Real-time PCR and Melting Curve Analysis for Reliable and Rapid Detection of SHV Extended-spectrum Beta-lactamases

Extended-spectrum beta-lactamases (ESBLs), e.g., ESBLs of the TEM or SHV type, compromise the efficacies of expanded-spectrum cephalosporins. An SHV non-ESBL that hydrolyzes only narrow-spectrum cephalosporins can be converted into an SHV ESBL through substitutions at three amino acid positions, 179, 238, or 238--240. In order to improve detection of SHV ESBLs, a novel method, based on real-time PCR monitored with fluorescently labeled hybridization probes and followed by melting curve analysis, was developed. It is able to (i) detect bla(SHV) genes with high degrees of sensitivity and specificity, (ii) discriminate between bla(SHV non-ESBL) and bla(SHV ESBL), and (iii) categorize the SHV ESBL producers into three phenotypically relevant subgroups. This method, termed the SHV melting curve mutation detection method, represents a powerful tool for epidemiological studies with SHV ESBLs. It even has the potential to be used in the diagnostic microbiology laboratory, because up to 32 clinical isolates can be processed in less than 1 h by starting with just a few bacterial colonies.