A High-Resolution Genomic and Phenotypic Analysis of Resistance Evolution of an Escherichia Coli Strain from a Critical Care Patient Treated with Piperacillin/tazobactam

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. 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 an Escherichia coli isolate that developed resistance to TZP during patient treatment. Our whole genome sequencing (WGS) analyses show that TZP resistance evolved via IS 26 -mediated duplication of a bla TEM-1 containing gene cassette on a plasmid, resulting in hyperproduction of TEM-1 β-lactamase. We demonstrate that ten copies of bla TEM-1 induce resistance greater than 32-times the MIC and exposure to TZP further increases amplification of bla TEM-1. Furthermore, in the absence of TZP, gene copy number of IS26 and bla TEM-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. Importance We investigated an Escherichia coli strain 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, bla TEM-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. We furthermore identify phenotypic changes that indicate host adaptation and mirror phenotypic changes observed in other species of opportunistic bacterial pathogens. 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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement AF was supported by a UKRI-Medical Research Council (MR/R015678/1) MRC/ CASE scholarship. EH acknowledges funding from Wellcome (217303/Z/19/Z) and the BBSRC (BB/V011278/1). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Both E. coli isolates used in this study were collected from the Royal Liverpool University Hospital (RLUH) (Liverpool, UK), as part of routine clinical diagnostic procedures. Informed consent was obtained from the patient to publish case details and carry out experimental work, and the study was approved by Liverpool School of Tropical Medicine Research Ethics Committee (Study number 22-074. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors and WGS can be found at NCBI under the bioproject ID PRJNA1061590.