Molecular genetic characteristics of resistome and virulome of carbapenem-resistant Klebsiella pneumoniae clinical strains.

The characteristics of resistome and virulome structure of four carbapenem-resistant Klebsiella pneumoniae clinical strains are present in the work. Two strains belonged to the sequence-type ST395, one strain - ST2262, one strain - to the new sequence-type 5816. The genes of fimbriae, enterobactin, beta-lactamase SHV type, resistance to fosfomycin fosA and transport of fluoroquinolones oqxAB in all Klebsiella strains chromosome structure were identified. The determinants of yersineobactin and aerobactin are enriched the virulome of ST395 NNKP315 and NNKP343 strains. The aerobactin genes are located on IncHI1B plasmids (IncHI1B/FIB) which highly homologous to the virulence pLVPK and pK2044 plasmids. IncR, IncL, IncQ plasmids carrying blaOXA-48, blaCTX-M-15, blaOXA-1, blaTEM-1, qnrS1, tetA, sul1, dfrA1, aac(6 ')-Ib-cr, catA1, catB3 etc. were identified in these strains. As a result of in silico analysis, an assumption about the localization of the blaOXA-48 in the structure of the IncHI1B plasmid of NNKP315 strain was made. This plasmid also contains the aminoglycosidases genes inserted into a class 1 integron In822. The mutations were found in the porin proteins OmpK35, OmpK36 and OmpK37 genes, which increases the carbapenem resistance. The virulome of NNKP16 (ST2262) strain additionally includes of the iron utilization system kfuABC chromosomal genes, and the virulome of NNKP15 (ST5816) strain contains of the capsular polysaccharide kvgAS and microcin E492 genes. Additional determinants of resistance were not identified in the resistome structure of K. pneumoniae NNKP16 and only the blaCTX-M-15 gene was found in the NNKP15 strain. The absence of acquired resistance genes seems to be due to the presence of the type I-E CRISPR-Cas system. Multiple drug resistance of the studied strains is associated with mutations identified in the gene structure of porin proteins OmpK36 and OmpK37, as well as the activity of efflux systems. It was showed the stop codon formation in the nucleotide sequence of the regulatory gene ramR to both strains, which can potentially provide overexpression of AcrAB efflux proteins.

[Experience of application of the PCR for identification of the causative agent of community acquired pneumonia in the military.]

Samples of sputum, blood, bronchoalveolar lavage, swabs from the oropharynx from 255 military personnel undergoing in-patient treatment with an x-ray confirmed diagnosis of community-acquired pneumonia (CAP) were examined by polymerase chain reaction (PCR). The comparison group was included 270 healthy recruits. The detection of S. pneumoniae, H. influenzaе, M. pneumoniae, C. pneumoniae, C. psittaci, L. pneumophila, Adenovirus, Herpes symplex I/II, Cytomegalovirus was carried out by PCR using AmpliSens commercial test systems (CRIE) and GenPak DNA PCR test (Isogen LLC, Moscow). The etiologic factor of CAP for military personnel is set in 94.1% of cases. S. pneumoniae was the dominant pathogen of CAP. A high level of S. pneumoniae carriage (86.3%) among military personnel was shown. The incidence of M. pneumoniae was 14.5±2.2%, and C. pneumoniae was 13.7±2.2%. The highest detection rates of C. pneumoniae and M. pneumoniae were obtained in patients with not severe CAP - 16.2±2.6% и 16.7±2.7% of cases, respectively. The frequency of detection of these pathogens in patients with severe CAP was significantly lower - 5.3±3.0% and 7.0±3.4%, respectively (p<0.05). The role of L. pneumophila and C. psittaci was negligible. The frequency of detection of adenoviruses was 14,1±2,2% of cases, in particular, in patients with severe CAP - in 36.8±6.4% of cases. A high frequency of bacterial-bacterial and bacterial-viral associations has been established. In etiological CAP diagnostic in military personnel PCR is a highly effective method, especially during periods of high morbidity The high level of S. pneumoniae carriage among military personnel and its dominant role in the etiologic structure of the CAP indicate the need for specific immunization of new recruits. The significant contribution of M. pneumoniae and C. pneumoniae to the incidence of CAP confirm the advisability of their inclusion in the algorithm for the examination of patients with CAP. The high frequency of association of microorganisms indicates the need to take this fact into account when prescribing antibiotic therapy.

