CN106467898A - 一种内生真菌菌株及其代谢产物提取方法和用途 - Google Patents
一种内生真菌菌株及其代谢产物提取方法和用途 Download PDFInfo
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Abstract
本发明涉及一种内生真菌菌株及其代谢产物提取方法和用途,属于微生物及微生物药物领域。经形态学特征观察及其rDNA ITS序列分析鉴定该菌株为镰孢菌Fusariumsp,其具有显著的抑菌活性。该菌株的代谢产物提取物对大肠杆菌、金黄色葡萄球菌、枯草杆菌等野生型菌株以及携带β‑内酰胺酶基因重组质粒的大肠杆菌或肺炎克雷伯菌等耐药菌株具有显著的抑菌活性,对临床分离的同时携带NDM‑1等多种β‑内酰胺酶基因的大肠杆菌、阴沟肠杆菌和肺炎克雷伯菌,以及耐甲氧西林金黄色葡萄球菌(MRSA)等“超级细菌”均具有显著的抑制活性。
Description
技术领域
本发明涉及一种内生真菌菌株及其代谢产物提取方法和用途,属于微生物及微生物药物领域。
背景技术
细菌耐药性在世界范围内日趋严重,涌现出了一些具有高度多重耐药能力的“超级细菌”。2010年世界著名医学杂志《The Lancet Infectious Diseases》(《柳叶刀-传染病》)报道了一种携带“新德里金属-β-内酰胺酶-1”(New Delhi metallo-β-lactamase-1,简写为NDM-1)耐药基因的新型“超级细菌”,该“超级细菌”能够耐受几乎所有的抗生素,仅对多粘菌素(colistin)和替加环素(tigecycline)敏感,有的菌株甚至也能够耐受这两种药物。该耐药基因可通过质粒在菌株间传递,从而使其快速传播和蔓延。此类细菌相继在包括我国在内的世界多国被发现,引起了人们极大的震动和关注。
NDM-1是一种新型β-内酰胺酶。产生β-内酰胺酶是细菌产生耐药性的主要机制之一,该类酶能够分解β-内酰胺类抗生素(青霉素类、头孢菌素类和碳青霉烯类等分子结构上含有β-内酰胺环的药物),从而使其失去活性。β-内酰胺酶种类繁多,目前临床危害严重的酶包括超广谱β-内酰胺酶、AmpC类β-内酰胺酶、丝氨酸碳青霉烯酶及金属β-内酰胺酶等,上述NDM-1即是一种金属β-内酰胺酶。细菌可同时携带多种β-内酰胺酶基因,有些菌株还连锁携带其它耐药基因,如对氨基糖苷类和喹诺酮类抗生素耐药的基因,从而导致多重耐药。
耐甲氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus,简写为MRSA)是另一类“超级细菌”。该类细菌早在上世纪六十年代已被发现,但至今尚未被控制,且其耐药性及危害愈来愈严重,已发展成为全球性致病菌,是院内感染最常见的病原菌之一,并逐渐向社区扩散。MRSA对绝大多数临床常用抗生素均有耐受性,包括β-内酰胺类、氨基糖苷类、氟喹诺酮类、四环素类、大环内酯类等药物。糖肽类抗生素万古霉素治疗MRSA感染的效果较好,但近年来出现了耐万古霉素的金黄色葡萄球菌菌株(Vancomycin-resistant S. aureus, 简写为VRSA)。另外利奈唑胺(linezolid)、达托霉素(daptomycin)及替加环素等也具有一定的疗效,但均有各自的不足。
上述耐药菌在临床上危害严重。面对这些耐药菌的感染,目前可选用的治疗方案和治疗药物非常有限,甚至无药可用,特别是对于免疫缺陷、年老体弱、大手术后、长期住院、存在严重基础疾病、长期大量应用广谱抗菌药物、入住ICU病房、多脏器功能不全、接受化疗或放疗等患者,治疗困难,病死率高。因此,研发新型抗生素刻不容缓。我们从药用植物十大功劳Mahoniafortunei的健康叶片中分离到一株内生真菌PF1167,体外实验表明,该菌株的代谢产物提取物对携带上述β-内酰胺酶基因的菌株及MRSA具有显著的抑制活性。该菌株易于培养,具有开发新抗生素的潜力。
