Poly (acetyl, arginyl) glucosamine disrupts Pseudomonas aeruginosa biofilms and enhances bacterial clearance in a rat lung infection model

Abstract

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 that has antimicrobial activity against a broad range of bacterial species, and also has mucolytic activity, which can normalize the rheological 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, and may warrant further evaluation as a therapeutic.

Introduction

Pseudomonas aeruginosa is a Gram-negative bacterium that is frequently implicated in respiratory infections, neutropenic sepsis and hospital-acquired infections [1–4]. P. aeruginosa is commonly isolated from the airways of patients with cystic fibrosis (CF) and other airway diseases impacted by chronic infection, including non-CF bronchiectasis and chronic obstructive pulmonary disease (COPD) [5, 6]. Given the growing global incidence of antibiotic-resistant infections by P. aeruginosa , the World Health Organization (WHO) recently classified this species as a critical priority for the research and development of new antimicrobials [7].

In the lung, P. aeruginosa is a particularly difficult pathogen to treat due to its propensity to form biofilms, which promote persistence in vivo in spite of antimicrobial therapy and immune effectors [1, 8–10]. The formation of airway biofilms by P. aeruginosa is a critical step in the development of chronic colonization of the bronchiectatic airway and is associated with worsened prognosis and increased utilization of antibiotics [11, 12]. Biofilms are highly complex three-dimensional ecosystems and less than 10 % of airway biofilm biomass is bacterium [10, 13]. The primary components of P. aeruginosa biofilm biomass are extracellular polymeric substances, including polysaccharides and alginate, which are secreted by P. aeruginosa [14]. In airway biofilms, additional components from the local airway environment are also involved, including gel-forming mucins and cellular debris such as extracellular DNA [13, 15, 16]. Together, these substances form a gel-like matrix scaffold that is thought to promote survival of P. aeruginosa and other airway pathogens within biofilm communities [14].

Recently, a polycationic glycopolymer, poly (acetyl, arginyl) glucosamine (PAAG, now being developed as SNSP113), has been shown to improve mucus viscoelasticity and mucociliary clearance in CF rats and ferrets [17]. In patients with CF, the architecture of the gel-forming mucin MUC5B is distorted due to deficient bicarbonate and an acidic pH that prevent calcium chelation and removal from the anionic mucin [17–19]. This excessive Ca²⁺ in the MUC5B limits normal mucus expansion and results in a more globular and viscous form of the protein that is intrinsically more adherent and less amenable for mucociliary transport [19, 20]. PAAG interferes with Ca²⁺ binding to MUC5B, restoring normal MUC5B architecture and improving its viscoelastic properties, which in turn enhance the function of the mucociliary clearance apparatus in vitro and in vivo [17]. There are known roles for calcium in maintenance of the integrity of the bacterial outer membrane [21, 22], as well as in bacterial attachment to surfaces [23] and formation of biofilms [24]. Studies have shown that calcium chelation disperses biofilms in vitro [25]. Supporting this notion, treatment with PAAG significantly diminished biofilm density for Burkholderia cepacia complex (Bcc) [26, 27]. Given these previous findings, in this study we sought to identify anti-microbial and anti-biofilm efficacy of PAAG against P. aeruginosa using laboratory and clinical isolate in vitro biofilm models, and respiratory infections in vivo.

Methods

Bacterial strains and growth conditions

The bacterial strains of P. aeruginosa utilized included a well-characterized non-mucoid P. aeruginosa strain (PAO1; ATCC#BAA-47) and P. aeruginosa PA529, a mucoid clinical isolate obtained from an adult CF patient at the University of Alabama at Birmingham (UAB) Cystic Fibrosis clinic. Bacterial strains were stored at −80 °C in tryptic soy broth (TSB) or Mueller–Hinton broth (MHB) containing 15 % (vol/vol) glycerol. All strains of P. aeruginosa were routinely cultured on tryptic soy agar (TSA) or in TSB unless stated otherwise. P. aeruginosa strains were streaked for colony isolation before inoculation into LB broth and shaking overnight at 37 °C, 200 r.p.m.

