Role of the multi-drug efflux systems on the baseline susceptibility to ceftazidime/avibactam and ceftolozane/tazobactam in clinical isolates of non-carbapenemase-producing carbapenem-resistant Pseudomonas aeruginosa

Abstract

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is one of the pathogens that urgently needs new drugs and new alternatives for its control. The primary strategy to combat this bacterium is combining treatments of beta-lactam with a beta-lactamase inhibitor. The most used combinations against P. aeruginosa are ceftazidime/avibactam (CZA) and ceftolozane/tazobactam (C/T). Although mechanisms leading to CZA and C/T resistance have already been described, among which are the resistance-nodulation-division (RND) efflux pumps, the role that these extrusion systems may play in CZA, and C/T baseline susceptibility of clinical isolates remains unknown. For this purpose, 161 isolates of non-carbapenemase-producing (Non-CP) CRPA were selected, and susceptibility tests to CZA and C/T were performed in the presence and absence of the RND efflux pumps inhibitor, Phenylalanine-arginine β-naphthylamide (PAβN). In the absence of PAβN, C/T showed markedly higher activity against Non-CP-CRPA isolates than observed for CZA. These results were even more evident in isolates classified as extremely-drug resistant (XDR) or with difficult-to-treat resistance (DTR), where CZA decreased its activity up to 55.2% and 20.0%, respectively, whereas C/T did it up to 82.8% (XDR), and 73.3% (DTR). The presence of PAβN showed an increase in both CZA (37.6%) and C/T (44.6%) activity, and 25.5% of Non-CP-CRPA isolates increased their susceptibility to these two combined antibiotics. However, statistical analysis showed that only the C/T susceptibility of Non-CP-CRPA isolates was significantly increased. Although the contribution of RND activity to CZA and C/T baseline susceptibility was generally low (two-fold decrease of minimal inhibitory concentrations [MIC]), a more evident contribution was observed in a non-minor proportion of the Non-CP-CRPA isolates affected by PAβN [CZA: 25.4% (15/59); C/T: 30% (21/70)]. These isolates presented significantly higher MIC values for C/T. Therefore, we conclude that RND efflux pumps are participating in the phenomenon of baseline susceptibility to CZA and, even more, to C/T. However, the genomic diversity of clinical isolates is so great that deeper analyzes are necessary to determine which elements are directly involved in this phenomenon.

Scalable and Chromatography‐Free Synthesis of Efflux Pump Inhibitor Phenylalanine Arginine β‐Naphthylamide for Its Validation in Wild‐Type Bacterial Strains

“…Thus, the originally discovered PAβN itself remains heavily utilized by laboratories as a classic tool compound and standard in EPI research, likely due in part to its commercial availability. While a full literature review of its use is beyond the scope of this report, we would like to highlight PAβN's recent use cases where it has been employed to inhibit efflux while other disease relevant parameters are investigated including virulence phenotypes, [16][17][18] biofilm formation and disruption, [19] the development of antibiotic resistance, [20][21][22][23] and quorum sensing; [24] it is also used as a standard for benchmarking newly developed EPIs. [25][26][27] Further, it is commonly used during the development process of new antibiotics; [28][29][30][31][32][33] indeed, our laboratory's interest in PAβN originated during our investigations of antibacterial compounds [34] and natural products [35] that lacked activity against P. aeruginosa.…”