CN113913318B - 一种同时携带四个喹诺酮类药物耐药突变位点的鼠伤寒沙门菌及其应用 - Google Patents
一种同时携带四个喹诺酮类药物耐药突变位点的鼠伤寒沙门菌及其应用 Download PDFInfo
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Abstract
本发明涉及耐药菌株技术领域,尤其涉及一种同时携带四个喹诺酮类药物耐药突变位点的鼠伤寒沙门菌及其应用。本发明的一种鼠伤寒沙门菌,同时携带四个喹诺酮类药物耐药突变位点,所述四个喹诺酮类药物耐药突变位点分别为:gyrA基因编码产物的第83位的丝氨酸突变为苯丙氨酸;gyrA基因编码产物的第87位的天冬氨酸突变为甘氨酸;parC基因编码产物的第80位丝氨酸突变为精氨酸;parE基因编码产物的第458位丝氨酸突变为脯氨酸。这些突变位点导致沙门菌菌株对喹诺酮类药物表现为高水平耐药。鉴于此,沙门菌Sal17241可做为筛选新型抗菌药物的模型材料,具有良好的应用前景。
Description
技术领域
本发明涉及耐药菌株技术领域,尤其涉及一种同时携带四个喹诺酮类药物耐药突变位点的鼠伤寒沙门菌及其应用。
背景技术
沙门菌(Salmonella spp)是公共卫生学上具有重要意义的人畜共患病病原菌,可引起人和动物的伤寒、副伤寒、胃肠炎和败血症等许多严重疾病,使全球遭受巨大的经济损失。目前,全世界已发现约2610种血清型。其中,鼠伤寒沙门菌(Salmonella entericasubsp.enterica serovar Typhimurium)是存在于畜禽及食品中的优势血清型,也是引起人类感染的主要血清型。
喹诺酮类药物,尤其是氟喹诺酮类药物,是治疗多重耐药沙门菌引起的可能危及生命的感染最后可选择的一类重要药物。随着这类药物在临床及生产上的广泛应用,细菌对这类药物的耐药问题日益严重。研究表明沙门菌对喹诺酮类药物的耐药性逐年上升,而鼠伤寒沙门菌耐药率普遍高于其他沙门菌。
沙门菌对喹诺酮类药物产生耐药性最主要的机制是喹诺酮药物作用靶位点DNA螺旋酶(编码基因:gyrA和gyrB)和拓扑异构酶Ⅳ(编码基因:parC和parE)发生基因突变,使酶发生结构改变,从而影响与喹诺酮药物的结合。gyrA、gyrB、parC和parE又称喹诺酮耐药决定区(quinolone resistance-determining regions,QRDR)。另外,质粒介导的喹诺酮类药物耐药机制(plasmid-mediated quinolone resistance,PMQR),包括QNR蛋白(编码基因:qnrA、qnrB、qnrC、qnrD、qnrS、qnrVC)、外排泵转运蛋白(编码基因:qepA和oqxAB),以及乙酰转移酶(编码基因:aac(6’)-Ib-cr)也是导致细菌对喹诺酮类药物敏感性下降的原因。
沙门菌对喹诺酮类药物的耐药程度通常与突变位点位置和数量有关。单一位点突变可引起低水平耐药,若2个或多个突变同时发生,则会导致高水平耐药菌株的产生。目前,已有关于沙门菌QRDR突变菌株的报道中,多以单一位点突变为主,多个突变同时发生而导致高水平耐药菌株的比较少见。
发明内容
本发明的目的在于克服现有技术的不足,提供一种同时携带四个喹诺酮类药物耐药突变位点的鼠伤寒沙门菌及其应用,本发明的鼠伤寒沙门菌可作为筛选功能微生物/新型抗菌药物的模型材料,具有良好的应用前景。
为实现上述目的,本发明采取的技术方案为:提供一种鼠伤寒沙门菌,所述鼠伤寒沙门菌同时携带四个喹诺酮类药物耐药突变位点,所述四个喹诺酮类药物耐药突变位点分别为:
gyrA基因编码产物的第83位的丝氨酸突变为苯丙氨酸;
gyrA基因编码产物的第87位的天冬氨酸突变为甘氨酸;