Minimum biofilm eradication concentration (MBEC)

To test the MBEC of PAAG against P. aeruginosa biofilms, overnight bacterial cultures were cultured to he logarithmic phase of growth and diluted to an approximate OD₆₀₀ of 0.25 (~10⁸ cells ml⁻¹) and added to wells of a 96-well microtitre dish. An MBEC High-throughput (HTP) Assay (Innovotech) lid with sterile peg inserts was added and the inoculated plates were incubated for 48 h at 37 °C on a shaker (3–5 r.p.m. min⁻¹). Pegs were then rinsed twice with phosphate-buffered saline (PBS) and placed in a 96-well flat-bottom microtitre plate containing 50–200 µg ml⁻¹ of PAAG formulated in sterile water and incubated at room temperature for 1 h. The plate lid was then transferred to a fresh plate containing PBS and sonicated to remove adherent bacteria. To calculate the MBEC, biofilms were serially diluted, plated onto MHB agar plates for viable colony counting.

Confocal laser scanning microscopy (CSLM) imaging and biofilm quantification

CLSM was performed using a Nikon A1R HD25 Confocal Laser Microscope (Nikon, Tokyo, Japan). Images were acquired and processed using NIS-elements 5.0 software. For the purpose of confocal imaging, P. aeruginosa PAO1 expressing the GFP-containing plasmid pSMC21 [28] was grown overnight in TSB and diluted to an OD₆₀₀ of 0.075 (~10⁷ cells ml⁻¹). P. aeruginosa were then cultured in TSB (0.3 ml) in 35 mm glass bottom well confocal Petri dishes (MatTek corp., Ashland, MA, USA) and biofilms were allowed to grow for 24 h at 37 °C in a humidified 5 % CO₂ incubator. Biofilms were treated with 0.1 ml vehicle (PBS) or PAAG for 3 h before imaging. At least five biological replicates were imaged for each condition, with the mean maximal biofilm thickness of three technical replicates used for statistical analysis. All representative images were projected using alpha-blending. Quantitative measures of biofilm structure were obtained from confocal Z-stack images using COMSTAT image analysis software.

CSLM imaging of LIVE/DEAD stained biofilms

To assess the impact of PAAG on the structure of mature P. aeruginosa biofilms, P. aeruginosa PAO1 was prepared as a broth culture in TSB for 24 h and then diluted to an OD₆₀₀ of 0.075 (~10⁷ cells ml⁻¹). Confocal dishes were inoculated with 0.3 ml of diluted culture, and biofilms were incubated at 37 °C in a humidified 5 % CO₂ incubator for 24 h. Biofilms were then treated for 3 h with vehicle or PAAG, essentially as described above, after which bacteria were stained using the LIVE/DEAD BacLight Bacterial Viability kit. Representative images were displayed using maximum intensity projection.

Influence of calcium on PAAG anti-biofilm activity

The experiment evaluated PAAG’s antibiofilm activity on P. aeruginosa biofilms in a calcium-rich environment. Biofilms were grown from an overnight culture of P. aeruginosa PAO1 or P. aeruginosa PA529 in TSB. Overnight cultures were adjusted to an approximate OD₆₀₀ of 0.25 (~10⁸ cells ml⁻¹) and then diluted 1 : 30 in TSB. The diluted culture (0.1 ml) was then added to each well of a 96-well microtitre dish in triplicate. Bacteria were grown in a stationary incubator for 48 h before washing with 1× PBS to remove planktonic bacteria. Then the biofilms were treated with PAAG 2–16 (100–500 µg ml⁻¹) alone or in combination with CaCl₂ (200 nM) and incubated at room temperature for 24 h. Biofilms treated with media were used as an untreated control. After 24 h, the wells were rinsed twice with PBS and dried for 2 h at 37 °C. The biofilms were stained with 1 % crystal violet for 15 min. Stained biofilms were washed twice with PBS and crystal violet was eluted in absolute methanol. After incubation for 5 min, the solubilized crystal violet was transferred into a fresh microtitre plate and the optical density (OD) was read at 590 nm. The average absorbance of biofilm-forming isolates was greater than the average absorbance of the negative control wells (+/−3 standard deviations), which confirmed biofilm formation.

Cell adherence after pretreatment with PAAG

Planktonic cultures of P. aeruginosa were grown in TSB for 24 h at 37 °C in a shaking incubator. Cultures were then reseeded and grown to mid-logarithmic phase (OD₆₀₀~=0.6), washed in PBS and diluted to ~10⁶ c.f.u. ml⁻¹ in F12K media. A549 immortalized human type II pneumocyte (ATCC#CCL-185) cells were grown as monolayers to 85–95 % confluence in 24-well tissue culture plates. The number of eukaryotic cells per well was determined by quantitation via haemocytometer; the average number of cells per well was 50 000 cells/well. PAAG (50–500 µg ml⁻¹) diluted in F12K medium or a medium-only control was added to each well and plates were incubated for 1 h at 37 °C with 5 % CO₂. Wells were then washed, and each well was inoculated with diluted bacterial culture. The infected monolayers were centrifuged (165 g for 5 min) and incubated at 37 °C with 5 % CO₂ for 1 h before being washed to remove any nonadherent bacteria. The wells were trypsinized (5 min) and lysed with 0.25 % Triton X-100 (5 min). Cell layers were removed from the surface by scraping before being serially diluted and spot-plated to quantify viable bacteria. All assays were performed in triplicate.

