Antibiotic sensitization of pathogenic Pseudomonas aeruginosa via negative hysteresis in the cell envelope

Antimicrobial resistance (AMR) is a major challenge for global health. Multi-drug treatments can help to reduce AMR and be implemented through the sequential administration of antimicrobials (Roemhild and Schulenburg 2019). Such sequential treatments can achieve high efficacy through negative cellular hysteresis: the antibiotic-induced sensitization of bacterial cells towards a subsequently administered antibiotic (Roemhild et al. 2018). However, to date, the exact characteristics of antibiotic sensitization via negative hysteresis are unknown. Therefore, our study aimed at providing fundamental insights into the nature of hysteresis and its possible applicability to treatment, focusing on the high-risk human pathogen Pseudomonas aeruginosa. We found that negative hysteresis is robustly expressed across the species P. aeruginosa, particularly upon switches from ß-lactam antibiotics to aminoglycosides, even if the ß-lactam is administered at sub-MIC levels. We further characterized the molecular basis of hysteresis by a transcriptomic analysis of wildtype Pseudomonas PA-14 and a PA-14 CpxS T163P mutant that is described here. We demonstrate that the Cpx envelope stress response system is centrally involved in inducible cellular sensitization. Furthermore, negative hysteresis and the Cpx system are linked in several cases to the expression of synergistic drug interactions. Overall, our study reveals negative hysteresis to be associated with ß-lactam-induced membrane stress, widely expressed across P. aeruginosa, thus identifying this phenomenon as a promising focus for effective antimicrobial therapy. Overall design: Pseudomonas aeruginosa PA14 and a PA14 CpxS T163P mutant were grown to mid-exponential phase (OD600 = 0.065) at 37 °C, and then either treated with the beta-lactam antibiotic carbenicillin at sub-MIC, Minimum Inhibitory Concentration, (50 mg/L) for hysteresis induction or not treated. After 30 min, cells were resuspended in antibiotic-free medium and harvested at three sequential time points (0, 30 and 60 min) to investigate the expression dynamics induced by short-term carbenicillin/Beta-lactam treatment that could explain induced sensitivity towards other antibiotics/negative hysteresis.