关键词: 鲍曼不动杆菌, 家蝇, 抗菌肽, 抗菌活性, 细胞膜, 活性氧

Abstract: Objective　To investigate the antibacterial activity of the antifungal peptide Mt6⁃21DLeu derived from Musca domestica against Acinetobacter baumannii (AB) and unravel its underlying mechanisms, so as to provide insights into development of novel agents against AB. Methods　The minimum inhibitory concentrations (MICs) of Mt6⁃21DLeu, M. domestica⁃derived antifungal peptide⁃1 (MAF⁃1A), and polymyxin B were determined against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and AB using the broth microdilution assay, and the antibacterial activity of Mt6⁃21DLeu and polymyxin B was dynamically assessed against AB over 24 hours with time⁃kill curves. The inhibitory effects of Mt6⁃21DLeu and polymyxin B on biofilm formation in AB at concentrations of 1/4 × MIC, 1/2 × MIC and MIC, and the eradication effects of Mt6⁃21DLeu and polymyxin B on mature biofilms in AB at concentrations of MIC, 2 × MIC, and 4 × MIC were evaluated using crystal violet staining. Structural changes in the cell membrane of AB were observed 3 hours post⁃exposure to Mt6⁃21DLeu at concentrations of MIC and 2 × MIC using scanning electron microscopy, and alterations in the cell membrane permeability of AB were analyzed 3 hours post⁃treatment with Mt6⁃21DLeu at concentrations of MIC and 2 × MIC by means of fluorescence microscopy and propidium iodide (PI) staining. Intracellular reactive oxygen species (ROS) levels in AB were measured 3 hours post⁃treatment with Mt6⁃21DLeu at concentrations of MIC, 2 × MIC, and 4 × MIC using flow cytometry. The survival of Caenorhabditis elegans exposed to Mt6⁃21DLeu at concentrations of MIC, 2 × MIC, and 4 × MIC was monitored for 7 consecutive days, and survival curves were plotted to evaluate the in vivo toxicity of Mt6⁃21DLeu. In addition, C. elegans infected with AB and treated with Mt6⁃21DLeu at a concentration of 4 × MIC served as the treatment group, and uninfected C. elegans served as the control group, while infected but untreated C. elegans served as the infection group. The in vivo antibacterial efficacy of Mt6⁃21DLeu at a concentration of 4 × MIC was evaluated by comparing the survival curves and bacterial load among the three groups.  Results　The MICs of MAF⁃1A were all >128 μg/mL against S. aureus, B. subtilis, E. coli, K. pneumoniae, P. aeruginosa, and AB. In contrast, the MICs of Mt6⁃21DLeu were >128, 32, 8, 8, 16, and 4 μg/mL against these strains, respectively, and the MIC of Mt6⁃21DLeu against AB was close to that of polymyxin B (2 μg/mL). Time⁃kill curve analysis showed that both Mt6⁃21DLeu at concentrations of MIC and 2 × MIC and polymyxin B at a concentration of MIC inhibited AB growth over the 24⁃hour study period. The biofilm biomass in AB was (52.38 ± 6.92)%, (40.88 ± 9.17)% and (14.77 ± 6.00)% post⁃exposure with Mt6⁃21DLeu at concentrations of 1/4 × MIC, 1/2 × MIC and MIC, (61.58 ± 7.35)%, (47.42 ± 5.51)% and (20.85 ± 10.48)% post⁃treatment with polymyxin B at concentrations of 1/4 × MIC, 1/2 × MIC and MIC, and (100.00 ± 15.92)% in the control group (only bacterial suspension), respectively (F = 68.38, P < 0.001), and pairwise comparisons indicated that Mt6⁃21DLeu and polymyxin B at all concentrations significantly inhibited biofilm formation as compared to the control group (all P values < 0.001). The mature biofilm biomass in AB was (73.44 ± 11.41)%, (72.56 ± 13.08)% and (49.65 ± 9.23)% post⁃exposure to Mt6⁃21DLeu at concentrations of MIC, 2 × MIC, and 4 × MIC, (84.38 ± 8.6)%, (72.31 ± 9.63)% and (58.85 ± 4.96)% post⁃treatment with polymyxin B at concentrations of MIC, 2 × MIC, and 4 × MIC, and (100.00 ± 6.36)% in the control group (F = 35.63, P < 0.001), and pairwise comparisons revealed that Mt6⁃21DLeu at all concentrations significantly eradicated biofilm biomass (all P values < 0.05); however, polymyxin B showed no clear⁃cut eradication effect at a concentration of MIC (P > 0.05). Scanning electron microscopy revealed pore formation and content leakage in the cell membrane of AB 3 hours post⁃treatment with Mt6⁃21DLeu at concentrations of MIC and 2 × MIC. Fluorescence microscopy showed that the proportions of PI⁃stained AB were (24.79 ± 11.51)% and (68.44 ± 15.80)% post⁃treatment with Mt6⁃21DLeu at concentrations of MIC and 2 × MIC, and (0.96 ± 0.94)% in the phosphate⁃buffered saline (PBS) treatment group (F = 105.90, P < 0.001), with the highest proportion of PI⁃stained AB seen post⁃treatment with Mt6⁃21DLeu at a concentration of 2 × MIC  (P < 0.05). Flow cytometry revealed that the relative intracellular ROS levels in AB were (652.00 ± 141.90), (694.33 ± 14.19), and (974.33 ± 160.02) 3 hours post⁃treatment with Mt6⁃21DLeu at concentrations of MIC, 2 × MIC and 4 × MIC, and (403.67 ± 86.56) in the PBS treatment group, respectively (F = 12.27, P < 0.05), with the highest intracellular ROS level measured following treatment with Mt6⁃21DLeu at a concentration of 4 × MIC (P < 0.05). Survival curve analysis revealed that Mt6⁃21DLeu posed no impact on C. elegans survival at concentrations of MIC ([χ2] = 0.02, P > 0.05), 2 × MIC ([χ2] = 0.06, P > 0.05) or 4 × MIC ([χ2] = 0.16, P > 0.05), and there was a significant difference in the survival period of C. elegans among the control group, the infection group, and the treatment group ([χ2] = 82.66, P < 0.05), with a significantly longer survival period in the treatment group than in the infection group ([χ2] = 45.00, P < 0.05). In addition, the log⁃transformed bacterial colony counts in C. elegans were (0.00 ± 0.00), (5.46 ± 0.03), and (3.91 ± 0.47) CFU/mL in the control group, the infection group, and the treatment group, respectively (F = 324.80, P < 0.001), and the log⁃transformed bacterial colony counts in C. elegans were significantly lower in the treatment group than in the infection group (P < 0.05). Conclusions　Mt6⁃21DLeu exerts potent antibacterial effects through disrupting the cell membrane integrity of AB and promoting intracellular ROS accumulation in AB, and exhibits promising potential for treatment of AB infections both in vivo and in vitro, which may serve as a candidate drug molecule against multidrug⁃resistant AB infections.

Key words: Acinetobacter baumannii, Musca domestica, Antimicrobial peptide, Antibacterial activity, Cell membrane, Reactive oxygen species