The emergence of antibiotic resistance has severely impaired the treatment of chronic respiratory infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa. Since the reintroduction of polymyxins as a last-line therapy against MDR Gram-negative bacteria, resistance to its monotherapy and recurrent infections continue to be reported and synergistic antibiotic combinations have been investigated. In this study, comprehensive in vitro microbiological evaluations including synergy panel screening, population analysis profiling, time-kill kinetics, anti-biofilm formation and membrane damage analysis studies were conducted to evaluate the combination of polymyxin B and meropenem against biofilm-producing, polymyxin-resistant MDR P. aeruginosa. Two phylogenetically unrelated MDR P. aeruginosa strains, FADDI-PA060 (MIC of polymyxin B [MIC_{polymyxin B}], 64 mg/L; MIC_meropenem, 64 mg/L) and FADDI-PA107 (MIC_{polymyxin B}, 32 mg/L; MIC_meropenem, 4 mg/L) were investigated. Genome sequencing identified 57 (FADDI-PA060) and 50 (FADDI-PA107) genes predicted to confer resistance to a variety of antimicrobials, as well as multiple virulence factors in each strain. The presence of resistance genes to a particular antibiotic class generally aligned with MIC results. For both strains, all monotherapies of polymyxin B failed with substantial regrowth and biofilm formation. The combination of polymyxin B (16 mg/L)/meropenem (16 mg/L) was most effective, enhancing initial bacterial killing of FADDI-PA060 by ~3 log₁₀ CFU/mL, followed by a prolonged inhibition of regrowth for up to 24 h with a significant reduction in biofilm formation (* p < 0.05). Membrane integrity studies revealed a substantial increase in membrane depolarization and membrane permeability in the surviving cells. Against FADDI-PA107, planktonic and biofilm bacteria were completely eradicated. In summary, the combination of polymyxin B and meropenem demonstrated synergistic bacterial killing while reinstating the efficacy of two previously ineffective antibiotics against difficult-to-treat polymyxin-resistant MDR P. aeruginosa.

Polymyxins are often the only effective antibiotics against the "Critical" pathogen Acinetobacter baumannii. Worryingly, highly polymyxin-resistant A. baumannii displaying dependence on polymyxins has emerged in the clinic, leading to diagnosis and treatment failures. Here, we report that arginine metabolism is essential for polymyxin-dependent A. baumannii. Specifically, the arginine degradation pathway was significantly altered in polymyxin-dependent strains compared to wild-type strains, with critical metabolites (e.g., L-arginine and L-glutamate) severely depleted and expression of the astABCDE operon significantly increased. Supplementation of arginine increased bacterial metabolic activity and suppressed polymyxin dependence. Deletion of astA, the first gene in the arginine degradation pathway, decreased phosphatidylglycerol and increased phosphatidylethanolamine levels in the outer membrane, thereby reducing the interaction with polymyxins. This study elucidates the molecular mechanism by which arginine metabolism impacts polymyxin dependence in A. baumannii, underscoring its critical role in improving diagnosis and treatment of life-threatening infections caused by "undetectable" polymyxin-dependent A. baumannii.

Klebsiella pneumoniae represents one of the most critical pathogens globally and bacteriophage (phage) therapy offers a promising alternative for treatment. Phage therapy poses challenges due to its high specificity and fast resistance development in bacteria. One promising option to overcome these challenges is combining phages with conventional antibiotics. This study investigates phage kinetics and pharmacodynamic interactions between novel phage pK8 and polymyxin B against a clinical multidrug-resistant K. pneumoniae. Time-kill assays with time-dissolved sampling of phages and bacteria were coupled with pharmacometric modelling to describe phage-bacteria kinetics and killing dynamics. Additionally, different interaction models were investigated to assess the observed synergy between phage pK8 and polymyxin B. Key findings reveal that while polymyxin B alone showed no effect and phage pK8 alone was not efficacious enough to prevent the regrowth of K. pneumoniae II-503, their combined application resulted in notable bactericidal effects up to complete eradication. This was particularly notable in scenarios with higher phage doses. The developed pharmacokinetic/pharmacodynamic model describes synergy as bacterial resensitisation to polymyxin B when combined with phage pK8. The study shows that phage pK8 and polymyxin B combination effectively combats a clinical polymyxin-resistant K. pneumoniae strain. These promising results pave the way for further in vivo studies to validate and refine treatment strategies for tackling multidrug-resistant infections.

Objectives: Antimicrobial resistance is a major global threat. Because of the stagnant antibiotic pipeline, synergistic antibiotic combination therapy has been proposed to treat rapidly emerging multidrugresistant (MDR) pathogens. We investigated antimicrobial synergy of polymyxin/rifampicin combination against MDR Acinetobacter baumannii . Methods: In vitro static time-kill studies were performed over 48 h at an initial inoculum of & SIM;10 7 CFU/mL against three polymyxin-susceptible but MDR A. baumannii isolates. Membrane integrity was examined at 1 and 4 h post-treatment to elucidate the mechanism of synergy. Finally, a semi-mechanistic PK/PD model was developed to simultaneously describe the time course of bacterial killing and prevention of regrowth by mono- and combination therapies. Results: Polymyxin B and rifampicin alone produced initial killing against MDR A. baumannii but were associated with extensive regrowth. Notably, the combination showed synergistic killing across all three A. baumannii isolates with bacterial loads below the limit of quantification for up to 48 h. Membrane integrity assays confirmed the role of polymyxin-driven outer membrane remodelling in the observed synergy. Subsequently, the mechanism of synergy was incorporated into a PK/PD model to describe the enhanced uptake of rifampicin due to polymyxin-induced membrane permeabilisation. Simulations with clinically utilised dosing regimens confirmed the therapeutic potential of this combination, particularly in the prevention of bacterial regrowth. Finally, results from a neutropenic mouse thigh infection model confirmed the in vivo synergistic killing of the combination against A. baumannii AB5075. Conclusion: Our results showed that polymyxin B combined with rifampicin is a promising option to treat bloodstream and tissue infection caused by MDR A. baumannii and warrants clinical evaluations. & COPY; 2023 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.