CN107574130B - 珊瑚球菌在捕食耐药菌和在制备抑制耐药菌药物中的应用 - Google Patents
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Abstract
本发明公开了珊瑚球菌在捕食耐药菌和在制备抑制耐药菌药物中的应用。本发明的五株粘细菌Myxococcus sp.GIM1.810，Myxococcus sp.GIM1.811，Corallococcus exiguus GIM1.813，Myxococcus sp.GIM1.815和Corallococcus coralloides GIM1.816能够捕食耐药菌，并且其还能够产生显著抑制耐药菌的活性天然产物，因此在对这些耐药菌进行生物防治或者开发有效抑制这些耐药菌的抗生素药物方面具有较好的实际应用价值。
Description
本申请是专利申请号：201510152005.0，发明名称：五株粘细菌在捕食耐药菌和在制备抑制耐药菌药物中的应用，申请人：广东省微生物研究所的分案申请。
技术领域：
本发明属于微生物领域，具体涉及珊瑚球菌在捕食耐药菌和在制备抑制耐药菌药物中的应用。
背景技术：
近些年来，随着旧有抗生素尤其是广谱抗菌素的广泛使用乃至滥用，各种耐药菌日益增多和复杂化。目前，细菌的耐药性已成为医疗事业和人类健康的重大威胁。近些年上市的新抗生素种类较少，对这些耐药菌可采取的手段很有限，它们的出现几乎是一种灾难。寻找和开发能高效抑制耐药菌的抗生素刻不容缓。
微生物天然产物一直是新抗生素的重要来源。但是，从传统的抗生素产生菌如链霉菌、枯草芽孢杆菌、假单胞菌等中发现新的抗生素越来越困难。同时，寻找新抗生素产生菌的研究又进展缓慢。近些年来，只有蓝细菌和粘细菌这两类微生物被开发为新的抗生素产生菌。
粘细菌是革兰氏阴性的土壤土著菌，同时也广泛分布于自然界中的各种环境。粘细菌分类地位特殊，虽然属于原核生物，但其很多特征与真核生物更为相似，具有复杂的社会性行为和形态发生，如多细胞信号传导和感应、共同协调运动、狼群式捕食行为、子实体结构的形成等。
粘细菌是自然界中重要的大分子降解者和微生物捕食者，在微生态平衡和地球生物圈的物质循环中扮演着重要的角色。粘细菌能够产生多种胞外裂解酶，如细胞裂解酶、核酸酶、酯酶、蛋白酶、多糖酶、淀粉酶和几丁质酶等。大多数粘细菌能裂解多种多样的细菌、真菌、酵母和藻类等微生物。已发现粘细菌能够裂解多种病原菌，具有重要的生物防治潜力。
粘细菌具有原核生物中最大的基因组，使得它们的次级代谢产物丰富多样。粘细菌中，能产生活性天然产物的比例很高。溶细菌的2000多株粘细菌中，可产生生物活性物质的达55％；溶纤维的700多株粘细菌中，可产生生物活性物质的达95％。粘细菌产生的活性物质具有结构新颖、种类多样、活性良好、作用机制复杂等特征，在药物开发，农业生产和生态治理等方面有广泛的应用潜力。粘细菌已经成为仅次于放线菌的第二大抗生素产生菌。目前已在粘细菌中发现了100余种全新结构的次级代谢产物和600多种新的结构衍生物，包括杂环、芳香环、多烯、大环、聚醚、生物碱和肽类。粘细菌中已发现的抗细菌类药物包括Corallopyronin A,Angiolam A,Thuggacins,Carolacton,Myxovirescins,Chondrochlorens等多种。
发明内容：
本发明的目的是提供五株粘细菌在捕食耐药菌和在制备抑制耐药菌药物中的应用。
本发明通过实验发现，Myxococcus sp.GIM1.810，Myxococcus sp.GIM1.811，Corallococcus exiguus GIM1.813，Myxococcus sp.GIM1.815和Corallococcuscoralloides GIM1.816对耐药沙门氏菌和耐药大肠杆菌都具有捕食作用。除Myxococcussp.GIM1.815对耐药金黄色葡萄球菌无捕食作用，其他如Myxococcus sp.GIM1.810，Myxococcus sp.GIM1.811，Corallococcusexiguus GIM1.813和Corallococcuscoralloides GIM1.816对耐药金黄色葡萄球菌都具有捕食作用。
