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

Abstract Piperacillin/tazobactam (TZP) is a widely used penicillin/β-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. β-lactamase activity in the presence and absence of tazobactam was determined to identify hyperproduction of β-lactamase and assess susceptibility to tazobactam inhibition. A previously unrecognized β-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%) β-lactamase production was increased and in 16 of these either strong alternative promoters or increased gene copy numbers of bla TEM-1 or bla SHV-1 were identified, 2) seven isolates (24%) produced bla OXA-1 , 3) three isolates (10%) produced inhibitor-resistant TEM-β-lactamases, and 4) a single isolate (3%) harboured a bla CTX-M gene as the only β-lactamase present. This β-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/β-lactamase inhibitor combinations but remained susceptible to cephalosporins. In conclusion, hyperproduction of bla TEM was the most prevalent mechanism of TZP-resistance underlying the TZP-R/3GC-S phenotype followed by production of bla OXA-1 and inhibitor-resistant TEM-β-lactamases. Furthermore, we identified a previously unrecognized CTX-M-β-lactamase, CTX-M-255 that was resistant to β-lactamase inhibitors.