Cytoplasmic membrane depolarization assay

Membrane depolarization of P. aeruginosa was measured using the membrane potential-sensitive fluorescent dye 3,3′-dipropylthiadicarbocyanine iodide (DiSC3-5) [29]. Overnight cultures of P. aeruginosa strains PAO1 and PA529 were pelleted by centrifugation (13 000 r.p.m. for 1 min) and washed three times with 5 mM HEPES (pH 7.8) buffer. Cells were then resuspended in 5 mM HEPES (pH 7.8) buffer with 0.2 mM EDTA to an OD₆₀₀ of 0.05 (45). DiSC3(5) and KCl were added to a final concentration of 0.4 µM and 0.1 M, respectively, and cultures were shaken (37 °C, 150 r.p.m.) for 20 min until fluorescence quenching was achieved. PAAG was added at a variety of concentrations (50–500 µg ml⁻¹) and fluorescence was measured at an excitation wavelength of 622 nm and an emission wavelength of 670 nm after 15 min of incubation. Treatment with Triton X-100 (0.1 % v/v) was used as the positive control for membrane depolarization. The experiment was performed in triplicate with three independent cultures.

Propidium Iodide (PI) uptake assay

Overnight cultures of P. aeruginosa were pelleted by centrifugation (13 000 r.p.m. for 1 min) and supernatants were discarded before resuspension in water to an OD₆₀₀ of 0.25 (~10⁸ cells ml⁻¹). PI (17 µg ml⁻¹) added to the wells of a 96-well plate containing ~10⁸ c.f.u. ml⁻¹ of bacterial suspension, essentially as described in previous studies [26]. All assays were performed at room temperature. Fluorescence was measured via SpectraMax Gemini XPS (Molecular Devices). Increasing concentrations of PAAG (50, 100, 250 and 500 µg ml⁻¹) were added to the wells. Cells treated with 0.1 % Triton X-100 were used as a positive control for membrane permeability. PI alone and PI on untreated cells were included as negative controls. Fluorescence was measured with an excitation wavelength of 535 nm and an emission wavelength of 625 nm every 10 min for 4 h. The experiment was performed in triplicate with three independent cultures.

Scanning electron microscopy (SEM)

For SEM imaging, planktonic cells of P. aeruginosa PAO1 or P. aeruginosa PA529 were grown for 24 h on silicon wafers placed in sterile 12-well plates. PAAG (250 µg ml⁻¹) was added and the plate was incubated for 1 h. The planktonic cells were fixed overnight with 2.5 % glutaraldehyde and 0.5 % paraformaldehyde in 0.1 M phosphate buffer and then rinsed with 0.1 M phosphate buffer (3×10 min/wash). The planktonic cells were then dehydrated gradually via sequential washes with 10, 25, 50, 75 and 95 % alcohol (5 min/wash) and 100 % alcohol (3×5 min/wash). Hexamethyldisilane (HMDS) was used for overnight drying. Samples were then sputter-coated with gold prior to SEM imaging.

Prevention of in vivo P. aeruginosa infection via pretreatment with nebulized PAAG

Sprague Dawley rats (Rattus norvegicus, age 6 months) were administered nebulized PAAG (250 µg ml⁻¹ ×20 ml over 45 min) in isotonic glycerol or glycerol vehicle control on days 1, 3, 5 and 7. On day 7 post-treatment, rats were infected intratracheally with P. aeruginosa (~10⁵ c.f.u./animal). Rats continued to receive nebulized PAAG or control for two doses at days 10 and 12 post-infection, at which time they were euthanized. The left lung of each mouse was harvested and homogenized in Dulbecco’s Modified Eagle Medium (DMEM,Gibco) for viable plate counting. Homogenate was plated on PIA and grown 24 h at 37 °C to obtain viable c.f.u. counts. The right lung of each animal was inflated with 10 % buffered formalin and stored at 4 °C for histological analysis. All animal procedures were performed using standard procedures according to American Veterinary Medical Association (AVMA) guidelines and were fully reviewed and approved by the UAB Institutional Animal Care and Use Committee (IACUC).

