A Nanoscale Characterization of the Interaction of a Novel Alginate Oligomer with the Cell Surface and Motility ofPseudomonas aeruginosa

Pseudomonas aeruginosa (PA) biofilm-associated infections are a common cause of morbidity in chronic respiratory disease and represent a therapeutic challenge. Recently, the ability of a novel alginate oligomer (OligoG) to potentiate the effect of antibiotics against gram-negative, multi–drug-resistant bacteria and inhibit biofilm formation in vitro has been described. Interaction of OligoG with the cell surface of PA was characterized at the nanoscale using atomic force microscopy (AFM), zeta potential measurement (surface charge), and sizing measurements (dynamic light scattering). The ability of OligoG to modify motility was studied in motility assays. AFM demonstrated binding of OligoG to the bacterial cell surface, which was irreversible after exposure to hydrodynamic shear (5,500 × g). Zeta potential analysis (pH 5–9; 0.1–0.001 M NaCl) demonstrated that binding was associated with marked changes in the bacterial surface charge (−30.9 ± 0.8 to −47.0 ± 2.3 mV; 0.01 M NaCl [pH 5]; P < 0.001). Sizing analysis demonstrated that alteration of surface charge was associated with cell aggregation with a 2- to 3-fold increase in mean particle size at OligoG concentrations greater than 2% (914 ± 284 to 2599 ± 472 nm; 0.01 M NaCl [pH 5]; P < 0.001). These changes were associated with marked dose-dependent inhibition in bacterial swarming motility in PA and Burkholderia spp. The ability of OligoG to bind to a bacterial surface, modulate surface charge, induce microbial aggregation, and inhibit motility represents important direct mechanisms by which antibiotic potentiation and biofilm disruption is affected. These results highlight the value of combining multiple nanoscale technologies to further our understanding of the mechanisms of action of novel antibacterial therapies

A New Class of Safe Oligosaccharide Polymer Therapy To Modify the Mucus Barrier of Chronic Respiratory Disease

The host- and bacteria-derived extracellular polysaccharide coating of the lung is a considerable challenge in chronic respiratory disease and is a powerful barrier to effective drug delivery. A low molecular weight 12–15-mer alginate oligosaccharide (OligoG CF-5/20), derived from plant biopolymers, was shown to modulate the polyanionic components of this coating. Molecular modeling and Fourier transform infrared spectroscopy demonstrated binding between OligoG CF-5/20 and respiratory mucins. Ex vivo studies showed binding induced alterations in mucin surface charge and porosity of the three-dimensional mucin networks in cystic fibrosis (CF) sputum. Studies in Humans showed that OligoG CF-5/20 is safe for inhalation in CF patients with effective lung deposition and modifies the viscoelasticity of CF-sputum. OligoG CF-5/20 is the first inhaled polymer therapy, represents a novel mechanism of action and therapeutic approach for the treatment of chronic respiratory disease, and is currently in Phase IIb clinical trials for the treatment of CF

OligoG CF-5/20 disruption of mucoid Pseudomonas aeruginosa biofilm in a murine lung infection model

Biofilm growth is a universal survival strategy for bacteria, providing an effective and resilient approach for survival in an otherwise hostile environment. In the context of an infection, a biofilm provides resistance and tolerance to host immune defenses and antibiotics, allowing the biofilm population to survive and thrive under conditions that would destroy their planktonic counterparts. Therefore, the disruption of the biofilm is a key step in eradicating persistent bacterial infections, as seen in many types of chronic disease. In these studies, we used both in vitro minimum biofilm eradication concentration (MBEC) assays and an in vivo model of chronic biofilm infection to demonstrate the biofilm-disrupting effects of an alginate oligomer, OligoG CF-5/20. Biofilm infections were established in mice by tracheal instillation of a mucoid clinical isolate of Pseudomonas aeruginosa embedded in alginate polymer beads. The disruption of the biofilm by OligoG CF-5/20 was observed in a dose-dependent manner over 24 h, with up to a 2.5-log reduction in CFU in the infected mouse lungs. Furthermore, in vitro assays showed that 5% OligoG CF-5/20 significantly reduced the MBEC for colistin from 512 μg/ml to 4 μg/ml after 8 h. These findings support the potential for OligoG CF-5/20 as a biofilm disruption agent which may have clinical value in reducing the microbial burden in chronic biofilm infections

The effect of alginate oligosaccharides on the mechanical properties of Gram-negative biofilms

