Biomedical subjects
Publications and source records attributed to A I Korotiaev.
The effect of R- and Hly-plasmid differing in phenotype, molecular size, conjugativity, stability and incompatibility properties on the cell cycle and nucleic acids content per cell has been studied in Escherichia coli. According to these properties the plasmids were divided into 3 categories. The possibilities of the autonomous plasmid replicon interactions with the host chromosome are discussed.
The Sfl2aI system of restriction-modification (RM) was revealed in the cells of Shigella flexneri encoded by pKMR114 plasmid belonging to the IncN incompatibility group. The genes for Sfl2aI RM system were cloned. The system was ascribed to the enzymes of the EcoRII specificity, as shown by in vivo and in vitro experiments. Restriction analysis of these genes' region and antigenic properties of the Sfl2aI endonuclease pointed to significant differences between this and EcoRII RM systems.
Two restriction-modification systems specified by two plasmids are discovered in the clinical species of Shigella. The plasmids are designated pKMR114 and pKMR115. Both are of 60.800 bp and belong to the IncN incompatibility group. The EcoRI, EcoRV, HindIII restriction patterns of both plasmid DNAs are identical. As shown by efficiency of plating of bacteriophage lambda vir on the strains harbouring plasmids encoding EcoRI, EcoRII, EcoRIII, EcoRIV, EcoRV systems and plasmids studied, the discovered plasmids control synthesis of EcoRII specificity enzymes. The main distinctive feature of the pKMR114 is the ability to decrease efficiency of plating of bacteriophage T4 having glycolised DNA.
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Physical maps of Enterobacteriaceae nonconjugative plasmids pKMR281 (Sm, Su, molecular weight of 6 kb) and pKMR285 (Sm, Su Tc, molecular weight of 9 kb) were constructed for endonucleases EcoRI, PstI, EcoRV, SmaI, BglII, SalI and PvuII. The genes controlling production of aminoglycoside-3''-phosphotransferase and dihydropteroate synthetase of type II and the genetic tetracycline resistance determinant of class A were localized on the plasmids. Heteroduplex analysis of plasmids pKMR281 and pKMR285 showed that plasmid pKMR281 was completely homologous to plasmid pKMR285. The site of plasmid pKMR285 nonhomology with respect to plasmid pKMR281 corresponded to the area containing the tetracycline resistance determinant. Plasmids pKMR281 and pKMR285 were compatible with the tester plasmids of 25 incompatibility groups of Enterobacteriaceae and probably constitute a new incompatibility group. It was shown that pKMR281 and pKMR285 type plasmids were widely distributed in clinical strains of E. coli and Shigella spp. isolated in the Krasnodar Region.
Antibiotic resistance of enterobacterial strains from population isolated in Krasnodar region is rather often controlled by the "plasmid" genes. The conclusion is based on using the colony hybridization with [32P]-DNA fragments of plasmids, carrying the genetic determinants of antibiotic resistance, as a method for antibiotic resistance, genes screening. Kanamycin resistance in the majority of strains is coded by APH (3') II gene, streptomycin resistance by APH (3") gene, chloramphenicol resistance by CATI, sulphonilamide resistance by DHPS type II gene. Tetracycline resistance of the studied enterobacterial strains is not connected with the widespread genetic determinants of a new class tetracycline resistance.
Shigella strains showed that in 43.9 per cent of the strains this feature was controlled by conjugative R plasmids. On the whole these were the plasmids allotting the bacterial cell with resistance simultaneously to SmTcCm (37.1 per cent) and SmTc (17.1 per cent). The plasmids with other phenotypes were less frequent: SmApCm, 11.4 per cent, TcApCm, 8.6 per cent; SmTcApCm, 8.6 per cent; Tc, 8.6 per cent; Sm, 2.9 per cent; Cm, 2.9 per cent and TcCm, 2.9 per cent. The incompatibility groups of 19 plasmids were determined: Inc I zeta, 28.6 per cent; Inc zeta I and Inc B, 14.2 per cent; Inc FII, 8.6 per cent and Inc I alpha, 2.9 per cent.
