Stress-Tolerant Dormant Bacterial Forms: Biological and Ultrastructural Properties of Moraxella catarrhalis and Kocuria rhizophila.

Dormant forms of causative agents of healthcare-acquired infections Moraxella catarrhalis and Kocuria rhizophila have been obtained. Dormant forms cells retained viability during long-term storage (≈107 CFU/ml after 2 months) under provocative conditions (lack of nutrient sources; temperature 20°C, oxygen access) were characterized by heat resistance, and acquired special ultrastructural organization typical of dormant forms (compacted nucleoid, thickened cell wall). They were also capable of forming alternative phenotypes (dominant and small colony variants) in a new cycle of germination in a fresh medium. These results demonstrate that the dormant forms can be responsible both for survival in the environment and persistence in the host organism.

Pseudomonas veronii strain 7-41 degrading medium-chain n-alkanes and polycyclic aromatic hydrocarbons.

Pollution of the environment by crude oil and oil products (represented by various types of compounds, mainly aliphatic, mono- and polyaromatic hydrocarbons) poses a global problem. The strain Pseudomonas veronii 7-41 can grow on medium-chain n-alkanes (C8-C12) and polycyclic aromatic hydrocarbons such as naphthalene. We performed a genetic analysis and physiological/biochemical characterization of strain 7-41 cultivated in a mineral medium with decane, naphthalene or a mixture of the hydrocarbons. The genes responsible for the degradation of alkanes and PAHs are on the IncP-7 conjugative plasmid and are organized into the alk and nah operons typical of pseudomonads. A natural plasmid carrying functional operons for the degradation of two different classes of hydrocarbons was first described. In monosubstrate systems, 28.4% and 68.8% of decane and naphthalene, respectively, were biodegraded by the late stationary growth phase. In a bisubstrate system, these parameters were 25.4% and 20.8% by the end of the exponential growth phase. Then the biodegradation stopped, and the bacterial culture started dying due to the accumulation of salicylate (naphthalene-degradation metabolite), which is toxic in high concentrations. The activity of the salicylate oxidation enzymes was below the detection limit. These results indicate that the presence of decane and a high concentration of salicylate lead to impairment of hydrocarbon degradation by the strain.

Next-Generation Antibiotics, Bacteriophage Endolysins, and Nanomaterials for Combating Pathogens.

This review presents various strategies to fight causative agents of infectious diseases. Species-specific programmable RNA-containing antibiotics open up new possibilities for creating next-generation of personalized drugs based on microbiome editing and can serve as a new tool for selective elimination of pathogenic bacterial species while keeping intact the rest of microbiota. Another promising approach in combating bacterial infections is genome editing using the CRISPR-Cas systems. Expanding knowledge on the molecular mechanisms of innate immunity has been actively used for developing new antimicrobials. However, obvious risks of using antibiotic adjuvants aimed at activation of the host immune system include development of the autoimmune response with subsequent organ damage. To avoid these risks, it is essential to elucidate action mechanisms of the specific ligands and signal molecules used as components of the hybrid antibiotics. Bacteriophage endolysins are also considered as effective antimicrobials against antibiotic-resistant bacteria, metabolically inactive persisters, and microbial biofilms. Despite significant advances in the design of implants with antibacterial properties, the problem of postoperative infections still remains. Different nanomodifications of the implant surface have been designed to reduce bacterial contamination. Here, we review bactericidal, fungicidal, and immunomodulating properties of compounds used for the implant surface nanomodifications, such as silver, boron nitride nanomaterials, nanofibers, and nanogalvanic materials.

[In vitro models of Mycobacterium tuberculosis dormancy, and in vivo models of latent tuberculosis infection.]

Modeling of tuberculosis infection is carried out in order to clarify various aspects of the tuberculosis pathogenesis, as well as the testing of new anti-tuberculosis drugs. The characteristic of in vitro models (n = 16) for Mycobacterium tuberculosis dormant state and in vivo models (n = 14) for the latent tuberculosis infection involving several animal species published to date are presented in this review. A brief description of the models and the results obtained by the authors are presented. The analysis of the published data reflects the list of methodological procedures that allow researchers to study the mechanism of the transition of M. tuberculosis cells to a dormant state and reverse to metabolically active state, as well as the process of conversion of active tuberculosis infection to a latent tuberculosis and reactivation.

[Diagnostic bacteriophage V32 as a tool for the rapid identification of Escherichia coli serogroup O157.]

