Within-patient evolution to piperacillin/tazobactam resistance in a clinical isolate of Escherichia coli due to IS 26 -mediated amplification of bla TEM-1B

A novel phenotype of and Klebsiella pneumoniae resistant to piperacillin/tazobactam (TZP), but susceptible to carbapenems and 3 generation cephalosporins has recently emerged. The resistance mechanism of this phenotype has been identified as hyperproduction of the β-lactamase , however the mechanism of hyperproduction in isolates lacking promoter region mutations is not well understood. We sought to understand this mechanism by focussing on a pair of isolates obtained from an individual patient across two infection episodes and displaying within-patient evolution to TZP resistance. Following confirmation that the two isolates were clonal, we found that the TZP-resistant isolate hyperproduced a β-lactamase but lacked mutations within β-lactamase promoter regions. Hybrid assembly of long and short sequencing reads of the two isolates revealed both harboured a novel IS-flanked composite transposon containing several antibiotic resistance genes, including , which was designated Tn. These resistance genes are also found to be present on a translocatable unit which had excised from Tn in the TZP-resistant isolate. By replicating the evolutionary event leading to TZP resistance we were able to observe excision of the translocatable unit from Tn following exposure to TZP and capture the TU in a plasmid containing a copy of IS. Subsequent amplification of the TU, and by extension , leads to β-lactamase hyperproduction and TZP resistance. Despite a significant increase in gene copy number (P value = <0.0001), we found that the TZP-resistant isolate was as fit as the susceptible ancestor. This mechanism of gene amplification, and the subsequent hyperproduction, of is an important consideration when using genomic data to predict resistance/susceptibility to TZP.