36种中药对罗非鱼源无乳链球菌体外抑菌及生物被膜消除效果
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摘要
为了筛选出对无乳链球菌(Streptococcus agalactiate)有抗菌效果的安全新药物,实验采用琼脂扩散法和微量稀释法测定了36种中药单体对3株罗非鱼致病性无乳链球菌的抑菌活性,测定了血根碱对无乳链球菌生长曲线和生物被膜成膜的影响。结果显示:受试菌株对血根碱、白屈菜红碱、厚朴酚、和厚朴酚和大黄酸极度敏感,抑菌圈均在20 mm以上,5种药物对无乳链球菌的最小抑菌浓度(MIC)为8～32μg/mL,表现出很强的抑制作用,其中血根碱对3株无乳链球菌的最小杀菌浓度(MBC)均为16μg/mL;生长曲线及生物被膜试验发现血根碱对无乳链球菌生长和生物被膜生成具有抑制作用。在不同血根碱浓度下,与其他浓度组相比当血根碱浓度达到16μg/mL及以上时,受试菌株生物被膜的生成率降低到2.76%以下,相同条件下,绿原酸作用下生物被膜生成率均在90%以上。
The antimicrobial activity of 36 natural compounds against 3 pathogenic Streptococcus agalactiate strains isolated from diseased tilapia by disc agar diffusion and broth micro-dilution method were investigaeted, to find out new safe drugs which have antimicrobial effect against. S. agalactiate. The effects of S. agalactiate growth curve and biofilm formation with sanguinarine were also determined. The result showed that all tested stains were extremely sensitive to sanguinarine, chelerythrine, magnolol, honokiol and rhein.The inhibitory zone of 5 compounds exceeded 20 mm. The minimal inhibitory concentration(MIC)of 5 drugs were ranged from 8 to 32 μg/mL. The minimum bactericidal concentration(MBC) of sanguinarine against 3 S. agalactiae strains were 16, 32 and 16 μg/mL, respectively. Further growth curve and biofilm test implied different sanguinarine concentrations had different antimicrobial effects on the growth and the biofilm formation of S. agalactiate. Compared with other concentrations, the biofilm formation of S. agalactiate was less than 2.76% once sanguinarine concentration reached to 16 μg/mL, and the biofilm formation rate of S. agalactiate was over 90% with chlorogenic acid at the same concentration gradient.
The antimicrobial activity of 36 natural compounds against 3 pathogenic Streptococcus agalactiate strains isolated from diseased tilapia by disc agar diffusion and broth micro-dilution method were investigaeted, to find out new safe drugs which have antimicrobial effect against. S. agalactiate. The effects of S. agalactiate growth curve and biofilm formation with sanguinarine were also determined. The result showed that all tested stains were extremely sensitive to sanguinarine, chelerythrine, magnolol, honokiol and rhein.The inhibitory zone of 5 compounds exceeded 20 mm. The minimal inhibitory concentration(MIC)of 5 drugs were ranged from 8 to 32 μg/mL. The minimum bactericidal concentration(MBC) of sanguinarine against 3 S. agalactiae strains were 16, 32 and 16 μg/mL, respectively. Further growth curve and biofilm test implied different sanguinarine concentrations had different antimicrobial effects on the growth and the biofilm formation of S. agalactiate. Compared with other concentrations, the biofilm formation of S. agalactiate was less than 2.76% once sanguinarine concentration reached to 16 μg/mL, and the biofilm formation rate of S. agalactiate was over 90% with chlorogenic acid at the same concentration gradient.
引文
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[26]郑曙明,黄建军,吴青,等.复方五倍子有效成分的分离鉴定及抑菌活性研究[J].水生生物学报,2010,34(1):57-64.
[27]卢静,王振宁,陈锐,等.几种中药单体和抗生素对嗜水气单胞菌及温和气单胞菌的体外抑菌活性研究[J].水生生物学报,2013,37(6):1128-1132.
[28]李笑春,黄建国,崔敏,等.三种中草药复方提取物的体外抑菌试验[J].甘肃畜牧兽医,2006,36(1):11-13.
[2]Edwards M S.Issues of antimicrobial resistance in group B Streptococcus in the era of intrapartum antibioticprophylaxis [J].Sem Ped Inf Dis,2006,17(3):149-152.
[3]Chen M,Li L P,Wang R,et al.PCR detection and PFGE genotype analyses of streptococcal clinicalisolates from tilapia in China [J].Vet Mic,2012,159(3-4):526-530.
[4]Hernande E,Figueroa J,Iregui C.Streptococcosis on a red tilapia,oreochromis sp,farm:a case study[J].J Fish Dis,2009,32( 3):247-252.
