Molecular characterization of selected enterococcus strains (previously streptococcus) using genotyping techniques.

Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.The genus Enterococcus comprises of a group of commensal organisms of the human gut which has been associated with cases of endocarditis and urinary tract infections. In the present study, 12 Enterococcus isolates were obtained from clinical specimens and characterized using genotyping techniques that have become an integral part of clinical research. There were three different genotyping methods used to identify the enterococci to species level and to determine the level of genetic diversity among the selected strains. These techniques were, randomly amplified polymorphic DNA-PCR (RAPD-PCR), 16S rDNA ribotyping analysis and pulse field gel electrophoresis (PFGE) respectively. The minimum inhibitory concentration (MIC) to penicillin and vancomycin were also determined using a disc diffusion assay and a microtitre plate dilution assay. All twelve strains were found to be vancomycin resistant enterococci (VRE) at a MIC value greater than 100μg/ml. Penicillin growth inhibition based on MIC values were categorized into three groups, susceptible (< 0.25 μg/ml), intermediate (≤ 3μg/ml) and resistant (≥ 4μg/ml) respectively. RAPD-PCR was performed using four random primers. Primers yielding the highest discriminative power were used for phylogenetic analysis. The phylogenetic analysis indicated that all 12 strains yielded clonal dissemination, therefore a low genetic diversity between them. The 16S rDNA of all strains were used to identify the enterococci at species level. The rDNA were sequenced and analysed using the NCBI BLAST algorithm and found to belong to three species of Enterococcus. These were E.faecalis, E.faecium and E.durans. PFGE analysis was performed by restriction of all 12 strain’s genomic DNA with the restriction enzyme SmaI. The PFGE patterns were divided into two groups with low genetic diversity. Compared with the RAPD PCR patterns PFGE gives a higher discriminatory power as a higher dissimilarity between the strains was observed. Similar penicillin MICs for each of the strains in the three categories are grouped together in the phylogenetic trees for both PFGE and RAPD-PCR. RAPDPCR is a sensitive, faster, specific and cost effective technique, PFGE analysis has given a higher discriminatory power, higher reproducibility of the results and the polymorphism seen in the patterns suggest that PFGE has a potential of being an essential tool in clinical diagnostics