发明内容
本发明为研制新型抗菌药物提供了一种内生真菌菌株及其代谢产物提取方法和用途,该菌株的代谢产物提取物对大肠杆菌、金黄色葡萄球菌、枯草杆菌等野生型菌株以及携带β-内酰胺酶基因重组质粒的大肠杆菌或肺炎克雷伯菌等耐药菌株具有显著的抑菌活性,对临床分离的同时携带NDM-1等多种β-内酰胺酶基因的大肠杆菌、阴沟肠杆菌和肺炎克雷伯菌,以及MRSA等“超级细菌”均具有显著的抑制活性。该菌株易于培养,具有开发新抗生素的潜力。
一种内生真菌菌株PF1167,经形态学特征观察及其rDNA ITS序列分析鉴定为镰孢菌Fusariumsp.,其微生物保藏编号为:CGMCC No.11815。保藏地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心,保藏日期:2016年1月08日。
本发明所述内生真菌菌株PF1167分离于十大功劳的健康叶片。该菌株的形态学特征如下:将内生真菌PF1167接种到马铃薯葡萄糖琼脂(Potato Dextrose Agar ,简写为PDA)培养基上,25℃下培养8天,可见白色菌落,菌丝绒毛状,菌落直径6.5cm,边缘规则,菌落背面初无色,培养7日后中部开始呈暗红色(图1-2),并逐渐加深;分生孢子梗不一,细长、简单,分枝不规则,分生孢子无色,不一,有大小型两种,大型分生孢子长棒形,小型分生孢子卵圆形,常聚生(图3-4)。提取该菌株的基因组总DNA,利用真菌通用引物ITS1(TCCGTAGGTGAACCTGCGG)和ITS4(TCCTCCGCTTATTGATATGC)对获得的基因组DNA进行PCR扩增,然后对PCR产物进行测序,获得该菌株的核糖体DNA 内转录间隔区序列(internaltranscribed spacer, ITS)数据。进入美国国家生物技术信息中心(National CenterforBiotechnology Information,NCBI)网站:http://www.ncbi.nlm.nih.gov/,对测序数据进行核酸Blast分析,结果表明所测序列与一株Fusariumsp.的ITS序列(登录号:KM513579.1)相似度达99%。应用MEGA 6.0软件构建系统发育树(图5),结果表明,该菌株与上述Fusariumsp.聚类于同一末端分支。结合菌株PF1167的形态学特征与ITS序列分析,鉴定该菌株为Fusarium属菌种,本菌株定名为Fusariumsp.PF1167。
将菌株PF1167接种于装有马铃薯葡萄糖液体培养基的瓶中,于25℃下静置培养21天,将培养物过滤,得上清液,用旋转蒸发仪将上清液浓缩,向浓缩液中加入3倍体积的无水乙醇,充分震荡后过滤,得上清液,将上清液浓缩干燥,溶于无菌水后制得代谢产物提取物。
所述的代谢产物提取物,具有抑菌活性。
所述的抑菌活性:被该菌株的代谢产物提取物抑制的菌株包括以下菌株:
(1)野生型大肠杆菌、金黄色葡萄球菌和枯草杆菌;
(2)携带β-内酰胺酶基因重组质粒的大肠杆菌或肺炎克雷伯菌菌株,上述基因包括临床危害严重的β-内酰胺酶基因:a. 超广谱β-内酰胺酶基因,包括TEM-10、TEM-12、TEM-26、SHV-5、SHV-12、SHV-18、OXA-2、OXA-10、OXA-48、CTX-M-3、CTX-M-14、CTX-M-15;b. Amc类β-内酰胺酶基因,包括DHA-1、CMY-2、FOX-5;c. 丝氨酸碳青霉烯酶基因,包括NMC-A、KPC-2、KPC-3;d.金属β-内酰胺酶基因,包括VIM-1和NDM-1;