Histological analyses

Inflated right lungs from infected rats were stored in 10 % neutral buffered formalin (Fisher Scientific, Waltham, MA, USA) at 4 °C until processing. Sections from each lobe of the right lung were trimmed and sent to the UAB Comparative Pathology Laboratory to be paraffin-embedded and haematoxylin and eosin (H and E) or periodic acid–Schiff (AB-PAS) stained. Images of stained lung sections were taken on a Leica LMD6 scope (Wetzlar, Germany) at 10× magnification. Semi-quantitative grading of all lung sections was performed by a board-certified veterinary pathologist in a blinded fashion (J.F.). For the H and E-stained sections, semi-quantitative histopathological scores were assigned using a scoring matrix based primarily on neutrophilic influx. Severity was rated on a scale of 0–4, where 0 represents no observable neutrophils, 1 represents ≤25 %, 2 represents 25–5 0%, 3 represents 50–75 % and 4 represents 75–100 % of the section affected of independently viewed fields of view. The density of PMNs ranged in affected fields was scored from 1 to 3, to represent mild, moderate and severe, respectively. The final histopathological score was calculated by multiplication of extent and severity scores.

Statistical analysis

Unless otherwise noted, graphs represent sample means±sem. Differences between group were analysed via one-way analysis of variance (ANOVA) assuming a Gaussian distribution with Dunnett’s multiple comparison tests unless otherwise stated. P-values <0.05 were considered statistically significant. Statistical analysis was performed using GraphPad Prism Version 6.0 (San Diego, CA, USA).

Results

PAAG treatment disrupts biofilm structure

PAAG has previously been demonstrated to have bactericidal effects on planktonic Pseudomonas aeruginosa , Staphylococcus aureus and Burkholderia cepacia [26, 27]. However, we wanted to address the effect of PAAG on preformed, mature P. aeruginosa biofilms. To determine the anti-biofilm activity of PAAG, biofilms of P. aeruginosa PAO1, and a mucoid clinical isolate, P. aeruginosa PA529, were allowed to grow and mature for 48 h and were subsequently exposed to varying concentrations of PAAG (0–500 µg ml⁻¹) for 1 h using a standard MBEC assay. Exposure of mature biofilms to PAAG at 50 µg ml⁻¹ or above resulted in a statistically significant reduction in viable bacterial counts for both P. aeruginosa PAO1 and P. aeruginosa PA529 (P<0.0001) (Fig. 1a, b). The effect of PAAG on preformed P. aeruginosa PAO1 (GFP+) biofilms was visualized using CSLM imaging. Exposure of the P. aeruginosa PAO1 biofilm to PAAG treatment resulted in biofilm reduction, as seen in representative confocal images (Fig. 1c, d). When quantified, there was a significant reduction in mean maximal biofilm thickness from (25.3 µm±4.9 µm for control versus 8.5 µm±3.5 µm for PAAG, P<0.01), and in total biomass (0.005 µm³/µm²±0.003 µm³/µm² for control versus 0.002 µm³/µm²±0.0009 µm³/µm² for PAAG, P<0.05). The surface area of PAAG-treated biofilms was also decreased, albeit below levels of statistical significance (3381 µm²±1595 µm² for control versus 1494 µm²±1.003 µm² for PAAG, P=0.0556) (Fig. 1e). These findings demonstrate that PAAG antibacterial efficacy against P. aeruginosa is preserved even in the setting of a well matured biofilm, and that PAAG induces destabilization of the usual biofilm architecture.

Fig. 1.

PAAG shows antimicrobial and antibiofilm effects against P. aeruginosa biofilms. Biofilms of (a) P. aeruginosa PAO1 and (b) P. aeruginosa PA529 were grown for 48 h on minimum biofilm eradication concentration (MBEC) plates before exposure to various concentrations of PAAG for 1 h. Viable cells were quantified as colony-forming units ml⁻¹. ****P<0.0001. (c, d) Effect of PAAG (200 µg ml⁻¹) as compared to untreated controls evaluated by confocal microscopy. (e) Maximal biofilm thickness, biomass and surface area of biofilms were measured using COMSTAT software. *P<0.05, **P<0.01.

PAAG shows antimicrobial effects on intact P. aeruginosa biofilms

Given our findings of antimicrobial activity of PAAG against planktonic P. aeruginosa and the ability of PAAG to disrupt the structural properties of P. aeruginosa biofilms, we next assessed the visualized antimicrobial effect of PAAG against a preformed biofilm of P. aeruginosa PAO1 using BacLight LIVE/DEAD (Molecular Probes) staining and CSLM imaging. Syto9 stained the viable bacteria (green) throughout the biofilm treated with PBS control (Fig. 2a). Treatment with PAAG resulted in significant bacterial killing throughout all levels of the biofilm, including at the deepest layers where the biofilm and glass–substratum interfaced (Fig. 2b).

