[Sensitivity of corynebacterium diphtheriae strains to antibacterial preparations].

AIM: Study the prevalence and mechanisms of resistance in circulating C. diphtheriae strains. MATERIALS AND METHODS: 664 C. diphtheriae strains isolated in 1987 - 2013 in various regions of Russia and sent to the reference center of Gabrichevsky Moscow Research Institute of Epidemiology and Microbiology were the object of the study. Antibiotic sensitivity of the strains was studied by disk-diffusion and E-test methods using 10 antimicrobial preparations. Nucleotide sequence analysis was carried out by using BLAST program and EMBL/GenBank database. RESULTS: Most of the studied strains turned out to be sensitive to all the antibacterial preparations used. 1.2% of C. diphtheriae strains turned out to be resistant to penicillin and 6.0% had intermediate level of resistance. 0.4 - 0.6% of the strains had intermediate level of resistance to macrolides, and 4.0 - 4.4% were resistant. 2.0% of the strains had multiple resistance. Erm(X)-specific PCR carried out in this study showed that all the C. diphtheriae strains resistant to macrolide antibiotics carry erm(X) gene. CONCLUSION: The results of the study indicate a fairly high level of prevalence for C. diphtheriae strains resistant to antibiotics.

[Genetic characteristics of Staphylococcus aureus No 6. strain--producer of secreted protein-containing compounds possessing protective properties].

AIM: Genotype characteristics of Staphylococcus aureus No 6: strain that is a producer of a protective protein complex. MATERIALS AND METHODS: Features of structure of 9 genes, that code synthesis of pathogenicity factors, of S. aureus--spa, coa, sea, seB, sec, pvl, tst-h, mecA and scc-mecA, that are responsible for synthesis of protein A, coagulase, enterotoxins A, B and C, Panton-Valentine toxin (PVL), heat shock syndrome protein, resistance to methicillin and staphylococci chromosomal cassette, respectively, were studied by amplification in PCR of the respective gene fragments with subsequent conduction of direct sequencing. RESULTS: The S. aureus No 6 strain under study possesses pvl gene fragments, as well as Spa and coagenes, detected in all the studied strains, that belong to t12507 and EMRSA-16 types, respectively. Sea, seb, sec genes responsible for.the synthesis of enterotoxins A, B and C were not detected in it, tst-h, mecA and scc-mecA gene fragments were not present. CONCLUSION: The detection of pvl gene fragment in the strain under study, on the one hand, and protective properties of the secreted protein-containing compound, on the other hand, give evidence in favor of the necessity of further analysis of extracellular proteome of S. aureus No 6.

[Multilocus sequencing of Corynebacterium diphtheriae strains isolated in Russia in 2002 - 2012].

AIM: Characterization of contemporary C. diphtheriae strains isolated in Russia by using multilocus DNA sequencing (MLST). MATERIALS AND METHODS: 28 toxigenic C. diphtheriae strains isolated in Russia in 2002-2012 and sent to diphtheria and pertussis reference center of Gabrichevsky Research Institute of Epidemiology and Microbiology were studied. C. diphtheriae strain genotyping was performed by using MLST based on atpA, dnaE, dnaK, fusA, leuA, odhA and rpoB gene fragments. Identification of alleles and ST was carried out according to EMBL/GenBank and PubMLST, eBurst approach was used for cluster analysis. RESULTS: By using MLST contemporary toxigenic C. diphtheriae strains isolated in Russia in 2002 - 2012 were characterized. 8 genotypes (ST41, ST5, ST8, ST28, ST25, ST44, ST-new1 and ST-new2) were identified, 3 among them were dominating--ST8, ST28 and ST-new1. Most of the toxigenic strains belong to biovar gravis and ST8. Among biovar mitis strains a higher heterogeneity by ST membership was noted, but with prevalence of ST28 strains. CONCLUSION: Use of MLST allowed to characterize contemporary circulating population of toxigenic C. diphtheriae strains isolated in Russia and showed perspective of application of this method for characterization of diphtheria causative agent population and detection of epidemically significant strains, as well as juxtaposing of them with genetic structure of foreign strains.

