A high-resolution genomic and phenotypic analysis of resistance evolution of an Escherichia coli strain from a critically unwell patient treated with piperacillin/tazobactam

Introduction
Resistance to the β-lactam/β-lactamase inhibitor (BL/BLI) combination antibiotic piperacillin/tazobactam (TZP) predominantly occurs via β-lactamase enzymes also leading to resistance to third-generation cephalosporins (3GCs). However, if β-lactamases inactive against 3GCs and inhibited by tazobactam are expressed at high levels leading to enzyme hyperproduction, the surplus enzyme escapes inhibition by tazobactam and inactivates the antibiotic piperacillin.

Hypothesis/Gap statement
Understanding this mechanism is clinically relevant as enzyme hyperproduction can emerge upon antibiotic administration, resulting in treatment failure despite initial resistance profiles supporting TZP use.

Aim
Our aim was to determine whether this was a case of within-patient evolution and by what mechanism or an acquisition of a second unrelated, more resistant, strain.

Methodology
Whole genome sequencing (WGS) was performed on the isolate to determine the genetic basis of resistance. We also assessed the impact of TZP exposure on the amplification of the blaTEM-1 gene and monitored the stability of gene copy number over five days in the absence of antibiotic pressure. In addition, we determined the MICs of ceftriaxone and TZP, with TZP MIC contextualised in relation to gene copy number and resistance levels.

Results
We report the identification of an Escherichia coli isolate that developed resistance to TZP during patient treatment but maintained sensitivity to ceftriaxone. We show that TZP resistance evolved via IS26-mediated duplication of a blaTEM-1 containing transposable unit on a plasmid, resulting in hyperproduction of TEM-1 β-lactamase, and that ten copies of blaTEM-1 induce resistance greater than 32-times the MIC. Furthermore, under experimental conditions, exposure to TZP further increases amplification of blaTEM-1, whereas, in the absence of TZP, gene copy number of IS26 and blaTEM-1 remains stable over five days, despite a 48,205 bp genome size increase compared to the pre-amplification isolate. We additionally detect phenotypic changes that might indicate host adaptation potentially linked to the additional genes that are amplified.

Conclusion
Our analysis advances the understanding of infections caused by isolates evolving β-lactamase hyperproduction, which represent a complex problem in both detection and treatment. As 40% of antibiotics active against WHO priority pathogens in the pre-clinical pipeline are BL/BLI combinations further investigations are of urgent concern.