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

<b>Introduction.</b> Resistance to the <i>β</i>-lactam/<i>β</i>-lactamase inhibitor (BL/BLI) combination antibiotic piperacillin/tazobactam (TZP) predominantly occurs via <i>β</i>-lactamase enzymes, also leading to resistance to third-generation cephalosporins (3GCs). However, if <i>β</i>-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.<b>Hypothesis/Gap statement.</b> 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.<b>Aim.</b> 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.<b>Methodology.</b> Whole-genome sequencing 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 <i>bla</i> _TEM-1 gene and monitored the stability of gene copy number over 5 days in the absence of antibiotic pressure. In addition, we determined the MICs of ceftriaxone and TZP, with TZP MIC contextualized in relation to gene copy number and resistance levels.<b>Results.</b> We report the identification of an <i>Escherichia coli</i> isolate that developed resistance to TZP during patient treatment but maintained sensitivity to ceftriaxone. We show that TZP resistance evolved via IS<i>26</i>-mediated duplication of a <i>bla</i> _TEM-1 containing transposable unit on a plasmid, resulting in hyperproduction of TEM-1 <i>β</i>-lactamase, and that ten copies of <i>bla</i> _TEM-1 induce resistance greater than 32 times the MIC. Furthermore, under experimental conditions, exposure to TZP further increases amplification of <i>bla</i> _TEM-1, whereas, in the absence of TZP, gene copy number of IS<i>26</i> and <i>bla</i> _TEM-1 remains stable over 5 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.<b>Conclusion.</b> Our analysis advances the understanding of infections caused by isolates evolving <i>β</i>-lactamase hyperproduction, which represents 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.