Genetic Diversity of Listeria Detected in the Production Environment of Meat Processing.

The safety of food production as concerns Listeria is the key to the sanitary wellbeing of manufactured products. Molecular-genetic methods for the analysis of Listeria, including whole-genome sequencing, are effective in monitoring persistent contaminants and in the epidemic investigation of cases of foodborne infections. They have been adopted in the European Union, United States, and Canada. In Russia, multilocus and whole-genome sequencing has proven itself in the analysis of clinical food isolates and Listeria from the environment. The objective of the study was molecular-genetic characterization of Listeria detected in the industrial environment of meat processing. To characterize the Listeria isolates, microbiological methods were used according to GOST (State Standard) 32031-2012, as well as multilocus sequencing, including the analysis of seven housekeeping genes and four virulence genes, as well as whole-genome sequencing. In swabs that were positive for the presence of Listeria spp. taken at two meat-processing plants in Moscow, Listeria monocytogenes constituted 81% and L. welshimeri 19%. The predominant genotype (Sequence Type, ST) of L. monocytogenes was ST8. The variety was supplemented with ST321, ST121, and ST2330 (CC9 (Clonal Complex 9)). L. welshimeri, which prevailed in the second production, was represented by ST1050 and ST2331. The genomic characteristics of L. welshimeri isolates confirmed that they have high adaptive capabilities both as concerns production conditions (including resistance to disinfectants) and the metabolic peculiarities of the gastrointestinal tract of animals. L. monocytogenes CC9 and CC121 are also correlated with food production in other countries. However, L. monocytogenes CC8 and CC321 can cause invasive listeriosis. The concordance in the internalin profile of the ST8 isolates from the industrial environment with the clinical isolates ST8 and ST2096 (CC8) is a cause for concern. The study showed the effectiveness of molecular-genetic methods in determining the diversity of Listeria detected in the production environment of meat processing, and laid the foundation for monitoring of persistent contaminants.

[Peculiarities of the influenza and ARVI viruses circulation during epidemic season 2019-2020 in some regions of Russia].

INTRODUCTION: The surveillance of influenza viruses in ARVI structure and study of their properties in epidemic season 2019-2020 in Russian Federation are actual for investigations due to tasks of Global Influenza Strategy initiated by WHO in 2019. MATERIAL AND METHODS: The data of epidemiological surveillance on influenza- and ARVI-associated morbidity and hospitalization in different age groups of population were analyzed; virological, genetic and statistical methods were used. RESULTS: Preschool children were involved in epidemic the most. Meanwhile, the highest rate of hospitalization was observed in patients of 18-40 years old. Influenza A(H1N1)pdm09 virus dominated in etiology of ARVI in hospitalized patients and pneumonia. The role of respiratory viruses in severe cases of pneumonia and bronchoalveolar syndrome in children was shown. The differences in spectrum of circulating viruses caused ARVI in different regions of Russia were found. Influenza A(H1N1)pdm09 and B/Victoria-like viruses were the main etiological agents that caused of epidemic; its activity among all ARVI was 7.3 and 8.0%, respectively. The differences in antigenic properties of influenza A(H3N2) and B epidemic strains compared to vaccine viruses were found. The populations of epidemic strains were presented by following dominant genetic groups: 6B1.A5/183P for A(H1N1)pdm09, 3С.2а1b+137F for A(H3N2) and V1A.3 line B/Victoria-like for B viruses. The good profile of epidemic strains susceptibility to anti-neuraminidase inhibitors has been saved. The most of the studied influenza strains had the receptor specificity characteristic of human influenza viruses. CONCLUSIONS: Obtained results identified the peculiarities of viruses caused the influenza and ARVI in epidemic season 2019-2020 in different regions of Russia. These results suggested the important role of influenza A(H1N1) pdm09 in severe cases and pneumonia in adults 18-40 years old. The continuing drift in influenza viruses was found, which, apparently, could not but affect the efficacy of vaccine prophylaxis and was also considered in the recommendations of WHO experts on the composition of influenza vaccines for the countries of the Northern Hemisphere in the 2020-2021 season.

Nanobodies Are Potential Therapeutic Agents for the Ebola Virus Infection.