(3)临床分离的同时携带包括NDM-1在内的多种β-内酰胺酶基因的大肠杆菌、肺炎克雷伯菌和阴沟肠杆菌;
(4)MRSA
经pH试纸测试,上述提取物pH值为中性。以苯/甲醇(4:1)作为展层剂,0.5M磷酸缓冲液(pH 7.0)处理过的新华3号滤纸条为载体对上述提取物进行纸层析,结果提示活性成分的Rf值在0.40-0.53之间。
本发明为研制新型抗菌药物,特别是新型抗耐药菌药物提供了一株容易培养、发酵成本低的内生真菌菌株,具有较高的开发价值和应用前景。
本发明工作得到了国家自然科学基金(No. 31300067)、江苏省高校自然科学研究重大项目(No.15KJA180002)和江苏省海洋生物技术重点实验室开放课题项目(No.2015HS003)的资助。
附图说明
图1 为菌株PF1167在PDA平板上的正面菌落形态照片。
图2为菌株PF1167在PDA平板上的反面菌落形态照片。
图3为菌株PF1167的产孢结构显微照片(放大倍数:600倍)。
图4为菌株PF1167的孢子形态显微照片(放大倍数:600倍)。
图5为基于菌株PF1167的ITS的系统发育分析。
图6为菌株PF1167代谢产物提取物活性成分的初步分离实验结果照片。
具体实施方式
下面结合具体实施例来进一步详细描述本发明,但这些实施例仅是范例性的,本发明的保护范围并不限于下述实施例。本领域的普通技术人员很容易根据本文说明对本发明进行复制或做出各种替换、修改或改变,这些复制、替换、修改或改变均在本发明的保护范围内。
实施例1:内生真菌菌株PF1167的分离、纯化及保存。
实验方法与步骤如下:
1. 样本的采集:采集十大功劳健康叶片,立即带回实验室用于内生真菌的分离。
2. 内生真菌的分离:将采集的叶片于流水下冲洗,去除表面的灰尘,晾干后将叶片剪切成约0.5×0.5 cm大小的小方块,按如下方法对其进行表面灭菌:先用70% 乙醇浸泡叶片小块样本l min,之后转入稀释40倍的84消毒液(爱特福基团)中浸泡10 min,最后用无菌水冲洗3次(每次1 min)。将表面灭菌的叶片小块接至含有PDA培养基的平板上,每一平板均匀放置5个小块,将上述平板置于25 ℃下培养3 - 7天,每日观察叶片边缘是否有菌丝长出。
3. 内生真菌的纯化:叶片边缘长出菌丝后,用无菌接种针切取大小为0.5 × 0.5cm的菌饼(菌丝及其下方培养基)转至新PDA培养基上,25 ℃下培养3 - 7天,观察菌落形态是否为纯菌落。如不是纯菌落,则从菌落边缘切取大小为0.5 × 0.5 cm的菌饼,按上述方法继续纯化,直至获得纯菌落。
4. 内生真菌的保藏:用无菌接种针从内生真菌纯菌落中切取5个大小为0.5 ×0.5 cm的菌饼,转至灭菌的装有石蜡油的冷冻管中,置于4 ℃冰箱中保存。
实施例2. 内生真菌菌株PF1167的鉴定。
1. 形态学鉴定:将内生真菌菌株PF1167接种至新PDA培养基上,置于25 ℃下培养7 - 30天,每日观察菌落正反面特征。待其生成孢子后,取一张载玻片,在其中央滴一滴乳酚油(配方:加热融化的苯酚20 mL;乳酸20 mL;甘油40 mL;水20 mL),挑取少许菌丝,置于乳酚油中,用接种针将菌丝拨散,盖上盖玻片,置于显微镜下观察其孢子及产孢结构形态特征,对照《真菌鉴定手册》(魏景超 1979)及《Illustrated genera of imeprfect fungi》(Barnett HL,Hunter BB 1998)进行形态学鉴定。菌落正反面形态见附图1、2,菌株孢子及产孢结构形态特征见附图3、4。根据其形态学特征,鉴定为:Fusarium属菌种。