因此，本发明的第一个目的是提供Myxococcus sp.GIM1.810，Myxococcussp.GIM1.811，Corallococcus exiguus GIM1.813，Myxococcus sp.GIM1.815或Corallococcus coralloides GIM1.816在捕食耐药菌中的应用。
优选，Myxococcus sp.GIM1.810，Myxococcus sp.GIM1.811，Corallococcusexiguus GIM1.813和Corallococcus coralloides GIM1.816在捕食耐药沙门氏菌、耐药大肠杆菌和耐药金黄色葡萄球菌中的应用；Myxococcus sp.GIM1.815在捕食耐药沙门氏菌和耐药大肠杆菌中的应用。
本发明通过实验发现：
Myxococcus sp.GIM1.810能够产生抑制S.aureus 11、S.aureus 46、SalmonellaCMCC51005、Salmonella 56、E.coli A16和E.coli D61-1的药物。
Myxococcus sp.GIM1.811能产生抑制Staphylococcus aureus ATCC8739、S.aureus 15、S.aureus 46、Salmonella CMCC51005、Salmonella 31、Salmonella 56、Escherichia coli ATCC8739、E.coli A16、E.coli A29、E.coli D57和E.coli D61-1的药物。
Corallococcus exiguus GIM1.813能产生抑制Staphylococcus aureusATCC8739、S.aureus 11、S.aureus 15和S.aureus 46的药物。
Myxococcus sp.GIM1.815能产生抑制Salmonella CMCC51005、Salmonella 31、Salmonella 47、Salmonella 56、Salmonella 59、Escherichia coli ATCC8739、E.coliA16、E.coli A29、E.coli D57和E.coli D61-1的药物。
Corallococcus coralloides GIM1.816能产生抑制Staphylococcus aureusATCC8739、S.aureus 11、S.aureus 15、S.aureus 46的药物。
因此，本发明的第二个目的是提供Myxococcus sp.GIM1.810，Myxococcussp.GIM1.811，Corallococcus exiguus GIM1.813，Myxococcus sp.GIM1.815、Corallococcus coralloides GIM1.816在制备抑菌药物中的应用。
进一步优选，Myxococcus sp.GIM1.810在制备抑制S.aureus 11、S.aureus 46、Salmonella CMCC51005、Salmonella 56、E.coli A16和E.coli D61-1的药物中应用。
Myxococcus sp.GIM1.811在制备抑制Staphylococcus aureus ATCC8739、S.aureus 15、S.aureus 46、Salmonella CMCC51005、Salmonella 31、Salmonella 56、大肠杆菌或耐药大肠杆菌的药物中的应用。
所述的耐药大肠杆菌为E.coli A16、E.coli A29、E.coli D57或E.coli D61-1。
Corallococcus exiguus GIM1.813在制备抑制金黄色葡萄球菌或耐药金黄色葡萄球菌的药物中的应用。所述的耐药金黄色葡萄球菌为S.aureus 11、S.aureus 15或S.aureus 46。
Myxococcus sp.GIM1.815在制备抑制沙门氏菌、耐药沙门氏菌、大肠杆菌或耐药大肠杆菌的药物中的应用。所述的耐药沙门氏菌为Salmonella 31、Salmonella 47、Salmonella 56、Salmonella 59，所述的耐药大肠杆菌E.coli A16、E.coli A29、E.coliD57和E.coli D61-1；
