A high-resolution genomic and phenotypic analysis of resistance evolution of anEscherichia colistrain from a critical care patient treated with piperacillin/tazobactam
Authors: Fraser Alice J. , Ball Robert , Cantillon Daire , Brettell Laura E. , Graf Fabrice E. , Munnoch John T. , Hoskisson Paul A. , Lewis Joseph M. , van Aartsen Jon J. , Parry Christopher M. , Heinz Eva , Edwards Thomas 
Published: January 2024

AbstractResistance 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. 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.We report the identification of anEscherichia coliisolate that developed resistance to TZP during patient treatment. Our whole genome sequencing (WGS) analyses show that TZP resistance evolved via IS26-mediated duplication of ablaTEM-1 containing gene cassette on a plasmid, resulting in hyperproduction of TEM-1 β-lactamase. We demonstrate that ten copies ofblaTEM-1 induce resistance greater than 32-times the MIC and exposure to TZP further increases amplification ofblaTEM-1. Furthermore, in the absence of TZP, gene copy number ofIS26andblaTEM-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 in the amplified cassette.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.ImportanceWe investigated anEscherichia colistrain obtained from the bloodstream of a hospitalised patient, that evolved resistance against the antimicrobials initially used as empirical treatment. Comparing the whole-genome sequences of the susceptible isolate with the evolved, resistant isolate showed duplications of the only encoded β-lactamase gene,blaTEM-1, resulting in increased enzyme production and resistance to TZP, a commonly prescribed BL/BLI combination antimicrobial. Despite the additional energy needed for increased enzyme production and retaining the additional copies of duplicated genes, we did not find growth differences under standard laboratory conditions and when using a high-throughput metabolic screen. We did however identify phenotypic changes that indicate host adaptation and mirror phenotypic changes observed in other species of opportunistic bacterial pathogens. In summary our findings highlight that BL/BLI combinations can lead to rapid within-patient evolution of antimicrobial resistance, which is of high relevance when considering the implementation of newly developed drugs, many of which belong to the BL/BLI class.