Fig. 2.

Antimicrobial effect of PAAG on P. aeruginosa PAO1 biofilms by LIVE/DEAD imaging. P. aeruginosa PAO1 biofilms treated with (a) vehicle control or (b) PAAG (250 µg ml⁻¹) were visualized by confocal imaging after LIVE/DEAD staining. Single channels of live bacteria (green) and dead bacteria (red) are represented, in addition to a merged image from a single z-series acquisition along with a representative image of the substratum layer.

PAAG treatment decreases P. aeruginosa biofilm formation on bronchoepithelial cells

PAAG significantly decreased P. aeruginosa biofilm and showed antimicrobial effects on bacteria grown on abiotic surfaces. However, the efficacy of PAAG in the disease-relevant context of bacterial biofilms grown on a respiratory epithelium has not yet been evaluated. Biofilms of P. aeruginosa PAO1 (GFP+) were cultured on confluent monolayers of CFTR −/− cystic fibrosis bronchoepithelial cells (CFBEs) for 6 h before treatment with PAAG (250 µg ml⁻¹). Biofilm formation was visualized via CLSM imaging and quantified using COMSTAT. Exposure of P. aeruginosa PAO1 biofilms grown on CFBEs to PAAG treatment again resulted in biofilm reduction, as seen in representative confocal images (Fig. 3a, b). Quantification of biofilm indicated a significant reduction in mean maximal biofilm thickness (32.7 µm±4.7 for control vs 12.2 µm±4.1 for PAAG, P<0.01), in total biomass (0.008 µm³/µm²±0.002 µm³/µm² for control versus 0.003 µm³/µm²±0.001 µm³/µm² for PAAG, P<0.01) and in surface area of the biofilm (3630 µm²±995 µm² for control versus 1694 µm²±700.5 µm² for PAAG, P<0.05) (Fig. 3c). These findings demonstrate that the ability of PAAG to disrupt and permeabilize Pseudomonas biofilms is retained in the clinically relevant context of co-culture with an epithelial cell layer.

Fig. 3.

PAAG maintains antibiofilm effects against biofilms on epithelia. Biofilms of P. aeruginosa PAO1 were grown on confluent monolayers of CFTR −/− bronchoepithelial cells (CFBEs) for 6 h before a 1 h treatment with PAAG (250 µg ml⁻¹). ****P<0.0001. The effect of PAAG was evaluated via confocal imaging of (a) untreated and (b) PAAG-treated biofilms. (c) Changes in biofilm structure, including maximal biofilm thickness, biomass and surface area of biofilms were measured using COMSTAT software. *P<0.05, **P<0.01.

Calcium chloride abrogates the effect of PAAG on P. aeruginosa biofilm dissolution

P. aeruginosa and other Gram-negative bacteria have their outer membrane coated with polyanionic lipopolysaccharide (LPS) molecules that provide structural integrity to the membrane of the bacteria by crosslinking with divalent cations, including calcium [30]. There is also precedent for calcium playing similar crosslinking roles for extracellular DNA or polysaccharide in biofilm matrix [23, 24]. Since our previous experiments demonstrated a potent biofilm removal effect by PAAG and because PAAG has previously been shown to displace calcium from mucins [17], we next sought to assess whether PAAG similarly causes calcium displacement to achieve biofilm dissolution. P. aeruginosa PAO1 or P. aeruginosa PA529 biofilms were treated with PAAG (100–500 µg ml⁻¹) with and without added CaCl₂ (200 nM). The remaining biofilm was quantified by crystal violet staining (absorbance at OD₅₉₀). PAO1 biofilms exposed to PAAG in a calcium-rich environment required a PAAG concentration of 500 µg ml⁻¹ to decrease biomass (OD₅₉₀=0.89±0.07 vs 0.38±0.07, P<0.01), whereas a PAAG concentration of 100 µg ml⁻¹ is sufficient without excess calcium (OD₅₉₀=0.89±0.07 vs 0.33±0.02, P<0.0001) (Fig. 4a). For biofilms of P. aeruginosa PA529, the addition of calcium abrogated the effects of PAAG at all concentrations tested (Fig. 4b). These findings suggest that dissolution of intact biofilms by PAAG may occur as a result of calcium displacement, which could affect either the stability of the biofilm matrix or the viability of bacteria present in the biofilm.