2. ITS序列分析:
a)实验所用的缓冲液的配制方法:
(1)1M Tris-HCl (pH 8.0) 缓冲液
称取12.11 g 三羟甲基氨基甲烷(Tris)放于烧杯中,加入约80 mL的去离子水,充分搅拌使其溶解。用浓盐酸调节pH至8.0,再将溶液定容至100 mL。高温高压灭菌后,于室温保存。
(2)0.1M EDTA缓冲液(pH 8.0)
称取3.72g 乙二胺四乙酸二钠(EDTA二钠)放于烧杯中,加入约80 ml去离子水中,充分搅拌使其溶解,调节pH至8.0,再将溶液定容至100 mL。高温高压灭菌后,于室温保存。
(3)10×TE 缓冲液配方
Tris-Cl缓冲液(pH 8.0) 100 mM/L
EDTA缓冲液(pH 8.0) 10 mM /L
调节酸碱度至所需pH值,分装后高压蒸汽灭菌20 min,置于冰箱中保存备用,用时将其稀释10倍。
(4)10% SDS溶液
称取10 g 十二烷基磺酸钠(SDS),溶于100 ml去离子水中,混匀备用。
(5)溴化乙锭(EB)溶液 (10 mg/mL)
在100 mL去离子水中溶解1 g EB。磁力搅拌数小时,以确保其完全溶解,然后用铝箔纸包裹容器或将溶液转移至棕色瓶中,室温保存。
b)菌株PF1167基因组总DNA的提取:将在PDA培养基上培养7天的内生真菌PF1167的菌丝从平板上小心刮取下来,用氯化苄法提取菌株基因组总DNA。步骤如下:①将菌丝放入1.5 ml 塑料小管中,加入450 μl TE缓冲液(pH 9.0),充分振荡混匀后加入150 μL 10%SDS缓冲液和450 μL氯化苄。②振荡混匀后50 ℃水浴1 h,期间每隔10 min振荡混合一次。③取出小管,13000 r/min离心10 min。离心完成后吸取上清液至一新小管中。④加入等体积的苯酚/氯仿/异戊醇(体积比为25:24:1)混合液,反复振荡混匀后13000 r/min离心10min,离心结束后吸取上清液至一新管中,重复此步骤一次。向取出的上清液中加入氯仿/异戊醇(体积比为24:1)混合液,反复振荡后13000 r/min离心10 min,吸出上清液转移至新小管。⑤加入1/10体积的醋酸钾溶液(5 M),再加入等体积的异丙醇后于-20 ℃过夜(或室温静置30min)。⑥取出上述小管,13000 r/min离心10 min,轻轻倒掉上清液,将离心管口放在卫生纸上吸取残留水分,保留沉淀部分。向含沉淀的小管中加入1 ml 70%乙醇,涡旋震荡,13000 r/min离心10 min,离心后小心倒掉小管中的乙醇,再重复此步骤2次。⑦将小管置于金属浴中,50 ℃烘干乙醇(不可残留任何乙醇),将沉淀溶于30 μl TE缓冲液(pH7.4)中,于-20 ℃保存、备用。
c)菌株PF1167的ITS序列扩增:采用真菌通用引物ITS1/TIS4及DreamTaq GreenPCR Master Mix(Thermo Scientific)进行其ITS序列的PCR扩增,引物序列分别为:
ITS1(5'to3'):TCCGTAGGTGAACCTGCGG;
TIS4(5'to3'):TCCTCCGCTTATTGATATGC
反应体系如下:
PCR的反应程序如下:95℃预变性5min,依次进入:95℃变性20s,45℃退火30s,68℃延伸1min;95℃变性20s,50℃退火30s,68℃延伸1min,然后开始进入循环:95℃变性20s,55℃退火30s,68℃延伸1min,共35个循环,最后68℃延伸10min。
