Multi-omics Profiling of Cross-resistance between Ceftazidime-Avibactam and Meropenem identifies common and Strain-specific Mechanisms in Pseudomonas aeruginosa Clinical Isolates

Pseudomonas aeruginosa is a highly clinically important pathogen due to its ability to infect various tissues and to develop multidrug resistance. Ceftazidime-Avibactam (CZA) is one of the last resort antibiotics used to treat multidrug resistant infections, however, the knowledge on the CZA resistance and cross-resistance mechanisms in P. aeruginosa is scarce. Here we performed laboratory evolution of six clinical isolates of P. aeruginosa to CZA and meropenem (MEM) combined with multi-omics profiling to identify resistance and cross-resistance mechanisms. Strains exposed to MEM exhibited a notably high resistance evolution rate, with only 22% (4/18) concurrently displaying cross-resistance to CZA. Conversely, resistance evolution to CZA was substantially lower in strains subjected to CZA (X/Y). While transcriptomic and proteomic analysis revealed highly heterogeneous strain response to antibiotic treatment with few commonly regulated genes and proteins, a simple machine learning model could separate CZA- and MEM-resistant from sensitive strains based on gene expression and protein abundances, revealing a few candidate genes potentially associated with antibiotic resistance. Whole-genome sequencing further underlined strain heterogeneity, since only two mutations were shared between >=3 strains: dacB mutation in CZA-treated strains and oprD mutation in MEM-treated strains. To validate some of these mechanisms, we performed CRISPR/Cas9 genome editing that identified mutations in dacB directly associated with CZA resistance; ongoing work focuses on genome editing of ampC, mexR/B, and arnA of strains subjected to CZA. This study contributes to a deeper comprehension of the intricate dynamics and molecular mechanisms that regulate CZA resistance in Pseudomonas aeruginosa.