Ebola fever is an acute, highly contagious viral disease with a mortality rate that can reach 90%. There are currently no licensed therapeutic agents specific to Ebola in the world. Monoclonal antibodies (MAbs) with viral-neutralizing activity and high specificity to the Ebola virus glycoprotein (EBOV GP) are considered as highly effective potential antiviral drugs. Over the past decade, nanobodies (single-domain antibodies, non-canonical camelid antibodies) have found wide use in the diagnosis and treatment of various infectious and non-infectious diseases. In this study, a panel of nanobodies specifically binding to EBOV GP was obtained using recombinant human adenovirus 5, expressing GP (Ad5-GP) for alpaca (Vicugna pacos) immunization, for the first time. Based on specific activity assay results, affinity constants, and the virus-neutralizing activity against the recombinant vesicular stomatitis virus pseudotyped with EBOV GP (rVSV-GP), the most promising clone (aEv6) was selected. The aEv6 clone was then modified with the human IgG1 Fc fragment to improve its pharmacokinetic and immunologic properties. To assess the protective activity of the chimeric molecule aEv6-Fc, a lethal model of murine rVSV-GP infection was developed by using immunosuppression. The results obtained in lethal model mice have demonstrated the protective effect of aEv6-Fc. Thus, the nanobody and its modified derivative obtained in this study have shown potential protective value against Ebola virus.

Variability of nonpathogenic influenza virus H5N3 under immune pressure.

Mutations arising in influenza viruses that have undergone immune pressure may promote a successful spread of mutants in nature. In order to evaluate the variability of nonpathogenic influenza virus A/duck/Moscow/4182-C/2010(H5N3) and to determine the common epitopes between it and highly pathogenic H5N1 avian influenza viruses (HPAIV), a set of escape mutants was selected due to action of MABs specific against A/chicken/Pennsylvania/8125/83(H5N2), A/Vietnam/1203/04(H5N1) and A/duck/Novosibirsk/56/05(H5N1) viruses. The complete genomes of escape mutants were sequenced and amino acid point mutations were determined in HA, NA, PA, PB1, PB2, M1, M2, and NP proteins. Comprehensive analysis of the acquired mutations was performed using the Influenza Research Database (https://www.fludb.org) and revealed that all mutations were located inside short linear epitopes, in positions characterized by polymorphisms. Most of the mutations found were characterized as substitutions by predominant or alternative amino acids existing in nature. Antigenic changes depended only on substitutions at positions 126, 129, 131, 145 and 156 of HA (H3 numbering). The positions 126, 145 and 156 were common for HA/H5 of different phylogenetic lineages of H5N1 HPAIV (arisen from A/goose/Guangdong/1/96) and low pathogenic American and Eurasian viruses. Additionally, mutation S145P increased the temperature of HA heat inactivation, compared to wild-type, as was proved by reverse genetics. Moreover, nonpathogenic A/duck/Moscow/4182-C/2010(H5N3) and H5N1 HPAI viruses have the same structure of short linear epitopes in HA (145-157) and internal proteins (PB2: 186-200, 406-411; PB1: 135-143, 538-546; PA: 515-523; NP: 61-68; M1: 76-84; M2: 45-53). These facts may indicate that H5 wild duck nonpathogenic virus could be used as vaccine against H5N1 HPAIV. Keywords: avian influenza virus; H5 hemagglutinin; escape mutants; genetic analysis; phenotypic properties; site-specific mutagenesis.

Characteristics of the Airway Microbiome of Cystic Fibrosis Patients.

Microbiota as an integral component of human body is actively investigated, including by massively parallel sequencing. However, microbiomes of lungs and sinuses have become the object of scientific attention only in the last decade. For patients with cystic fibrosis, monitoring the state of respiratory tract microorganisms is essential for maintaining lung function. Here, we studied the role of sinuses and polyps in the formation of respiratory tract microbiome. We identified Proteobacteria in the sinuses and samples from the lower respiratory tract (even in childhood). In some cases, they were accompanied by potentially dangerous basidiomycetes. The presence of polyps did not affect formation of the sinus microbiome. Proteobacteria are decisive in reducing the biodiversity of lung and sinus microbiomes, which correlated with the worsening of the lung function indicators. Soft mutations in the CFTR gene contribute to the formation of safer microbiome even in heterozygotes with class I mutations.

Genetic features of highly pathogenic avian influenza viruses A(H5N8), isolated from the European part of the Russian Federation.

Highly pathogenic avian influenza viruses (HPAIV) A(H5N8) of group B (Gochang1-like) have emerged in the Tyva Republic of eastern Russia in May 2016. Since November 2016, HPAIV A(H5N8) has spread throughout the European part of Russia. Thirty-one outbreaks were reported in domestic, wild and zoo birds in 2017. The present study aimed to perform a comparative analysis of new HPAIV A(H5N8) strains. Phylogenetic analysis revealed four genetically distinct subgroups in HPAIV A(H5N8) from the 2016-2017 season. Russian strains consisted of three subgroups with differences between isolates from Tyva, Siberia (Chany Lake), and the European part of Russia. Strains from the European part of Russia showed the beginnings of divergent evolution. Slight differences of the Voronezh strains were suggested by sensitivity to antiviral compounds. Testing for host-specific mutations in sequenced strains revealed the absence of mutations associated with possible increased tropism/virulence in mammalian species, including humans. Only one residue of polymerase basic-1, 13P, is discussed, because the L13P mutation increased complementary RNA synthesis in mammalian cells. We concluded that the evolution of HPAIV A(H5N8) is continuous. Surveillance in Russia revealed new cases of HPAIV A(H5N8) and led to the elaboration of prevention strategies, which should be implemented.

