Objectives: We aimed to conduct a comprehensive genomic analysis of ceftolozane/tazobactam (C/T) resistance mechanisms in Pseudomonas aeruginosa by combining novel institutional data with publicly available sequencing data. Methods: We analyzed 1,682 P. aeruginosa isolates, comprising 339 isolates from Alfred Hospital (Melbourne, Australia) and 1,343 isolates from six public datasets. All isolates underwent whole-genome sequencing and C/T broth microdilution (BMD) susceptibility testing. We assessed previously reported intrinsic and acquired resistance mechanisms. We then conducted a genome-wide association study (GWAS) and machine learning analysis to identify novel genes associated with resistance. We then evaluated the impact of mutations in these genes on MIC values and ceftolozane binding affinity. Results: Among 1,682 P. aeruginosa isolates representing 527 distinct sequence types, 343 (20.4%) were C/T-resistant. Carbapenemase genes were detected in 206/1,682 (12.2%) isolates. Mutations in previously reported resistance-associated genes (ftsI, mpl, ampD, ampC, ampR, oprD) were more frequent in resistant isolates but were also found in almost all susceptible isolates. Successive mutations conferred additive increases in MIC. Combined GWAS and machine learning analyses a priori identified five key genes significantly associated with resistance: ftsI, ampR, ampC, PA3329, and PA4311. Molecular docking simulation revealed that the R504C mutation in penicillin-binding protein 3 (PBP3), which is encoded by ftsI, reduced binding contacts and hydrogen bonds with ceftolozane, significantly decreasing binding affinity (P=0.016). Conclusions: Our analysis of 1,682 P. aeruginosa genomes demonstrated complex pathways to C/T resistance and showed that ftsI may play an underappreciated role. We discovered two previously unidentified genes associated with C/T resistance, whose function remains to be determined.

N.M. has received research support from GlaxoSmithKline, unrelated to the current study. A.Y.P. has received research funding from MSD through an investigator-initiated research project. All other authors declare no conflict of interest.

This work was supported by the National Health and Medical Research Council of Australia (Emerging Leader 1 Fellowship APP1176324 to N.M. and Practitioner Fellowship APP1117940 to A.Y.P) and the Australian Medical Research Future Fund (FSPGN000048)

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Ethics committee of Alfred Hospital gave ethical approval for this work

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The in-house sequence files have been deposited in NCBI project ID PRJNA1220180