d)PCR反应产物的确认:配置1% 琼脂糖凝胶,取PCR产物和GeneRuler 1kb PlusDNA Ladder各5 μl上样,在110v、160mA条件下电泳30 min。电泳结束后将凝胶置于EB溶液(0.5 μg/ml)中染色30 min,然后置于凝胶成像系统中观察条带。如出现500 bp - 700 bp条带,则初步判断扩增成功。
e)PCR反应产物的测序:将PCR产物送生工生物工程上海(股份)有限公司进行测序。
f) 数据分析:将菌株PF1167的ITS序列在NCBI网页(http://www.ncbi.nlm.nih.gov/)上进行在线比对分析(blastn),找到与其最相似的已知序列。用软件MEGA 6.0对目的序列进行系统发育分析,用邻位连接法(Neighbor-Joining, NJ)构建系统发育树,系统发育树的评估用Bootstrap检验,共进行1000 次重复。系统发育树见附图5,结果表明所测序列与一株Fusariumsp.的ITS序列(登录号:KM513579.1)相似度达99%。系统进化树结果表明,该菌株与上述Fusariumsp.聚类于同一末端分支。
根据ITS序列分析,鉴定该菌株为:Fusarium属菌种,与形态学鉴定结果一致。结合两种鉴定结果,该菌株鉴定为Fusariumsp.,本株定名为Fusariumsp.PF1167。
实施例3. 内生真菌PF1167代谢产物的体外抑菌实验。
一、实验方法(微量肉汤二倍稀释法)
1)涉及的细菌菌种:① 20种携带β-内酰胺酶基因重组质粒的大肠杆菌或肺炎克雷伯菌菌株;② 3种携带NDM-1等多种β-内酰胺酶基因的临床分离菌株;③MRSA;④ 野生型大肠杆菌、金黄色葡萄球菌和枯草杆菌。
2) 菌种的活化:将各菌种从-80 ºC冰箱中取出,分别划线接种于Luria-Bertani(LB)培养基平板上。其中对于携带β-内酰胺酶基因重组质粒的菌株,所用的培养基中含有相应的抗生素,所含抗生素的类别与浓度与其质粒选择标记一致,具体见表1。对于携带NDM-1等多种β-内酰胺酶基因的临床分离菌株,所用培养基含终浓度为100 μg/ml的氨苄青霉素。将各平板置于37 ºC培养箱中过夜培养。
3) 菌液的配制:从上述各LB平板上分别挑取单菌落至含LB液体培养基的试管中。其中,对于携带β-内酰胺酶基因重组质粒的菌株,所用的培养基中含有相应的抗生素,所含抗生素的类别与浓度与其质粒选择标记一致,具体见表1;对于携带NDM-1等多种β-内酰胺酶基因的临床分离菌株,所用培养基含终浓度为100 μg/ml的氨苄青霉素。将各试管置于摇床上,于37 ºC下振荡培养,每隔一段时间取出少量菌液用酶标仪测定其在波长600 nm处的光密度 (optical density,OD600) 值,当菌液OD600 值为1时取出菌液,用LB培养液稀释5,000倍,备用。
4) 提取物的制备:将菌株PF1167接种于含20 ml马铃薯葡萄糖液体培养基的三角瓶中,于25 ℃下静置培养21天,将培养物过滤,得上清液,用旋转蒸发仪将上清液浓缩。向浓缩液中加入3倍体积的无水乙醇,充分震荡后过滤,得上清液。将上清液浓缩干燥,获得粗提物。准确称量粗提物,将其溶于无菌水中,配成100 mg/ml的溶液,然后依次2倍稀释,配成系列梯度溶液,最低浓度至0.1 mg/ml。配制溶液的过程中,所用的移液器吸头及塑料小管均在灭菌后使用。用pH试纸测试提取物的酸碱度。
5) 抑菌实验:在无菌96-孔板的各孔中分别加入10 μL各浓度提取物溶液和90 μL稀释后的菌液,混匀。另取3个孔分别加入10 μL无菌水和90 μL稀释后的菌液作为对照,立即测定各孔菌液OD600值。将96-孔板置于37 ºC下恒温培养18~20 h,然后再次测定各孔菌液OD600值。根据每个孔菌液OD600值的变化计算提取物的抑菌率。抑菌率的计算公式如下:
