can develop resistance by mutation, transfection or biofilm formation. Resistance was induced in by growth in sub-inhibitory concentrations of ciprofloxacin for 30 days. The ability of the antimicrobials to disrupt biofilms was determined using crystal violet and live/dead staining. Effects on the cell membranes of biofilm cells were evaluated by measuring release of dyes and ATP, and nucleic acids. None of the strains developed resistance to AMPs while only ATCC 25923 developed resistance (128 times) to ciprofloxacin after 30 passages. Only peptides reduced biofilms of ciprofloxacin-resistant cells. The antibiofilm effect of melimine with ciprofloxacin was more (27%) than with melimine alone at 1X MIC ( < 0.001). Similarly, at 1X MIC the combination of Mel4 and ciprofloxacin produced more (48%) biofilm disruption than Mel4 alone ( < 0.001). Combinations of either of the peptides with ciprofloxacin at 2X MIC released ≥ 66 nM ATP, more than either peptide alone ( ≤ 0.005). At 2X MIC, only melimine in combination with ciprofloxacin released DNA/RNA which was three times more than that released by melimine alone ( = 0.043). These results suggest the potential use of melimine and Mel4 with conventional antibiotics for the treatment of biofilms.