[The molecular typing of clinical isolates Klebsiella pneumoniae producing beta-lactamases of extended specter of action.]

The carbapenem resistant isolates of K. pneumoniae (4 strains) isolated from wound discharge of patients of burn department were analyzed with the purpose of molecular typing. The whole genome sequencing was implemented using highly productive sequenator MiSeq (Illumina, USA). Belonging of three isolates K. pneumoniae to sequence-type 395-serotype K2 is established. It is demonstrated that one isolate has a unique combination of sequence-type 23-serotype K57. The genetic determinants of main factors of pathogenicity and anti-microbial resistance are established and their localization is determined. All strains were characterized by availability of genes determining manifestation of invasive characteristics (fimbriae type I and III, proteinssiderofors), conditioning fast propagation in huma tissues. The genes responsible for synthesis of invasini and toxic substances such as α-hemolysin, enterohemolysin, shiga-like enterotoxins, cytotoxic necrotic factor, and hypermucoid phenotype regulator were not detected. In the structure of chromosome genes coding synthesis of ß-lactamases of group SHV-1 and genes of resistance to quinolones and phosphomycin were detected. In the structure of mobilom a set of genes of resistance was detected including determinants of ß-lactamases of larger specter of action CTX-M-15 and serin carbapenemases OXA-48 (ST395 isolates), in strain ST23 - only CTX-M-55. Previously, producers of CTX-M-55 of cefalosporinases among Russian isolates of Klebsiella pneumoniae were not detected. In all strains genes of ß-lactamases OXA-1 and TEM-1, genes of resistance to aminoglycoside, fluoroquinolone, phoenicols, sulfonamides and trimethoprim are present. Additionally, in one isolate were detected genes determining resistance to macrolid. The obtained results provide supplementary information about molecular genetic characteristic of carbapenem resistant strains of Klebsiella pneumoniae circulating in the Russian Federation.

[Metagenomic studies and infectious diseases diagnostics].

Principles of mass parallel sequencing, otherwise called next generation sequencing (NGS), appeared at the beginning of 2000s and were realized in dozens of NGS platforms. High performance and sequencing speed of NGS platforms opened wide horizons for scientists in the field of genomic studies, including metagenomic, first of all related to studies of structure of various microbiocenoses. Dozens of studies dedicated to studies of microbiome and virome of various biotopes of humans in normal state and pathology by using NGS platforms have appeared, forming novel conceptions on pathogenesis and epidemiology ofvarious infectious diseases. Significant cost reduction of the analysis facilitates expansion of sphere of application for NGS technologies not only in the field of fundamental, but also applied microbiologic studies, including etiologic diagnostics of infectious diseases. Due to the increase of the number of cases of infectious diseases, that do not have a typical clinical presentation, use of metagenomic approach is of particular importance, allowing to carry out detection of a wide spectrum of causative agents of bacterial, viral and parasitic infections. Technologic features of mass parallel sequencing platform, main methods of metagenomic studies and bioinformatics approaches, used for the analysis of data obtained, are presented in the review. Studies on healthy human microbiome and in pathology are described; possibilities and perspectives of metagenomic approach application in diagnos- tics and system of epidemiologic control of infectious diseases are examined.

NGS Technology in Monitoring the Genetic Diversity of Cytomegalovirus Strains.

