猴耳环水提物对水产病原菌的抑菌活性及其机制

Inhibitory activity and mechanism of aqueous extract from Pithecellobium clypearia against aquatic pathogenic bacteria

摘要:

抗菌药物的过度使用导致细菌耐药性和水产品药物残留问题日益严重，寻找抗菌药物替代品成为迫切需求。猴耳环作为一种具有抗菌和抗炎活性的中草药，尚未在水产病害防控中得到充分研究。【目的】评估猴耳环水提物对水生动物病原菌的抑菌活性，并探究其作用机制，为水产抗菌药物开发提供新思路。【方法】采用微量肉汤法评估107株水生动物病原菌的耐药性，分析猴耳环水提物的抑菌活性，测定猴耳环水提物处理后的无乳链球菌和副溶血弧菌的胞外K⁺含量及其超微结构变化。【结果】107株病原菌对磺胺类药物的耐药率高达67.29%，46.73%的病原菌表现出多重耐药性，其中气单胞菌的耐药性最严重。猴耳环水提物在12.50 mg/mL浓度下对所有病原菌表现出抑制作用，尤其对气单胞菌最小抑菌浓度(minimal inhibitory concentration,MIC)值低至0.39 mg/mL，并且对同一菌属不同耐药特征的菌株MIC值相近。与原始株相比，水提物对人工诱导舒伯特气单胞菌恩诺沙星耐药株的抑菌效果更显著，原始株和耐药株的MIC分别为0.78 mg/mL和0.20 mg/mL。此外，猴耳环水提物处理后细菌胞外K⁺浓度显著增加，细菌细胞膜结构受损，提示猴耳环可能通过破坏菌膜结构来发挥其抑菌作用。【结论】本研究得出了猴耳环对水产病原菌具有一定的体外抑菌效果，在防治水生动物细菌病方面具有进一步研究和开发的巨大潜力。

关键词:病原菌;耐药性;猴耳环;体外抑菌;杀菌机制

Abstract:

The aquaculture industry has rapidly expanded in recent years in China,whereas it faces the challenge brought by bacterial diseases.Antibacterial agents have been the primary tools to combat these diseases.However,prolonged and haphazard usage of antibacterial agents in aquaculture has exacerbated antimicrobial resistance and led to severe antimicrobial residues.Considering these challenges,scholars worldwide have been exploring natural alternatives,such as Chinese herbal medicines.Among them,Pithecellobium clypearia stands out due to its antibacterial,antiviral,and anti-inflammatory properties,coupled with its safety and lack of antimicrobial resistance.Nonetheless,the potential of P.clypearia in the prevention and control of aquatic diseases remains underexplored.[Objective]This study evaluated the in vitro inhibitory activity of P.clypearia aqueous extract against aquatic pathogenic bacteria,including an artificially induced antimicrobial-resistant strain.Additionally,we investigated changes in bacterial cell membrane permeability and observed cellular alterations by transmission electron microscopy to elucidate the mechanism of the extract.Our findings are expected to pave the way for developing P.clypearia as an environmentally friendly antibacterial agent,reducing antibacterial agent dependency,and mitigating pathogen resistance in aquaculture.[Methods] We employed the microbroth method to assess the antimicrobial resistance of 107 pathogen strains attacking aquatic animals and analyzed the inhibitory activity of P.clypearia aqueous extract against aquatic pathogenic bacteria,including an artificially induced antimicrobial-resistant bacterial strain.Furthermore,we determined the extracellular K⁺ content and ultrastructural changes in Streptococcus agalactiae and Vibrio parahaemolyticus after treatment with the extract.[Results]The resistance rate of 107 pathogen strains to sulfonamides was as high as 67.29%,and 46.73% of the strains showed multidrug resistance,among which Aeromonas sp.showed the most severe resistance.The aqueous extract (12.50 mg/mL) of P.clypearia exerted inhibitory effects on all the pathogenic bacteria,especially on Aeromonas sp.with the minimum inhibitory concentration (MIC) as low as 0.39 mg/mL.The MICs of the extract were similar for the strains belonging to the same genus but with different antimicrobial resistance characteristics.The aqueous extract of P.clypearia showed stronger inhibitory effect on the artificially induced enrofloxacin-resistant strain of A.schubertii than on the original strain,with the MICs of 0.78 mg/mL on the original strain and 0.20 mg/mL on the resistant strain.In addition,the treatment with P.clypearia aqueous extract significantly increased the extracellular K⁺ concentration,leading to damage to the bacterial cell membrane structure,leakage of intracellular contents,and vacuolation of cytoplasm,which suggested that P.clypearia exerted the antibacterial effect by destroying the bacterial membrane structure.[Conclusion]P.clypearia exerts in vitro inhibitory effects on aquatic pathogenic bacteria and demonstrates great potential for further research and development in the prevention and treatment of bacterial diseases in aquatic animals.The antibacterial mechanism of P.clypearia appears to involve disrupting bacterial cell membranes.The application of P.clypearia in aquaculture promises to reduce antibiotic dependency and pathogen resistance,paving the way for a healthy and sustainable aquaculture industry.

Key words:pathogenic bacterium;antimicrobial resistance;Pithecellobium clypearia;antibacterial activityin vitro;bactericidal mechanism