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  • Title: Antibacterial activity of closantel against methicillin-resistant Staphylococcus aureus and itsbiofilm.
    Author: Tang H, Zhu Y.
    Journal: Zhong Nan Da Xue Xue Bao Yi Xue Ban; 2024 Apr 28; 49(4):611-620. PubMed ID: 39019790.
    Abstract:
    OBJECTIVES: The antimicrobial resistance of Staphylococcus aureus (S. aureus) has become a challenge in the treatment of infectious diseases. It is of great clinical value to discovery effective antimicrobial agents against multi-drug resistant S. aureus and its biofilms. This study aims to explore the antibacterial activity of the antiparasitic drug closantel against methicillin-resistant S. aureus and its biofilms through drug repurposing. METHODS: The sensitivity of S. aureus to closantel was assessed using microbroth dilution and disk diffusion methods. The bacteriostatic and bactericidal activities of closantel were determined by time-kill curves and colony count. Scanning electron microscopy combined with SYTOX Green and DiSC3(5) fluorescence probes were used to study the bactericidal mechanism of closantel. The influence of resistance was assessed by continuous exposure to sub-inhibitory concentrations of closantel. The anti-biofilm activity was evaluated using 96-well plates and crystal violet staining, and cytotoxicity was measured using the CCK-8 assay. RESULTS: The minimal inhibitory concentration (MIC) of closantel for both methicillin-sensitive and methicillin-resistant S. aureus ranged from 0.125 to 1.000 μg/mL. Disk diffusion tests showed that 80 μg of closantel created an inhibition zone, which increased in diameter with higher drug amounts. Sub-inhibitory concentrations (0.031 μg/mL) of closantel significantly inhibited S. aureus proliferation, reducing bacterial turbidity from 0.26±0.00 to 0.11±0.01 (t=16.06, P<0.001), with stronger inhibition at higher concentrations. Closantel at 0.25×MIC inhibited S. aureus proliferation for 12 hours, while 1×MIC inhibited it for over 24 hours, with the number of viable bacteria decreasing as the drug concentration increased. Mechanistic studies indicated that closantel effectively disrupted the integrity of S. aureus cell membranes, significantly increasing SYTOX Green and DiSC3(5) fluorescence intensity. Even after 25 days of continuous exposure to sub-inhibitory concentrations of closantel, no resistance developed. Closantel at 0.0625 μg/mL significantly inhibited biofilm formation, reducing it from 1.29±0.16 to 0.62±0.04 (t=11.62, P<0.001), showing a clear dose-dependent effect. Closantel at 2 μg/mL also significantly eradicated established biofilms, reducing biofilm mass from 1.62±0.34 to 0.51±0.39 (t=4.84, P<0.01). Additionally, closantel exhibited extremely low cytotoxicity, with half-maximal lethal concentrations for HepG2 liver cancer cells and normal LO2 liver cells both exceeding 64 μg/mL. CONCLUSIONS: Closantel exhibits strong antibacterial activity against S. aureus and its biofilm with low cytotoxicity against human cells, making it a promising candidate for new therapeutic strategies against S. aureus-related infections. 目的: 金黄色葡萄球菌(以下简称“金葡菌”)的耐药性已成为感染性疾病治疗中的难题，研究针对多重耐药金葡菌及其生物膜的抗菌药物具有重要的临床价值。本研究将药物重新利用，探讨抗寄生虫药物氯氰碘柳胺对耐甲氧西林金葡菌及其生物膜的抗菌活性。方法: 通过微量肉汤稀释实验和纸片扩散实验检测金葡菌对氯氰碘柳胺的敏感性。采用时间-抑菌/杀菌曲线结合平板稀释菌落计数检测氯氰碘柳胺的抑菌效率和杀菌活性；扫描电镜联合荧光探针SYTOX Green和DiSC3(5)研究氯氰碘柳胺对金葡菌的杀菌机制；亚抑菌浓度的氯氰碘柳胺连续作用检测其对金葡菌耐药的影响；96孔板结合结晶紫染色法检测氯氰碘柳胺的抗生物膜活性；CCK-8试剂盒检测氯氰碘柳胺的细胞毒性。结果: 氯氰碘柳胺对甲氧西林敏感和耐药金葡菌的最低抑菌浓度(minimal inhibitory concentration，MIC)均为0.125~1.000 μg/mL。