Novel glycopolymer sensitizes Burkholderia cepacia complex isolates from cystic fibrosis patients to tobramycin and meropenem

Abstract

Burkholderia cepacia complex (Bcc) infection, associated with cystic fibrosis (CF) is intrinsically multidrug resistant to antibiotic treatment making eradication from the CF lung virtually impossible. Infection with Bcc leads to a rapid decline in lung function and is often a contraindication for lung transplant, significantly influencing morbidity and mortality associated with CF disease. Standard treatment frequently involves antibiotic combination therapy. However, no formal strategy has been adopted in clinical practice to guide successful eradication. A new class of direct-acting, large molecule polycationic glycopolymers, derivatives of a natural polysaccharide poly-N-acetyl-glucosamine (PAAG), are in development as an alternative to traditional antibiotic strategies. During treatment, PAAG rapidly targets the anionic structural composition of bacterial outer membranes. PAAG was observed to permeabilize bacterial membranes upon contact to facilitate potentiation of antibiotic activity. Three-dimensional checkerboard synergy analyses were used to test the susceptibility of eight Bcc strains (seven CF clinical isolates) to antibiotic combinations with PAAG or ceftazidime. Potentiation of tobramycin and meropenem activity was observed in combination with 8-128 μg/mL PAAG. Treatment with PAAG reduced the minimum inhibitory concentration (MIC) of tobramycin and meropenem below their clinical sensitivity breakpoints (≤4 μg/mL), demonstrating the ability of PAAG to sensitize antibiotic resistant Bcc clinical isolates. Fractional inhibitory concentration (FIC) calculations showed PAAG was able to significantly potentiate antibacterial synergy with these antibiotics toward all Bcc species tested. These preliminary studies suggest PAAG facilitates a broad synergistic activity that may result in more positive therapeutic outcomes and supports further development of safe, polycationic glycopolymers for inhaled combination antibiotic therapy, particularly for CF-associated Bcc infections.

Fig 1. Schematic of the set-up for a three-dimensional checkerboard technique.

Diagram depicts the design of the 96-well test plates to include 3 different antibiotics. Each plate has a fixed concentration of PAAG or ceftazidime. Each plate also has a standard checkerboard of meropenem vs tobramycin. The arrows indicate increasing concentrations of each antimicrobial drug. The diagram is adapted from Stein et. al., 2015 [38].

Fig 2. Isobole analysis of synergistic antibiotic activity.

(A) B. multivorans EH4, (B) B. multivorans AU10398, (C) B. cenocepacia AU10321. The lowest FIC index values of the combinations were reported as median +/- standard deviation. The graphs on the right column shows isoboles of Bcc strains exhibiting synergistic effects with the triple combination antibiotic treatment of meropenem, tobramycin and PAAG. The graphs on the left column exhibits isoboles of Bcc strains with a triple combination antibiotic treatment of meropenem, tobramycin and ceftazidime. The FIC values were chosen from the lowest concentration of antibiotics where addition of PAAG that could potentiate the effect of the antibiotics. Dotted line refers to FIC 0.5.