[Genotypic differences between Corynebacterium diphtheriae biovar gravis and mitis strains].

AIM: Study structure ofa genetic determinant of amylase activity (amygene) in Corynebacterium diphtheriae biovar gravis and mitis strains. MATERIALS AND METHODS: 87 C. diphtheriae strains (31 gravis biovar strains and 56 mitis biovar strains) as well as C. diphtheriae PW8 strain were analyzed to detect structural features of C. diphtheriae strains of various biovars. 10 pairs of primers were used in PCR that flank mutually overlapping regions within DIP0357 locus as well as additional primers that flank DIP0353-DIP0354, DIP0357 and DIP0358 loci. RESULTS: All the C. diphtheriae biovar gravis strains were established to contain a full-size DIP0357 locus (amy gene) whereas in all the mitis biovar strains this genome fragment is absent. All the studied C. diphtheriae biovar gravis strains do not have significant changes within DIP0354-DIP0357 loci (amy gene) whereas in genome of 57 studied C. diphtheriae biovar mitis strains the major part of this fragment including the complete nucleotide sequence of amy gene is absent. CONCLUSION: C. diphtheriae biovar gravis strains have a genetically determined ability to produce amylase that can be viewed as an additional pathogenicity factor giving microorganisms wider capabilities to colonize the mucous membrane of oropharynx.

[The quality control of nutrient medium in laboratory bacteriologic diagnostics of diphtheria].

The article deals with the results of studying the growth characteristics of nine nutrient mediums for primary plating of pathologic material. It is demonstrated that for successful and proper functioning of bacteriologic laboratory providing bacterial analysis for diphtheria the permanent quality control is needed to monitor the nutrient mediums for primary plating. The quality control is applied to evaluate the growth characteristics on such criteria as germination of isolated culture, intensity of its growth in 24 and 48 hours, optimal size of colonies characterized by their cultural characteristics, inhibiting activity concerning concurrent microflora.

[Characteristics of Bordetelia pertussis strains isolated from pertussis patients in Moscow by using multilocus sequencing].

AIM: Genotyping of B. pertussis strains isolated from pertussis patients in Moscow. MATERIALS AND METHODS: 53 strains of B. pertussis isolated from pertussis patients in Moscow in 2007 - 2010 as well as 3 vaccine strains currently used in Russia for the production of DTP vaccine were studied by multilocus sequencing (MLST) based on allele combinations of ptxA, ptxC and tcfA genes. RESULTS: A genetic characteristic of B. pertussis strains isolated from pertussis patients in Moscow by using MLST is presented. Allele profile analysis of the studied B. pertussis strains was performed, 4 sequence types (ST) were identified--ST1, ST2, ST3 and ST5, most of the circulating strains (86.7%) were shown to belong to ST5, equal percentage of cases (5.7%)--to ST2 and ST3, and 1.9%--to ST1, while 2 vaccine production strains belong to ST2 and 1 - to ST1. CONCLUSION: Clonal structure of contemporary Moscow strains was shown to be different from strain structure used for the production of DTP vaccine.

[Molecular genetic features of structure of gene coding tracheal colonization factor in Bordetella pertussis strains].

AIM: Comparative analysis of structure of tcfA gene coding tracheal colonization factor of Bordetella pertussis strans isolated in Moscow from patients with pertussis. MATERIALS AND METHODS: Ninety-seven strains of B. pertussis isolated in different periods of pertussis infection epidemic process (1948 - 1989--from collection of Gabrichevsky Moscow Research Institute of Epidemiology and Microbiology; 1990 - 2007--isolated in Moscow from patients with pertussis) were studied. Primers for amplification of tcfA gene region with size 945 n.p. were used. Amplicons obtained in PCR were used for sequencing. Nucleotide sequences of tcfA gene types of B. pertussis strains were matched to EMBL/GenBank database. RESULTS: Sequencing of tcfA gene fragments revealed two sequence variants. Ninety-six of 97 studied B. pertussis strains had the same sequence variant--variant 1. The one strain was characterized by other nucleotide sequence--variant 2, which differed from variant 1 by presence of insertion g in position 396 that led to reading frame shift. CONCLUSION: The structure of tcfA gene circulating population of B. pertussis strains is homogenous and is characterized by presence of "vaccine" allele dominating in majority of countries in the world.