纸片扩散实验发现80 μg的氯氰碘柳胺即可出现抑菌圈，且随着药量增加抑菌圈直径明显增大。亚抑菌浓度(0.031 μg/mL)的氯氰碘柳胺可显著抑制金葡菌的增殖，使细菌浊度从0.26±0.00减少到0.11±0.01(t=16.06，P<0.001)，且随着氯氰碘柳胺浓度的升高，其抑菌活性越强。0.25×MIC的氯氰碘柳胺可抑制金葡菌增殖12 h，1×MIC的氯氰碘柳胺可抑制金葡菌增殖长达24 h以上，且活菌数量随着药物浓度升高而减少。机制研究结果表明氯氰碘柳胺可有效破坏金葡菌细胞膜的完整性，使SYTOX Green和DiSC3(5)的荧光强度明显升高。即使经过亚抑菌浓度氯氰碘柳胺的25 d连续作用，仍未产生耐药性。0.062 5 μg/mL的氯氰碘柳胺即可显著抑制金葡菌生物膜的形成，使其形成量从1.29±0.16减少到0.62±0.04(t=11.62，P<0.001)，且呈现明显的剂量依赖性。2 μg/mL的氯氰碘柳胺还可显著清除已形成的生物膜，使生物膜量从1.62±0.34减少到0.51±0.39(t=4.84，P<0.01)。此外，氯氰碘柳胺的细胞毒性极低，其对肝癌细胞HepG2和正常肝脏细胞LO2的半数抑制浓度均大于64 μg/mL。结论: 氯氰碘柳胺对金葡菌及其生物膜具有强抗菌活性且对人体细胞毒性低，有望为金葡菌相关感染提供新的治疗策略。. OBJECTIVE: The antimicrobial resistance of Staphylococcus aureus (S. aureus) has become a challenge in the treatment of infectious diseases. It is of great clinical value to discovery effective antimicrobial agents against multi-drug resistant S. aureus and its biofilms. This study aims to explore the antibacterial activity of the antiparasitic drug closantel against methicillin-resistant S. aureus and its biofilms through drug repurposing. METHODS: The sensitivity of S. aureus to closantel was assessed using microbroth dilution and disk diffusion methods. The bacteriostatic and bactericidal activities of closantel were determined by time-kill curves and colony count. Scanning electron microscopy combined with SYTOX Green and DiSC3(5) fluorescence probes were used to study the bactericidal mechanism of closantel. The influence of resistance was assessed by continuous exposure to sub-inhibitory concentrations of closantel. The anti-biofilm activity was evaluated using 96-well plates and crystal violet staining, and cytotoxicity was measured using the CCK-8 assay. RESULTS: The minimal inhibitory concentration (MIC) of closantel for both methicillin-sensitive and methicillin-resistant S. aureus ranged from 0.125 to 1.000 μg/mL. Disk diffusion tests showed that 80 μg of closantel created an inhibition zone, which increased in diameter with higher drug amounts. Sub-inhibitory concentrations (0.031 μg/mL) of closantel significantly inhibited S. aureus proliferation, reducing bacterial turbidity from 0.26±0.00 to 0.11±0.01 (t=16.06, P<0.001), with stronger inhibition at higher concentrations. Closantel at 0.25×MIC inhibited S. aureus proliferation for 12 hours, while 1×MIC inhibited it for over 24 hours, with the number of viable bacteria decreasing as the drug concentration increased. Mechanistic studies indicated that closantel effectively disrupted the integrity of S. aureus cell membranes, significantly increasing SYTOX Green and DiSC3(5) fluorescence intensity. Even after 25 days of continuous exposure to sub-inhibitory concentrations of closantel, no resistance developed. Closantel at 0.0625 μg/mL significantly inhibited biofilm formation, reducing it from 1.29±0.16 to 0.62±0.04 (t=11.62, P<0.001), showing a clear dose-dependent effect. Closantel at 2 μg/mL also significantly eradicated established biofilms, reducing biofilm mass from 1.62±0.34 to 0.51±0.39 (t=4.84, P<0.01). Additionally, closantel exhibited extremely low cytotoxicity, with half-maximal lethal concentrations for HepG2 liver cancer cells and normal LO2 liver cells both exceeding 64 μg/mL. CONCLUSION: Closantel exhibits strong antibacterial activity against S. aureus and its biofilm with low cytotoxicity against human cells, making it a promising candidate for new therapeutic strategies against S. aureus-related infections.
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