The evolution of TEM-1 extended-spectrum β-lactamases in E. coli by cephalosporins

Abstract Objectives This study was conducted to examine the molecular mechanisms responsible for evolution of TEM-type extended-spectrum β-lactamases (ESBLs), following selective pressure from four third-generation cephalosporins; ceftazidime, cefotaxime, ceftriaxone and ceftibuten. In addition, the approach selective enrichment for ESBL detection in environmental samples was investigated. Methods By the use of experimental evolution, resistant variants were isolated and mutations in TEM-1 were examined by DNA sequencing. Using E-tests and disc diffusion assays, resistance levels and development cross-resistance were determined for ESBL producers. Selective plating with or without prior growth in selective broth was used to examine the approach of selective enrichment for ESBL detection. Results The third-generation cephalosporins ceftazidime, cefotaxime and ceftriaxone selected for ESBL, while ceftibuten did not select for ESBL. All ESBL variants additionally remained susceptible towards ceftibuten. DNA sequencing of the TEM-1 coding sequence of mutants, revealed mutations that not previously had been isolated through selection. This indicates that the potential for ESBL evolution is much broader than can be inferred from sequence analysis of clinical samples alone. Results: also indicate that selective enrichment for the enhanced detection of ESBL producers may give unreliable results due to the selection of spontaneous mutations in the narrow-spectrum β-lactamases such as TEM-type ESBL producers. Conclusions These results help explain the molecular changes responsible for evolution of TEM-type ESBLs, meanwhile question the appropriate use of selective enrichment for detection of ESBLs in environmental samples.