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

Background:
blaTEM-1 encodes a narrow-spectrum b-lactamase that is inhibited by b-lactamase inhibitors and commonly present in Escherichia coli. Hyperproduction of blaTEM-1 may cause resistance to penicillin/b-lactamase
inhibitor (P/BLI) combinations.

Objectives:
To characterize EC78, an E. coli bloodstream isolate, resistant to P/BLI combinations, which contains extensive amplification of blaTEM-1 within the chromosome.

Methods:
EC78 was sequenced using Illumina and Oxford Nanopore Technology (ONT) methodology. Configuration of blaTEM-1 amplification was probed using PCR. Expression of blaTEM-1 mRNA was determined usingquantitative PCR and b-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 blaTEM-1B as the only acquired b-lactamase preceded by the WT P3 promoter and present at a copy number of 182.6 with blaTEM-1B bracketed by IS26 elements. The chromosomal location of the IS26-blaTEM-1B amplification was confirmed by ONT sequencing. Hyperproduction of blaTEM-1 was confirmed by increased transcription of blaTEM-1 and b-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 blaTEM amplification of isolates resistant to P/BLI combinations identified an additional strain containing an IS26-associated amplification of a blaTEM gene.

Conclusions:
IS26-associated amplification of blaTEM 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.