Corallococcus coralloides GIM1.816在制备抑制金黄色葡萄球菌或耐药金黄色葡萄球菌的药物中的应用。所述的耐药金黄色葡萄球菌为S.aureus 11、S.aureus 15或S.aureus 46。
本发明的五株粘细菌Myxococcus sp.GIM1.810，Myxococcus sp.GIM1.811，Corallococcus exiguus GIM1.813，Myxococcus sp.GIM1.815和Corallococcuscoralloides GIM1.816能够捕食耐药菌，并且其还能够产生显著抑制耐药菌的活性天然产物，因此在对这些耐药菌进行生物防治或者开发有效抑制这些耐药菌的抗生素药物方面具有较好的实际应用价值。
附图说明：
图1是五株粘细菌对耐药菌的捕食效果。
具体实施方式：
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
实施例1.粘细菌对耐药菌的捕食作用研究
本实施例的五株粘细菌分别为：Myxococcus sp.GIM1.810(保藏编号为GIM1.810)，Myxococcus sp.GIM1.811(保藏编号为GIM1.811)，CorallococcusexiguusGIM1.813(保藏编号为GIM1.813)，Myxococcus sp.GIM1.815(保藏编号为GIM1.815)和Corallococcus coralloides GIM1.816(保藏编号为GIM1.816)，上述五株粘细菌都保藏于广东省微生物菌种保藏中心，其保藏编号见菌种名后面的括号内，该保藏中心的菌株是对外销售的，因此任何人都可以从该保藏中心购买到该菌株。
本实施例中所用耐药菌株分别为：耐药金黄色葡萄球菌三株(S.aureus 11、S.aureus 15、S.aureus 46)，沙门氏菌四株(Salmonella sp.31、Salmonella sp.47、Salmonella sp.56、Salmonellasp.59)，大肠杆菌四株(E.coli A16、E.coli A29、E.coliD57、E.coli D61-1)。均由华南农业大学兽医学院药理与毒理学实验室提供，具体耐药信息详见表1。
表1耐药菌菌株MIC值
注：μg/L；MIC临界值表示大于临界值即为耐相应抗生素菌株；大肠杆菌及沙门氏菌共用一个临界值；高于临界值菌株，即菌株抗相应抗生素，均用加粗标示。
1、分别接种上述五种粘细菌到CYE培养液(10mM MOPS(3-(N-吗啡啉)丙磺酸)，10g/L酪蛋白胨，5g/L酵母提取物，8mM MgSO4，PH 7.6，溶剂为水，灭菌消毒备用)中，150rpm、30℃培养3d，而后用MMC缓冲液(10mM MOPS，4mM MgSO4，2mM CaCl2，PH 7.6，溶剂为水，灭菌消毒备用)冲洗稀释至1×1011cell/ml。
2、接种耐药菌株到LB培养基中，150rpm、37℃培养到对数期，而后用MMC缓冲液冲洗稀释至1×109cell/mL。
3、将20μL耐药菌菌液滴到CFL固体培养基(10mM MOPS，1mM KH2PO4，8mM MgSO4，0.2g/L(NH4)2SO4,0.2g/L柠檬酸钠，0.2g/L丙酮酸钠，0.1g/L酪胨，15g/L琼脂，pH 7.6，溶剂为水，灭菌消毒备用)上，待干燥后，在其边缘处滴加1μL粘细菌菌液(与墨汁按体积比2：1混匀)，两个菌落边缘相距约3mm。将平板置于32℃培养，3、5、7、9d后观察捕食现象。
具体结果如图1所示，从图1可以看出，本实施例的Myxococcus sp.GIM1.810，Myxococcus sp.GIM1.811，Corallococcus exiguus GIM1.813，Myxococcus sp.GIM1.815和Corallococcus coralloides GIM1.816对耐药沙门氏菌(图中只显示了Salmonellasp.31，其他耐药沙门氏菌是相同的结果)和耐药大肠杆菌(图中只显示了E.coli A16，其他耐药大肠杆菌是相同的结果)都具有捕食作用。除Myxococcus sp.GIM1.815对耐药金黄色葡萄球菌无捕食作用，其他如Myxococcus sp.GIM1.810，Myxococcus sp.GIM1.811，Corallococcus exiguus GIM1.813和Corallococcus coralloides GIM1.816对耐药金黄色葡萄球菌(图中只显示了S.aureus 11，其他耐药金黄色葡萄球菌是相同的结果)都具有捕食作用。