[A direct accelerated method for detection of a whooping cough pathogen].

The developed direct method for the laboratory diagnosis of pertussis, which is based on isothermal amplification technologies, has a high (100%) specificity and sensitivity (102 m.cl.), can detect the pathogen of the disease just in the clinical sample from a patient within 7-8 hours after start of the study. The clinical trials conducted at Infectious Diseases Hospital One (Moscow) on examination of 103 patients (63 patients with the clinical diagnosis of pertussis and 40 with other respiratory tract diseases) provided evidence its high specificity and diagnostic efficiency as compared with a bacteriological test, the efficiency in different clinical types of the disease and during examinations of patients in different periods after the onset of the disease, as well as during examinations of patients with suspected pertussis and pertussis-like diseases.

[Polymorphism of tox and dtxR genes in circulating strains of Corynebacterium diphtheriae].

Polymorphism of tox and dtxR genes responsible for diphtheria toxin synthesis was revealed. Seven point mutations in tox gene were detected; study of their combinations allowed to determine 10 allelic variants of the tox gene in C. diphtheriae strains. Majority of mutations did not lead to changes in substitutions in amino acid sequence of diphtheria toxin. In tox gene from 2 strains of mitis biovar, ribotype "Otchakov" isolated in Saint-Petersburg, mutation in position 1252 (G --> C), which corresponds to change of glycine on arginine in amino acid sequence of diphtheria toxin (G393R), was identified. Mutation localizes in R domain of fragment B of diphtheria toxin. In the dtxR gene 16 point mutations were registered; study of their combinations allowed to determine 10 allelic variants of the dtxR gene. Two mutations led to amino acid substitutions in regulatory protein DtxR: in position 640 (C --> A), which corresponds to change of leucine on isoleucine (L2141), and in position 440 (C --> T), which corresponds to change of alanine on valine (A147V). Mutation A147V is characteristic for all strains of epidemic clonal group (strains of biovar gravis, ribotype "Sankt-Peterburg/Rossija", enzyme types of complex 8), dominated in Russia during diphtheria epidemic at 1990s. Strains of this group were characterized by high level of diphtheria toxin production.

[Dynamics of changes in pathogenic characteristics of Bordetella pertussis strains].

AIM: To study pathogenic characteristics of B. pertussis strains isolated from patients during different periods of pertussis infection epidemic process. MATERIALS AND METHODS: Strains of B. pertussis isolated in Moscow during 1967 - 1971, 1980 - 1985, and 2001 - 2005 were studied. Nutrient media: Bordet-Gengou blood agar, casein-charcoal agar. ANIMALS: mice - F1 hybrids (CBA x C57BL6). Pathogenic characteristics of strains were studied by assessment of virulence (LD50), leukocytosis-stimulating (LS units) and histamine-sensitizing (HSD50) activities of cultures. Genotyping was performed using standard equipment and reagents for DNA isolation, amplification, sequencing and detection of results. RESULTS: On the sample of 164 strains, pathogenic and genotypic characteristics of B. pertussis populations circulated during 1967 - 1971, 1980 - 1985, and 2001 - 2005. Majority of B. pertussis strains isolated in 1967 - 1971 and strains circulated during current phase of epidemic process were virulent (80.75% and 81.8% respectively) and had significant leukocytosis-stimulating and histamine-sensitizing activity, whereas strains isolated from patients with pertussis in 1980 - 1985 characterized by lower virulence and toxicity. Genotyping showed strains carrying "non-vaccine" allele ptxA1, which emerged in the middle of 1970s, totally displaced strains with "vaccine" alleles ptxA2 and ptxA4. CONCLUSION: Adaptive changes of B. pertussis driven by increased vaccination coverage involve both ptxA gene and pathogenic characteristics of infectious agent in the range of genotypically homogenous population with domination of strains, which have high levels of virulence and toxicity.

