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 β-lactamase that is inhibited by β-lactamase inhibitors and commonly present in Escherichia coli. Hyperproduction of bla_TEM-1 may cause resistance to penicillin/β-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 β-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 β-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 β-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.