(M1230-01-05) Immobilization of Depolymerase Coating with Lay Down Bactericidal Activity on Biomedical Implants

Purpose: Biomedical implants, such as catheters, prosthetics, and tracheal tubes, have revolutionized medicine, but they are increasingly threatened by the risk of bacterial infections, particularly by antibiotic-resistant bacterial strains. Despite the use of bio-inert materials, implant infection is still one of the most frequent and severe complications for healthcare-associated infections, causing significant morbidity and mortality and a socioeconomic burden.[1] Bacterial adhesion and subsequent biofilm formation play pivotal roles in the pathogenesis of implant infections. Surface modification is an effective strategy to mitigate implant-related infection.[2] In this study, we explored the feasibility of using a mussel-inspired polydopamine (PDA) coating technique to immobilize bacteriophage-encoded depolymerases, which is an emerging class of antivirulence agents capable of degrading the bacterial capsular polysaccharides (CPS) and exopolysaccharides (EPS), on various implant materials for the mitigation of implant infections.

Methods: A depolymerase, Dpo71, encoded by the Acinetobacter baumannii phage vB_AbaM-IME-AB2 and an MDR strain of A. baumannii, MDR-AB2, isolated from a pneumonia patient were selected as the model depolymerase and model bacteria. Figure 1 sketched the protocols employed to immobilize recombinant Dpo71 onto implant surface via the PDA approach using an established protocol [3]. Briefly, dopamine (3 mM) was dissolved in Tris-HCl solution (pH = 8.6, 10 mM). Titanium (Ti) plates, polydimethylsiloxane (PDMS) plates, or glass discs were immersed in the dopamine solution overnight at 37 ºC. After removing residual reactants by washing with deionized water thrice, they were activated by EDC-NHS mixture for 30 min, followed by incubation with different concentrations of Dpo71 solutions (DPO1: 0.01 mg/mL; DPO2: 0.02 mg/mL; DPO3: 0.1 mg/mL) for 12 h at 4 °C for surface coating. The morphologies of surface were characterized by atomic force microscope (AFM). Water contact angle (WCA) was detected using a DropMeter A-200 contact angle system. The surface coverage of FITC labeled Dpo71 on the Ti surface was examined using a fluorescence microscope. The antibacterial and antibiofilm activities of the coated materials were also evaluated in the absence and presence of human serum.

Results: The recombinant Dpo71 depolymerase with excellent CPS/LPS degrading activity (Figure 2a) was successfully immobilized onto various material surfaces with surface coverage increase with increasing concentration of Dpo71 (Figure 2b). The surface also became more hydrophilic (Figure 2c), providing a more favorable environment for enzyme stabilization to retain its activity. The surface topologies of various surfaces were visualized by AFM (Figure 2d). Surfaces modified with higher concentration of Dpo71 were rougher, because of its higher surface coverage as noted with the FITC labelled Dpo71 (Figure 2e). The antibacterial and antibiofilm activities of the immobilized Dpo71 were confirmed that the coated materials could effectively kill planktonic bacteria and remove biofilms in the presence of serum as depicted in Figure 3.

Conclusion: The mussel-inspired PDA functional coating has been confirmed to be a feasible approach in immobilizing depolymerase onto implant surfaces. The potent antibacterial and antibiofilm activities of the coated implants demonstrated great potential of this approach in mitigating bacterial-associated implant infections.

References: 1. Abebe, A.; Tener, M.; Waller, S.; el Atrouni, W., Catheter-Related Bloodstream Infections Review. Hospital Medicine Clinics 2014, 3(1), E32-E49.
2. Neoh, K.G.; Li, M.; Kang, E.T.; Chiong, E.; Tambyah, P.A., Surface modification strategies for combating catheter-related complications: recent advances and challenges. Journal of Materials Chemistry B 2017, 5(11), 2045-2067.
3. Alves, D.; Sileika, T.; Messersmith, P.B.; Pereira, M.O., Polydopamine-Mediated Immobilization of Alginate Lyase to Prevent P. aeruginosa Adhesion. Macromolecular Bioscience 2016, 16(9), 1301-1310.

Acknowledgements: This work was supported by the Health and Medical Research Fund, the Health Bureau, Government of the Hong Kong Special Administrative Region (Ref: 22210142).


Figure 1. Schematic illustration of the fabrication procedure of depolymerase-based coatings that endow surface with antibacterial activity.


Figure 2. Characterization of the DPO coatings. (a) The ability of depolymerases degrading CPS/LPS extracts. (b) photos of DPO coating on different materials (Ti, PDMS, and glass) (c) WCA analysis showed that DPO coatings increased the hydrophilicity of surface. (d) AFM images of surfaces of the uncoated Ti surface as the control, and the PDA, DPO1 (0.01 mg/mL), DPO2 (0.02 mg/mL), and DPO3 (0.1 mg/mL) coatings. (e) Fluorescence microscope images of the uncoated Ti surface, PDA, DPO1, DPO2, and DPO3 coatings.


Figure 3. Antibacterial activity of DPO coatings. (a) Antibacterial activity of uncoated, PDA-coated, Dpo71-coated (DPO3) surfaces with different serum concentrations (0%, 5%, 10%, 15%, 20%). (b) The antibacterial kinetics of uncoated (control), PDA-coated, Dpo71-coated (DPO3) surfaces with 20% serum. (c) Antimicrobial activity of DPO coatings with different surface coverage in 20% serum. (d) Morphology of bacteria on the uncoated, PDA-coated, and Dpo71-coated surfaces in the absence/presence of serum. (Scale bar = 1 μm). (e) Antibiofilm activity of the uncoated surface (control), and the PDA and DPO coatings with 20% serum under confocal microscopy. (Scale bar = 200 μm)