Five plasmids detected in the clinical strains of Shigella were studied. The plasmids had similar phenotypes of antibiotic resistance and allotted streptomycin and tetracycline resistance to the host cell. None of them allotted the bacterial cells with sensitivity to phages specific for the Inc groups F, N, P and I. The molecular weights of the plasmids ranged from 47 to 50 MD. The studies on the incompatibility showed that pKMR 227, pKMR 228-1, pKMR 231, pKMR 242-1 and pKMR 245 plasmids simultaneously belonged to two Inc groups: Inc I zeta and Inc B. Two plasmids, i.e. R 805a and R 861a simultaneously belonging to the two Inc groups: Inc I zeta and Inc B are described in the literature. pKMR 231 and pKMR 242-1 plasmids detected by the authors differ from them by the antibiotic resistance phenotype. The other three pKMR plasmids in addition differ from them by belonging to Fin.
Nonconjugative R-plasmids pBS76 and pBS94 (Sm Su), pBS95 and pBS96 (Sm Su Ap) isolated from clinical strains of Pseudomonas aeruginosa and plasmids pKMR281-pKMN284 (Sm Su), pKMR285-pKMR286 (Sm Su Tc) isolated from clinical strains of enterobacteria have been studied. Restriction maps of these plasmids are presented in the paper with some of plasmid genes for antibiotic resistance localized on them. The resistance determinants of plasmids pBS95 and pBS96 are shown to be included in transposon Tn3612 analogous to Tn3. Plasmids pBS76, pBS94-96 are of the wide host range and belong to incompatibility group P4 (IncQ). Plasmids pKMR281-pKMR286 are mutually incompatible and share the conspicuous DNA homology. They are inherited only by enterobacteria and are compatible with IncQ plasmids but in contrast to them are mobilized by RP4 plasmid with lower frequency.
One hundred and eighty-six clinical strains of Enterobacteriaceae isolated in the Krasnodar region and Krasnodar in 1982 from patients with acute intestinal diseases were studied. 137 strains of Shigella were investigated in detail. It was shown that S. flexneri, S. sonnei, S. boydii and S. dysenteriae accounted for 77, 15, 5 and 2 per cent, respectively. 78.1 per cent of the wild Shigella strains were resistant to antibiotics and in 43.9 per cent of the strains this property was controlled by conjugative R plasmids. 11 variants of the antibiotic resistance spectra were revealed. However, strains of SmTcApCm, SmTc and SmTcAp phenotypes were the most frequent. Among the resistant strains 80.3 per cent were resistant to tetracycline, 75.7 per cent to streptomycin, 50.4 per cent to ampicillin, 31.7 per cent to chloramphenicol, 1.8 per cent to trimethoprim, and 0.9 per cent to kanamycin. The investigation showed that the strains isolated in one region usually had similar phenotypes of antibiotic resistance. The frequency of the plasmid transfer varied from 10(-1) to 10(-7). However, the majority of the plasmids were transferred at a frequency of 10(-4)-10(-5). Only one of the investigated plasmids had a capacity for transmitting fd phage sensitivity to the host cells. 33 plasmids belong to the fi+ type and the others belong to the fi- type. The majority of the plasmids have molecular weights of 46 to 50 mD.
KMR plasmids controlling antibiotic resistance and the capacity for production of the colonization antigen were identified in wild strains of E. coli (026, 0126, 0124) and S. sonnei isolated from patients with acute intestinal diseases. The strains of E. coli 026 and E. coli 0126 carried p KMR207-1 plasmid determining resistance to chloramphenicol and tetracycline and the adhesive properties. The molecular weight of the plasmid is 98 mD. The strain of S. sonnei carried p KMR 208-1 plasmid responsible for resistance to streptomycin, chloramphenicol and tetracycline and the adhesive properties. The molecular weight of this plasmid is 98 mD. The resistance to streptomycin and tetracycline and the capacity for the synthesis of the colonization antigen in E. coli 0214 was controlled by p KMR209 plasmid with the molecular weight of 2.66 mD. The restriction analysis suggests that p KMR207a-1 and p KMR 207b-1 plasmids detected in E. coli of different serotypes were identical, since they could be broken with BamH1 endonuclease into equal numbers of fragments similar by their molecular weights. p KMR207-1 and p KMR208-1 plasmids differed in their sensitivity to BamH-1 endonuclease. However, they were broken into 6 fragments similar by their molecular weights. p KMR207-1 and p KMR208-1 plasmids are probably closely related but not identical.
pKMR207-1, pKMR208-1, pKMR209, pKMR210 and pKMR212 plasmids controlling simultaneously antibiotic resistance and capacity for synthesis of the colonization antigen were detected in wild strains of E. coli of various serotypes. The properties of compatibility of plasmids pKMR to each other and their attitude to the known incompatibility groups were studied. It was shown that pKMR207-1, pKMR208-1, pKMR210 and pKMR212 plasmids were incompatible. These plasmids had the incompatibility properties with respect to R388 (Inc FI) plasmid and probably belonged to the incompatibility group Inc. FI. pKMR209 plasmid was compatible with pKMR207-1, pKMR208-1, pKMR210 and pKMR212 plasmids and belonged to another incompatibility group.