[Regularities of the ubiquitous polyhostal microorganisms selection by the example of three taxa].

The investigation of the bacterial populations' heterogeneity contributes to the control of natural foci, causative agents of nosocomial infections, to the analysis of the microbial evolution. Multilocus sequence typing (MLST) was employed for the analysis of the diversity and features of the distribution of polyhostal ubiquitous microorganisms of the genera Burkholderia, Leptospira, and Listeria, which belong to three bacterial phyla: Proteobacteria, Spirochaetes, and Firmicutes. According to the bacterial samples analysis microbial genotypes prevalent and unique to Russia were identified; their occurrence in different Federal Regions was investigated; their similarity with global spread genotypes was appreciated. Obtained results allowed identifying common regularities of the selection of the microorganisms capable to cause the diseases of human and animals. The formation of genotypes that are most pathogenic for the host was demonstrated for all groups of bacteria. Leptospira spp. and Listeria monocytogenes strains with these genotypes have been circulating for a long time, being supported by natural foci. The formation of a wide variety of genotypes with different pathogenicity was demonstrated in the local geographic areas. In Russia, the zonal difference in all three groups of bacteria is most clearly traced to the Far Eastern Federal Region. The results are thought to contribute to analyzing the factors of selection and the phylogeny of the taxa under study.

[Search for RNA of the hepatitis E virus autochthonous for Russia in the most likely infection sources].

The research carried out for 30 years from the moment of hepatitis E virus (HEV) discovery has proved the presence of the autochthonous HEV in non-endemic areas: Europe and Russia. Monitoring of the HEV antibodies (anti-HEV) among the Russian population has revealed regions with increased seroprevalence that testifies to high probability of local HEV infection in these areas. Contact with HEV can represent special danger for patients of the risk groups. In this work, the blood sera testing was carried out in order to assess the anti-HEV presence among these contingents (groups). Seropositive sera from the patients from the regions with high anti-HEV seroprevalence, risk groups patients, samples with high probability of HEV occurrence including the animals as possible reservoir, have been used for RNA extraction. The developed system of HEV RNA detection both in real-time RT-PCR and in a nested PCR variant has confirmed its sensitivity to the synthetic reference templates and positive control samples in commercial test system (Genesig, Great Britain). HEV RNA was absent in all tested samples. This indicates a low frequency of the autochthonous HEV carriage occurrence. Sampling enlargement to tens of thousands persons is necessary for significant HEV RNA detection.

[Characterization of genotypes for Burkholderia cepacia complex strains isolated from patients in hospitals of Russian Federation].

88 cultures of microorganisms referred to the Burkholderia cepacia complex (Bcc) during initial identification were analyzed by multilocus sequencing (Multilocus Sequence Typing, MLST). 13 genotypes (sequence type, ST) were detected, 9 of them (708, 709, 710, 711, 712, 714, 727, 728, 729) were identified for the first time. Two new alleles for the gene trpB (357, 358), one of the genes atpD (306) and gltB (352) were detected and registered. It was found that strains of 2 genotypes (711, 712) belong to the species B. multivorans, 1 (ST102) - B. contaminans, 1 (ST51) - B. stabilis, 1 (ST729) - B. vietnamiensis. Most strains of the sample, representing 8 genotypes (208, 241, 728, 727, 708, 709, 710, 714), belong to the species B. cenocepacia. Identified genotypes differ in the global spread of the world: 4 genotype (51, 102, 208, 241) have intercontinental distribution, 1 (712) - intra. It is shown that strains causing nosocomial infections, in most cases refer to genotypes 728 and 708. Epidemiologically significant in respect of patients with cystic fibrosis should recognize genotype 709, detected in strains isolated from patients in seven federal districts (FD) of Russia. The Bcc strains of genotypes 241 (B. cenocepacia) and 729 (B. vietnamiensis) were isolated from the patients of the Far Eastern FD. They are not typical for other FD Russia. The possibility of concomitant infection in cystic fibrosis patient with two genotypes 709 - epidemiologically significant and 708 - nosocomial, was indicated. The long-termpersistence of a single genotype strain in the organism of patients with cystic fibrosis was demonstrated as for Bcc species B. cenocepacia (ST 709), so for B. multivorans (ST712). The possibility of transferring the strain Bcc, typical for nosocomial environment to patient with cystic fibrosis at surgery was observed.

