Vol 9, No 2 (2019)

Here we review currently available data showing that innate immune signs predisposing to recurrent and invasive pneumococcal infections were identified in children. Streptococcus pneumoniae (pneumococcus) belongs to Grampositive bacteria being the major cause of morbidity and mortality in infants, especially in developing countries and in communities with low socioeconomic status. Due to the lack of anti-pneumococcal vaccination, the significant proportion of pneumococcus carriers develop non-invasive (pneumonia, otitis media, sinusitis) and severe invasive (bacteremia/septicemia, meningitis) pneumococcal infection. A great deal of diverse factors related to pneumococcus biological features (virulence factors) as well individualized host-specific immunity are implicated in efficient bacterial penetration across the mucous membranes. The TLR signaling system plays a crucial role in the human nonspecific defense upon the first encounter with the pathogen. Various TLRs comprise the first pattern recognition receptor fami ly ever described which sense ligands derived from the outer bacterial wall. The complement system is the ancient innate immunity component mainly involved in intravascular elimination of bacterial agents. In addition, the complement proteins serve as a bridge between innate and adaptive immunity, ensuring optimal conditions for B- and T-cell maturation and differentiation. Because pneumococcus secretes the IgA protease, a local protective effects related to IgA antibodies might not be so prominent. Therefore, B-cell immunodeficiency and impaired complement system hold a lead place among congenital causes resulting in severe and recurrent pneumococcal infections in children. Thus, based on available data, we concluded that impaired B-cell function, the complement components deficiency as well as receptor-recognition receptors (TLR-2, -9, -4, MYD88 adapter protein, TLR cascade enzymes: IRAK4, NEMO, NOD-like receptors: NOD2, NLRP3; C-type lectins: MBL, Dextin-2, and, possibly, ficoline) play the most important role among congenital immunodeficiencies predisposing to invasive and recurrent pneumococcal infections play the most important role among congenital immunodeficiencies predisposing to invasive and recurrent pneumococcal infections, and should be used as a rationale for immunological surveillance and organizing immunogenetics screening in these patients. 

Gastric and duodenal recurrent inflammatory diseases have a high prevalence, but the role played by microbes in its development remained unclear. However, the data published in 1983 by Marshall and Warren about isolating Helicobacter pylori from the stomach mucosa of the patient with gastritis and proposing relevant cultivation methods was the turning point in investigating etiology of the upper digestive tract inflammatory disorders. Moreover, it was shown that the majority of H. pylori spp. are found within the gastric lumen upon colonization, whereas around 20% of them are attached to the epithelial cells in the stomach. In addition, effects of interacting H. pylori with gastric epithelium and activation of some defense mechanisms due to bacterial colonization and spreading were analyzed. It was found that along with triggering pro-inflammatory response induced by proteins VacA as well as phosphorylated/unphosphorylated CagA, wherein the latter is able to induce a set of protective reactions H. pylori disrupts intercellular contacts, affects epithelial cell polarity and proliferation, and activates SHP-2 phosphatase resulting in emerging diverse types of cellular responses. The activation mechanisms for the mitogen-activated protein kinase (MAPK) pathway were discussed. The ability of H. pylori to regulate apoptosis, particularly via its suppression, by expressing ERK kinase and protein MCL1 facilitating bacterial survival in the gastric mucosa as well as beneficial effects related to bacterial circulation on gastric epithelial cell survival elicited by anti-apoptotic factors were also examined. Of note, persistence of H. pylori are mainly determined by activating transcriptional factors including NF-κB, NFAT, SRF, T-cell lymphoid enhancing factor (TCF/LEF), regulating activity of MCL1 protein, in turn, being one of the main anti-apoptotic factors, as well as induced production of the migration inhibitory factor (MIF). The role of VacA cytotoxin in triggering epithelial cell apoptosis via caspase-mediated pathways was also considered. Infection with H. pylori is accompanied by release of proinflammatory cytokine cocktail detected both in vitro and in vivo. In particular, bacterial urease activating transcriptional factor NF-κB was shown to play a crucial role in inducing cytokine production. Moreover, such signaling pathways may be activated after H. pylori is attached to the cognate receptor in the gastric epithelial surface by interacting with CD74 and MHC class II molecules. Finally, a role for various CD4⁺ T cell subsets, particularly type 17 T helper cells (Th17) in inducing immune response against H. pylori antigens in gastric mucosa was revealed were also discussed. 