[5]肖辉,苏振霞,单娟娟,等.16种中草药提取物对嗜水气单胞菌的体外抑菌实验[J].水生态学杂志,2009,(3):53-56.
[6]Protera C.Forging a link between biofilms and disease[J].Science,1999,283(5409):1837-1839.
[7]王文博,汪建国,李爱华,等.中草药应用于水产养殖的研究概况[J].中国水产,2006,(7):60-61.
[8]向枭,周兴华.中草药添加剂在水产养殖上的应用[J].粮食与饲料工业,2000,(3):27- 29.
[9]金珊,王国良,赵青松,等.中草药对海水养殖鲈鱼病原菌的抑菌效果研究[J].海洋科学,1999,(5):8-10.
[10]Mo W Y,Lun C H,Choi W M,et al.Enhancing growth and non-specific immunity of grass carp and Nile tilapia by incorporating Chinese herbs(Astragalus membranaceus and Lycium barbarum) into food waste based pellets[J].Environ Pollut,2016,219:475-482.
[11]Christybapita D,Divtagnaneswari M,Micheal R D.Oral administration of Eclipta alba leaf aqueous extract enhances the non-specific immune responses and disease resistance of Oreochromis mossambicus.[J].Fish Shellfish Immunol,2007,23(4):840-852.
[12]Wu C C,Liu C H,Chang Y P,et al.Effects of hot-water extract of Toona sinensis on immune response and resistance to Aeromonas hydrophila in Oreochromis mossambicus[J].Fish Shellfish Immunol,2010,29(2):258-263.
[13]李仪奎.中药药理实验方法学[M].上海:上海科学技术出版社,1991.
[14] Rodriguez-Tudela J L,Barohiesi F,Bille J,et al.Method for the derermination of minimurm inhibitory concentration (MIC) by broth dilution of fermentative yeasts[J].Chin Microb Infec,2019,9(8):i-viii.
[15]吴静,陈瑛,贾桂珍.中草药对猪链球菌生物被膜形成的影响[J].畜牧与兽医,2015,47(1):106-108.
[16]Pérez-Giraldo C,Cruz-Villalón G,Sánchez-Silos R,et al.In vitro,activity of allicin against Staphylococcus epidermidis,and influence of subinhibitory concentrations on biofilm formation[J].J Appl Microbiol,2003,95(4):709-711.
[17]Wink M,Schmeller T,Latz -Bruning B,et al.Modes of action of allel-ochemical alkaloids:Interaction with neuror-eceptors DNA and other molecular targets [J].J Chem Ecol,1998,24(11):1881-1937.
[18]Lenfeld J,Krotil M,Marsalek E,et al.Antiinflammatory activity of quaternary benzophenanthridine alkaloids from chelidonium majus[J].Planta Med,1981,43(2):161-169.
[19]胡海军.血根碱及白屈菜红碱抑菌和杀螨活性构效关系[D].陕西杨凌:西北农林科技大学,2008:37.
[20]陈俭清,周永辉,杨艳北,等.中药水提物对体外猪链球菌生物被膜作用的试验[J].中国兽医杂志,2016,(11):14-16.
[21] 史祺云,程威,金琨琪,等.鱼腥草素钠对金黄色葡萄球菌生物被膜的抑制作用[J].黑龙江畜牧兽医,2014,(7):14-16.
[22]王静慧.血根碱对细菌生物被膜的作用研究[D].杭州:浙江工商大学,2012:47.
[23]Costerton J W.Introduction to biofilm [J].Int J Antimicrob Agents,1999,11:217-221.
[24]Ramos C,Licht T R,Stermberg C,et al.Monitoring bacterial growth activity in biofilms from laboratory flow chambers,plant rhizosphere,and animal intestine[ J].Methods Enzymol,2001,337:21-42.
[25]曹凤娇.芦荟大黄素抑制金黄色葡萄球菌生物被膜形成的分子机制研究[D].长春:吉林农业大学,2017:35.
[26]郑曙明,黄建军,吴青,等.复方五倍子有效成分的分离鉴定及抑菌活性研究[J].水生生物学报,2010,34(1):57-64.
[27]卢静,王振宁,陈锐,等.几种中药单体和抗生素对嗜水气单胞菌及温和气单胞菌的体外抑菌活性研究[J].水生生物学报,2013,37(6):1128-1132.
[28]李笑春,黄建国,崔敏,等.三种中草药复方提取物的体外抑菌试验[J].甘肃畜牧兽医,2006,36(1):11-13.