Fig. 4.

Biofilm dissolution by PAAG is abrogated by Ca²⁺. P. aeruginosa SUS116 biofilms were exposed to 200 µg ml⁻¹ of PAAG and either sterile water or calcium chloride. Co-exposure of the biofilms to PAAG and calcium chloride resulted in reduced biofilm dissolution compared to co-exposure with sterile water (87.2 % reduction in sterile water vs 58.9 % reduction in 150 mM calcium chloride, P<0.0001).

PAAG prevents P. aeruginosa bacterial adhesion on A549 cell surface

Calcium displacement is known to interfere with adherence of P. aeruginosa to respiratory epithelia [31]. Given the findings that PAAG treatment resulted in bacterial killing at the substratum, we next sought to determine whether PAAG altered P. aeruginosa attachment to immortalized lung pneumocytes (A549) using a cell adherence assay. Both P. aeruginosa PAO1 and P. aeruginosa PA529 adhered to A549 cells treated with vehicle/untreated control (UT). However, pretreatment with PAAG resulted in a dose-dependent reduction decrease in the adherence of P. aeruginosa (Fig. 5a, b). PAAG at a concentration of 250 µg ml⁻¹ resulted in a 3 log reduction in the number of bacteria on the surface of A549 cells for both strains of P. aeruginosa tested (P<0.0001).

Fig. 5.

Pretreatment with PAAG reduced adherence of P. aeruginosa to A549 cells. A549 immortalized lung carcinoma cells were pretreated for 1 h with PAAG (0–500 µg ml⁻¹), washed and then incubated with ~10⁶ cells P. aeruginosa PAO1 (a) or P. aeruginosa Pa529 (b). Subsequently, to estimate the number of bacteria attached, A549 cells were exposed to the detergent Triton X-100 and washed, and the lysate was serially diluted in sterile PBS and plated onto tryptic soy agar (TSA) plates to determine the number of c.f.u. per well. Untreated A549 cells incubated with ~10⁶ P. aeruginosa PAO1 or P. aeruginosa Pa529. ****P<0.0001, ***P=0.0001, compared to the untreated control of the respective strains.

PAAG exposure results in bacterial membrane depolarization and permeability

Calcium displacement has been shown to compromise the integrity of the bacterial membrane. We hypothesized that this might be the mechanism by which PAAG produces antimicrobial effects on P. aeruginosa [21]. DiSC3, a membrane potential-sensitive dye was used to evaluate the depolarization potential of PAAG on P. aeruginosa membranes. PAAG treatment resulted a rapid increase in DiSC3 fluorescence, reflecting depolarization of the electrical potential gradient (Fig. 6a, b). Exposure to PAAG at concentrations equal to or greater than their MIC led to rapid permeabilization of the cytoplasmic membranes in both strains of P. aeruginosa , depolarizing the electric potential gradient resulting in the release of diSC3 and a consequent increase in fluorescence. Outer membrane permeabilization of the P. aeruginosa isolates was determined via propidium iodide (PI) uptake assay. PAAG also rapidly permeabilized the outer membrane of both Pseudomonas isolates tested in a concentration-dependent manner, as indicated by an increase in PI fluorescence. Untreated cells demonstrated no change in PI fluorescence intensity (Fig. 6c, d).

Fig. 6.

PAAG disrupts P. aeruginosa membrane integrity. Depolarization of the cytoplasmic membrane of (a) PAO1 and (b) PA529 by PAAG was measured using the membrane potential-sensitive dye, DiSC3. Results are represented as a percentage of the detergent Triton X-100 (TX-100) compared to the untreated control (UT) of the respective strains. ****P<0.0001. The ability of PAAG to disrupt the integrity of the outer membrane of (c) P. aeruginosa PAO1 and (d) P. aeruginosa PA529 was evaluated with the fluorescent dye propidium iodide (PI) and measured as relative fluorescent units (r.f.u.). Changes in fluorescence were measured via spectrophotometer at 617 nm.

PAAG exposure causes morphological changes in P. aeruginosa

Following exposure to PAAG (250 µg ml⁻¹), morphological changes were observed on the otherwise smooth outer bacterial surfaces of both P. aeruginosa PAO1 and PA529. The morphological changes are indicative of membrane disruption, leaving a jagged appearance on the surface, resulting in the collapse of the bacterial structure (Fig. 7). This is consistent with rapid uptake of the cell-impermeable dye, propidium iodide, observed in Fig. 6.

Fig. 7.