Modern molecular genetic methods, massive parallel sequencing in particular, allow for genotyping of various pathogens with the aim of their epidemiological marking and improvement of molecular epidemiological surveillance of actual infections, including cytomegalovirus infection. The aim of the study is to evaluate the next-generation sequencing (NGS) technology for genotyping clinical isolates of cytomegalovirus (CMV). Materials and Methods: The object of the study were samples of biological substrates (leukocyte mass, saliva, urine) taken from patients who underwent liver and kidney transplantation. Detection of CMV DNA was carried out by a real-time PCR using commercial diagnostic AmpliSense CMV-FL test systems (Central Research Institute for Epidemiology, Moscow, Russia). DNA extraction was performed using DNA-sorb AM and DNA-sorb V kits (Central Research Institute for Epidemiology) in accordance with manufacturer's manual. The quality of the prepared DNA library for sequencing was assessed by means of the QIAxcel Advanced System capillary gel electrophoresis system (QIAGEN, Germany). Alignment and assembly of nucleotide sequences were carried out using CLC Genomics Workbench 5.5 software (CLC bio, USA). The sequencing results were analyzed using BLAST of NCBI server. Results: CMV DNA samples were selected for genotyping. The two variable genes, UL55(gB) and UL73(gN), were used for CMV genotype determination, which was performed using NGS technology MiSeq sequencer (Illumina, USA). Based on the exploratory studies and analysis of literature sources, primers for genotyping on the UL55(gB) and UL73(gN) genes have been selected and the optimal conditions for the PCR reaction have been defined. The results of sequencing the UL55(gB) and UL73(gN) gene fragments of CMV clinical isolates from recipients of solid organs made it possible to determine the virus genotypes, among which gB2, gN4c, and gN4b were dominant. In some cases, association of two and three CMV genotypes has been revealed. Conclusion: The application of the NGS technology for genotyping cytomegalovirus strains can become one of the main methods of CMV infection molecular epidemiology, as it allows for obtaining reliable results with a significant reduction in research time.

[Study of prevalence of rare and difficult to cultivate causative agents of inflammatory diseases of respiratory organs].

AIM: Study the prevalence of Mycoplasma pneumoniae, Chlamydophila pneumoniae, Chlamydophila psittaci, Legionella pneumophila, Moraxella catarrhalis, Cytomegalovirus, Herpes simplex I/II virus (HSV I/II) in individuals of various age groups with varying inflammatory broncho-pulmonary diseases. MATERIALS AND METHODS: 384 adults and 1001 children with clinically confirmed diagnoses were examined by PCR method: community-acquired pneumonia, acute bronchitis, bronchial asthma, ARD/ARVD, as well as 127 healthy children and 52 healthy adults. Sputum, smears from posterior fornix of pharynx, blood, saliva from children of the first year of life were used as material for the study. RESULTS: Wide prevalence of M. pneumoniae and C. pneumoniae among adults and M. pneumoniae among children older than 1 year with inflammatory diseases of respiratory organs was established. C. psittaci, L. pneumophila, M. catarrhalis occurred in isolated cases in both adults and children. Active replication of herpes group viruses was detected in patients of all age groups with inflammatory broncho-pulmonary diseases, and in children Cytomegalovirus replication predominated, in adults--HSV I/II. CONCLUSION: High frequency of prevalence of M. pneumoniae and C. pneumoniae in inflammatory diseases of respiratory tract was established, giving evidence of reasonability and necessity of examination of patients with various nosologic forms of diseases for these species of microorganisms with the aim of effective etiotropic therapy.

An Algorithm for the Selection of Probes for Specific Detection of Human Disease Pathogens Using the DNA Microarray Technology.

The aim of the study was to develop an algorithm for the selection of discriminating probes to identify a wide range of causative agents of human infectious diseases. Materials and Methods: The algorithm for selecting the probes was implemented in the form of the disprose (DIScrimination PRObe SElection) computer program written in the R language. Additionally, third-party software was used: the BLAST+ and ViennaRNA Package programs. The developed algorithm was tested by selecting specific probes for detecting Chlamydophila (Chlamydia) pneumoniae - an atypical bacterial pathogen causing community-acquired pneumonia (CAP). Nucleotide sequences for analysis were downloaded from the NCBI databank. Results: An algorithm for the selection of specific probes capable of detecting human infectious pathogens has been developed. The algorithm is implemented in the form of the disprose modular program, which allows for performing all stages of the probe selection process: loading the nucleotide sequences and their metadata from available databanks, creating local databases, forming a pool of probes, calculating their physicochemical parameters, aligning the probes and sequences contained in local databases, processing and evaluating the alignment results. The algorithm was successfully tested and its performance was confirmed by selecting a set of probes for the specific detection of Chlamydophila pneumoniae. The specificity of the selected probes calculated in silico indicated a low risk of their nonspecific binding and a high potential of using them as molecular genetic diagnostic tools (DNA microarrays, PCR). Conclusion: An algorithm for the selection of specific probes detecting a wide range of human pathogens in clinical biomaterial has been developed and implemented in the form of the disprose modular program. The probes selected using this program can serve as the functional basis of DNA-oriented microarrays able to identify causative agents of polyetiological diseases, such as CAP. Due to the flexibility and openness of the program, the scope of its application can be expanded.

