Drug susceptibilities and mechanisms of drug resistance Among salmonella typhimurium isolates from persons in Western Kenya

Abstract

Drug resistant microorganisms are a major worldwide health issue, as a number of important human pathogens have now acquired mechanisms that make them largely resistant to most currently available treatment regimens. The action of antimicrobial compounds can be negated at a number of points, including enzymatic inactivation, the employment of alternative metabolic pathways to bypass their activity, sequestration, reduced uptake, and alteration of the target site to render it not susceptible to the effect of otherwise toxic substances. This study emphasized on the molecular characterization of 20 antibiotic resistant S typhimurium strains in Western Kenya collected from two health care centers. Consent to undertake research study in Mukumu and Maseno Mission hospitals was obtained from the respective hospital authorities. S typhimurium was isolated by conventional microbiological culture methods in 34 stool specimens from randomized inpatients and outpatients having diarrhoea, and fever of 38°C and above Phenotypic and genotypic identification of the respective S typhimurium strains was performed using serotyping, biochemical testing and distinct molecular tests including 16S rRNA sequencing. Antimicrobial screening was done by agar-disc-diffusion, E-test and automated VITEK R 2 for a total of 22 antimicrobials. The presence of tetracycline, ampicillin, streptomycin, gentamicin, sulfamethoxazole and kanamycin resistance genes was studied as well. The diversity of the strains was analyzed using Pulsed Field Gel Electrophoresis (PFGE), fluorescence Amplified Fragment Length Polymorphism (fAFLP) and Multi-Locus-Variable-Number-Tandem regions (MLVNTR). Out of the 45 isolates obtained from selected hospitals in Kenya, 37 were S typhimurium, 8 were S typhi. However in Germany, 13 of the isolates were identified as S typhi; 20 isolates were identified as S typhimurium. Four strains could not be clearly identified. Malic dehydrogenase (mdh) a house keeping gene fragment of 261bp fragment for Salmonella typhimurium was amplified for all 20 isolates. All the 20 isolates were positive for S typhimurium but negative for S enteritidis. Four of the 20 strains selected for 16S rRNA were found to be 99% homologous to the S typhimurium LT2 sequence. Microrestriction PFGE analysis by XbaI gave patterns A, 72.7%; patterns B, C, D and E were 4.5%. Among the typed phages, three isolates were type 1, and eight, were type 2 and five were type 3. The phage DT 104 was not detected among the strains under study. VI MA·~·-~·-·'-I·N:··l\VERSITY '-' ••.•••• ",-, f· >, S.G. S. L'B'RARY Integron analysis indicated 9 strains positive for class I integron with six isolates having l.2Kb fragment and three isolates displaying 0.8Kb fragment. Four plasmid profiles (i) 70, 55, l.8, l.4Kb (ii) 70, l.8, l.4Kb (iii) 70, 4.6, Kb(iv) 60Kb were observed .Profile type (ii) was the most abundant followed by type (i), (iv) .and type (iii) respectively. Salmonella plasmid virulence factor (spv) and invasive gene (inv) was amplified for all strains. Genetic polymorphism, was generaly not observed except for afew isolates that displayed polymorphism. The bigA gene was the most variable with repeat lengths of 33bp. S. typhimurium drug resistant to ampicillin, streptomycin, sulfamethoxazole-trimethoprim, chloramphenical, amikacin,kanamycin, gentamicin, tetracycline and ciprofloxacin was observed respectively both by agar disc diffusion and VITEK R2 respectively. For clarification we used E -test strip (AB Biodisk, Solna., Sweden) for tetracycline and the results confirmed the agar diffusion test in Germany. For further confirmation molecular methods were usedto analyze resistance genes within the isolates. In this regard, the presence of tetA, tetB, tetC, tetD, tetE, tetG were determined. The obtained results indicated one S. typhimurium and two S. enteritidis positive samples were tetracycline resistant and harbour tetracycline resistance gene tetA (256bp) responsible for tetracycline drug efflux pump. tetB was observed in a few S. typhi isolates. S. typhimurium molecular antimicrobial results showed the presence of resistance gene to Grm , aadB , blapsEl , bla TEM , aadA , strB . Both phenotypic and genotypic cephalosprins, peniciIIins, tetracycline and aminoglycosides antimicrobial drug tests were done and the results indicate that the isolates harbour genes encoding for streptomycin, ampicilin, and gentamicin. Conclusion arrived is that both the phenotypic and genotypic methods of characterization, as well as the expansion of genotyping to a broader range of genes, are essential to understand antibiotics resistance as weII as the epidemiologic relationship of Salmonella isolates as has been shown by this study. The generated data clearly show that S. typhimurium strains from Western Kenya show heterogeneity and are also different from strains which originate from other parts of Kenya and around the world. It is also concluded from the study that phage type DT 104 is not the sole source of multiple antibiotic resistance in S. typhimurium isolates since none of the isolates had the phage though antibiotic resistance was observed. Antimicrobial susceptibility testing showed that 100% of S. typhimurium isolates were resistant to ampicillin, and in most cases, this resistance was inassociation with choramphenical, streptomycin, sulphonamides and tetracycline resistance. Conclusion also ViI drawn is that, most of the isolates had a pentadrug resistance to ampicilin, amikacin, chloramphenical, streptomycin, sulfamethoxazole-trimethoprim, and tetracycline. Antimicrobial resistance was fairly low as compared to the world resistance patterns and more studies need to be done to establish the reason. S. typhimurium strains were tetracycline resistant and possessed tetracycline resistance gene tetA responsible for tetracycline drug efflux pump. In conclusion, there is increasing evidence that the role of efflux pumps in antibiotic resistance in S. typhimurium is significant. Therefore the effect of efflux pumps needs to be considered in the design of future antibiotics and the role of inhibitors assessed in order to maximize the efficacy of current and future antibiotics.