PAAG causes phenotypic changes to bacterial cell morphology. SEM images were taken of planktonic cultures of P. aeruginosa PAO1 (a, c) and P. aeruginosa PA529 (b, d) treated with vehicle control (a, b) or PAAG (c, d) for 60 min. Images representative of that observed in multiple independent samples.

In vivo pre-treatment with PAAG decreases P. aeruginosa infection

Having demonstrated that PAAG has a bactericidal effect on biofilms of P. aeruginosa and reduces bacterial adhesion to an epithelial monolayer in vitro, we next sought in vivo confirmation of PAAG efficacy. We used a model of acute pulmonary infection by P. aeruginosa PA529 in Sprague Dawley rats following pre-exposure to PAAG or vehicle control and concurrent treatment until euthanasia at 48 h post-infection (Fig. 8a). This model sought to identify whether PAAG can impair P. aeruginosa adhesion to an intact airway epithelium and reduce bacterial burden. Using this model, rats treated with PAAG demonstrated a significantly lower bacterial burden of P. aeruginosa in lung homogenate (2.38 log c.f.u. ml⁻¹ in treated rats vs 4.86 log c.f.u. ml⁻¹ in untreated controls, P<0.05) (Fig. 8b). Lung histology revealed rats treated with control exhibited significant peri-bronchiolar inflammation and neutrophilic infiltrate following P. aeruginosa infection, whereas this finding was abrogated in the PAAG treatment group. Histopathological analysis indicated significant inflammation in all the infected rats, with slightly diminished neutrophilic counts and peri-bronchial infiltration in rats treated with PAAG (Fig. 8c).

Fig. 8.

PAAG pretreatment inhibits Pseudomonas aeruginosa infection in vivo. Sprague Dawley rats were inoculated intratracheally with P. aeruginosa PA529 (~10⁵ c.f.u.), treated daily with nebulized PAAG (250 µg ml⁻¹x20 ml over 45 min) or vehicle control (1% glycerol×20 ml over 45 min) and then euthanized 48 h later for analysis. (a). The number of animals that grew P. aeruginosa (grey bars) or were sterile (black bars) *P<0.05 by Fisher’s exact test. (b). Bacterial load as measured by c.f.u. isolated post-treatment from vehicle control vs PAAG-treated rats (~10⁶ vs ~10⁵, *P<0.05 by unpaired t-test). Dotted line represents initial inoculation level of 10⁵ CFUs. (c). H and E (top) or AB/PAS staining of representative lung histology sections from animals without P. aeruginosa inoculation as compared to those with P. aeruginosa inoculation and either vehicle or PAAG treatment. Red arrow designates peribronchiolar neutrophilic inflammation and yellow arrow designates neutrophilic inflammation in the parenchyma. Less severe peribronchiolar and parenchymal neutrophilic inflammation was evident in PAAG-treated tissue. AB/PAS-stained sections demonstrate reduced mucus expression in PAAG treated rats in the peribronchial region.

Discussion

The global threat of rising antibiotic-resistant infections has led to an emergent need to identify and develop new antimicrobials with novel and multimodal mechanisms of action [7]. P. aeruginosa causes a wide variety of opportunistic infections, including respiratory infections in patients with CF. P. aeruginosa has a large and complex genome and rapidly develops antibiotic resistance against multiple classes of antibiotics, making treatment difficult [3, 8, 11, 12, 32]. P. aeruginosa persists within multicellular biofilm communities, which are inherently resistant to antibiotics via multiple mechanisms, including impaired antibiotic penetration, expression of antibiotic-inactivating enzymes, reduced metabolic activity of biofilm-embedded bacteria and augmented inter-bacterial genetic transfer [8, 12, 17, 18]. Given the implications of biofilms impairing antibiotic efficacy, taking advantage of mechanisms that combat biofilm structural integrity and prevent bacterial biofilm formation is of substantial scientific and clinical importance.

The study highlights the bactericidal potential of PAAG against laboratory isolate P. aeruginosa within planktonic cultures or as a biofilm. The study also exemplifies its potential to disrupt and permeabilize the structure of mature biofilms formed by these isolates. PAAG has been studied and proven to be an effective bactericidal agent against planktonic cultures of Pseudomonas aeruginosa, Staphylococcus aureus and Burkholderia cepacia [26, 27]. PAAG was also found to effectively remove preformed mature P. aeruginosa biofilms both qualitatively and quantitatively. Visual evidence of biofilm removal using CSLM confirmed a significant reduction in biofilm thickness following exposure to PAAG (Fig. 1), accompanied by increased bacterial killing within the deepest layers of the biofilm. Visualization also confirmed impaired biofilm growth at the level of the substratum, suggesting diffusion of PAAG through the biofilm layers. This observation was of particular significance, as it suggested that PAAG has the potential to reduce biofilm integrity, potentially by dissolution and dispersal of the biofilm matrix (Fig. 2).