[Molecular genetic method of rapid detection of Bordetella pertussis strains are possessed of different ptx genes].

Features of structure of different B. pertussis genes are studied in many countries of the world, and, first of all, ptxA gene, which encodes main protective antigen of the microbe--pertussis toxin. Starting from 1980s, B. pertussis strains with new "non-vaccine" allele ptxA1 gradually displaced strains with old "vaccine" alleles--ptxA2 and ptxA4, and now the formers dominate in circulating bacterial population. Molecular genetic method of rapid identification of B. pertussis strains, based on the differences in ptxA gene structure, was developed. The method using phenomenon of endonuclease restriction can be applied for differentiation of B. pertussis from B. parapertussis strains in diagnostic purposes.

[Modern strains of Bordetella pertussis: immunobiological properties and vaccine improvement].

Strains of B. pertussis isolated from patients in Moscow in 2001-2005 as well as strains included in locally produced diphtheria-tetanus-whole cell pertussis (DTP) vaccine were studied. Nucleotide sequences in genes of pertactin and S1-subunit of pertussis toxin of isolated strains, their immunobiological properties and opportunity to use for producing of the acellular pertussis vaccine were determined. Genes of pertactin and S1-subunit of pertussis toxin in the isolated wild strains differed from the same genes in strains included in the local DTP vaccine. Majority of the isolated strains belonged to serotype 1.0.3 and were markedly virulent.

[Molecular genetic characteristics of the B. pertussis strains isolated in different periods of the pertussis epidemic process].

Despite the fact that the mass immunization of the children population with the DPTs vaccine has been carried out in the Russian Federation since 1959, the pertussis infection persists to be one of the pressing problems for the children population. Although the vaccination coverage of the children population with pertussis vaccines is high in Russia, at present time the pertussis incidence rates are increasing among schoolchildren and remain high among infants younger than 12 months old. Many researchers believe that the variability of the genetic structure of the pertussis causative agent may be one of the causes of increasing pertussis incidence rates. This investigation provides the molecular genetic characteristics of 97 B. pertussis strains isolated in pertussis patients in Moscow in different periods of pertussis epidemic process since the 1950s up to present time. It shows the changes in the structures of genes, which are encoding the main protective antigens of the pertussis microbe that are the pertussis toxin (ptxS1) and the pertactin (pm). The structurre of the ptxS1 and pm gene of the B. pertussis vaccine strains was compared with the structures of these genes in the B. pertussis strains isolated from the pertussis patients at present time and also in past years. All B. pertussis strains isolated in the prevaccination period (1948-1959) and most strains (95%) isolated during the first twenty years of the mass immunization in Russia are characterized by the presence of the so called "vaccine" alleles of the pertussis toxin and pertactin genes that are ptxS1 B or ptxS1 D and pm 1 alleles that corresponds to the genetic structure of the vaccine producing strains. In the early 1970s the B. pertussis strains of another toxin and pertactin genetic structures with so-called "non-vaccinal" alleles ptxS1 A and pm 3 (pm 2 since 1980s) began to appear. The B. pertussis strains with "non-vaccinal" alleles have completely displaced the "old" strains. At present time in Moscow the pertussis disease is caused by the B. pertussis strains bearing ptxS1 A and pm 2 or pm 3 alleles of pertussis toxin and pertactin genes. There was no correlation between the genotype and serotype. Thus, the structure of the B. pertussis toxin and pertactin genes in strains which have been isolated since the 1980s up to now differs from the structure of these genes in strains which are used for producing DPTs vaccine. The data obtained in this investigation suggest that the genetic structure specificity of circulating B. pertussis strains that are producing the disease at present time should be used as one of the criteria for selecting vaccine producing strains.