[The express diagnostic of microorganisms affecting respiratory tract of patients with mucoviscidosis].

The shared bacteria Burkholderia capacia complex and Achromobacter sp. infect the respiratory tract of patients with mucoviscidosis brining on disorders of respiratory patency. Burkholderia capacia complex is characterized by transmissivity and higher lethality of patients infected by Burkholderia. Hence, the importance of differentiation of these phenotypically similar microorganisms is obvious. The developed express technique of diagnostic includes the separation of DNA from phlegm amplification and sequenation was fragments of genes recA, gltB, gyrB, 16S rDNA. The evaluation of products of amplification of genes recA, gltB makes it possible to differentiate Burkholderia capacia complex and Achromobacter sp. The analysis of successions of recA, gltB, gyrB makes it possible to identify genotype of Burkholderia capacia complex on the basis of data of allele profiles of strains of Burkholderia capacia complex circulating in Russia. The succession of gene 16S rDNA makes it possible to determine the taxonomic position of microorganism dominating in phlegm and not belonging to Burkholderia capacia complex or Achromobacter sp. The real time polymerase chain reaction in presence of intercalating dye Sybr Green I, DMSO and D(+)-trehalose makes it possible to differentiate Burkholderia capacia complex from other microorganisms infecting respiratory tract of patients with mucoviscidosis. This approach provides additional reduction of diagnostic duration and decrease possibility of contamination.

[Accumulation of the bvg- Bordetella pertussis a virulent mutants in the process of experimental whooping cough in mice].

The duration of the persistence and dynamics of accumulation of insertion bvg- Bordetella pertussis mutants were studied in lungs of laboratory mice after intranasal and intravenous challenge by virulent bacteria of the causative agent of whooping cough. The capability of the virulent B. pertussis bacteria to long-term persistence in the body of mice was tested. Using the real-time PCR approximately hundred genome equivalents of the B. pertussis DNA were detected in lungs of mice in two months after infection regardless of the way of challenge. Using the bacterial test bacteria were identified during only four weeks after challenge. Bvg- B. pertussis avirulent mutants were accumulated for the infection time. The percentage of the avirulent bacteria in the B. pertussis population reached 50% in 7-9 weeks after challenge. The obtained results show that the laboratory mice can be used for study of the B. pertussis insertion mutant formation dynamics in vivo and confirm the hypothesis about insertional bvg- B. pertussis virulent mutants accumulation during development of pertussis infection in human.

[Persistence of Pseudomonas aeruginosa strains in patients of Federal Scientific Center of Transplantology and Artificial Organs].

AIM: Study genetic diversity of P. aeruginosa strains persisting in patients of Federal Scientific Center of Transplantology and Artificial Organs, and main factors facilitating persistence of strains in the hospital. MATERIALS AND METHODS: 136 P. aeruginosa strains isolated from patients of the center for 3 years 6 months were genotyped by RAPD-PCR and MLST methods and studied for antibiotics resistance and presence of integrons. RESULTS: Genetic diversity of strains persisting in hospital was established. Strains of main genotypes ST235, ST446, ST598 were isolated from patients of various surgical departments. Patients were shown to be colonized by these strains during stay in reanimation and intensive therapy department (RITD) of the hospital. Strains of dominant genotype 235 were isolated from 47% of examined patients during more than 3 years. Only genotype 235 strains contained integron with cassettes of antibiotics resistance genes blaGES5 and aadA6 in the genome. CONCLUSION: The data obtained show that over the period of observation in the center 1 clone of P. aeruginosa that belonged to genotype 235 dominated. This clone was endemic for this hospital and in the process of prolonged persistence became more resistant to antibiotics. Colonization of patients with these strains occurs in RITD. This confirms the necessity of constant monitoring of hospital microflora for advance detection of potentially dangerous epidemic hospital strains able to cause hospital infections.

[Refinement of taxonomic position of Lactobacillus genus probiotic strains by 16S rDNA and rpoA gene sequencing].

