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Non-tuberculosis

Pseudomonas aeruginosa is a common opportunistic pathogen that can cause chronic infections in multiple disease states, including respiratory infections in patients with cystic fibrosis (CF) and non-CF bronchiectasis. Like many opportunists, P. aeruginosa forms multicellular biofilm communities that are widely thought to be an important determinant of bacterial persistence and resistance to antimicrobials and host immune effectors during chronic/recurrent infections. Poly (acetyl, arginyl) glucosamine (PAAG) is a glycopolymer which has antimicrobial activity against a broad range of bacterial species, and also has mucolytic activity which can normalize rheologic properties of cystic fibrosis mucus. In this study, we sought to evaluate the effect of PAAG on P. aeruginosa bacteria within biofilms in vitro, and in the context of experimental pulmonary infection in a rodent infection model. PAAG treatment caused significant bactericidal activity against P. aeruginosa biofilms, and a reduction in the total biomass of preformed P. aeruginosa biofilms on abiotic surfaces, as well as on the surface of immortalized cystic fibrosis human bronchial epithelial cells. Studies of membrane integrity indicated that PAAG causes changes to P. aeruginosa cell morphology and dysregulates membrane polarity. PAAG treatment reduced infection and consequent tissue inflammation in experimental P. aeruginosa rat infections. Based on these findings we conclude that PAAG represents a novel means to combat P. aeruginosa infection, which may warrant further evaluation as a therapeutic.

Burkholderia cepacia complex (Bcc) lung infections in cystic fibrosis (CF) patients are often associated with a steady decline in lung function and death. The formation of biofilms and inherent multidrug resistance are virulence factors associated with Bcc infection and contribute to increased risk of mortality in CF patients., Burkholderia cepacia complex (Bcc) lung infections in cystic fibrosis (CF) patients are often associated with a steady decline in lung function and death. The formation of biofilms and inherent multidrug resistance are virulence factors associated with Bcc infection and contribute to increased risk of mortality in CF patients. New therapeutic strategies targeting bacterial biofilms are anticipated to enhance antibiotic penetration and facilitate resolution of infection. Poly (acetyl, arginyl) glucosamine (PAAG) is a cationic glycopolymer therapeutic being developed to directly target biofilm integrity. In this study, 13 isolates from 7 species were examined, including Burkholderia multivorans, Burkholderia cenocepacia, Burkholderia gladioli, Burkholderia dolosa, Burkholderia vietnamiensis, and B. cepacia. These isolates were selected for their resistance to standard clinical antibiotics and their ability to form biofilms in vitro. Biofilm biomass was quantitated using static tissue culture plate (TCP) biofilm methods and a minimum biofilm eradication concentration (MBEC) assay. Confocal laser scanning microscopy (CLSM) visualized biofilm removal by PAAG during treatment. Both TCP and MBEC methods demonstrated a significant dose-dependent relationship with regard to biofilm removal by 50 to 200 μg/ml PAAG following a 1-h treatment (P

Antibiotic treatments often fail to completely eradicate a bacterial infection, leaving behind an antibiotic-tolerant subpopulation of intact bacterial cells called persisters. Persisters are considered a major cause for treatment failure and are thought to greatly contribute to the recalcitrance of chronic infections. Pseudomonas aeruginosa infections are commonly associated with elevated levels of drug-tolerant persister cells, posing a serious threat to human health. This study represents the first time a novel large molecule polycationic glycopolymer, poly (acetyl, arginyl) glucosamine (PAAG), has been evaluated against antibiotic and carbonyl cyanide m-chlorophenylhydrazone induced P. aeruginosa persisters. PAAG eliminated eliminated persisters at concentrations that show no significant cytotoxicity on human lung epithelial cells. PAAG demonstrated rapid bactericidal activity against both forms of induced P. aeruginosa persister cells resulting in complete eradication of the in vitro persister cells within 24 h of treatment. PAAG demonstrated greater efficacy against persisters in vitro than antibiotics currently being used to treat persistent chronic infections such as tobramycin, colistin, azithromycin, aztreonam, and clarithromycin. PAAG caused rapid permeabilization of the cell membrane and caused significant membrane depolarization in persister cells. PAAG efficacy against these bacterial subpopulations suggests it may have substantial therapeutic potential for eliminating recurrent P. aeruginosa infections.

The incidence of multidrug-resistant (MDR) organisms, including methicillin-resistant Staphylococcus aureus (MRSA), is a serious threat to public health. Progress in developing new therapeutics is being outpaced by antibiotic resistance development, and alternative agents that rapidly permeabilize bacteria hold tremendous potential for treating MDR infections. A new class of glycopolymers includes polycationic poly-N (acetyl, arginyl) glucosamine (PAAG) is under development as an alternative to traditional antibiotic strategies to treat MRSA infections. This study demonstrates the antibacterial activity of PAAG against clinical isolates of methicillin and mupirocin-resistant Staphylococcus aureus. Multidrug-resistant S. aureus was rapidly killed by PAAG, which completely eradicated 88% (15/17) of all tested strains (6-log reduction in CFU) in ≤ 12-hours at doses that are non-toxic to mammalian cells. PAAG also sensitized all the clinical MRSA strains (17/17) to oxacillin as demonstrated by the observed reduction in the oxacillin MIC to below the antibiotic resistance breakpoint. The effect of PAAG and standard antibiotics including vancomycin, oxacillin, mupirocin and bacitracin on MRSA permeability was studied by measuring propidium iodide (PI) uptake by bacterial cells. Antimicrobial resistance studies showed that S. aureus developed resistance to PAAG at a rate slower than to mupirocin but similar to bacitracin. PAAG was observed to resensitize drug-resistant S. aureus strains sampled from passage 13 and 20 of the multi-passage resistance study, reducing MICs of mupirocin and bacitracin below their clinical sensitivity breakpoints. This class of bacterial permeabilizing glycopolymers may provide a new tool in the battle against multidrug-resistant bacteria.

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.