection of such variants.

[Surviving Forms in Antibiotic-Treated Pseudomonas aeruginosa].

Survival of bacterial populations treated with lethal doses of antibiotics is ensured by the presence of very small numbers of persister cells. Unlike antibiotic-resistant cells, antibiotic tolerance of persisters is not inheritable and reversible. The present work provides evidence supporting the hypothesis of transformation (maturation) of persisters of an opportunistic pathogen Pseudomonas aeruginosa revealed by ciprofloxacin (CF) treatment (25-100 µg/mL) into dormant cystlike cells (CLC) and non-culturable cells (NC), as was described previously for a number. of non-spore-forming bacteria. Subpopulations of type 1 and type 2 persisters, which survived antibiotic treatment and developed into dormant forms, were heterogeneous in their capacity to form colonies or microcolonies upon germination, in resistance to heating at 70 degrees C, and in cell morphology Type 1 persisters, which were formed after 1-month incubation in the stationary-phase cultures in the medium with decreased C and N concentrations, developed in several types of surviving cells, including those similar to CLC in cell morphology. In the course of 1-month incubation of type 2 persisters, which were formed in exponentially growing cultures, other types of surviving cells developed: immature CLC and L-forms. Unlike P. aeruginosa CLC formed in the control post-stationary phase cultures without antibiotic treatment, most of 1-month persisters, especially type 2 ones, were characterized by the loss of colony-forming capacity, probably due to transition into an uncultured state with relatively high numbers of live intact cells (Live/Dead test). Another survival strategy of P. aeruginosa populations was ensured by a minor subpopulation of CF-tolerant and CF-resistant cells able to grow in the form of microcolonies or regular colonies of decreased size in the presence of the antibiotic. The described P. aeruginosa dormant forms may be responsible for persistent forms in bacteria carriers and latent infections and, together with antibiotic-resistant cells, are important as components of test systems to assay the of efficiency of potential pharmaceuticals against resistant infections.

[Effect of Inherent Immunity Factors of Development of Antibiotic Tolerance and Survival of Bacterial Populations under Antibiotic Attack].

Effect of human inherent immunity factors of, a gene-encoded antibacterial peptide indolicidin (Ind) and a cytokine interleukin 1 (IL1) on formation of antibiotic-tolerant persister cells surviving in the presence of ciprofloxacin (Cpf, 100 µg/mL) and ampicillin (Amp, 100 µg/mL) in submerged bacterial cultures (Staphylococcus aureus FGA 209P, Escherichia coli K12, and Pseudomonas aeruginosa PAO1) was studied. While Ind in physiological concentrations (0.3 and 3.0 µg/mL) introduced to the lag- or exponential-phase cultures of test organisms exhibited no reliable effect on population growth, the number of persisters increased at 3.0 µg/mL. Bactericidal Ind concentrations (9 µg/mL) suppressed S. aureus growth (-0.1% of surviving cells) with subsequent recovery due to development of the more antibiotic-tolerant white variant. Treatment with Cpf after Ind addition resulted in mutual potentiation of their antimicrobial activity, with the number of S. aureus persisters 2 to 3 orders of magnitude lower than in the case of the antibiotic alone. IL1, another immunity factor, when introduced (0.1-1 ng/mL) to the exponentially growing S. aureus culture (but not to the lag phase culture) had a temporary growth-static effect, with the number of persisters surviving Cpf treatment (100 µg/mL) increasing by 1 to 2 orders of magnitude. Electron microscopy revealed significant alterations in the outer cell envelope layer of surviving S. aureus cells, which should be associated with their changed antigenic properties. Thus, the factors of human inherent immunity have a dose-dependent effect on the growth of bacterial populations. In combination with antibiotics, they exhibit synergism of antimicrobial action (indolicidin) and minimize (indolicidin) or increase (interleukin 1) the frequency of formation of persister cells responsible for survival of a population subjected to an antibiotic attack.

[Effect of Stress on Emergence of Antibiotic-resistant Escherichia coli Cells].

Effect of sublethal doses of physical and chemical stressors (heat shock for 2 h at 45 degrees C and addition of C12-alkylhydroxybenzene, a microbial alarmone) on development of resistance to the subsequent lethal antibiotic attack and the role of the time interval between these treatments were studied on a submerged batch culture of Escherichia coli 12. The interval sufficient for the development of stress response provides for development of temporary adaptive resistance to the antibiotic attack, resulting in increased number of surviving persister cells. The interval below the time required for the stress response potentiates cell death and results in a decreased number of persisters. Heterogeneity of the fractions (10(-4) to 10(-2)% of the intial CFU number) surviving lethal doses of an antibiotic (a mpicillin or ciprofloxacin) was found. Apart from a low number of antibiotic-resistant cells (up to 0.005% of surviving cells), the fractions contained antibiotic-tolerant forms, such as temporarily resistant metabolically adapted cells, long-term persisters, and the cells of slowly growing SCV variants with small colonies (d ≤ 1 mm). Persisters are hypothesized to act as precursors for cystlike dormant cells (CLC), in which the cell differentiation stage is completed and the processes of cell ametabolism (transition to the anabiotic state) are still incomplete.

[Microbial dormancy and prevention of healthcare-associated infections].

Healthcare-associated infections (HCAI) remain one of the most challenges of modern health care and assume increasing social and medical significance. The specific features of HCAI are frequent recurrences and inefficiency of antibiotic therapy, a reason for which is antibiotic resistance in microorganisms. The review discusses antibiotic resistance, a form of antibiotic tolerance (AT), and its role in the development of HCAI. It also describes essential differences between AT and antibiotic tolerance at the cellular and molecular genetic levels. Relationships between AT and dormancy of microorganisms, pathogens of HCAI, are discussed. The paper gives the data available in the literature on how AT occurs in HCAI pathogens and discusses the diagnosis of this condition. It also analyzes the literature data on pharmacological attempts to overcome AT and discusses novel approaches to antibiotic therapy for HCAI.