Molecular Epidemiology of Extended Spectrum Β-Lactamase- Producing Enterobacteriaceae at the Komfo Anokye Teaching Hospital

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Bacterial isolates of the Enterobacteriaceae at the Komfo Anokye Teaching Hospital (KATH) have become resistant to almost all the commonest antimicrobial drugs, including the third generation cephalosporins, but the cause of which is unknown. Worldwide reports however have it that many such resistances to the cefotaxime, ceftriaxone and caftazidime for example are often due to the production of extended spectrum β–lactamases (ESBLs) by the organisms. This study therefore was undertaken to determine the prevalence and the genotypes of ESBL isolates and the phylogenetic clones of clinical isolates of E. coli. A total of 405 isolates comprising E. coli, 156(38.5), Klebsiella pneumoniae 234(57.8%) and Klebsiella oxytoca 15(3.7%) were studied. These were non-duplicate isolates collected from February to April 2008 and again from March to July 2009. The isolates were obtained from blood, urine, sputum, wound and others (pus, aspirates etc). Antimicrobial susceptibility of the isolates was determined using Kirby-Bauer disc diffusion method. ESBL phenotypes were determined by the double disc synergy method using cefpodoxime with co-amoxiclav, and then confirmed by the combined disc method. The ESBL genotypes, BlaTEM, BlaCTX-M and BlaSHV were determined using duplex PCR. ECOR type of ESBL positive E. coli isolates were determined. Out of the 405 isolates tested 234(57.8%) were ESBL producers. Among the E. coli were 77(49.4%) ESBL producers, 144(61.5%) were ESBL producers among Klebsiella pneumoniae and 13(86.7%) were among Klebsiella oxytoca. BlaTEM was the commonest, with prevalence of 96.2%, followed by BlaCTX-M (94.4%) and BlaSHV (32.5%) with as much as 151(64.5%) possessing two genes and 70(29.9%) of the isolates possessing three genes respectively. There were more ESBL producers among in-patients (64.4%) than there were in out-patients (39.7%), but the difference was not statistically significant (P= 0.2374). ECOR type of E. coli isolates belong to group B1 (74.0%) and group B2 (26.0%), no A or D genotypes were detected. A subset of 29 ESBL-producing isolates were further genotyped using multilocus sequence typing and all belonged to the same sequence type (ST88), which is a member of the B1 phylogroup. Representative ST88 isolates were further characterized for virulence factors that are associated with E. coli pathotypes (EAEC, EHEC, EPEC, ETEC, and ExPEC). Four fimbrial genes typically associated with ExPEC virulence were identified. Phylogenetic relationship among the isolates of ECOR collection and the ESBL ST88 producing clone from KATH was determined using a representative of the closest common ancestor as an outgroup for a dendogram. These data suggest that antibiotic use at KATH or in the community selected for the emergence and spread of a resistant ExPEC clone.
A thesis submitted to the School of Medical Sciences, Department of Clinical Microbiology, Kwame Nkrumah University of Science and Technology Kumasi in partial fulfilment of the requirements for the award of the degree (PHD IN CLINICAL MICROBIOLOGY), 2011