Molecular Investigation of the Mechanisms of Resistance to Ceftazidime/Avibactam in Carbapenem-Resistant, Non-Carbapenemase-Producing Pseudomonas aeruginosa

Abstract

Background: Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infections, particularly in immunocompromised patients. However, treatment options are increasingly limited due to rising antimicrobial resistance (AMR), including resistance to ceftazidime-avibactam (CZA), a critical β-lactam/β-lactamase inhibitor combination. While such resistance is often linked to metallo-β-lactamases (MBLs), the detection of CZA-resistant isolates lacking MBL genes suggests the presence of alternative resistance mechanisms. Recent evidence suggests that the overexpression of efflux pumps, such as MexAB-OprM, and the hyperproduction of chromosomally encoded AmpC β-lactamase variants (PDCs) contribute to these factors. Understanding these pathways is essential for developing effective treatment strategies. Aim: This study aimed to investigate the molecular mechanisms of CZA resistance in non-carbapenemase-producing P. aeruginosa, with a specific focus on the role of the MexAB-OprM efflux system and the AmpC β-lactamase (PDC). Methods: A total of 448 clinical PSA isolates were screened for susceptibility to CZA. Of these, 104 (23.2%) were found to be resistant. Whole-genome sequencing (WGS) was performed on 35 randomly selected resistant isolates, among which 4 lacked MBL genes (e.g., blaVIM, blaNDM, blaIMP). These four were subjected to broth microdilution (BMD) testing against 10 antimicrobial agents belonging to different classes, in addition to CZA. Moreover, BMD against CZA was performed under four conditions: CZA alone, in the presence of phenylalanine-arginine β-naphthylamide (PAβN), an efflux pump inhibitor, with cloxacillin, an AmpC inhibitor and with both inhibitors. MIC values were recorded to assess the contribution of these mechanisms. Results: All four non-MBL-producing isolates were classified as extensively drug-resistant (XDR) and harbored mutations in ampD, ampR, and mexR, along with PDC variants (PDC-1, PDC-11, or PDC-12). MIC values decreased in three isolates following exposure to PAβN, and in one isolate with cloxacillin, changing the status of these isolates from resistant to susceptible, which points to the contribution of MexAB-OprM and AmpC to CZA resistance in these isolates, respectively. The dual inhibition condition showed no additional reduction beyond PAβN or cloxacillin alone. These isolates belonged to the high-risk sequence types ST244, ST357, and ST1047. 3 Conclusion: This study highlights the multifactorial basis of CZA resistance in non-MBL-producing P. aeruginosa, with efflux pump overexpression and AmpC derepression having significant roles. These findings underscore the importance of routine molecular characterization of resistant P. aeruginosa isolates and highlight the therapeutic potential of targeting efflux and AmpC-mediated resistance mechanisms.