Activity of Antimicrobial Peptides and Ciprofloxacin against Pseudomonas aeruginosa Biofilms

Pseudomonas aeruginosa is increasingly resistant to conventional antibiotics, which can be compounded by the formation of biofilms on surfaces conferring additional resistance. P. aeruginosa was grown in sub-inhibitory concentrations of the antimicrobial peptides (AMPs) melimine and Mel4 or ciprofloxacin for 30 consecutive days to induce the development of resistance. Antibiofilm effect of AMPs and ciprofloxacin was evaluated using crystal violet and live/dead staining with confocal microscopy. Effect on the cell membrane of biofilm cells was evaluated using DiSC(3)-5 dye and release of intracellular ATP and DNA/RNA. The minimum inhibitory concentration (MIC) of ciprofloxacin increased 64-fold after 30 passages, but did not increase for melimine or Mel4. Ciprofloxacin could not inhibit biofilm formation of resistant cells at 4× MIC, but both AMPs reduced biofilms by >75% at 1× MIC. At 1× MIC, only the combination of either AMP with ciprofloxacin was able to significantly disrupt pre-formed biofilms (≥61%; p < 0.001). Only AMPs depolarized the cell membranes of biofilm cells at 1× MIC. At 1× MIC either AMP with ciprofloxacin released a significant amount of ATP (p < 0.04), but did not release DNA/RNA. AMPs do not easily induce resistance in P. aeruginosa and can be used in combination with ciprofloxacin to treat biofilm.