Genotypic identification of tetracycline resistance genes in Salmonella Typhimurium from man and bovine
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Tetracyclines are still the second most commonly used antibiotics worldwide, after penicillins. However, their efficacy against a variety of bacterial and rickettsial disease agents has been reduced by the high frequency of microbial resistance. Initially, organisms isolated from patients seemed to be resistant to one antibiotic. From the 1950's multiple-drug-resistant organisms were noted. The objectives of the present study were: (1) To determine the minimum inhibitory concentrations of oxytetracycline hydrochloride to S. typhimurium strains isolated from man and cattle in Kenya. (2) To identify tetracycline resistance genes belonging to the types A, B or C amoDg the strains. (3) To determine the location of these genes in the S. typhimurium strains. (4) To determine the total plasmid content of the strains for use as epidemiological tools. Salmonella typhimurium strains from man and cattle were examined for tetracycline resistance genes using synthetic probes A, B, and C Ninety-seven (97) strains isolated from either blood, stool, urine, pus or cerebrospinal fluid of patients at Kenyatta National Hospital during the period 1988-90, and 17 strains isolated from cases of calf diarrhea reported to the Department of Veterinary Pathology and Microbiology, University of Nairobi, were used in this study. The determination of minimum inhibitory concentration (M.LC) was done using the agar dilution method. Strains with M.LC >5Ilg/ml were categorized as resistant to oxytetracycline. Ninety-three percent (93%) of the human strains and 83% of the bovine strains were resistant. In general, resistant bovine strains had higher M.LC.s than human strains. Ten percent (10%) of the strains were sensitive to oxytetracycline. Plasmid isolation was carried out by the principle based upon the disruption of the bacterial cell wall by treatment with lysozyme, lysis of the internal cell membranes with detergent, and denaturation of chromosomal DNA by alkaline pH. Plasmid DNA was recovered by ethanol precipitation in the cold. Plasmids of various molecular weights were separated by electrophoresis. Total plasmids were classified into 6 plasmid profile groups on the basis of molecular weights. Fourteen (14) strains lost plasmids during subculturing and subsequent plasmid isolation procedures. The major (heavy) plasmids were 65, 63(60), 46, and 36 megadaltons (Mda). Plasmid profile analysis indicated that group 2 <major plasmids 65 and 46 megadaltons) contained 37(40%) of the human strains and 4(25%) of the bovine strains. Colony and Southern blot hybridization were carried out using 'Y 32P-labelled synthetic tetracycline resistance probes A, B, and C on synthetic nylon filters. Colony blot hybridization results indicated 22 strains (19 human and 3 bovine) were positive for tetracycline resistance determinant type A, 7 and 5 human strains tested positive for presence of tetracycline resistance determinants type Band C respectively. Sixty percent (60%) of the resistant strains were negative for resistance determinants A, B or C and may belong to other tetracycline resistance determinants. Three (3) human strains had determinants encoding both type A and B tetracycline resistance. Southern blot hybridization results revealed a 65 (also the 63 and 60) Mda plasmids and a smaller 5.2 Mda plasmid as responsible for carrying the resistance determinants A, Band C encoding tetracycline resistance. An apparently cryptic plasmid 4.0 MDa was found in all the human and bovine strains that had plasmids. This plasmid may be an important epidemiological marker. There was no consistent connection between plasmid profile groups and M.I.e. figures for the strains. However, plasmid profile group 2 (major plasmids 65 and 46 Mda) contained 60% of all strains that had the resistance determinant type A. This same group showed 25% similarity in total plasmid content of human and bovine strains and may serve to emphasize the possibility of clonal origin of human and bovine S. typhimurium strains. All the strains that tested positive for presence of tetracycline resistance determinant type Band Chad M.I.e. figures >64Jlg/ml. However, there was no consistent relationship between M.Le. figures and resistance determinant type A. The findings of this study indicate that plasmid profiling is a useful tool for characterizing strains from common sources and the spread of such strains and their antimicrobial resistance determinants. A remarkably high level of resistance by human and bovine strains to oxytetracycline was observed. There exisfs a potential cross-transfer of resistance among human and bovine populations as evidenced by the results of the Southern blot hybridization. The emergence and transmission of resistance factors, even among the once tetracycline-sensitive microbial strains, may result in treatment failure and subsequent rise in treatment costs.