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Authors: EZEAMAGU, C. O.
Keywords: Methicillin resistance
mecA gene
staphylococcus species
Plasmid profile
Issue Date: Mar-2014
Abstract: Methicillin-resistant staphylococcus (MRS) infections are of global concern in healthcare institutions and community settings with significant morbidity and mortality due to multi-drug resistance challenges. In Nigeria, most methicillin resistance detection was based on phenotypic method with scanty reports on molecular characterisation of MRS. In this study, molecular techniques were used to determine the presence of methicillin resistant gene (mecA) with its associated resistance determinants (vanA and blaZ) and plasmid profile of staphylococci isolated from clinical and community samples. Staphylococcus species from clinical (55) and community (53) samples were isolated from air, selected waste water drainages and human swabs (eye, semen, ear, high vagina swab, throat, urethra, wound, nostril, skin) in the University of Ibadan and University College Hospital. They were identified using standard microbiological procedures. The isolates identity was confirmed to genus level using 16S-rRNA specific primers and identified to species level by PCR-Restriction Fragment length Polymorphism supplemented with PCR species-specific-primers. The isolates were phenotypically screened for resistance to methicillin and other antibiotics by agar diffusion method. Multiplex PCR was used to assess presence of mecA and the resistant determinants while Simplex PCR was used to determine the origin of mecA isolates by detecting the presence of Panton-Valentine Leukocidin (PVL). Plasmid profiles of clinical (35) and community (19) isolates with multiple drug resistance were determined using standard procedures. Data was analysed by descriptive statistics. The organisms were identified as S. epidermidis (92.6 %), S. aureus (6.5 %) and S. xylosus (0.9 %). Phenotypic resistance to methicillin was 72.7 and 62.3 % in clinical and community isolates respectively. In the clinical isolates of S. epidermidis, 30.9, 32.7, 34.5, 40.0, 41.8, 60.0, 76.4, and 89.1 % were resistant to Chloramphenicol, Vamcomycin, Streptomycin, Erythromycin, Gentamycin, Tetracycline, Cotrimoxazole and Cloxacillin respectively. Correspondingly, in community isolates of S. epidermidis, 28.3, 3.8, 32.1, 50.9, 26.4, 58.5, 90.6 and 92.5 % were resistant to these antibiotics. In the clinical isolates of S. aureus, 3.6, 5.5, 5.5, 7.3, 7.3, 7.3, 9.1 and 9.1 % were resistant to Vamcomycin, Erythromycin, Chloramphenicol, Streptomycin, Gentamycin, Tetracycline, Cotrimoxazole and Cloxacillin respectively. In community isolates, 1.9 % S. aureus were resistant to Cotrimoxazole, Chloramphenicol, Erythromycin, Gentamycin and Streptomycin while 3.8 % were resistant to Cloxacillin. Among clinical isolates of S. xylosus, 1.8 % was resistant to all the antibiotics except Chloramphenicol and Streptomycin. All the strains lacked vanA gene, while only clinical isolates (3.6 %) had mecA when its specific primers were used and 5.5 % using its regulatory element specific primers in PCR. The blaZ gene was found in 16.4 % of clinical and 1.8 % of community isolates. There was no PVL in the isolates with mecA. Plasmid size of 23.13kb was found in 94.3 % of clinical and 84.2 % of community isolates. The detection of blaZ gene in community isolates showed that such resistance determinants predominantly found in clinical isolates are also emerging in the community isolates. Hence, setting up antibiotic surveillance system is necessary to minimize this trend.
Description: A thesis in the Department of Microbiology Submitted to the Faculty of Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY of the UNIVERSITY OF IBADAN
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