Piperacillin-Tazobactam Resistance Mechanisms in Escherichia coli and Identification of a CTX-M-255 β-Lactamase Selectively Conferring Resistance to Penicillin/β-Lactamase Inhibitor Combinations

Piperacillin/tazobactam (TZP) is a widely used penicillin/{beta}-lactamase inhibitor combination with broad antimicrobial activity. Recently, Escherichia coli strains resistant to TZP but susceptible to third generation cephalosporins (TZP-R/3GC-S isolates) have been increasingly identified. Here, we investigated resistance mechanisms underlying the TZP-R/3GC-S phenotype in clinical E. coli isolates. A total of 29 TZP-R/3GC-S E. coli isolates were retrieved from urinary cultures and subjected to whole genome sequencing. Resistance to TZP was confirmed by minimum inhibitory concentration determination. {beta}-lactamase activity in the presence and absence of tazobactam was determined to identify hyperproduction of {beta}-lactamase and assess susceptibility to tazobactam inhibition. A previously unrecognized {beta}-lactamase was identified and cloned to determine its resistance profile. Four different resistance mechanisms underlying the TZP-R/3-GC phenotype were identified: 1) In 18 out of 29 isolates (62%) {beta}-lactamase production was increased and in 16 of these either strong alternative promoters or increased gene copy numbers of blaTEM-1 or blaSHV-1 were identified, 2) seven isolates (24%) produced blaOXA-1, 3) three isolates (10%) produced inhibitor-resistant TEM-{beta}-lactamases, and 4) a single isolate (3%) harboured a blaCTX-M gene as the only {beta}-lactamase present. This {beta}-lactamase, CTX-M-255, only differs from CTX-M-27 by a G239S amino acid substitution. In contrast to CTX-M-27, CTX-M-255 conferred resistance to penicillin/{beta}-lactamase inhibitor combinations but remained susceptible to cephalosporins. In conclusion, hyperproduction of blaTEM was the most prevalent mechanism of TZP-resistance underlying the TZP-R/3GC-S phenotype followed by production of blaOXA-1 and inhibitor-resistant TEM-{beta}-lactamases. Furthermore, we identified a previously unrecognized CTX-M-{beta}-lactamase, CTX-M-255 that was resistant to {beta}-lactamase inhibitors.