[Epidemic process of pertussis in Moscow].

Materials reflecting the dynamics of pertussis morbidity during the period of 1958 - 2003 under the conditions of prolonged mass immunization of the child population with adsorbed DPT vaccine are presented. The planned vaccination of children led to the decrease of pertussis morbidity during the first 10 years, but groundless abstentions from vaccination during the 1980s - 1990s contributed to a sharp rise in morbidity among children of younger age groups. During the recent four years a rise in pertussis morbidity was registered in 2000 (71.79 per 100,000 of the population), followed by the most significant for the last 20 years drop in morbidity in 2002--down to 9.89. But in 2003 the growth of morbidity was again registered (38.67). Recently periodic rises and drops in morbidity occurred simultaneously with the increased coverage of children of younger age groups with vaccination. In recent years changes in the age structure of patients were observed: the specific proportion of school children increased (in 2003 morbidity rates in children aged 6 - 10 years were 288.6 - 270.7), simultaneously high morbidity among children aged up to one year (274.9) was registered. The specific proportion of pertussis-affected children aged above 7 years reached 65%. From the late 1990s until present in 87.1% of cases strains of serotype 1.0.3 prevailed in the population of B. pertussis strains. But in recent years the circulation of strains 1.2.3, spread in the prevaccination period and having toxicity similar to that of strains of serotype 1.0.3, while exceeding them in virulence, in sufficiently high proportion (7.0% in 2002) was noted. This was indicative of the possibility of the unfavorable development of the epidemic process of pertussis infection.

[Corynebacterium diphtheriae nontoxigenic strain carrying the gene of diphtheria toxin]. / Kharakteristika netoksigennykh shtammov Corynebacterium diphtheriae, nesushchikh gen difteriinogo toksina.

Among 828 C. diphtheriae nontoxigenic cultures isolated in different region of Russia in 1994-2002, 114 cultures (13.8%) had the gene of diphtheria toxin (gene tox) and were thus called nontoxigenic tox-carrying (NTTC) strains. All NTTC strains were found to belong to biovar mitis and formed neither normal, nor "defective" diphtheria toxin. The most of NTTC strains (94%) belonged to ribotype "Moskva", not occurring among C. diphtheriae toxigenic strains. The incapacity of NNTC strains of forming diphtheria toxin was caused by mutation: the deletion of one nucleotide which led to the shift of the open reading frame and to the formation of the stop codon. The results of these studies are indicative of the fact that a sufficiently homogeneous and isolated group of C. diphtheriae nontoxigenic strains is spread in Russia. These strains carry the nonexpressing gene of diphtheria toxin and are of no epidemic importance in diphtheria infection.

[Genetic structure of Corynebacterium diphtheriae strains isolated in Russia during epidemics of various intensity]. / Geneticheskaia struktura shtammov Corynebacterium diphtheriae, vydelennykh v Rossii pri raznoi intensivnosti épidemicheskogo protsessa difterii.

The genetic structure of C. dipthteriae toxigenic strains isolated in Russia during the period of more than 50 years was analysed. The use of the method of ribotyping made it possible to register 17 C. diphtheriae ribotypes. The study revealed that the genetic structure of C. diphtheriae population varied in the dynamics of the epidemic process: each epidemic cycle characterized by predominant spread of epidemic strains of definite biovars and ribotypes. Thus, C. diphtheriae strains of biovar gravis, ribotype M11, dominated in the 40-60 years and C. diphtheriae strains of biovar mitis, closely related ribotypes M1 and M1v, dominated in the 80 years. During the last epidemic rise of diphtheriae morbidity in the 90 s C. diphtheriae strains of biovar gravis, closely related ribotypes G1 and G4, dominated among circulating strains. The proportion of these ribotypes began to increase 3 years before the rise of morbidity. The data of microbiological monitoring are recommended for use in the prognostication of the development of the epidemic process of diphtheria infection.