Bacteriophage V32, a representative of bacterial viruses of the Myoviridae family Ounavirinae subfamily, is proposed for search and identification of E. coli O157 serogroup, including Shiga-toxin producing E. coli O157:H7 (STEC O157:H7), among cultures of enterobacteria from the primary seeding of the material studied. Phage genome containes a linear double-stranded DNA of 87875 base pairs with G/C-content of 38.9% and includes 132 open reading frames (ORF). In the genome, there are no determinants of antibiotic resistance, virulence genes of STEC and other well-known pathogroups of E. coli. It has been established that phage V32 has lytic activity against all studied cultures of E. coli O157 serogroup (n=183) isolated from people and farm animals in various regions of the Russian Federation, as well as in Japan and Italy. At the same time, the phage lyses only 6 of 182 strains (3.3%) of E. coli not belonging to the O157 serogroup and is not active against strains of other enterobacteria. That is, the phage has a high specificity. The use of bacteriophage V32 as a diagnostic tool is a highly efficient, fast, cheap and simple method for identifying E. coli serogroup O157, including the serotype E. coli O157: H7, in any bacteriological laboratory without special equipment and special training of performers.

[The characteristic of diarrheagenic Escherichia separated from children aged under 5 years old in Yaroslavl.]

The diarrheagenic bacteria coli take a significant place among agents of acute intestinal infections in children aged under 5 years. The main danger among these pathogens is represented by both enterotoxigenic E. coli causing enteritis and enterocolitis accompanied by acute dehydration diarrhea and Escherichia producing shiga-toxin being agents of hemorrhagic colitis and hemolytic uremic syndrome. The fast and proper identification of agents of these two groups of pathogens is an important task of bacteriologists to be resolved for successful treatment of patient because tactics of therapy of enterotoxigenic diarrhea and hemorrhagic colitis and hemolytic uremic syndrome significantly differ. The high capacity of Escherichia coli to form populations resistant to anti-microbial medications, including pan-resistant ones, also is a serious problem for science and public health. The object of study was a collection of isolates of E. coli (n = 112), separated from 112 children aged under 5 years with clinical manifestations of acute intestinal infection, food toxic infection hemocolitis and diarrhea of obscure etiology in Yaroslavl in 2015-2017. Initially, the strains of E. coli were tested using diagnostic agglutinating coli-serums and then using reagents' kit «AmpliSens®Escherichiosis-FL¼ for detection and differentiation DNAof diarrheagenic bacteria coli and also with specific oligonucleotide primers to genes of virulence and O-serum group belonging. The obtained data permitted to determine belonging of analyzed strains of E. coli to four sub-groups: ЕНЕС (n = 9), EPEC (n = 17), ETEC (n = 1) и EAgEС (n = 1). All of them were agents of genes of pathogenicity specific for every pathogroup. The most numerous group EPEC was represented by strains of five serogroups with dominating among them serogroup O26 (9 strains). Therefore, studying collection of strains of diarrheagenic Escherichia isolated during 2015-1017 in Yaroslavl from children aged under 5 years with acute intestinal infections permitted to demonstrate efficiency of application of molecular genetic methods of analysis for characterizing E. coli i.e. establishment their serogroups, detection of genes of virulence and attributing to pathogroups.

Immune response to Vi-antigen of Salmonella typhi is dependent on introduction of positive charge and shape of charged group into polysaccharide.

A model of a controlled conversion of polysaccharide Vi-antigen of S. typhi into zwitterionic antigen is proposed. The immunological properties of modifications of this antigen conjugated to a protein support were studied.

[The loop mediated isothermal amplification of DNA: principle of method and perspectives of application in molecular diagnostic of cholera: publications review].

The article considers characteristics of technology of reaction of loop mediated isothermal amplification of DNA (LAMP), issues of optimization of reaction and perspectives of its application as a quick highly-specific test in molecular diagnostics of infectious diseases and monitoring of contamination of environment objects with pathogens. The analysis of publications data concerning application of LAMP in diagnostics of cholera testifies high diagnostic value. The LAMP supports possibility of direct rapid detection of toxin-producing strains of Vibrio cholerae in clinical samples. This technique also provides identification of determinants of cholera vibrio in pure culture, samples from environment objects and food products. The research studies established exceeding of parameters of sensitivity and specificity of LAMP as compared with polymerase chain reaction that permits considering LAMP as a perspective technique for express-analysis of clinical material from patients with suspicion on cholera. The LAMP technique can be also used in screening studies of environment objects. The development of test-systems based on application of this technology is required.

The use of loop-mediated isothermal DNA amplification for the detection and identification of the anthrax pathogen.