Tularemia is an anthropozoonotic infection caused by Francisella tularensis. In clinical and sanitary-epidemiological practice, traditional diagnostics methods in tularemia are based on serological assays for detecting specific antibodies, allowing to diagnose it and estimate durability of patients’ immunity after vaccination. Previously, it was shown that specific serum antibodies in patients recovered after tularemia, unlike to those vaccinated with the live tularemia vaccine F. tularensis 15 NIIEG, can interact with specific epitopes on lipopolysaccharides isolated from strains of various subspecies — F. tularensis (Ft) and F. novicida (LPS Fn), while LPS Fn-specific immunoglobulins are lacked in the blood of vaccinated individuals. A set of experiments on identifying antibodies with similar specificity in laboratory animals of various species — mice, guinea pigs and rats with differed sensitivity to tularemia, administered with live tularemia vaccine strain as well as virulent F. tularensis strains to simulate vaccine-mediated and infectious processes, respectively was conducted. A methodical approach has been developed that allows to analyze humoral response in modelled infectious process in animals highly sensitive to tularemia such as BALB/c mice and guinea pigs that consisted of preliminary immunization with live tularemia vaccine followed by infection with virulent F. tularensis strains. It was shown that induction of specific anti-LPS Ft antibodies occured in these animal species, both after vaccination and infection with virulent strains. It was noted that, unlike guinea pigs and rats, mice both during vaccination and infection were characterized by significantly lower titers of LPS Ft-specific antibodies. However, no specific interaction between mouse serum and LPS Fn might be detected. Moreover, two types of immunoglobulins with different antigen specificities to the LPS Ft and LPS Fn epitopes were detected by dot-blot analysis in guinea pigs immunized with live tularemia vaccine, followed by infection with a virulent strain. In addition, antibodies to LPS Fn were also detected in the serum of rats infected with virulent, but not vaccine-based, F. tularensis strains. Thus, previous experimental data on the production of immunoglobulins with different antigenic specificity were confirmed in an experimental tularemia modelled in rats and guinea pigs that demonstrated a diagnostic significance and feasibility of using LPS Fn to confirm tularemia infection in humans. 

Chronic hepatitis B is a severe liver disease caused by persistent infection of hepatitis B virus in human hepatocytes. Chronic hepatitis B is one of the most common diseases in the world. According to recent estimations, more than 250 million people are chronically infected and more than 1 million of people die annually due to consequences of chronic hepatitis B: liver cirrhosis and hepatocellular carcinoma. The key factor of hepatitis B virus persistency is a special form of viral genome called circular covalently closed DNA. Current therapeutics suppress viral replication but have no effect on circular covalently closed DNA as it exists in the nuclei of hepatocytes as a minichromosome and is not accessible for therapeutics. Commonly, viral reactivation occurs after cessation of treatment. Therefore, duration of antiviral treatment is supposed to be indefinitely long. One of the most promising approaches to target circular covalently closed DNA is the technology of site-specific nucleases CRISPR/Cas9 from Streptococcus pyogenes. A short guide RNA recruits an SpCas9 protein to the viral genome and induces generation of DNA double strand breaks. However, there are several limitations of CRISPR/Cas9 hampering translation of this technology into the clinic. First, efficacy of CRISPR/Cas9 needs to be improved. Second, CRISPR/Cas9-mediated off-target mutagenesis represents a menacing problem which has to be addressed. To overcome these limitations, several approaches have been devised to improve CRISPR/Cas9 activity (modification of guide RNAs) and reduce off-target mutagenesis (a Cas9 protein with enhanced specificity, eSpCas9). In this study, we compared antiviral activity of a classic SpCas9 with an eSpCas9 system as well as analyzed effects of gRNAs modification on anti-HBV effects. Here, we demonstrated that SpCas9 has the highest antiviral potency, reducing transcription and replication of HBV over 90%. Hepatitis B virus covalently closed circular DNA declined over 90% post CRISPR/Cas9 transfection. Although it was previously shown that modified guide RNAs increase nucleolytic activity of CRISPR/Cas9, our results indicated that this modification impairs antiviral activity of CRISPR/Cas9. To conclude, CRISPR/Cas9 effectively suppress viral replication and transcription per se. Described modifications do not potentiate antiviral activity of CRISPR/Cas9 system and should not be used for development of future therapeutics. The best strategy to improve CRISPR/Cas9 efficacy is to design new highly effective guide RNAs. 

