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, 14 (7), e0217205
eCollection

Synergistic Effects of LFchimera and Antibiotic Against Planktonic and Biofilm Form of Aggregatibacter Actinomycetemcomitans

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Synergistic Effects of LFchimera and Antibiotic Against Planktonic and Biofilm Form of Aggregatibacter Actinomycetemcomitans

Marie Rossini Carmela T Lachica et al. PLoS One.

Abstract

Adjunctive use of antibiotics in periodontal treatment have limitations and disadvantages including bacterial resistance. Antimicrobial peptides (AMPs) are potential new agents that can combat bacterial infection. In this study, antimicrobial activity of different concentrations of conventional antibiotics minocycline (MH), doxycycline (DOX), and antimicrobial peptides LL-37, LL-31, Lactoferrin chimera (LFchimera) and Innate Defense Regulator Peptide 1018 (IDR-1018) against Aggregatibacter actinomycetemcomitans ATCC 43718 were determined using colony culturing assay. Subsequently, in vitro activity of the most effective drug and peptide combination was evaluated by checkerboard technique. Impact of the drug and peptide co-administration on biofilm at different stages, i.e., during adhesion and 1-day old biofilm was compared to each of the agents used alone. Results revealed that the killing effects of all AMPs range from 13-100%. In contrast, MH and DOX at 1 and 5 μM showed no killing activity and instead stimulated growth of bacteria. DOX has better killing activity than MH. LFchimera displayed the strongest killing amongst the peptides. Checkerboard technique revealed that combining DOX and LFchimera yielded synergism. Confocal laser scanning microscopy further showed that the combination of DOX and LFchimera caused significant reduction of bacterial adhesion and reduction of biomass, average biofilm thickness and substratum biofilm coverage of 1-day old biofilm compared to DOX and LFchimera alone. In conclusion, LFchimera alone and in combination with DOX exhibited strong antibacterial and anti-biofilm property against A. actinomycetemcomitans. The findings suggest that LFchimera should be considered for development as a new potential therapeutic agent that may be used as an adjunctive treatment for periodontitis.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Killing activity of antimicrobial agent.
Effect of the different concentrations of the 2 antibiotics and the 4 peptides against A. actinomycetemcomitans. Bacterial suspensions were incubated with 1, 5, 10, 20, 40 and 50 μM of each agent and processed as described in materials and methods. Data are the mean ± SD of triplicates from three independent experiments. *P < 0.01 compared with both MH and DOX, #P < 0.01 compared with other peptides, +P < 0.05 compared with MH.
Fig 2
Fig 2. Effect of antimicrobial agent on adhesion state of biofilm-forming.
Confocal laser scanning micrographs of A. actinomycetemcomitans attached cells on glass coverslips after 30 min incubation in 1mM PPB (A), 1.4 μM DOX (B), 0.125 μM LFchimera (C) and combination of 1.4 μM DOX and 0.125 μM LFchimera (D). Bacteria were stained with LIVE/DEAD BacLight Bacterial Viability kit. Green color indicates live bacteria and red color indicates dead bacteria. Images were viewed at 630x magnification. (E) Bacterial count from 20 random areas of 2 coverslips for each sample. Data presented are the mean ± SD of adherent bacteria. *P < 0.01 compared with control and other agents.
Fig 3
Fig 3. Effect of antimicrobial agent on biofilm formation.
CLSM 3-D reconstruction of 1-day old A. actinomycemcomitans ATCC 43718 biofilm after 15 min exposure to 1 mM PPB (A), 1.4 μM DOX (B), 0.125 μM LFchimera (C), combination of 1.4 μM DOX and 0.125 μM LFchimera (D), 13 μM DOX (E) and 1.5 μM LFchimera (F). The biofilms were stained with FITC-ConA. Green color indicates exopolysaccharide of biofilm matrix. CLSM-COMSTAT analysis comparing the effect of 1 mM PPB, 1.4 μM DOX, 0.125 μM LFchimera and the combination on biomass (G), average biomass thickness (H) and substratum coverage (I). CLSM-COMSTAT analysis comparing the effect of 1mM PPB, 13 μM DOX, 1.5 μM LFchimera and the combination on biomass (J), average biomass thickness (K) and substratum coverage (L). Values are means ± SD from 2 independent experiments. *P < 0.01 compared with control and other agents.

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