AIM: Revision of the species identification of collection lactobacilli strains based on 16S rDNA and rpoA gene sequencing. MATERIALS AND METHODS: 52 lactobacilli cultures that present mostly Gamaleya Research Institute of Epidemiology and Microbiology (GIMC) collection were studied. 16S rDNA gene fragments were amplified by using Lb16a, Lb16b, 16S-midford, 16S-midrev primers. 2 different reverse primers were used for the analysis of rpoA gene depending on lactobacilli species. DNA fragments sequencing was performed with 3130 Genetic Analyzer (Applied Biosystems/Hitachi) with primers used for amplification. RESULTS: The effectiveness of sequencing of 2 targets for differentiation of species within lactobacilli phylogenetic groups was shown. Species diversity was demonstrated for GIMC lactobacilli strain collection that includes members of 9 species. All the strains marked previously as L. acidophilus were determined to belong to L. helveticus. Strains belonging to recently discovered L. farraginis species that has promising application in agriculture were detected. CONCLUSION: Genetic passports of original strains of 9 species of lactobacilli that are promising for further research.

[Development of Staphylococcus Haemolyticus multilocus sequencing scheme and its use for molecular-epidemiologic analysis of strains isolated in hospitals in Russian federation in 2009-2010].

AIM: Development of Staphylococcus haemolyticus strain typing method based on multilocus sequencing for resolving problems of molecular epidemiology. MATERIALS AND METHODS: 102 strains of coagulase negative staphylococci (CNS) isolated in hospitals of various specialization in N. Novgorod and Moscow were studied. Species identification of strain was performed by using tuf gene fragment sequencing, S. haemolyticus strain differentiation--by MLST results. eBURST approach was used for cluster analysis of MLST data; structural changes in tagatose-6-phosphate kinase were studied by using InterProScan platform and SWISS-MODEL site programs; MLST scheme gene allele variability analysis was performed by using MEGA4.0 program package. RESULTS: In the 102 strains sampled CNS was detected in 28 strains of the S. haemolyticus species. The MLST scheme developed for the first time for S. haemolyticus including mvaK, rphE, tphK, gtr, arcC, triA, aroE genes allowed the differentiation of the sampled strains by 11 genotypes. Strains with ST 3, 8, 6, 1, 4, 5 and 11 differed by highest epidemiologic significance. Cluster and phylogenetic analysis of the data obtained showed a high adaptive ability of the nosocomial S. haemolyticus strains. Multiresistance to antibacterial preparations was detected in the analyzed strains. CONCLUSION: The MLST method developed was effective in the differentiation of S. haemolyticus strains that circulate in hospitals and threaten both neonates and hospitalized adult patients.

[Estimation of species diversity of coagulase-negative staphylococci isolated in hospitals of Russian Federation in 2009-2010].

AIM: Comparative analysis of species diversity of sample of coagulase-negative staphylococci (CNS) isolated in hospitals of different specializations. MATERIALS AND METHODS: For identification of 102 CNS strains, biochemical systems manufactured by NPO "Diagnostic Systems", VITEK 2 Compact, and BBL Crystal as well as sequencing of fragments of tuf and gap genes were used. RESULTS: Greater differentiating capability of genotyping compared with phenotyping methods for species identification of staphylococci was demonstrated. Six CNS species were identified in the sample: S. epidermidis, S. haemolyticus, S. hominis, S. warneri, S. capitis, and S. pasteuri. The largest species diversity was noted for strains from maternity hospitals in Nizhny Novgorod and Kulakov Scientific Center for Obstetrics, Gynecology and Perinatology. Strains isolated from blood of patients in Bakulev Center for Cardiovascular Surgery were represented mostly by S. epidermidis and S. haemolyticus. Differences in species diversity of CNS--causative agents of neonatal conjunctivitis and omphalitis--were observed. CONCLUSION: Two species of CNS: S. epidermidis and S. haemolyticus pose special threat as nosocomial pathogens both in hospitals for adults and obstetrical facilities. Additionally, in neonatal units it is necessary to control such species as S. warneri, S. capitis, S. pasteuri.

[Analysis of prevalence and variability of Legionella pneumophila and Legionella spp. strains on the basis of study of allelic profiles].

AIM: To analyze prevalence and variability of Legionella strains isolated in town Verkhnaya Pyshma located in Sverdlovsk region during prophylactic surveillance of potentially dangerous water objects in 2007 - 2008. MATERIALS AND METHODS: Sequencing of mip gene was conducted for identification of species of Legionella. Multi-locus sequence typing was used for describing of allelic profiles of Legionella pneumophila strains. RESULTS: Five firstly identified on Russian territory strains of Legionella species were deposited in institute's collection. Sixty-three strains of L. pneumophila belonging to 28 sequence types were characterized. Relation between strains isolated in industrial building and from water supply system was demonstrated. CONCLUSION: Observations made on the basis of study of L. pneumophila strains isolated from cooling stacks of industrial plants confirmed potential danger of these objects as a source of dissemination of Legionella infection.