Clinically Relevant Concentrations of Polymyxin B and Meropenem Synergistically Kill Multidrug-Resistant Pseudomonas aeruginosa and Minimize Biofilm Formation

Abstract

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 [MICpolymyxin B], 64 mg/L; MICmeropenem, 64 mg/L) and FADDI-PA107 (MICpolymyxin B, 32 mg/L; MICmeropenem, 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.

An attacin antimicrobial peptide, Hill_BB_C10074, from Hermetia illucens with anti-Pseudomonas aeruginosa activity

“…Even at the lowest concentration tested (5 µg/mL) Hill_BB_C10074 completely disrupted the P. aeruginosa membrane. Notably, Hill_BB_C10074 (5 µg/ ml) possessed a greater membrane disruptive power than the membrane-targeting clinical antibiotic polymyxin B which has been shown to have an minimum inhibitory concentration of 64 µg/ml [35]. Hill_BB_C10074 also inhibited the growth of the highly virulent clinical strain P. aeruginosa Pa14 that was first isolated from an infected wound of a burn patient [36].…”

“…SLBs were exposed to varying concentrations of 6xHis-Hill_BB_C10074 (5-500 µg/mL), the Buffer control (500mM Tris pH 8.0), daptomycin (0.25-2.5 µg/mL, supplemented with 3 mM CaCl 2 co-factor) or polymyxin B (2.8-280 µg/mL). Concentration ranges of antibiotic controls were selected to encompass clinical relevance [35,47]. SLBs were exposed for 20-minutes then washed in the PBS buffer supplemented with the electrolyte 2 mM MgCl 2 .…”

An Efficient Approach for the Design and Synthesis of Antimicrobial Peptide-Peptide Nucleic Acid Conjugates

“…Flow cytometry was employed to examine the membrane disorganization in A. baumannii 5075R and 5075D following treatments of LASP-072, LASP-097, LASP-132 (0.040 mM, 1, 24 h) ( Zhu et al, 2020 ; Wickremasinghe et al, 2021 ). Samples were assessed with an ACEA NovoCyte ® high-performance benchtop flow cytometer (ACEA Biosciences, Santa Clara, CA, United States) using blue (BL) and violet (VL) laser detection channels.…”

“…Flow cytometry was employed to examine the membrane disorganization in A. baumannii 5075R and 5075D following treatments of LASP-072, LASP-097, LASP-132 (0.040 mM, 1, 24 h) (Zhu et al, 2020;Wickremasinghe et al, 2021). Samples…”

PEG-Functionalized Magnetite Nanoparticles for Modulation of Microbial Biofilms on Voice Prosthesis

“…Through the developed transient “cracks”, a variety of molecules may pass, promoting the uptake of the perturbing peptide itself and resulting in cell death [ 46 , 47 ]. Nonetheless, as it happens with any antibiotic, the administration of high doses of polymyxin has led to the emergence of drug resistance, threatening the utility of this increasingly important last-line therapeutic option [ 45 , 48 ]. Therefore, synergistic therapies must be sought.…”