抑制率(%)= [1- (样品OD600增加量/对照OD600增加量)]×100
以抑菌率等于或超过90%时的提取物浓度为最低抑菌浓度(minimum inhibitoryconcentration,MIC)。上述实验重复3次,根据实验结果确定提取物的MIC。用同样的方法选测不同抗生素对上述细菌菌株的MIC。
二、实验结果:
1.提取物的pH 值
经测试,提取液pH值为中性。
2.提取物对携带β-内酰胺酶基因重组质粒的菌株的抑制作用
提取物对测试的20种携带临床危害严重的β-内酰胺酶基因的菌株均有抑制作用,对其中18株细菌的MIC等于或低于2.5mg/ml,对另外2株细菌(A10和A11)的MIC为5mg/ml(表1)。氨苄青霉素(ampicillin,简写为AMP)、卡那霉素(kanamycin,简写为KAN)及庆大霉素(gentamicin,简写为GM)对上述菌株的抑菌实验结果证明测试菌株含有正确的重组质粒,同时表明上述菌株普遍对AMP具有耐受性,有些菌株对KAN或GM也有不同程度的耐受性(表1)。
表1提取物对携带β-内酰胺酶基因重组质粒的菌株的抑制作用
注:“-”表示测试浓度范围内(0.1-100μg/ml)无抑菌作用。
3.提取物对携带NDM-1等多种β-内酰胺酶基因的临床分离菌株的抑制作用
我们测试了提取物及AMP、KAN、GM、头孢他啶(ceftazidime,简写为CAZ)、美罗培南(meropenem,简写为MEM)和替加环素(tigecycline,简写为TGC)等6种抗生素对3种临床分离菌株的MIC,结果表明:三种临床分离菌株具有很强的耐药性,其中18H3和18H4能够耐受所有测试的抗生素,18H2能够耐受除替加环素外的所有测试抗生素。对于上述耐药菌,菌株PF1167代谢产物提取物均具有抑制作用(表2)。
表2提取物对携带多种β-内酰胺酶基因的临床分离菌株的MIC(μg/ml)
注:“-”表示测试浓度范围内(0.1-100μg/ml)无抑菌作用。
4.提取物对MRSA及野生型金黄色葡萄球菌、枯草杆菌及大肠杆菌的抑制作用。
提取物对MRSA及野生型金黄色葡萄球菌、枯草杆菌及大肠杆菌均有抑制作用,其中对MRSA的抑制作用略低于野生型金黄色葡萄球菌。作为对照,我们同时测定了AMP对上述菌株的抑菌活性,从表3可以看出,AMP对野生型金黄色葡萄球菌、枯草杆菌及大肠杆菌的具有较强的抑制作用,但MRSA对AMP不敏感。
表3提取物对MRSA及野生型金黄色葡萄球菌、枯草杆菌及大肠杆菌的MIC
注:“-”表示测试浓度范围内(0.1-100μg/ml)无抑菌作用。
实施例4 菌株PF1167代谢产物提取物活性成分的初步分离实验。
1.实验方法:
剪取新华3号滤纸条(1.5×17 cm),用0.5 M磷酸缓冲液(pH 7.0)处理后晾干,将菌株PF1167代谢产物提取液(100 mg/ml)点样于滤纸条上,点样量为80 ul,用展层剂苯/甲醇(4:1)展开,当展层剂迁移至距原点15 cm处取出,将滤纸剪成3段(每段5 cm),分别贴于涂布有A20菌液的LB平板上,置于37 ℃培养过夜后观察抑菌圈位置。
2. 实验结果:
实验结果如图6所示,可见抑菌圈位于距原点6-8 cm处,提示提取液有效成分在上述条件下的Rf值在0.40-0.53之间。
SEQUENCE LISTING
<110> 江苏师范大学
<120> 一种内生真菌菌株及其代谢产物提取方法和用途
<130> 2016
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 529
