Prevalence of uropathogens, and molecular characterization of extended spectrum beta-lactamase producing escherichia coli isolates in Brong-Ahafo Regional Hospital-Sunyani

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May, 2015
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Antimicrobial resistance is a growing international problem. β-lactam antibiotics especially are heavily depended on and this has led to increase in the incidence of Extended-Spectrum Beta-lactamase (ESBL) worldwide, especially in developing countries. However, data on the existence and magnitude of ESBLs in urinary pathogens in local settings is limited. The aim of this study was to determine the prevalence of Uropathogens, and molecularly characterize ESBL producing Escherichia coli from urine samples, in a regional hospital. The study was a cross sectional one conducted at the Brong- Ahafo Regional Hospital, Sunyani in Ghana. Bacterial isolates were obtained from urine samples from both hospitalized and non-hospitalized patients. Isolates were tested for antimicrobial susceptibility on Mueller-Hinton Agar plates (Oxoid GmbH, Wesel, Germany) by the Kirby Bauer disc diffusion method. The results were expressed as susceptible or resistant according to the criteria recommended by the manufacturer or Clinical and Laboratory Standards Institute (CLSI). E coli isolates were further screened for ESBLs using cefotaxime, cefpodoxime and ceftazidime antibiotic discs and ESBLs production confirmed using CLSI’s combined disc method. Conventional PCR was used to detect bla CTX-M, and bla TEM type ESBLs and visualization of PCR products done by agarose gel electrophoresis. A total of 200 urinary pathogens were obtained between January and December 2014. Greater proportion of the Uropathogens were resistant to all the antimicrobials used. One hundred and seventy-two (172, 96.6%) isolates were resistant to tetracycline and 144(90.5%) isolates were resistant to Ampicillin/sulbactam. One hundred and fifty (150, 93.2%) isolates were readily susceptible to Amikacin. Among the third generation cephalosporins, ceftizoxime achieved 50% sensitivity and 20.6% and 16.7% for cefotaxime and ceftazidime respectively against all isolates. The isolates also showed v strong resistance to the fluoroquinolones. Nalidixic acid (80.8%); ciprofloxacin (74.1%); ofloxacin (65.4%) and levofloxacin (64.6%). Chloramphenicol and gentamicin achieved 23.4% and 24.1% sensitivity respectively. Of the 200 isolates, 51(26%) isolates were E. coli. Of the 51 E. coli isolates 43(84.3%) were ESBL producers and 8 (15.7%) were ESBL negative. The ESBL producing E. coli were significantly more resistant to all Antibiotics compared to other strains (p<0.0001). Lower resistance was observed in Amikacin and Ceftizoxime. Multi drug resistance was found to be more in ESBL producers than non ESBL producers. Molecular studies of the ESBLs revealed 26 (66.7%) of the E coli carried Bla TEM genes. Three (3) E. coli isolate showed extra band indicating a different gene other than the bla TEM. Twenty-three (23.1%) of the isolates were phenotypically positive for ESBLs but negative for both bla TEM and bla CTX-M genes. Twenty-eight (28, 71.8%) of isolates haboured bla CTX-M, while 22 representing 56.4% carried both bla TEM and CTX-M genotypes. The phenotypes and different bla genes in E. coli isolates implicated in UTIs in non-hospitalized and hospitalized patients is worryingly high in the Brong-Ahafo Regional Hospital. Data on Culture and susceptibility testing should guide therapy and surveillance studies for β-lactamase producers in developing countries in order to preserve the efficacy of β- lactam antibiotics.
Thesis submitted to the Department of Clinical Microbiology, Kwame Nkrumah University of Science and Technology, in partial fulfillment of the requirements for the degree of Master of Philosophy School of Medical Sciences, College of Health Sciences.