The influence of a novel, safe antibiofilm therapy on the mechanical properties of Pseudomonas aeruginosa and Acinetobacter baumannii biofilms in vitro was characterized. A multiscale approach employing atomic force microscopy (AFM) and rheometry was used to quantify the mechanical disruption of the biofilms by a therapeutic polymer based on a low-molecular weight alginate oligosaccharide (OligoG). AFM demonstrated structural alterations in the biofilms exposed to OligoG, with significantly lower Young’s moduli than the untreated biofilms, (149 MPa vs 242 MPa; p < 0.05), a decreased resistance to hydrodynamic shear and an increased surface irregularity (Ra) in the untreated controls (35.2 nm ± 7.6 vs 12.1 nm ± 5.4; p < 0.05). Rheology demonstrated that increasing clinically relevant concentrations of OligoG (<10%) were associated with an increasing phase angle (δ) over a wide range of frequencies (0.1–10 Hz). These results highlight the utility of these techniques for the study of three-dimensional biofilms and for quantifying novel disruption therapies in vitro

Overcoming Drug Resistance with Alginate Oligosaccharides Able To Potentiate the Action of Selected Antibiotics

The uncontrolled, often inappropriate use of antibiotics has resulted in the increasing prevalence of antibiotic-resistant pathogens, with major cost implications for both US and European healthcare systems. We describe the utilization of a low molecular weight oligosaccharide nanomedicine (OligoG) based on the biopolymer alginate, which is able to perturb multi-drug resistant (MDR) bacteria by modulating biofilm formation/persistence and reducing resistance to antibiotic treatment; evident using conventional and robotic MIC screening and microscopic analyses of biofilm structure. OligoG increased the efficacy of conventional antibiotics (up to 512-fold) against important MDR pathogens including Pseudomonas, Acinetobacter and Burkholderia spp., appearing to be effective with several classes of antibiotic (i.e. macrolides, β-lactams, tetracyclines). Using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) increasing concentrations of alginate oligomer (2, 6 and 10%) were shown have a direct effect on the quality of the biofilms produced and on the health of the cells within that biofilm. Biofilm growth was visibly weakened in the presence of 10% OligoG as seen by decreased biomass and increased intercellular spaces, with the bacterial cells themselves becoming distorted and uneven due to apparently damaged cell membranes. This study demonstrates the feasibility of reducing the tolerance of wound biofilms to antibiotics with the use of specific alginate preparations

Germ tube assays.

<p>(A) Light microscopy images of <i>Candida albicans</i> (CCUG 39343) cells grown with/without the presence of OligoG, (Scale bar is 100 µm). (B) Percentage of <i>Candida</i> cells producing hyphae for four different strains grown for 2 hours in the presence of OligoG (0, 0.2, 0.5, 2, 6 and 10%). <i>Candida glabrata</i> as a non-hyphae producer was the negative control. *indicates significantly different from the control, (<i>P</i><0.05).</p

Scanning electron microscopy of <i>Candida tropicalis</i> 519468 (x 1.20 K) treated with 2% OligoG with/without fluconazole (FLC).

<p>FLC was used at concentrations of 0.5, 1 and 2 µg/mL (equivalent to ‘below’, ‘at’ and ‘above’ the MIC value respectively). Scale bar is 40 µm.</p

AFM imaging of <i>Candida tropicalis</i> 519468 grown on polystyrene with/without 2% OligoG and/or fluconazole (FLC).

<p>FLC was used at 1 mg/L (equivalent to the MIC) with more rounded cells (post OligoG treatment), more flattened cells (post fluconazole treatment) and both flattened, and “wrinkled” cells (post combination treatment) apparent. Z scale of 7.5 µm. Scale bar is 15 µm.</p

Strains used for susceptibility testing and their source.

1<p>Resistant to 5-flucytosine, fluconazole, itraconazole.</p>2<p>Recommended by CLSI as reference strains for antifungal susceptibility testing.</p><p>Strains used for susceptibility testing and their source.</p

MIC of antifungals alone and with increasing concentrations of OligoG (2, 6, 10%) for a range of <i>Candida</i> spp.

<p>*MIC values of fluconazole for <i>C. tropicalis</i> strains (519468 and T2.2) were determined at 36 h.</p><p>Bold numbers indicate a Fractional Inhibitory Concentration Index (FICI)≤0.5; indicative of synergy <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112518#pone.0112518-Odds1" target="_blank">[25]</a>.</p><p>MIC of antifungals alone and with increasing concentrations of OligoG (2, 6, 10%) for a range of <i>Candida</i> spp.</p