The Genome Structure of Ciprofloxacin-Resistant Mycoplasma Hominis Clinical Isolates.

The genome structure of three ciprofloxacin-resistant Mycoplasma hominis clinical isolates was studied using next-generation sequencing on the Illumina platform. The protein sequences of the studied Mycoplasma strains were found to have a high degree of homology. Mycoplasma hominis (M45, M57, MH1866) was shown to have limited biosynthetic capabilities, associated with the predominance of the genes encoding the proteins involved in catabolic processes. Multiple single-nucleotide substitutions causing intraspecific polymorphism of Mycoplasma hominis were found. The genes encoding the efflux systems - ABC transporters (the ATP-binding cassette superfamily) and proteins of the MATE (multidrug and toxic compound extrusion) family - were identified. The molecular mechanism of ciprofloxacin resistance of the Mycoplasma hominis M45 and M57 isolates was found to be associated with the Ser83Leu substitution in DNA gyrase subunit A. In the Mycoplasma hominis MH1866 isolate it was related to the Lys144Arg substitution in topoisomerase IV subunit A.

[Comparative evaluation of biological properties of Salmonella typhimurium strains of different origin and genetic nature of resistance to antibiotics]. / Sravnitel'noe izuchenie biologicheskikh svoistv shtammov Salmonella typhimurium razlichnogo proiskhozhdeniia i geneticheskoi prirody ikh rezistentnosti k antibiotikam.

The results obtained in the comparative study of the biological characteristics of Salmonella typhimurium strains of different origin are presented. The circulation of two biovariants differing in a number of biological characteristics, mainly in their susceptibility to antibiotics, has been shown. R-plasmids, mostly with the markers of resistance to tetracycline and with a molecular weight of 64 Md, have been isolated from "hospital" and "sporadic" strains possessing multiple antibiotic resistance.

[The use of a molecular hybridization method for studying the enterotoxigenic properties of Salmonella]. / Primenenie metoda molekuliarnoi gibridizatsii dlia izucheniia énterotoksigennykh svoistv sal'monell.

The occurrence of the tox gene among 320 Salmonella strains of 23 serovars, differing in their origin, sensitivity to antibiotics, the presence of R-plasmids and a number of biochemical properties, has been studied by the method of DNA-DNA hybridization in situ. Essential differences in the occurrence of the tox gene have been detected both among S. typhimurium hospital strains and strains isolated in sporadic diseases, from the environment, from animals and among salmonellae belonging to different serovars. The direct correlation between the presence of the enterotoxigenicity gene and plasmids controlling resistance to antibiotics in Salmonella strains has been established. The expediency of using the method of gene probing for the study of the enterotoxigenic properties of salmonellae has been substantiated.

[Outer membrane proteins in the characterization of salmonellae of various origins]. / Belki naruzhnoi membrany v kharakteristike sal'monell razlichnogo proiskhozhdeniia.

Outer membrane proteins have been studied in 76 Salmonella strains isolated from various sources and differing in their sensitivity to antibiotics, the presence of R-plasmids and a number of enzymatic properties. The outer membranes have been isolated by the modified method of L. W. Coulton and D. T. F. Wan. The study of the isolated proteins has been carried out by means of gel and disc electrophoresis in polyacrylamide gel with sodium dodecyl sulfate according to K. Weber and M. Osborn. The composition of the proteinograms thus obtained has revealed the presence of essential differences between hospital strains and cultures isolated from animals and from the environment in sporadic infections, as well as between strains belonging to different serovars. The possibility of using the characterization of outer membrane proteins of salmonellae in epidemiological investigations is discussed.

[An experimental study of a lactoglobulin preparation against opportunistic bacteria and salmonellae]. / Eksperimental'noe izuchenie preparata laktoglobulina protiv uslovno-patogennykh bakterii i sal'monell.

