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.

Structure and gene cluster of the O-polysaccharide of Yersinia rohdei H274-36/78.

A branched O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Yersinia rohdei H274-36/78 and found to contain d-rhamnose, d-mannose, and 3,6-dideoxy-4-C-[(S)-1-hydroxyethyl]-d-xylo-hexose called yersiniose A (Yer). Partial acid hydrolysis of the O-polysaccharide eliminated Yer residues to give a modified linear polysaccharide. Studies by sugar analysis and 1H and 13C NMR spectroscopy, including computational NMR analysis, enabled structure elucidation of a hexasaccharide repeating unit of the O-polysaccharide having two Yer residues attached as monosaccharide side chains. The O-antigen gene cluster of Y. rohdei H274-36/78 located between JUMPStart and galF genes contained putative genes for synthesis of precursors of two O-antigen constituents, GDP-d-Man and GDP-d-Rha, whereas genes responsible for synthesis of CDP-Yer were within the chromosome outside the O-antigen gene cluster. Glycosyltransferase genes and ABC 2 transporter genes were present in the O-antigen gene cluster, and hence the structure established is consistent with the polysaccharide synthesis gene content of the genome.

[The study of patterns of development of resistance of Staphylococcus aureus to triclosan].

The article presents the results of studying molecular genetic mechanisms of development of resistance to antiseptic Triclosan in strain Staphylococcus aureus АТСС25923. The modifcations of strain S. aureus АТСС25923 (Tr1, Tr2, Tr1С и Tr2С) are obtained resistant to 64 mg/l of Triclosan and stably preserving the given characteristic under cultivation in absence of selective pressure. The strain S. aureus Tr1was characterized by slightly delayed growth and the strain S. aureus Tr2 was characterized by growth velocity comparable with initial strain. In the Triclosan-resistant strains a mutation C284T in gene fabI was detected resulting in amino-acid replacement A95V in enzyme enoyl-acyl protein reductase FabI, triclosan target. Besides, in these strains a stably inheriting mutation was detected in genes associated with transport of substances in cell: hypothetical transport protein HlyC/CorC family transporter, protein-transporter of ions of Na+, K+, Li+ and alkali of Na+/H+ antiporter subunit F, membrane hypothetical protein and ATP-binding protein. It is demonstrated that resistance to triclosan in staphylococci is associated with acquirement of point mutations in genes of enoyl-acyl protein reductase and also in other genes related to transport of substances in bacterial cell.

[ON THE ORIGIN OF HYPERVIRULENCE OF THE CAUSATIVE AGENT OF PLAGUE].

The attempt to combine Yersinia pseudotuberculosis and Yersinia pestis into one species has been unsupported by microbiologists due to the specific features of the epidemiology and clinical presentations of their induced diseases and to basic differences in their virulence. Pseudotuberculosis is predominantly a relatively mild human intestinal infection transmitted through contaminated food and plague is an acute generalized disease with high mortality, which is most frequently transmitted by the bites of infected fleas. Y. pestis hypervirulence, the ability of single bacteria to ensure the development of predagonal bacteriemia in rodents, which is sufficient to contaminate the fleas, is one of the main events during pathogen adaptation to a new ecological niche. By analyzing the data of molecular typing of the representative kits of naturally occurring Y. pestis isolates, the authois consider the issues of formation of intraspecies groups with universal hypervirulence, as well as biovars that are highly virulent only to their major host. A strategy for searching for selective virulence factors, the potential molecular targets for vaccination and etiotropic treatment of plague, is discussed.

A small-scale experiment of using phage-based probiotic dietary supplement for prevention of E. coli traveler's diarrhea.

Traveler's diarrhea (TD) is caused by Escherichia coli in 30% of cases. We have developed a phage cocktail for prophylaxis of TD caused by E.coli, Shigella flexneri, Shigella sonnei, Salmonella enterica, Listeria monocytogenes or Staphylococcus aureus, and investigated its effectiveness against infection caused by the non-pathogenic Lac (-) strain of E.coli K12 C600 in animal and human trials. On the 6th day of both animal and human trials E. coli K12 C600 strain was detected in titer of 104 CFU/g of mice feces and 106 CFU/g of human feces in the control (untreated) groups, while it was not detected in the samples of either of the study (phage-treated) groups. These results have great significance because the original coliphages included in the cocktail have a broad host-range including ETEC, EAEC and EHEC strains which cause severe cases of TD.

