Изучение гетерогенности по признаку токсинообразования музейных штаммов Corynebacterium diphtheriae

The heterogeneity of toxigenic signs on the inside straining level of 32 museum strains of C. diphtheriae have been studied by Flec-test and PCR with the pair of specific primers for tox-gene. Four strains were isolated in Moscow in 1954-1959, one strain was isolated in Horlovka of the Donetsk region in 1961, other 5 strains in Kharkiw in 1963 (3 strains) and in 1991 (2 strains), and 22 strains in Lviv region in 1986 2000. Fourteen strains belong to gravis variant, 17 strains to mitis variant and one strain intermedius variant. The PW-8 industrial strain and NCTC 10648 gravis tox+ standart strain have been studied as well the purpose of comparison. Fifteen strains were toxigenic and 17 strains were non-toxigenic. The 8 non-toxigenic strains contain non-expression tox-gene. The strains for investigation were received from 2 sources: 1) the Museum of Microorganisms (State Institution) belonging to the I. I. Mechnikov Microbiological and Immunological Institute of the NAMSciences of Ukraine and 2) Lviv Research Institute of Epidemiology and Hygiene belonging to the Ministry of Health of Ukraine. Before PCR test we have reindentified the strains with the aim of stydying the toxin formation using Flec-test. The strains of C. diphtheriae have been replated in selective medium so as to have isolated colonies in a thermostate for 40-48 hrs. Synchronization of cultures has been achieved by means of "cold shock". Toxin formation, toxinogenicity and molecular genetic typing has been studied in 50 colonies of each strain. A commercial kit "DNAexpress" ("Lytex", Russia) has been used to express the chromosomal DNA. PCR with 2 specific primers for the gene of diphtheria toxin has been conducted using a commercial test system manufactured by "Amply-Sens" (Russia). A DNA-fragment has been amplified by 360 nucleotide pairs. Genotyping has been performed by PCR with arbitrary primer № 45 (5'GGATCCAAAACGACGGCCAGT-3') (Mokrousov I. V et al, 1996) (random amplification of polymorphic DNA PCR). PCR has been performed by Saiki et al. (1988) modification method, volume 25 mcl. Amplification has been conducted in the following parameters: t° 92°C for 3 mins, then 35 cycles at 92°C for 50 secs, 54°C for 60 secs, and 70°C for 60 secs, synthesis at 70°C for 3 mins. The obtained material has undergone by electrophoresis in a 2 % agarose gel with 1 % solution ethidium bromide with DC of 180 V for 3 3,5 hrs in a horizontal electrophoresis device. Only 7 (46,7 %) toxigenic strains were stable as to the toxigenic signs. Other 8 (53,3%) strains lost their toxigenic function in the replating on the elective mediums. One in eight non toxigenic strains with tox-gene had toxigenic function. It was determined that loss of toxigenic signs was not accompanied by a loss tox-gene by PCR with the pair of specific primers. These strains can restore toxigenic signs and cause disease. The inside straining level heterogeneity was determined due to genotyping by PCR with the arbitrary primers (random amplification of polymorphic DNA PCR). It has been found that 8 C. diphtheriae strains have genetic polymorphism. Probably, spontaneous genic conversion occurs.