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

摘要： 目的　探究家蝇抗真菌衍生肽Mt6⁃21DLeu对鲍曼不动杆菌的抗菌活性及其潜在机制，为开发新型抗鲍曼不动杆菌药物提供实验依据。方法　采用微量液体稀释法检测Mt6⁃21DLeu、家蝇抗真菌肽⁃1A（musca　domestica　antifungal　peptide⁃1A， MAF⁃1A）、多黏菌素B对金黄色葡萄球菌、枯草芽孢杆菌、大肠埃希菌、肺炎克雷伯菌、铜绿假单胞菌、鲍曼不动杆菌的最小抑菌浓度（minimum inhibitory concentration，MIC），通过时间⁃杀菌曲线动态评估Mt6⁃21DLeu和多黏菌素B在24 h内对鲍曼不动杆菌的抑菌效果，采用结晶紫染色法分别检测浓度为1/4 × MIC、1/2 × MIC、MIC的Mt6⁃21DLeu和多黏菌素B对鲍曼不动杆菌生物被膜的抑制作用以及浓度为MIC、2 × MIC、4 × MIC的Mt6⁃21DLeu和多黏菌素B对鲍曼不动杆菌成熟生物被膜的清除效果。采用扫描电镜观察经浓度为MIC、2 × MIC的Mt6⁃21DLeu作用3 h时鲍曼不动杆菌细胞膜结构变化，采用荧光显微镜结合碘化丙啶（propidium iodide，PI）染色分析浓度为MIC、2 × MIC的Mt6⁃21DLeu作用3 h后鲍曼不动杆菌细胞膜通透性的变化，采用流式细胞术检测浓度为MIC、2 × MIC、4 × MIC的Mt6⁃21DLeu作用鲍曼不动杆菌菌体3 h时鲍曼不动杆菌细胞内活性氧（reactive oxygen species，ROS）水平。此外，连续7 d监测经浓度为MIC、2 × MIC、4 × MIC的Mt6⁃21DLeu作用后秀丽隐杆线虫的生存状态，绘制生存曲线评估Mt6⁃21DLeu的体内毒性。以感染鲍曼不动杆菌且以4 × MIC浓度的Mt6⁃21DLeu治疗的秀丽隐杆线虫为治疗组，未感染鲍曼不动杆菌的秀丽隐杆线虫为对照组，感染鲍曼不动杆菌但未予治疗的秀丽隐杆线虫为感染组，通过比较各组秀丽隐杆线虫生存曲线及体内带菌量评估浓度为4 × MIC的Mt6⁃21DLeu的体内抗菌效果。结果　MAF⁃1A对金黄色葡萄球菌、枯草芽孢杆菌、大肠埃希菌、肺炎克雷伯菌、铜绿假单胞菌、鲍曼不动杆菌的MIC均> 128 μg/mL，Mt6⁃21DLeu对上述各菌的MIC依次为> 128、32、8、8、16、4 μg /mL，其对鲍曼不动杆菌的MIC接近多黏菌素B（2 μg/mL）。时间⁃杀菌曲线分析显示，浓度为MIC、2 × MIC的Mt6⁃21DLeu和浓度为MIC的多黏菌素B均可在24 h内抑制鲍曼不动杆菌生长。经浓度为1/4 × MIC、1/2 × MIC、MIC的Mt6⁃21DLeu和多黏菌素B处理后，鲍曼不动杆菌生物被膜含量依次为（52.38 ± 6.92）%、（40.88 ± 9.17）%、（14.77 ± 6.00）%，（61.58 ± 7.35）%、（47.42 ± 5.51）%和（20.85 ± 10.48）%；仅含鲍曼不动杆菌悬液的生长对照组生物被膜含量为（100.00 ±15.92）%；各组差异有统计学意义（F = 68.38，P < 0.001）；两两比较结果显示，不同浓度的Mt6⁃21DLeu和多黏菌素B均可抑制鲍曼不动杆菌生物被膜形成（P均< 0.001）。此外，浓度为MIC、2 × MIC、4 × MIC的Mt6⁃21DLeu和多黏菌素B处理后，鲍曼不动杆菌生物被膜含量依次为（73.44 ± 11.41）%、（72.56 ± 13.08）%、（49.65 ± 9.23）%，（84.38 ± 8.6）%、（72.31 ± 9.63）%、（58.85 ± 4.96）%，仅含鲍曼不动杆菌悬液的生长对照组生物被膜含量为（100.00 ± 6.36）%，差异有统计学意义（F = 35.63，P < 0.001）；两两比较结果显示，不同浓度的Mt6⁃21DLeu均可清除鲍曼不动杆菌生物被膜（P均< 0.05），但浓度为MIC的多黏菌素B对鲍曼不动杆菌生物被膜无显著清除作用（P > 0.05）。扫描电镜观察显示，浓度为MIC及2 × MIC的Mt6⁃21DLeu作用3 h后，鲍曼不动杆菌细胞膜出现孔洞及内容物泄漏；荧光显微镜观察显示，经浓度为MIC及2 × MIC的Mt6⁃21DLeu处理后PI染色的鲍曼不动杆菌比例分别为（24.79 ± 11.51）%和（68.44 ± 15.80）%，而仅加入磷酸缓冲盐溶液（phosphate⁃buffered saline，PBS）的鲍曼不动杆菌悬液中PI染色的鲍曼不动杆菌细胞比例为（0.96 ± 0.94）%，差异有统计学意义（F = 105.90，P < 0.001）；其中经浓度为2 × MIC的Mt6⁃21DLeu处理后PI染色的鲍曼不动杆菌细胞比例最高（P < 0.05）。流式细胞术检测结果显示，浓度为MIC、2 × MIC、4 × MIC的Mt6⁃21DLeu作用于鲍曼不动杆菌菌体3 h时，鲍曼不动杆菌细胞内ROS相对丰度为（652.00 ± 141.90）、（694.33 ± 14.19）、（974.33 ± 160.02），而仅加入PBS的鲍曼不动杆菌菌液中ROS相对丰度为（403.67 ± 86.56），差异有统计学意义（F = 12.27，P < 0.05）；经浓度为4 × MIC的Mt6⁃21DLeu处理后鲍曼不动杆菌细胞内ROS相对丰度最高（P 均< 0.05）。生存曲线分析结果显示，浓度为MIC、2 × MIC、4 × MIC的Mt6⁃21DLeu对秀丽隐杆线虫寿命无影响（[χ2] = 0.02、0.06、0.16，P > 0.05）；对照组、感染组、治疗组秀丽隐杆线虫生存时间差异有统计学意义（[χ2]  = 82.66，P < 0.05），其中治疗组较感染组生存时间延长（[χ2]  = 45.00，P < 0.05）。3组秀丽隐杆线虫菌落浓度对数值分别为（0.00 ± 0.00）、（5.46 ± 0.03）、（3.91 ± 0.47） CFU/mL，差异有统计学意义（F = 324.80，P < 0.001）；治疗组线虫体内带菌量较感染组减少（P < 0.05）。结论　Mt6⁃21DLeu可通过破坏鲍曼不动杆菌细胞膜完整性、促进细胞内ROS蓄积发挥高效抗菌作用，且在体内外均表现出鲍曼不动杆菌感染治疗潜力，可作为一种抗多重耐药鲍曼不动杆菌感染的候选药物分子。

关键词: 鲍曼不动杆菌, 家蝇, 抗菌肽, 抗菌活性, 细胞膜, 活性氧