The results of detection and identification of Bacillus anthracis strains in loop-mediated isothermal DNA amplification (LAMP) reaction performed under optimized conditions with original primers and thermostable DNA polymerase are presented. Reproducible LAMP-based detection of chromosomal and plasmid DNA targets specific for B. anthracis strains has been demonstrated. No cross reactions with DNA from bacterial strains of other species of the B. cereus group were detected. The development of tests for anthrax-pathogen detection based on the optimized reaction of loop isothermal DNA amplification is planned. These tests will be convenient for clinical studies and field diagnostics due to the absence of requirements for sophisticated equipment.

[Aptamers in the Treatment of Bacterial Infections: Problems and Prospects].

Aptamers are short single-stranded oligonucleotides which are selected via targeted chemical evolution in vitro to bind a molecular target of interest. The aptamer selection technology is designated as SELEX (Systematic evolution of ligands by exponential enrichment). SELEX enables isolation of oligonucleotide aptamers binding a wide range of targets of interest with little respect for their nature and molecular weight. A number of applications of aptamer selection were developed ranging from biosensor technologies to antitumor drug discovery. First aptamer-based pharmaceutical (Macugen) was approved by FDA for clinical use in 2004, and since then more than ten aptamer-based drugs undergo various phases of clinical trials. From the medicinal chemist's point of view, aptamers represent a new class of molecules suitable for the development of new therapeutics. Due to the stability, relative synthesis simplicity, and development of advanced strategies of target specific molecular selection, aptamers attract increased attention of drug discovery community. Difficulties of the development of next-generation antibiotics basing on the conventional basis of combinatorial chemistry and high-throughput screening have also amplified the interest to aptamer-based therapeutic candidates. The present article reviews the investigations focused on the development of antibacterial aptamers and discusses the potential and current limitations of the use of this type of therapeutic molecules.

[CONSTRUCTION AND PROPERTIES OF THE FRANCISELLA TULARENSIS VACCINE STRAIN WITHOUT ONE COPY OF THE IGLC GENE AND WITHOUT RECA GENE].

The live vaccine based on the Francisella tularensis subsp. holarctica vaccine strain 15 NIIEG line is used in Russia against tularemia. This vaccine is highly effective, but fairly unstable. Therefore, development of stable live tularemia vaccine with minimal side effect is rather urgent. The method of allel removal in the F. tularensis vaccine strain was used to remove one copy of the iglC gene, which is required to provide intracellular production of the vaccine strain, as well as removal of the recA gene. The latter is crucial for homological recombination. pGM5 suicide vector based on pHV33 bireplicon plasmid was constructed to provide replacement of intact F. tularensis chromosome segments by modified segments. Modified chromosome segments contain F. Tularensis DNA fragment without iglC structural gene segment 545 p. b. (in pGMΔiglC plasmid), as well as DNA fragment containing no recA structural gene segment 1060 p.b. (pGMΔrecA plasmid). The constructed 15/23-1ΔrecA mutant, in contrast to the vaccine strain 15, was capable of reproducing in the macrophage-like cells J774A.1 line, whereas the efficiency of the reproduction was 8-10 times less. BALB/c mouse responded to immunization by the 15/23-1ΔrecA strain by smaller weight decrease (-2%) as compared to the strain 15 (-14%). Bacteria of the 15/23-1ΔrecA strain were virtually incapable of germinating from the BALB/c murine spleen 14 days after invasion, whereas bacteria of the strain 15 were found in the murine organs even after 21 days. The F. tularensis 15/23-1ΔrecA strain having smaller reaction ability can be used as a basis for construction of stable live safe tularemia vaccine.

[THE CONDITION AND TENDENCIES OF DEVELOPMENT OF CLINICAL AND SANITARY MICROBIOLOGY IN THE RUSSIAN FEDERATION AND PROBLEM OF IMPORT SUBSTITUTION].

The import substitution becomes one of the strategic tasks of national economy as a result of prolongation of economic sanctions concerning the Russian Federation of part of the USA, EU countries, Japan and number of other countries. It is not proper to be limited in import substitution only by goods because in conditions ofsanctions when access toforeign technologies is complicated Russia is needed to substitute foreign technologies by national designs in faster manner One of directions of effective import substitution is localization of production of laboratory equipment and consumables for clinical and sanitary microbiology on the territory ofthe Russian Federation and countries of Customs union. In Russia, in the field ofdiagnostic of dangerous and socially significant infections, all components for import substitution to implement gene diagnostic, immune diagnostic. bio-sensory and biochip approaches, isolation and storage of live microbial cultures, implementation of high-tech methods of diagnostic are available. At the same time, national diagnostic instrument-making industry for microbiology is factually absent. The few devices of national production more than on 50% consist of import components. The microbiological laboratories are to be equipped only with import devices of open type for applying national components. The most perspective national designs to be implemented are multiplex polimerase chain reaction test-systems and biochips on the basis of national plotters and readers. The modern development of diagnostic equipment and diagnostic instruments requires supplement of national collections of bacterial and viral pathogens and working-through of organizational schemes of supplying collections with strains. The presented data concerning justification of nomenclature of laboratory equipment and consumables permits to satisfy in fill scope the needs of clinical and sanitary microbiology in devices, growth mediums, consumables of national production and to refuse import deliveries without decreasing quality of microbiological analysis. This approach will ensure appropriate response to occurring challenges and new biological dangers and maintenance of biosecurity of the Russian Federation at proper level.

