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

Resistance to the -lactam/-lactamase inhibitor (BL/BLI) combination antibiotic piperacillin/tazobactam (TZP) predominantly occurs via -lactamase enzymes, also leading to resistance to third-generation cephalosporins (3GCs). However, if -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. 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. 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. 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 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. We report the identification of an isolate that developed resistance to TZP during patient treatment but maintained sensitivity to ceftriaxone. We show that TZP resistance evolved via IS-mediated duplication of a containing transposable unit on a plasmid, resulting in hyperproduction of TEM-1 -lactamase, and that ten copies of induce resistance greater than 32 times the MIC. Furthermore, under experimental conditions, exposure to TZP further increases amplification of , whereas, in the absence of TZP, gene copy number of IS and 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. Our analysis advances the understanding of infections caused by isolates evolving -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.