Evaluation of phenotypic and genotypic extended-spectrum beta-lactamase detection methods

The emergence and spread of resistance in Enterobacteriaceae is a growing concern in human medicine today. Enterobacteriaceae producing extended-spectrum β-lactamases (ESBLs) have become efficient at inactivating β-lactam antibiotics especially the newer third generation cephalosporins. In addition, ESBL producing Enterobacteriaceae are frequently resistant to other groups of commonly used non-β-lactam antibiotics such as the fluoroquinolones. Reliable, rapid and low cost methods to detect ESBLs in clinical microbiology laboratories are therefore required. The aim of this project was to evaluate the phenotypic and genotypic detection methods for ESBLs and to examine the optimum antimicrobial agent(s) for ESBL detection. A comparison with the CLSI susceptibility test and the ESBL screen test was performed using a number of clinical isolates of E. coli and Klebsiella spp. suspected to contain ESBLs. Two confirmatory tests, the double disc synergy test and the combination disc test for ESBLs were also compared. Single and multiplex PCR assays were established using primers for the TEM-, SHV- and OXA-type β-lactamases. The results of this study show that ESBL screening is required in routine laboratories for successful detection of ESBLs. The best indicator cephalosporin for detection of ESBLs in E. coli was cefpodoxime whilst the best indicators for detection of ESBLs in K. pneumoniae were cefpodoxime and ceftazidime. The combination disc confirmatory test demonstrated the highest rate of detection. The multiplex PCR assay was found to be a rapid and cost-effective method for ESBL detection. However, nucleotide sequencing is required to confirm ESBL production amongst these organisms