[CONTEMPORARY CONCEPTION OF IMMUNE RESPONSE ACTIVATION MECHA- NISM BY CONJUGATED POLYSACCHARIDE VACCINES].

Vaccination remains the most effective method of control of spread of a whole range of infections of both viral and bacterial nature. Many bacterial pathogens (Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae) carry polysaccharide capsule on the surface, that is one of the elements of protection from host organism immune system. At the same time, vaccination with bacteria exopolysaccharides (EPS) ensures infection neutralization. Effectiveness of such vaccine prophylaxis is limited by age of the vaccinated, intensity and duration of the immunity, development of secondary immune response. EPS conjugation with protein antigens was known for a long time to ensure activation of T-cell immunity against EPS and formation of secondary immune response. However, detailed studies of mechanism of immunity modulation by a protein partner as part of a glycoconjugate has not been carried out. T-lymphocyte activation was traditionally thought to occur exclusively due to peptide presentation, that are products of processing of protein component of the conjugate. Recently, information, accumulated in the field of natural carbohydrate, glycolipid and glycoprotein antigen presentation to T-cells, has generated interest in studying mechanisms of cell immunity activation by conjugated vaccines. Progress in this field, as well as development of novel chemical and biochemical, including combinative technologies of synthesis and study of these molecules, opens new opportunities for detailed understanding of mechanism of action for conjugated vaccines and creation of glycoconjugates with increased effectiveness of protective action.

[Molecular-genetic characterization of shiga-toxin producing Escherichia coli isolated during a food-borne outbreak in St. Petersburg in 2013].

UNLABELLED: Shiga toxin-producing Escherichia coli (STEC) food-borne infections are reported worldwide and represent a serious problem for public healthcare. In the Russian Federation there is little information on epidemiology and etiology of STEC-infections as well as on molecular-genetic peculiarities of STEC pathogens. OBJECTIVE: Our aim was to describe a food-borne outbreak as hemorrhagic colitis (HC) along with hemolytic uremic syndrome (HUS), enterocolitis, and acute gastroenteritis in children in St. Petersburg in 2013. METHODS: Epidemiological, microbiological, molecular-genetic and bioinformatic methods were applied. RESULTS: Objects to study were clinical specimens, milk and food samples, as well as STEC strains isolated during the outbreak. The outbreak of food-borne infection was found to be caused by STEC-contaminated raw milk as confirmed by epidemiological analysis, detection of STEC DNA and isolation of relevant pathogens in milk and sick children fecal specimens. The whole-genome sequencing revealed two groups ofpathogens, E. coli O157:H7 and E. coli O101:H33 among collected strains. Group I strains were attributed to the previously known sequence type ST24, while group II strains belonged to the previously non-described sequence type ST145. In strain genomes of both groups there were identified nucleotide sequences of VT2-like prophage carrying stx2c gene, plasmid enterohemolysin gene, and gene of the STEC main adhesion factor intimin. Gene of intimin gamma was identified in E. coli O157:H7 strains and intimin iota 2 in E. coli O101:H33 strains. The latter previously was identified only in enteropathogenic E. coli (EPEC) strains. CONCLUSION: The additional knowledge of epidemiology and biology of STEC pathogens would assist clinicians and epidemiologists in diagnosing, treating and preventing hemorrhagic colitis.

[Features of biofilm morphology, formed by Corynebacterium diphtheriae gravis tox+ strains].