Helicobacter pylori represents a pathogen causing chronic infection in around a half of the global human population, which manifestations vary from asymptomatic infection to developing gastritis and peptic ulcer. The data accumulated suggest that overt clinical types of this infection are associated with lost immunoregulation and increased pro-inflammatory cell-mediated immune response triggered by H. pylori-specific T helper cells. Here, we examined the degree of peripheral blood CD4⁺ T cell maturity and related expression of chemokine receptors involved in migration to gastrointestinal tract (CCR9 and CCR6), as well as Тand B-cell zones of lymphoid organs (CCR7 and CXCR5). It was shown that overt H. pylori-infection was coupled to changes in expression pattern of chemokine receptors on T helper cells. In particular, percentage of mature CD4⁺CD45RO⁺ T cells bearing CCR9 and immature CD4⁺CD45RO^– Т cells expressing CXCR5 was increased in peripheral blood of patients with chronic gastroduodenitis. However, increased amount of activated mature CD4⁺CD45RO⁺ICOS⁺ T cells was observed in patients with chronic gastroduodenitis comorbid with peptic ulcer that was also associated with elevated amount of mature CCR6⁺ T helpers (mainly CD4⁺CD45RO⁺CCR7^– CCR6⁺ cells) and follicular T helper cells as well as emerging minor CD4⁺CD45RO⁺CXCR5⁺CCR6^+.T cell subset, not affecting CD4⁺CCR9⁺ Т cells. Thus, the data obtained evidence that tissue-specific T-helper cell migration is controlled separately in of H. pylori-associated diseases. 

In theRussian Federation, Y. pestis NIIEG strain-based live attenuated vaccine is used for immunization against plaque on epidemiological indications, displaying high efficiency. However, individual fluctuations in adaptive immunity after vaccination necessitate conducting a search for genes underlying variability of developing immune response. Of note, HLA (Human Leukocyte Antigen) gene polymorphism plays an important role in this process. In our study, we identified HLA class II haplotypes for HLA-DRB1, HLA-DQA1, and HLA-DQB1 in 120 residents of theterritoryofPre-Caspiannatural plague focus, who were immunized against plague. In addition, level of TNFα production correlated with detecting allelic groups HLA-DRB1*04 (р = 0.05) and DRB1*12 (р = 0.01). The data obtained show that HLA class II gene polymorphism can affect the level of cytokine secretion in response to plague immunization. Examining the genes regulating immune factor production will allow to get better insight into the mechanisms underlying immune response variations after vaccination as well as contribute to predicting immunogenicity and efficiency of developing vaccine preparations. 

