| dc.description.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.
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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
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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. | en_US |