The results of this study highlight the potential of PAAG to kill P. aeruginosa in biofilms, as well as disrupt its biofilm structure. Of significance, bactericidal effects were demonstrated using the laboratory isolate P. aeruginosa PAO1 as well as a mucoid CF clinical isolate, P. aeruginosa PA529. Visual evidence of biofilm removal by PAAG using CSLM demonstrated that biofilm thickness was significantly reduced following exposure to PAAG (Fig. 1) and was accompanied by increased bacterial killing, including in the deepest layers of the biofilm. These studies also demonstrated bacterial killing and impaired biofilm growth at the level of the substratum, suggesting diffusion of the PAAG through the layer. This observation was of particular significance, as it suggested that PAAG has the potential to reduce biofilm integrity, potentially by dissolution and dispersal of the biofilm matrix (Fig. 2).

In the airways of patients with CF and other forms of bronchiectasis, biofilms are composed of a gel-like matrix of anionic extracellular polymeric substances, including mucin, extracellular DNA and polysaccharides [12, 14]. PAAG, a novel polycationic glycopolymer, has been demonstrated to normalize the structure of the airway mucin MUC5B in models of the CF airway. MUC5B linearization, a process that occurs normally in the setting of intact anion transport but is dysregulated in CF, contributes to abnormalities in mucus rheology and delayed mucociliary clearance [17]. Given our previously published description on the effect of PAAG on the displacement of Ca²⁺ from MUC5B [17] and the known role of calcium in biofilm formation by P. aeruginosa , we suspected and demonstrated that PAAG exhibited Ca²⁺-dependent anti-biofilm activity. To that end, we compared biofilm dissolution as a result of PAAG in varying levels of calcium. The presence of calcium was found to negate the effect of PAAG on biofilm dissolution, suggesting that a similar mechanism of calcium displacement is necessary for PAAG effects on P. aeruginosa biofilms.

In addition, the outer-cell membranes of P. aeruginosa (and other Gram-negative bacteria) are dependent on Ca²⁺ for maintaining structural integrity interaction with negatively charged LPS on the outer membrane [30]. Because of the impact of PAAG to displace Ca²⁺ in polymeric structures of mucins, we hypothesized that PAAG could also disrupt the cell membranes of the P. aeruginosa isolates tested. Experiments exploring cell membrane disruption using a propidium iodide probe showed that PAAG rapidly permeabilized the P. aeruginosa cell membrane in a dose-dependent manner. Furthermore, membrane depolarization studies demonstrated that PAAG caused rapid depolarization of the membrane as well. The effect of PAAG on the morphology of bacteria was also visualized using SEM imaging. SEM imaging provides visual evidence that exposure of planktonic P. aeruginosa cells to PAAG results in the deformation and disruption of the bacterial membrane.

Preventing and impairing bacterial adhesion to the epithelium is critical in preventing biofilm formation. Given the effects of PAAG on substratum viability and bacterial adhesion to A549 epithelial cells, in addition to anti-bacterial properties, we sought in vivo confirmation of reduced bacterial viability and colonization. Rats that received nebulized PAAG prior to and immediately following P. aeruginosa inoculation demonstrated decreased rates of P. aeruginosa colonization and reduced mean colony counts. Further, lung histology demonstrated that PAAG pretreatment resulted in decreased lung injury and neutrophilic inflammation. Given the prior findings that PAAG normalizes CF mucus rheology, this finding may be of additional clinical importance as neutrophilic inflammation is a key component in the vicious cycle of the development of bronchiectasis and chronic infection state [33].

P. aeruginosa is associated with the development of biofilm-associated infections in a variety of clinical scenarios, including medical device infections and chronic infections, such as chronic airway colonization in CF, bronchiectasis and other airway diseases. Novel mechanisms that result in the prevention or dissolution of biofilms may alter the landscape of medical management of these infections. PAAG is a potential therapeutic that has previously been shown to normalize CF mucin structure and enhance mucociliary clearance. This study builds upon that finding by demonstrating the potent antimicrobial and anti-biofilm effects of PAAG. Future studies evaluating the effect of PAAG on airway inflammation, in an environment in which P. aeruginosa infection is chronic, such as the CF lung, are warranted.