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

RESEARCH ARTICLE Novel glycopolymer sensitizes Burkholderia cepacia complex isolates from cystic fibrosis patients to tobramycin and meropenem Vidya P. Narayanaswamy1, Scott Giatpaiboon1, Shenda M. Baker1, William P. Wiesmann1, John J. LiPuma2, Stacy M. Townsend1* 1 Synedgen, Inc., Claremont, California, United States of America, 2 University of Michigan, Department of Pediatrics and Communicable Diseases, Ann Arbor, Michigan, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Narayanaswamy VP, Giatpaiboon S, Baker SM, Wiesmann WP, LiPuma JJ, Townsend SM (2017) Novel glycopolymer sensitizes Burkholderia cepacia complex isolates from cystic fibrosis patients to tobramycin and meropenem. PLoS ONE 12(6): e0179776. https://doi.org/ 10.1371/journal.pone.0179776 Editor: Abdelwahab Omri, Laurentian, CANADA Received: March 17, 2017 Accepted: June 2, 2017 Published: June 29, 2017 Copyright: © 2017 Narayanaswamy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. * 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. Funding: The funders, through Synedgen Inc., provided support in the form of salaries for authors SMT, VPN, SG, SMB, and WPW, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ’author contributions’ section. PLOS ONE | https://doi.org/10.1371/journal.pone.0179776 June 29, 2017 1 / 12 Glycopolymer sensitizes Burkholderia to antibiotic treatment Competing interests: I have read the journal’s policy and the authors of this manuscripts have the following competing interests: VNP SG SMT SMB and WPW are paid employees of Synedgen, SMT SMB and WPW have ownership and patents affiliated with Synedgen, and SMB and WPW are Board members. The glycopolymer used in this study is protected by US Patent number 8,119,780 B2 and others pending in development of drug products to treat cystic fibrosis associated lung infections and related indications. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Introduction Burkholderia cepacia complex (Bcc) is a group of genotypically diverse strains currently consisting of 20 species and are the causative agent of severe lower respiratory infections in cystic fibrosis patients [1]. Colonization of the lung with Bcc is associated with an increased risk of accelerated pulmonary decline, early death, and often excludes Bcc infected patients from consideration for lung transplantation [2, 3]. Furthermore, Bcc respiratory infections in CF patients frequently lead to exacerbations, causing a significant and rapid decline in lung function that is often not reversible [4]. Bcc have the ability to resist and adapt to antibiotic treatment and adverse environmental conditions, making it virtually impossible to eradicate from the CF lung [5–7]. Antimicrobial therapies for infection caused by Bcc are severely limited by the broad-spectrum resistance exhibited by most strains [8, 9]. Meropenem and tobramycin are two commonly used antimicrobial agents that are generally recommended to treat CF pulmonary exacerbation [10], however only a few reports describing treatments for CF patients infected with Bcc have been published [4, 11–14]. Though these antimicrobial agents cause a reduction in bacterial density, clinical improvement in lung function was not observed [11]. Opportunities exist for developing new, more effective therapeutic strategies potentially involving the use of multiple antibiotic therapies to treat chronic lung infections associated with CF. Development of alternate antibacterial strategies to potentiate the antimicrobial activity of conventional antibiotics have become increasingly important due to the emerging threat of multi-drug resistant infection. Poly (acetyl, arginyl) glucosamine (PAAG), is a recently discovered novel class of glycopolymer therapeutics that demonstrate broad antibacterial activity across a spectrum of drug resistant antibiotics and in many cases, has shown synergy with antibiotics in vitro [14]. A wide range of pathogenic bacteria associated with CF disease, including methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and nontuberculous mycobacteria (NTM), are sensitive to PAAG alone or in combination with antibiotics [15]. PAAG is a polycationic polysaccharide and is observed to be biocompatible with minimal eukaryotic cytotoxicity [16]. Many antimicrobial peptides and polyethylenimines (PEI) are also polycationic, but have some degree of cytotoxicity that limits their use [14]. Rapid permeabilization of bacteria (...truncated)