<212> DNA
<213> Fusarium sp.PF1167
<400> 1
gggctagggc ttcactccca acccctgtga cataccaatt gttgcctcgg cggatcagcc 60
cgctcccggt aaaacgggac ggcccgccag aggaccccta aactctgttt ctatatgtaa 120
cttctgagta aaaccataaa taaatcaaaa ctttcaacaa cggatctctt ggttctggca 180
tcgatgaaga acgcagcaaa atgcgataag taatgtgaat tgcagaattc agtgaatcat 240
cgaatctttg aacgcacatt gcgcccgcca gtattctggc gggcatgcct gttcgagcgt 300
catttcaacc ctcaagccct cgggtttggt gttggggatc ggcgagccct tgcggcaagc 360
cggccccgaa atctagtggc ggtctcgctg cagcttccat tgcgtagtag taaaaccctc 420
gcaactggta cgcggcgcgg ccaagccgtt aaacccccaa cttctgaatg ttgacctcgg 480
atcaggtagg aatacccgct gaacttaagc atatcaaaag ccggaggaa 529
Claims (4)
1.一种内生真菌菌株,其特征在于:为内生真菌菌株PF1167,保藏编号为CGMCCNo.11815;保藏单位:中国微生物菌种保藏管理委员会普通微生物中心。
2.一种内生真菌菌株PF1167的代谢产物提取方法,其特征在于:将菌株PF1167接种于装有马铃薯葡萄糖液体培养基的瓶中,于25℃下静置培养21天,将培养物过滤,得上清液,用旋转蒸发仪将上清液浓缩,向浓缩液中加入3倍体积的无水乙醇,充分震荡后过滤,得上清液,将上清液浓缩干燥,溶于无菌水后制得代谢产物提取物。
3.根据权利要求2所述的代谢产物提取物,其特征在于:具有抑菌活性。
4.根据权利要求3 所述的抑菌活性,其特征在于:被该菌株的代谢产物提取物抑制的菌株包括以下菌株:
野生型大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphilococcus aureus)和枯草杆菌(Bacillus subtilis);
携带β-内酰胺酶基因重组质粒的大肠杆菌或肺炎克雷伯菌(Klebsiella pneumoniae)菌株,上述基因包括如下临床危害严重的β-内酰胺酶基因:
超广谱β-内酰胺酶基因,包括TEM-10、TEM-12、TEM-26、SHV-5、SHV-12、SHV-18、OXA-2、OXA-10、OXA-48、CTX-M-3、CTX-M-14、CTX-M-15;
Amc类β-内酰胺酶基因,包括DHA-1、CMY-2、FOX-5;
丝氨酸碳青霉烯酶基因,包括NMC-A、KPC-2、KPC-3;
金属β-内酰胺酶基因,包括VIM-1、NDM-1;
临床分离的同时携带包括NDM-1在内的多种β-内酰胺酶基因的大肠杆菌、肺炎克雷伯菌和阴沟肠杆菌(Enterobacter cloacae);
耐甲氧西林金黄色葡萄球菌(Methicillin-resistant S. aureus, MRSA)。
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