Piperacillin/tazobactam resistance in a clinical isolate of Escherichia coli due to IS26-mediated amplification of blaTEM-1B

Abstract

A phenotype of Escherichia coli and Klebsiella pneumoniae, resistant to piperacillin/tazobactam (TZP) but susceptible to carbapenems and 3rd generation cephalosporins, has emerged. The resistance mechanism associated with this phenotype has been identified as hyperproduction of the β-lactamase TEM. However, the mechanism of hyperproduction due to gene amplification is not well understood. Here, we report a mechanism of gene amplification due to a translocatable unit (TU) excising from an IS26-flanked pseudo-compound transposon, PTn6762, which harbours bla_TEM-1B. The TU re-inserts into the chromosome adjacent to IS26 and forms a tandem array of TUs, which increases the copy number of bla_TEM-1B, leading to TEM-1B hyperproduction and TZP resistance. Despite a significant increase in bla_TEM-1B copy number, the TZP-resistant isolate does not incur a fitness cost compared to the TZP-susceptible ancestor. This mechanism of amplification of bla_TEM-1B is an important consideration when using genomic data to predict susceptibility to TZP.

Fig. 1. Hyperproduction of TEM-1B in the piperacillin/tazobactam-resistant isolate is mediated by an increase in copy number.

a Increase in nitrocefin hydrolysis by the piperacillin/tazobactam (TZP)-resistant isolate (black line) compared to the TZP-susceptible (pink line) isolate, as measured at an optical density of 450 nm (OD₄₅₀)_, due to hyperproduction of a β-lactamase in the TZP-resistant isolate. All error bars represent the standard error of the mean (n = 3). b Comparison of the fold change in copy number of the antimicrobial resistance genes present on the pseudo-compound transposon of the TZP-susceptible isolate (pink circles)/translocatable unit (TU) of the TZP-resistant isolate (black circles) as assessed by qPCR of a single DNA extract compared to the housekeeping gene uidA. All error bars represent the standard error of the mean (n = 4). Source data are provided as a Source Data file.

Fig. 2. Schematics of the pseudo-compound transposon PTn6762, translocatable unit and positions of primers used in this study.

Schematic showing (a) the characterisation of PTn6762 (HP = hypothetical protein) and the position of the primer pairs to detect (b) the junctions of the PTn6762 in the chromosome, (c) the presence of the tandem array of TUs in the chromosome and (d) the junctions of the insertion of the TU into pHSG396:IS26.

Fig. 3. Copy number of antimicrobial resistance genes present on the translocatable unit increased following in vitro replication of the evolutionary event leading to piperacillin/tazobactam resistance.

Fold change in copy number of all the antimicrobial resistance genes found on the pseudo-compound transposon compared to the housekeeping gene uidA following growth of the piperacillin/tazobactam (TZP)-susceptible isolate in the absence of antibiotics (pink circles), TZP-susceptible isolate in the presence of 8/4 µg/ml TZP (black circles), TZP-susceptible isolate transformed with pHSG396 plasmid containing IS26 in the presence of 8/4 µg/ml TZP and 35 µg/ml chloramphenicol (green circles) and TZP-susceptible isolate transformed with pHSG396 plasmid containing IS26 in the presence of 35 µg/ml chloramphenicol only (purple circles). All error bars represent the standard error of the mean (n = 3). Source data are provided as a Source Data file.

Fig. 4. Gene amplification did not incur a fitness cost in the piperacillin/tazobactam-resistant isolate compared to the piperacillin/tazobactam-susceptible isolate.

Relative fitness of the piperacillin/tazobactam (TZP)-resistant isolate compared to the TZP-susceptible isolate and the TZP-susceptible isolate grown in the presence of TZP compared to the TZP-susceptible isolate grown in the absence of TZP, assessed comparatively in LB broth (LB, black circles), iso-sensitest broth (ISO, pink squares) and M9 (green triangles). All error bars represent the standard error of the mean (n = 3). Source data are provided as a Source Data file.

Fig. 5. Proposed mechanism of hyperproduction of TEM-1B mediated by IS26.

Schematic of the proposed mechanism of amplification leading to hyperproduction of TEM-1B; (1) a pseudo compound transposon, PTn6762, is present on the chromosome flanked by two copies of IS26. (2) PTn6762 is excised from the chromosome, (3) which then forms a TU which (4) re-inserts into the chromosome adjacent to the chromosomally located IS26 (5) creating a tandem array of the TU and increasing the copy number of bla_TEM-1B.