Extensive Gene Amplification as a Mechanism for Piperacillin-Tazobactam Resistance in Escherichia coli

Although the TEM-1 β-lactamase (Bla(TEM-1)) hydrolyzes penicillins and narrow-spectrum cephalosporins, organisms expressing this enzyme are typically susceptible to β-lactam/β-lactamase inhibitor combinations such as piperacillin-tazobactam (TZP). However, our previous work led to the discovery of 28 clinical isolates of Escherichia coli resistant to TZP that contained only bla(TEM-1). One of these isolates, E. coli 907355, was investigated further in this study. E. coli 907355 exhibited significantly higher β-lactamase activity and Bla(TEM-1) protein levels when grown in the presence of subinhibitory concentrations of TZP. A corresponding TZP-dependent increase in bla(TEM-1) copy number was also observed, with as many as 113 copies of the gene detected per cell. These results suggest that TZP treatment promotes an increase in bla(TEM-1) gene dosage, allowing Bla(TEM-1) to reach high enough levels to overcome inactivation by the available tazobactam in the culture. To better understand the nature of the bla(TEM-1) copy number proliferation, whole-genome sequence (WGS) analysis was performed on E. coli 907355 in the absence and presence of TZP. The WGS data revealed that the bla(TEM-1) gene is located in a 10-kb genomic resistance module (GRM) that contains multiple resistance genes and mobile genetic elements. The GRM was found to be tandemly repeated at least 5 times within a p1ESCUM/p1ECUMN-like plasmid when bacteria were grown in the presence of TZP.