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Abstract
Approximately 80% of microbial infections including device and non-device-associated infection are biofilms mediated. Pseudomonas aeruginosa and Staphylococcus aureus frequently colonise medical devices and cause biofilm related infections. Antimicrobial peptides (AMPs), owing to the minimal possibility of development of bacterial resistance and broad-spectrum activity at low concentrations, can be used to prevent such infections. The present study investigates how two AMPs melimine and Mel4 interact with P. aeruginosa and S. aureus and prevent biofilm formation. AMPs were immobilized on glass using azido benzoic acid linker. Subsequent experiments examining membrane interactions and its impact on the leakage of cellular contents were performed and compared with free peptides. The mechanism of action of both free and immobilized peptides against P. aeruginosa was predominantly membrane disruption via depolarization and permeabilization. Disruption of cell membranes was associated with release of ATP and DNA/RNA. Like P. aeruginosa, depolarization of the cytoplasmic membrane of S. aureus occurred more slowly when the AMPs were bound to glass. Loss of membrane depolarization resulted in slower release of ATP. Similar to P. aeruginosa, free or immobilised melimine permeabilized the cytoplasmic membrane of S. aureus and released nucleic acid. Mel4 when either free or bound transiently permeabilized the cytoplasmic membrane of S. aureus, this effect was not associated with release of nucleic acids. The mechanism of action of free and immobilized Mel4 was involved in release of autolysins along with minimum effect on cell membrane of S. aureus. Both AMPs either in solution or surface immobilized followed similar sequence of events of membranes interaction against P. aeruginosa and S. aureus. However, membranes disruption kinetics was slower for immobilized peptides at their minimum inhibitory concentration but was faster at equivalent concentrations. Neither P. aeruginosa nor S. aureus could develop resistance to melimine and Mel4. Both AMPs inhibited biofilm formation and disrupted pre-formed biofilms of ciprofloxacin resistance and sensitive isolates of P. aeruginosa and S. aureus and showed enhanced antibiofilm effect in combination with ciprofloxacin.
This study has provided a significant increase in the understanding of the mechanism of action of AMPs bound to surfaces and against biofilms of these two medically important bacteria.