Tick-borne encephalitis (TBE) is a natural focal viral neuroinfection vastly spread in the mid-latitude climate zone of theEurasia. Lethal cases and disability related to TBE determine its high epidemiological significance as well as underlie undiminished attention to prevent it in endemic territories. At the same time, TBE epidemiologic features significantly differ in various geographic areas. Importantly, Republic Sakha (Yakutia) is not among TBE endemic areas in theRussian Federation. However, in the last decade an increased incidence of ixodic tick bites was registered in the Republic Sakha. A pattern of pathogen-specific population immunity is one of the most valuable criteria for assessing magnitude of epidemic process in TBE foci. Hence, our study was aimed at assessing natural herd immunity TBEV in Republic Sakha associated with elevated incidence of tick bite visits. Here, we analyzed the data regarding the rate of tick attacks in the period of 2001–2007 in Republic Sakha. Residents and individuals unvaccinated against TBEV in the city ofYakutskas well as various administrative regions were examined to record the peak incidence of tick attacks. It was found that a range of the administrative regions recording with registered of ixodic tick attacks and elevated incidence of tick bites was expanded. Moreover, the top incidence of tick bites was annually recorded in the south regions of the republic Aldanian, Neryungri, Olyekminsk, Leninsk, Khangalassky districts, city ofYakutskand its suburbs. Furthermore, TBEV-specific immunoglobulins were detected in 5.7±0.68% of the residents examined suggesting about contacts with the pathogen. In addition, the peak count of seropositive people (8.9±1.85%) was identified in Namsky district located in the central part of the republic. Overall, detection rate of TBEV-specific antibodies among general adult population (6.9±0.95%) was significantly higher compared to those observed in children (3.9±0.89%, р < 0.05). no significant differences in urban (5.8±1.12%) vs. rural population (5.63±0.84%) was found. 

Pertussis infection remains a high-priority issue both for Russian health care and abroad. A rise of pertussis incidence in various human age groups instigates a search for new ways to fight this infection and improve methods for its laboratory diagnostics. By taking into consideration a short-term effect induced by acellular and whole-cell vaccines, a feasibility of introducing the second or even the third pertussis revaccination is vigorously debated. Objective of the study was to analyze the experience and effectiveness of acellular pertussis vaccines in countries, which use the second and third pertussis revaccination in the National Immunization Schedule in order to have an insight into adjusting strategy and tactics of pertussis immunization In Russia. Analyzing pertussis prevalence demonstrated that despite a wide immunization coverage pertussis incidence in the last years (2008–2015) was increased in a large number of countries in the European region, as well as inAustralia,CanadaandUSA. However, the reasons for elevated pertussis incidence have not been clarified yet. On one hand, it may be accounted for by low vaccination coverage in adolescents and adults; weakened immune protection after vaccination; genetic changes in Bordetella pertussis; shortened durability of protective immunity in children vaccinated with acellular vs. whole-cell vaccine; improved monitoring and morbidity reporting, as well as improved laboratory diagnostics due to shifting from serological and bacteriological to molecular genetic assays. In an attempt to solve this issue, researchers from several countries collaborate to discuss and develop a strategy to reduce pertussis incidence. ForRussia, the most important is to empower and/or improve existing infant immunization strategy in order to provide wide coverage with the four dose pertussis vaccine for decreasing the risk of pertussis morbidity and mortality. It is worth noting the “cocoon” strategy given the high risk of pertussis infection in children of the first months of life. We believe that forRussiait is worth investigating an opportunity of using children 2–3 months of life an acellular vaccine as the first vaccination, which is expected to increase the coverage of this cohort and allow to increase proportion of children who might complete vaccination by 5 months of age. At the same time, more reasonable might be to preserve a number of age groups for pertussis vaccination in the current National Immunization Schedule, as expanding age limits for vaccination might put a risk at increasing pertussis morbidity in older individuals, which could be hard to diagnose. 

