Activity of Antimicrobial Peptides and Ciprofloxacin against Pseudomonas aeruginosa Biofilms

Abstract

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.

Keywords: P. aeruginosa; antibiotic resistance; antimicrobial peptides; biofilm; ciprofloxacin; combination therapy.

Figure 1

Growth curves for P. aeruginosa ATCC 27853 at sub-MIC of the antimicrobial peptides (AMPs) melimine and Mel4 or ciprofloxacin (Cipro). The positive control was growth in the absence of any antimicrobial.

Figure 2

Change in resistance of P. aeruginosa ATCC 27853 to ciprofloxacin, melimine or Mel4 following serial passage at sub-MIC for 30 days. The MIC of melimine and Mel4 did not change, and values overlap at the bottom of the figure.

Figure 3

Inhibition of biofilm formation of P. aeruginosa ATCC 27853. Biofilm formation of the ciprofloxacin-resistant (A) or sensitive (B) cells of P. aeruginosa ATCC 27853 was inhibited by various concentrations of melimine, Mel4 and ciprofloxacin alone or in combination. The strain was made resistant to ciprofloxacin by sub-passage for 30 days at a sub-MIC concentration (Section 4). * represent significant (p < 0.001) decreases compared to the negative control (bacteria grown in the absence of antibiotics). # indicates significant (p < 0.001) decrease for the combinations compared to melimine or Mel4 alone while ## indicates p = 0.051 compared to Mel4 alone. Means (±SD) of three independent repeats in triplicate. Negative control = bacteria grown in the absence of antimicrobials, Cipro = ciprofloxacin.

Figure 4

Disruption of pre-established biofilm of P. aeruginosa ATCC 27853. Biofilms of the ciprofloxacin-resistant (A) and sensitive (B) cells of P. aeruginosa ATCC 27853 were disrupted at various concentrations by melimine, Mel4 and ciprofloxacin alone or in combination. * represents significant (p < 0.001) decrease compared to the negative control (biofilm treated with buffer). # indicates significants (p < 0.001) decrease for the combinations compared to melimine or Mel4 alone. Means (±SD) of three independent repeats in triplicate. Negative control = bacteria grown in the absence of antimicrobials. Cipro = ciprofloxacin.

Figure 5

Representative confocal laser scanning microscopy images of biofilms of the ciprofloxacin-resistant isolate of P. aeruginosa ATCC 27853 after treatment with AMPs and ciprofloxacin alone or in combination. The antibiofilm effects were evaluated at 4× the MIC of all antimicrobials after incubation for 24 h. The biofilms of P. aeruginosa were stained with SYTO-9 (excited at 488 nm, green live cells) and propidium iodide (excited at 514 nm, red dead cells). The cells exposed to ciprofloxacin alone when excited at 514 nm had a red-ish color indicating some of the cells had taken up the propidium iodide, which was confirmed in the insert showing the biofilm excited at 514 nm.

Figure 6

Cell membrane depolarization of pre-formed (24 h) biofilm cells. Cell membrane depolarization of P. aeruginosa ATCC 27853 (A) by melimine and ciprofloxacin alone or in combination, and (B) by Mel4 and ciprofloxacin alone or in combination against pre-formed (24 h) biofilms. Means (±SD) of three independent repeats in triplicate. Cipro = ciprofloxacin, DMSO = dimethyl sulfoxide.

Figure 7

Leakage of (A) ATP and (B,C) nucleic acid from pre-formed biofilm cells of P. aeruginosa ATCC 27853. Leakage of ATP or nucleic acid from pre-formed (24 h) biofilms of P. aeruginosa ATCC 27853 following treatment for 3 h with either of the two peptides and ciprofloxacin alone or in combination. The strain was made resistant to ciprofloxacin by passage for 30 days at a sub-MIC concentration (0.25 µg/mL). * represent significant (p = 0.001) and ** represent significant (p = 0.002) increases in the amount of ATP or nucleic acid release compared to the negative control. # represent significant (p = 0.044) increase in the release of ATP of the combination of melimine and ciprofloxacin compared to melimine alone, and ## represent a significant (p = 0.039) increase in the release of ATP of the combination of Mel4 and ciprofloxacin compared to Mel4 alone.