AIM: Study the structure of homogenous microbial communities of Corynebacterium diphtheriae gravis tox+ strains during formation of biofilms in vitro. MATERIALS AND METHODS: Object of study--typical and biofilm cultures of C. diphtheriae gravis tox+ museum and circulating strains. Intensity of biofilm formation was evaluated by OD on microplate reader at 540 nm wave length studying 120 and 720 hour cultures. S-450 (Hitachi, Japan) scanning electron microscope was used. RESULTS: The peak of exopolysaccharide matrix (EPS) formation, that is formed in the process of biofilm formation, by museum strain takes place at earlier terms of cultivation (120 hours) than circulating (720 hours). An inverse correlation was established during analysis of bacterial cells of museum and circulating strains of C. diphtheriae during biofilm formation between them and intensity of EPS formation. At maximum EPS content, that took place at various terms of cultivation of the 2 studied strains of diphtheria causative agent, a reduction of corynebacteria cells was observed. CONCLUSION: Bacterial biofilms of museum and circulating strains of C. diphtheriae and patterns of dynamics of EPS reflect, probably, adaptive abilities of the causative agent, that determine its competitiveness in the fight for adhesion sites, resistance to factors of natural immunity and as a result--prolonged persistence in the organism of bacterial carriers.

[Francisella tularensis strains molecular typing using the multiple-locus variable-number tandem repeat analysis].

Variable-number tandem repeat analysis (VNTR) of 25 loci was used for molecular typing of the Francisella tularensis strains isolated from different regions of Russia and the former Soviet Union. This approach allowed us to subdivide F. tularensis subspecies and determine genotype diversity with regard to the geographical prevalence. All 25 loci were examined for their ability to discriminate subspecies and local geographical group. 42 genotypes among the 58 investigated F. tularensis subsp. holarctica isolates were found using cluster VNTR analysis.

[Molecular genetic identification of Staphylococcus aureus strain, caused a foodborne illness outbreak in St. Petersburg in 2013].

BACKGROUND: Staphylococcus aureus is one of the most important human pathogens and causes over 100 nosologicalforms of diseases. The lack of data on the spread of S. aureus genetic types specific for different forms of staphylococcal infections in Russia makes it difficult to timely identify and control strains of this epidemiologically dangerous bacterial pathogen. OBJECTIVE: The aim of the study was to carry out a molecular genetic research of S. aureus isolates obtained during a widespread foodborne illness outbreak among builders at the Pulkovo airport in St. Petersburg in 2013. METHODS: The ability of the isolates to produce staphylococcal enterotoxins was studied by immunoenzyme techniques. Gene typing was carried out by sequence-specific primer-based PCR, as well as by sequencing genomic nucleotide sequences of two independent isolates of the pathogen. RESULTS: An enterotoxin A gene in genomes of S. aureus isolates etiologically associated with the outbreak was identified. The production of enterotoxin A by the isolates was shown. According to the complex analysis all isolates producing staphylococcal enterotoxins were identical and constituted the S. aureus strain, sequence-type ST30 and spa-type t2509. The genome of the identified S. aureus strain carried a set of various staphylococcal toxins. The full genome sequence among other techniques revealed high levels of similarity between genomes of the strain under study and well-known reference strain S aureus MRSA 252. CONCLUSION: The complete molecular genetic study of the S. aureus strain involved into the widespread foodborne illness outbreak was first carried out in Russia, allowing of further using the strain as a Russian reference strain to study potential epidemic outbreaks in the Russian Federation.

Enzymes of SPZ7 phage: isolation and properties.

Bacteriophage enzyme preparations exolysin and endolysin were studied. Exolysin (a phage-associated enzyme) was obtained from tail fraction and endolysin from phage-free cytoplasmic fraction of disintegrated Salmonella enteritidis cells. A new method for purification of these enzymes was developed, and their molecular masses were determined. The main catalytic properties of the studied enzymes (pH optimum and specificity to bacterial substrates) were found to be similar. Both enzymes lyse Escherichia coli cells like chicken egg lysozyme, but more efficiently lyse S. enteritidis cells and cannot lyse Micrococcus luteus, a good substrate for chicken egg lysozyme. Similar properties of exolysin and endolysin suggest that these enzymes are structurally similar or even identical.

Biological properties and structure of the lipopolysaccharide of a vaccine strain of Francisella tularensis generated by inactivation of a quorum sensing system gene qseC.

A knockout mutant with a deletion in a quorum sensing system gene qseC was generated from the vaccine strain Francisella tularensis 15 by site-directed mutagenesis. The variant with the inactivated gene qseC differed from the parental strain in growth rate on solid nutrient medium but had the same growth dynamics in liquid nutrient medium. The mutation abolished almost completely the resistance of the vaccine strain to normal rabbit serum and its ability to survive in macrophages; in addition, the strain lost the residual virulence. A significant phenotypic alteration was observed in the lipopolysaccharide of F. tularensis. Particularly, the mutant strain synthesized no noticeable amount of the lipopolysaccharide with the high-molecular-mass O-polysaccharide, presumably as a result of impairing biosynthesis of the repeating unit, namely, a loss of the ability to incorporate a formyl group, an N-acyl substituent of 4-amino-4,6-dideoxy-D-glucose.