Resistance to piperacillin/tazobactam in Escherichia coli resulting from extensive IS26-associated gene amplification of blaTEM-1

Background: bla(TEM-1) encodes a narrow-spectrum beta-lactamase that is inhibited by beta-lactamase inhibitors and commonly present in Escherichia coli. Hyperproduction of bla(TEM-1) may cause resistance to penicillin/beta-lactamase inhibitor (P/BLI) combinations. Objectives: To characterize EC78, an E. coli bloodstream isolate, resistant to P/BLI combinations, which contains extensive amplification of bla(TEM-1) within the chromosome. Methods: EC78 was sequenced using Illumina and Oxford Nanopore Technology (ONT) methodology. Configuration of bla(TEM-1) amplification was probed using PCR. Expression of bla(TEM-1) mRNA was determined using quantitative PCR and beta-lactamase activity was determined spectrophotometrically in a nitrocefin conversion assay. Growth rate was assessed to determine fitness and stability of the gene amplification was assessed by passage in the absence of antibiotics. Results: Illumina sequencing of EC78 identified bla(TEM-1B) as the only acquired beta-lactamase preceded by the WT P3 promoter and present at a copy number of 182.6 with bla(TEM-1B) bracketed by IS26 elements. The chromosomal location of the IS26-bla(TEM-1B) amplification was confirmed by ONT sequencing. Hyperproduction of bla(TEM-1) was confirmed by increased transcription of bla(TEM-1) and beta-lactamase activity and associated with a significant fitness cost; however, the array was maintained at a relatively high copy number for 150 generations. PCR screening for bla(TEM) amplification of isolates resistant to P/BLI combinations identified an additional strain containing an IS26-associated amplification of a bla(TEM) gene. Conclusions: IS26-associated amplification of bla(TEM) can cause resistance to P/BLI combinations. This adaptive mechanism of resistance may be overlooked if simple methods of genotypic prediction (e.g. gene presence/absence) are used to predict antimicrobial susceptibility from sequencing data.