实施例2.粘细菌的代谢产物对耐药菌的抑制作用研究
1、在VY/2培养基(鲜酵母5.0g/L，CaCl2·2H20 1.0g/L，VB120.0005g/L，PH 7.2)中分别接种五株粘细菌Myxococcus sp.GIM1.810，Myxococcus sp.GIM1.811，Corallococcusexiguus GIM1.813，Myxococcus sp.GIM1.815和Corallococcuscoralloides GIM1.816，150rpm，30℃培养7d。
2、4000rpm离心，分别收集菌体和发酵液上清。发酵液用等体积的乙酸乙酯萃取12h。菌体用丙酮浸泡后超声破碎，然后用乙酸乙酯萃取12h。萃取液旋转蒸发后，用甲醇溶解萃取物，发酵液和菌体破碎液萃取物分别溶解成50mg/mL和100mg/mL浓度。
3、接种耐药菌到LB液体培养基中，150rpm，37℃培养到对数期。按1:100比例与50℃左右的LB琼脂培养基(液体状态)混合，摇匀后每个平皿加20mL。在平板上粘附已滴加5μL萃取液的6mm直径滤纸片。37℃培养18～24h后测定抑菌圈直径。
4、五株粘细菌粗提物对耐药菌的抑制效果见表2。
表2.五株粘细菌粗提物的抑菌圈直径
B：发酵液粗提物；C：菌体粗提物；抑菌圈直径单位：mm；—，表示粗提物对相应耐药菌没有活性；
表中所用耐药菌同实施例1中相同，其中Staphylococcus aureus ATCC8739、Salmonella CMCC51005、Escherichia coli ATCC8739分别为金黄色葡萄球菌、沙门氏菌和大肠杆菌的标准菌株。
从表2可以看出，Myxococcus sp.GIM1.810能够产生抑制S.aureus 11、S.aureus46、Salmonella CMCC51005、Salmonella 56、E.coli A16和E.coli D61-1的药物。
Myxococcus sp.GIM1.811能产生抑制Staphylococcus aureus ATCC8739、S.aureus 15、S.aureus 46、Salmonella CMCC51005、Salmonella 31、Salmonella 56、Escherichia coli ATCC8739、E.coli A16、E.coli A29、E.coli D57和E.coli D61-1的药物。
Corallococcus exiguus GIM1.813能产生抑制Staphylococcus aureusATCC8739、S.aureus11、S.aureus 15和S.aureus 46的药物。
Myxococcus sp.GIM1.815能产生抑制Salmonella CMCC51005、Salmonella 31、Salmonella47、Salmonella 56、Salmonella 59、Escherichia coli ATCC8739、E.coliA16、E.coli A29、E.coli D57和E.coli D61-1的药物。
Corallococcus coralloides GIM1.816能产生抑制Staphylococcus aureusATCC8739、S.aureus 11、S.aureus 15、S.aureus 46的药物。
Claims (4)
1.Corallococcus coralloides GIM1.816在制备捕食耐药沙门氏菌、耐药大肠杆菌和耐药金黄色葡萄球菌药物中的应用。
2.Corallococcus coralloides GIM1.816在制备抑制金黄色葡萄球菌药物中的应用。
3.根据权利要求2所述的应用，其特征在于，所述的金黄色葡萄球菌是耐药金黄色葡萄球菌。
4.根据权利要求3所述的应用，其特征在于，所述的耐药金黄色葡萄球菌为S.aureus11、S.aureus 15或S.aureus 46。
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