The nucleotide composition of chromosome and plasmid DNA free of hybrid RNA isolated from resting Escherichia coli cells preliminary cultivated with the help of [14C] uracil has been studied. It has been established that DNA contains [14C]uracil side by side with adenine, thymine, guanine and cytosine. It confirms the presence of RNA fragments in the composition of bacterial DNA which are connected with it covalently.
DNA content in bacterium Bacillus subtilis, Pseudomonas aeruginosa. Proteus vulgaris, Vibrio proteus at different growth condition has been studied. It has been shown that during the cultures growth DNA content is various independently of culture age and as a rule exceeds one chromosome weight and depends on growth condition.
The clinical isolate of Sh. flexneri 1b, resistant to 5 antibiotics and sulfonamides, has been studied by the method of conjugation and found to have a group of transfer-suppressed pKMR-plasmids: pKMR 204-1 (Ap Sm Tc Cm Km Su), pKMR 204-2 (Sm Km Su), pKMR 204-5 (Km Su) and pKMR 204-7 (Sm Tc Cm Km Su), whose molecular weight was 99, 71.2, 73.8 and 59.5 Md respectively. The treatment of the plasmids with restriction endonuclease BamHI has revealed that plasmids pKMR 204-2 and pKMR 204-5 are definitely related to plasmid KMR 204-1, being its deletion mutants. At the same time plasmids pKMR 204-1 and pKMR 204-7 differ in their sensitivity to endonuclease BamHI and stably coexist within the same cell, thus seeming to belong to different compatibility groups.
After the transfer of prototype plasmids R6K (IncX), R387 (IncK), R27 (IncH1) and T (IncN) to E. coli M nalr the appearance of histidine-dependent mutants (R27, T), histidine-leucine-dependent mutants (R6K), methionine-proline-dependent mutants (R387) was observed among the resulting transconjugates. The mutations of E. coli M nalr R+ cells induced by the introduction of the plasmids were accompanied by the transformation of the cells from the S-form into the R-form. In contrast to the prototrophs E. coli M nalr, the auxotrophs carrying plasmids R6K, R27, T acquired sensitivity to phage T7, and the methionine-proline-dependent mutant became sensitive to phages T and T7. The above-mentioned plasmids rendered E. coli M cells capable of synthetizing the donor pili. But the adsorption of phages T3 and T7 on the auxotrophic cells, both with and without plasmids, occurred due to their interaction with the cell-wall receptors.
The morphological properties, cell cycle and DNA content in rifampicin- and nalidixic acid-resistant mutants of E. coli M without plasmids and with R plasmids R6K and pKMR200 were studied. It was shown that the cells of the strains both containing the plasmids and containing no plasmids had elongated bacteria in their populations. Such bacteria were found to be nonseparated rods, 2-4 in number. The plasmids had an effect on the cell cycle period and DNA content: the presence of either R6K or pKMR200 induced lowering of the generation time and DNA content as compared to those in the strains without plasmids.
pKMR-plasmids controlling the antibiotic resistance and adhesive properties were isolated from clinical strains of E. coli O26 and O124, and Sh. sonnei. Two of them, i.e. pKMR 207 and pKMR 208 were conjugative. On conjugation they jointly transferred the features of the antibiotic resistance and capacity for production of the colonization antigen. The studies on transformation of E. coli K 12 802 with the plasmid DNA of E. coli O124 showed that the antibiotic resistance and colonization properties in E. coli O124 were controlled by the nonconjugative plasmid pKMR 209. It was found that plasmids pKMR 207 and pKMR 208 had the fi(-)-phenotype. None of the plasmids allotted the host cells sensitivity to the donor specific phages of the incompatibility groups F, N, P, W, and I. Probably, the plasmids did not belong to these incompatibility groups. When the cells of E. coli K 12 802 were transformed with the plasmid DNA of the wild strain to the hemolytic strain of S. typhimurium with multiple antibiotic resistance, 3 pKMR 210 plasmids with different markers of the antibiotic resistance were detected in the transformants. One of the plasmids controlled both the drug resistance and the capacity for production of hemolysin. The ability of the detected pKMR plasmids to inhibit fertility and relation to the donor specific phages was studied.