In this study, we propose a chronobiological method for examining inter-microbial interactions in bacterial and fungal associations in female reproductive tract. Fungal and bacterial species were isolated in 45 women of reproductive aged 19–35, with regular menstrual cycle, applying no hormonal contraceptives, without previous gynecological surgery, abortions, miscarriages with vaginal eubiosis and dysbiosis in history. Sexually transmitted diseases (HIV infection, syphilis, gonorrhea, trichomoniasis, chlamydiosis) were excluded in all subjects. Proliferation rate, morphogenesis and phospholipase activity were examined within the 48-hour period every 4 hours, in winter time, Moon phase IV. The data obtained were assessed by using Student’s t-test, Wilcoxon test, and least squares method. All subjects were divided into the groups: group 1 — women with vaginal eubiosis, group 2 — women with vaginal dysbiosis. It was shown that in all subjects experimental parameters of C. albicans cultures showed a diurnal dynamics characterized in healthy women by circadian rhythms with a single peak of activity. However, in women with vaginal dysbiosis C. albicans was characterized by significant ultradian (around 12 hours long) rhythms with two peaks of biological activity. Concurrence and consistency in manifested physiological functions related to clinical isolates was coupled to temporal pattern of distributed biological resources in fungi depending on course of infectious process. It was found that in vaginal eubiosis exometabolites released by dominant associated microbiota did not significantly change microbiota-related amplitude-phase parameters. The data obtained evidenced that temporal pattern of parameters related to C. albicans from healthy individuals was stable and independent on bacterial metabolites. In contrast, dominant microsymbiont in vaginal dysbiosis inhibited fungi-related rhythms, which might be important in establishing lactobacillus-associated biotope colonization resistance. Effects of metabolites released by the associated microbiota typical to dysbiosis was revealed by increased mesor, amplitude, preserved biorhythm spectral pattern in examined properties as well as amplitude-phase characteristics indicating at enhanced or sustained C. albicans adaptive potential. Therefore, the amplitude-phase parameter of C. albicans physiological activity served as a marker of opposite (enhanced/weakened) effect of microsymbiont survival described in “microbial dominant-associate” pairs. 

Remaining unmanageable,Norwalkvirus infection is clearly tended to be recorded at higher rate, including adult patients. In many cases, clinical picture of adult acute diarrhea in patients positive for Norwalk virus in clinical samples vs. pathogenetically-caused norovirus infection differs, thereby justifying comparison of clinical and laboratory data. A cohort retrospective study with 146 hospitalized adult patients suffering from acute moderate diarrhea positive for fecalNorwalkvirus was performed. Along with standard laboratory tests (culturing, serologic for detecting 4-fold increase in titer between paired serum samples, ELISA), detection of diarrhea-linked agents included PCR kit Amplisens® AII-bacto-screen-FL Lab (Interlabservice). The data obtained demonstrated that in adult patients with acute diarrhea 54.1% of cases were positive forNorwalkvirus as well as for other enteric pathogens, including bacteria found in 36.3% of cases. Moreover, clinical samples of patients with acute diarrhea hospitalized at least on day 4 vs. day 3 after the onset were significantly more often (by 1.5-fold) positive for association between Norwalk virus and other acute enteric infection agents mainly due to astrovirus (р = 0.03; PCR data) and Shigella spp. (culture-based and serologic methods; р = 0.03). In addition,Norwalkvirus was associated with rotavirus, but not other enteric pathogens, at 2.1-fold higher rate in clinical samples from patients treated vs. untreated with antimicrobials before hospitalization. Finally, clinical samples positive for Norwalk virus from patients with vs. without developed colitis syndrome were at higher risk of developing virus-bacterial enteric infection detecting Salmonella spp. at 7.6and 3-fold higher rate verified by culture-based and PCR assay, respectively. Importantly, patients with vs. without hemorrhagic colitis Salmonella spp. verified primarily a culture-based method was detected by 11-fold more frequently (p = 0.01). 

In this article data concerning coherent fluctuation nephelometry (CFN) use in clinical microbiology is presented. CFN-analyzer allows to solve two important problems – fast urine screening for bacteriuria within 2-4 hours and antibiotic susceptibility testing within 3-6 hours. Altogether more than 650 urine samples were tested, and the effectivity of CFN-analyzer for preliminary selection of samples for further analysis was shown. Method allows to detect negative samples, reducing the number of urine analyses by 70-80%. Simultaneous analysis of growth curves and concentration of microorganisms shows high sensitivity and specificity (95.2% и 96.9%). Also more than 250 antibiotic susceptibility tests were performed using CFN-analyzer to show its effectiveness for determination of resistant properties of both pure cultures and urine microflora without isolation of bacteria. The agreement with traditional methods was from 84% to 88%. The use of CFN-analyzer with express methods of identification of microorganisms (chromogenic nutrient broths or mass-spectrometry) allows to make full urine analysis within 1-2 days. In the future CFN-analyzer gives an opportunity to screen different human biological liquids, and finds an application for other microbiological tasks, including standardization and speeding-up in sanitary bacteriology.