A preclinical study of seven batches of lactoglobulin, a new biological preparation against opportunistic bacteria and salmonellae, has been carried out. High antibacterial activity of the preparation with respect to the virulent forms of Salmonella typhimurium, Salmonella enteritidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus vulgaris, Proteus mirabilis has been established. The preparation has been shown to be safe and nontoxic. The 4-year term of its storage at a temperature of 6 degrees +/- 4 degrees C has been substantiated.

[Drug resistance of opportunistic enterobacteria isolated from various sources]. / Lekarstvennaia ustoichivost' uslovno-patogennykh énterobakterii vydelennykh iz razlichnykh istochnikov.

Resistance of 159 strains of opportunistic enterobacteria to 9 antibacterial drugs was studied. The strains were isolated from man and cattle. It was shown that the overwhelming majority of the isolates (93 per cent) were polyresistant irrespective of the genus. There was a high frequency of the strains resistant to the widely used antibiotics such as chloramphenicol (73 per cent), ampicillin (73.6 per cent) and rifampicin (95.6 per cent) and sulfanilamides (99.3 per cent). Gentamicin and nalidixic acid proved to be the most active against the cultures: 11.9 and 10 per cent of the resistant strains, respectively. The strains of enterobacteria isolated from different sources had a sensitivity to the antibiotics. Multiple antibiotic resistance to at least 5 drugs, variability of the spectra and high resistance were more characteristic of the isolates from the animals. The necessity of a rational use of antibacterial drugs in veterinary is indicated.

[A comparative study of two commercial systems for the enzymatic identification of enterobacteriaceae]. / Sravnitel'noe izuchenie dvukh kommercheskikh sistem dlia énzimoidentifikatsii énterobakterii.

Two commercial test systems: Entero-Test manufactured in Czechoslovakia and made in the USSR are compared. Species identification of 33 newly isolated strains of gram-negative bacteria isolated from the wounds of subjects injured in the earthquake in Armenia was carried out. The advantages and shortcomings of the examined systems are analyzed. Species identification of Enterobacteriaceae by the two systems coincided in 97.2% of the examined cultures.

[Genetic determinants of antibiotic resistance in bacteria of the genus Salmonella]. / Geneticheskie determinanty rezistentnosti k antibiotikam bakterii roda Salmonella.

Distribution of genetic determinants of resistance to streptomycin, kanamycin, chloramphenicol, tetracycline, sulfanilamides and trimethoprim in strains of Salmonella isolated from studied. The majority of the resistant strains carried the genes of aminoglycoside-3"-phosphotransferase, type I aminoglycoside-3'-phosphotransferase, type I chloramphenicol acetyltransferase and type II dihydropteroate synthetase. Tetracycline resistance in the strains was often due to the class B tetracycline resistance genetic determinants. It was suggested that the resistance mechanisms controlled by these genes provided higher levels of resistance to the above drugs in Salmonella as compared to the other mechanisms. Plasmid resistance genes were detected in more than 90 per cent of the clinical strains and in 35 per cent of the sporadic strains of S. typhimurium. The antibiotic resistance of the Salmonella strains of other serovars was not as a rule controlled by the plasmid genes.

[Significance of the antibiotic resistance of the causative agents of salmonellosis in its epidemiology and the procedure for controlling the infection]. / Znachenie antibiotikorezistentnosti vozbuditelei sal'monelleza v épidemiologii i taktika bor'by s infektsiei.

Antibiotic resistance of a wide set of strains (1501) of different serovars (69) was studied and the nature of the resistance was determined. Virulent bacteriophages designed with regard to the biological properties of the isolates were considered as possible agents for the control of antibiotic-resistant microorganisms. It was shown that multiple resistance to antibacterial drugs was mainly characteristic of the serovar of S. typhimurium. In Gorky and its region, strains carrying R plasmids determining the resistance to tetracycline and chloramphenicol with a molecular weight of 58-64 Md predominated. The antibiotic-resistant strains were dangerous from the epidemiological point of view. The use of the bacteriophages is advisable for the treatment of patients, sanation of bacterial carriers or decontamination of disease sources and prophylactic phaging with regard to the epidemiological indications for preventing group diseases.