[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.

[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.

[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.

[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.

[The use of DNA fingerprint analysis for the differentiation of populations of toxigenic Corynebacterium diphtheriae]. / Primenenie fingerprintnogo analiza DNK dlia differentsiatsii populiatsii toksigennykh Corynebacterium diphtheriae.

13 C. diphtheriae strains were used as a model to establish the conditions of making the fingerprint analysis of chromosomal DNA. These strains, subdivided into 7 groups in accordance with the character of their restriction splitting, were mostly isolated from territorially close sources and belonged to the same phagotype. Probably, C. diphtheriae DNA has strain variations manifested by an unequal number and location of the sites of the recognition of specific endonucleases, which may be used for the intraspecific differentiation of C. diphtheriae.

[The coagglutination reaction for detecting diphtheria toxin]. / Reaktsiia koaggliutinatsii dlia vyiavleniia difteriinogo toksina.

High-titer antidiphtheria antitoxic rabbit serum has been obtained, and on the basis of this serum a coagglutinating diagnosticum has been developed. The sensitivity of the test has been found to depend on the content of antitoxic antibodies in the serum and on its purity. Diagnostica prepared from native serum containing 500 I. U./ml (a titer of 1:51, 200 in the passive hemagglutination test) permit the detection of 0.02-0.03 Lf/ml of diphtheria toxin. A decrease in antibody titer to 5-25 I. U./ml leads to a drop in sensitivity to 0.2-2 Lf/ml. The use of LgG fraction and pure antibodies increases the sensitivity of the test to 0.002-0.003 Lf/ml. The possibility of detecting toxin in Corynebacterium diphtheriae strains is shown.

[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.