The article presents results of research related to the selection of an effective method for isolating bacteriophages active against bacteria of the genus Flavobacterium. The number of bacterial strains used in the work — 3. All strains cultures were obtained from the museum of the department of microbiology, virology, epizootiology and veterinary-sanitary examination of federal state budgetary educational institution of higher education Ulyanovsk «State Agricultural University named after PA Stolypin» and have tinctorial and biochemical properties characteristic for this genus (and species). The studies consisted of 4 series of experiments. The first series of experiments was connected with the approbation of a technique for isolating a bacteriophage without using an influencing factor. As a result, the desired bacteriophages were not isolated. The second series of experiments consisted in testing the isolation method using an inducing factorultraviolet irradiation. The distance to the objects of irradiation is0.3 m. Exposure time: 1 min, 3 min, 5 min, 10 min, 15 min. The third series of experiments was also associated with the use of inducing factor X-ray irradiation. In the course of the work, three operating modes were determined, each of which differed in the duration of the exposure, the periodicity of the exposure, and the dose received. As a result of studies related to the use of inducing factors, the desired bacteriophages were not identified. The last series of experiments was to allocate bacteriophages from the objects of the natural places сommemorative (possible) existence of the «owner» (the studied microorganism) and viruses (bacteriophage desired). Three phage isolates were isolated from environmental objects: FL-j-1, FL-pec-2, FL-aq-3. Bacteriophage FL-j-1 is active against a strain of F. johnsoniae VKM B-1426 bacteriophage FL-pec-2 is active against strain F. pectinovorum VKM B-1171 bacteriophage FL-aq-3 strain active against F. aquatile VKPM B-8534. Further studies may be related to study properties of the isolated phages: study of the specificity in respect of this kind of sensitivity study, the study and higher lytic activity, selection of the most promising strains phages further selection of selected phages, as well as studying the possibility of bacteriophages from use in the method of the reaction increase titer phage for the purpose of indicating the microorganism being studied. 

At present, a rise of antimicrobial resistance requires that susceptibility of infectious agents to antimicrobial agents could be accurately evaluated as related errors may lead to selecting improper therapeutics provoking spread of drug resistance. Pathogen sensitivity to antimicrobial agents is commonly determined by a disc diffusion method. A quality of nutrient medium used in assays plays a crucial role influencing final results. In Russia, it turned out that regulatory documents such as the nationwide guidelines and clinical recommendations outlining methodology for antimicrobial susceptibility testing underlay availability in domestic market few nutrient media, including Mueller–Hinton Agar, AGV medium etc. exhibiting sometimes unsatisfactory quality. To harmonize such methodology with international requirements, theStateResearchCenterfor Applied Microbiology and Biotechnology has developed a technology and promoted manufacture of Russia-made Mueller–Hinton agar satisfying requirements of EUCAST documents, clinical guidelines, and ISO/TS 16782:2016. The main objective of this study was to compare quality of new agar product with five similar foreign media while examining 11 test strains by disc diffusion method. As a result, some of nutrient media available to the Russian market turned out to be off-standard: not all of them satisfy to the EUCAST requirements and clinical guidelines since diameter distribution for growth inhibition recommended by EUCAST for quality control does not fit into permissible range. Moreover, susceptibility of P. aeruginosa ATCC 27853 to aminoglycosides, fluoroquinolones, Meropenem, as well as S. aureus ATSS 25923 and E. faecalis ATCC 29212 to tigecycline was assessed with certain mistakes. The data obtained by us were analyzed in accordance to the new document ISO/TS 16782:2016 “Clinical laboratory testing — criterion for acceptable lots of dehydrated Mueller–Hinton agar and broth for antimicrobial susceptibility testing”, not approved yet In Russia. To determine potential reason for deviation of data from reference range, we measured concentration of bivalent metals in all nutrient media examined by atomic emission spectrometry with inductively coupled plasma. We determined new patterns affecting reliability of results on microbial antibiotic susceptibility. A need to check intralaboratory quality control of nutrient media was emphasized.