(T0930-01-06) Combatting Pseudomonas aeruginosa Biofilms: A Strategy with Glutathione-Conjugated Hydrogels for Disruption and Biofouling Mitigation

Purpose: Antibiotic-resistant biofilms often induce chronic inflammation and disrupt the orderly progression of wound healing cascades. Encapsulating therapeutics within polymeric matrices has been shown to improve their efficacy, permeation, and retention at the infection site. Extending this approach, antimicrobial hydrogels offer a multifaceted solution, maintaining a moist wound environment, absorbing tissue exudates, inhibiting bacterial growth, and facilitating the delivery of multiple therapeutic agents, thereby actively engaging in wound healing processes. This study builds upon our earlier work on glutathione-conjugated poly(ethylene glycol) [GSH-PEG] hydrogels, designed for encapsulating and controlling the release of charge-bearing therapeutic molecules [1]. Here, we examine the efficacy of meropenem delivered through these hydrogels against Pseudomonas aeruginosa (P. aeruginosa) biofilms. Furthermore, we assess the interaction between these hydrogels and biofilms, particularly in terms of surface biofouling.

Methods: P. aeruginosa isolates [ATCC27853, AR0440, and AR0514] (10⁹ CFU (colony forming unit)/ mL) were cultured for 24 hours, then treated with meropenem (0 - 1,000 µg/mL) for another 24 hours to assess biofilm reduction via crystal violet staining. Once meropenem's effective concentration was determined, hydrogels were fabricated through photo-initiated radical polymerization using GSH and PEG diacrylate [1]. These hydrogels were then loaded with or without meropenem in phosphate-buffered saline (PBS), UV sterilized, and applied to 24-hour-old biofilms. Antibiofilm efficacy was measured via the crystal violet assay after 24 hours. Hydrogels were then processed for viable biofilm quantification by sonication, followed by serial dilution in PBS. Plated aliquots were incubated, and CFUs were counted after 24 hours.

Results: Meropenem exhibited variable effects on the biofilm biomass across three strains of P. aeruginosa. For ATCC 27853, there was a significant reduction in biofilm biomass at 5 µg/mL compared to the untreated control, with no additional benefits observed at higher concentrations. Conversely, AR0440 showed an increase in biofilm biomass at lower concentrations (0.5 and 5 µg/mL) and required higher concentrations (100 µg/mL and above) to significantly reduce biomass compared to the control. Notably, AR0514 did not exhibit any significant reduction in biofilm biomass at any tested concentration, underscoring its potential resistance to meropenem (Figure 1). These findings emphasize that the biofilm inhibitory effect of meropenem is not solely concentration-dependent and varies significantly among strains. Meropenem-loaded GSH-PEG hydrogels showed reductions of 82.5%, 83.1%, and 61.0% versus untreated controls for ATCC 27853, AR0440, and AR0514, respectively. They also showed a substantial reduction in surface biofouling for biofilms of P. aeruginosa strains compared to drug-free hydrogels. The reduction was particularly striking for ATCC27853, resulting in no CFUs after plating and incubation, indicating a complete inhibition of bacterial growth. For the other two strains, the reduction was significant, with p-values < 0.0001, reflecting a substantial decrease in surface biofouling compared to drug-free hydrogels (Figure 2). These findings demonstrate the outstanding capability of GSH-PEG hydrogels for biofilm eradication, laying a solid foundation for their future application in the in vivo management of chronic wound infections.

Conclusion: GSH-PEG’s promising features permit flexible meropenem delivery while reducing surface biofouling and warrant further investigation as a customizable platform for management of biofilm-infected chronic wounds.

References: [1] Andrianopoulou A, Sokolowski K, Wenzler E, Bulman ZP, Gemeinhart RA. Assessment of antibiotic release and antibacterial efficacy from pendant glutathione hydrogels using ex vivo porcine skin. J Control Release. 2024; 365:936-949.


Figure 1. Quantification of biofilm biomass by crystal violet staining following 24-hour exposure of P. aeruginosa biofilms to meropenem across a concentration range of 0.5, 5, 100, 200, 500, and 1,000 µg/mL. The data are presented as mean ± standard deviation from three independent experiments. **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.


Figure 2. (A) Schematic diagram illustrating the experimental design used to assess the effectiveness of meropenem-loaded GSH-PEG hydrogels in the eradication of P.aeruginosa biofilms and their anti-biofouling properties. (B) Biofilm biomass quantification via crystal violet staining after 24 hours of treatment with PBS, blank GSH-PEG hydrogel, meropenem-loaded GSH-PEG hydrogel, and free meropenem on P. aeruginosa isolates. (C) Quantification of viable bacteria adhered to hydrogels after incubation with biofilms. The data are presented as mean ± standard deviation from three independent experiments. ****: p < 0.0001, #: value was below detection limit.