[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.

[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.

Role of Structure-Based Changes due to Somatic Mutation in Highly Homologous DNA-Binding and DNA-Hydrolyzing Autoantibodies Exemplified by A23P Substitution in the VH Domain.

Anti-DNA autoantibodies are responsible for tissue injury in lupus. A subset of DNA-specific antibodies capable of DNA cleavage can be even more harmful after entering the living cells by destroying nuclear DNA. Origins of anti-DNA autoantibodies are not fully understood, and the mechanism of induction of DNA-cleaving activity remains speculative. The autoantibody BV04-01 derived from lupus-prone mouse is the only DNA-hydrolyzing immunoglobulin with known 3D structure. Identification and analysis of antibodies homologous to BV04-01 may help to understand molecular bases and origins of DNA-cleaving activity of autoantibodies. BLAST search identified murine anti-DNA autoantibody MRL-4 with sequences of variable region genes highly homologous to those of autoantibody BV04-01. Despite significant homology to BV04-01, not only MRL-4 had no DNA-cleaving activity, but also reversion of its unusual P23 mutation to the germline alanine resulted in a dramatic loss of affinity to DNA. Contrary to this effect, transfer of the P23 mutation to the BV04-01 has resulted in a significant drop in DNA binding and almost complete loss of catalytic activity. In the present paper we analyzed the properties of two homologous autoantibodies and mutants thereof and discussed the implications of unusual somatic mutations for the development of autoantibodies with DNA-binding and DNA-hydrolyzing activity.

[Determination of critical kanamycin concentration for Mycobacterium tuberculosis in the MGIT Bactec 960 system].

The critical concentration of canamycin for the MGIT Bactec 960 system, which has been determined on a panel of the tho--roughly characterized genetically heterogeneous clinical Mycobacterium tuberculosis isolated in the Central Region of Russia, is equal to 1.25 microg/ml.

[Determining drug resistance of the clinical strains of Mycobacterium tuberculosis to pyrazinamide].

Description of the pyrazinamide-resistant clinical strains of M. tuberculosis derived from sputum of patients treated in TB clinics in Tula was made (June, 2001 - July, 2002). It was demonstrated that 30.3% (n = 91) strains were resistant to pyrazinamide. It was found out that these strains were resistant to other antituberculosis drugs in most cases. The method of PCR-sequencing was used to find the mutations in the gene pncA determining resistance to pyrazinamide. 44 different types of mutations localized in 28 codons were detected. The predominance of the mutations in 57 (13.2%), 63 (7.7%), 97 (7.7%), 12 (6.6%), 103 (6.6%) codons and in -11 (6.6%) promoter ofp ncA was observed in the pyrazinamide-resistant strains. Several new mutations determining resistance of the clinical strains of M. tuberculosis to pyrazinamide were described. A high correlation between resistance of mycobacteria to pyrazinamide and activity of pyrazinamidase was observed.

[Structure of deletions detected in the genomes of clinical Mycobacterium tuberculosis strains].

Deletions were analyzed in the genomes of four Mycobacterium tuberculosis strains isolated from the sputum of patients living in the Central Region of Russia. The strains of the Beijing family were found to have deletions affecting 40 open reading frames (ORF) and to amount to 0.7% of the genome H37Rv. Genome deletions in a strain from the Haarlem family affected 20 ORF and accounted for 0.26% of the genome H37Rv. Six of the eight deletions were located at the site of preferred cloning on the insertion element IS6110. Prophage phiRv1-associated deletion sequence 149 is the only common to the strains of both families. The deletion ends were evenly distributed among the intergenic and coding chromosome regions with an insignificant preference of the latter. The authors revealed a new deletion in strain 1540 belonging to the Haarlem family and a two-component deletion in the region RvD2. The deletions detected in the genomes of Russian Beijing strains were typical of strains from South-East Asian countries.