Biopolymer delivered recombinant phage endolysin as a treatment option for methicillin resistant Staphylococcus aureus mediated wound infection in rats

Abstract

In silico analysis and biochemical characterization showed that LysMR-5 had modular architecture consisting of 3 domains N-terminal CHAP, amidase-2, and C-terminal SH3b_5 domain. The 3D structure predictions using MODELLER and Circular Dichroism spectroscopy revealed that LysMR-5 is a mixture of and#945; helix and and#946;-sheets. Biochemical characterization revealed that optimum conditions for maximum LysMR-5 activity were similar to physiological conditions (37°C and pH 7.4). Antibacterial assays revealed that LysMR-5 has high antibacterial activity against S. aureus, at low concentration of 15µg/ml it caused 4 log unit bacterial reduction within 30 min of incubation. LysMR-5 loaded Alg-Chi NPs with small size (276 nm), PDI (0.34) and good stability were formulated. LysMR-5 was efficiently released from nanoparticles following a biphasic release behavior and Korsmeyer-Peppas model release kinetics. On in vivo application treatment with LysMR-5 significantly reduced the severity and duration of excision wound infection in rats, although treatment with LysMR-5 loaded Alg-Chi NPs was superior to free LysMR-5 treatment. However, maximum therapeutic efficacy was observed in case of combination therapy of LysMR-5 along with fusidic acid ointment (FAO), as better results in terms of reduction in wound bioburden, associated inflammation (in terms of MPO activity and cytokine levels) and faster re-epithelization of wound in shorter time period was obtained, confirming the potential of delivery system and combination therapy in enhancing antibacterial potential of endolysin. Hence, on the basis of findings of this study it is established that LysMR-5 has features conducive for a promising antimicrobial agent and Alg-Chi NPs are promising delivery vehicles for endolysins, making them promising therapeutic option for controlling drug-resistant infections.