parC基因编码产物的第80位丝氨酸突变为精氨酸;
parE基因编码产物的第458位丝氨酸突变为脯氨酸。
作为本发明所述鼠伤寒沙门菌的优选实施方式,所述gyrA基因的NCBI登录号为AE006468 REGION:2373710..2376422,所述parC基因的NCBI登录号为AE006468 REGION:3336954..3339227,所述parE基因的NCBI登录号为AE006468 REGION:3343969..3345861。
作为本发明所述鼠伤寒沙门菌的优选实施方式,所述鼠伤寒沙门菌是鼠伤寒沙门菌(Salmonella enterica subsp.enterica serovar Typhimurium)Sal17241,其分类命名为鼠伤寒沙门菌(Salmonella enterica subsp.enterica serovar Typhimurium),已于2020年9月30日保藏于广东省微生物菌种保藏中心,保藏地址:广州市先烈中路100号大院59号楼5楼,保藏编号:GDMCC No:61226。
本发明发现一株同时携带四个喹诺酮类药物耐药突变位点gyrAS83F、gyrAD87G、parCS80R和parES458P的鼠伤寒沙门菌,该菌株的gyrA基因编码产物发生双突变:Ser83→Phe(丝氨酸突变为苯丙氨酸,简写为S83F)、Asp87→Gly(天冬氨酸突变为甘氨酸,简写为D87G),同时parC基因编码产物发生单位点突变(Ser80→Arg)(丝氨酸突变为精氨酸,简写为S80R),以及parE基因编码产物发生单位点突变(Ser458→Pro)(丝氨酸突变为脯氨酸,简写为S458P)。这是首次发现四个喹诺酮类药物耐药突变位点gyrAS83F、gyrAD87G、parCS80R和parES458P同时存在于一株鼠伤寒沙门菌菌株中,该菌株在国内外均未见报道。同时,该菌株不具有任何质粒介导的喹诺酮类药物耐药基因。因此,这些突变位点导致该菌株对喹诺酮类药物表现为高水平耐药,萘啶酮酸、环丙沙星、左氧氟沙星和莫西沙星对沙门菌Sal17241菌株最小抑菌浓度分别为8192μg/mL、32μg/mL、16μg/mL、8μg/mL。
本发明的沙门菌Sal17241,为革兰氏阴性、短杆菌,API 20E鉴定为沙门菌,符合率为89.4%。其生化特征如下:精氨酸双水解酶、赖氨酸脱羧酶、鸟氨酸脱羧酶阳性、分解柠檬酸盐、产生硫化氢,发酵葡萄糖、甘露醇、山梨醇、鼠李糖、密二糖、***糖,尿素酶、苯丙氨酸脱氨酶、氧化酶、ONPG试验、吲哚试验、VP试验均为阴性,不能液化明胶,不发酵肌醇、蔗糖、苦杏仁苷,具沙门菌典型生理生化特征。血清抗原式鉴定为1,4,5,12:i:1,2,为典型的鼠伤寒沙门菌血清型。
沙门菌Sal17241可培养于LB、BHI和NA培养基中。
本发明同时提供一种微生物菌剂,所述微生物菌剂包含所述的鼠伤寒沙门菌。
本发明还提供所述的鼠伤寒沙门菌在作为筛选新型抗菌药物的模式菌株中的应用。
本发明的有益效果:
本发明提供了一种同时携带喹诺酮类药物四个耐药突变位点gyrAS83F、gyrAD87G、parCS80R和parES458P的沙门菌——沙门菌Sal17241。多个突变位点同时存在导致该菌株对喹诺酮类药物表现为高水平耐药,萘啶酮酸、环丙沙星、左氧氟沙星和莫西沙星对沙门菌Sal17241菌株最小抑菌浓度分别为8192μg/mL、32μg/mL、16μg/mL、8μg/mL。鉴于此,沙门菌Sal17241可作为筛选功能微生物/新型抗菌药物的模型材料,具有良好的应用前景。
附图说明
图1:本发明沙门菌Sal17241菌株的菌落形态图。
图2:本发明沙门菌Sal17241菌株的镜检观察形态图。
图3:本发明沙门菌Sal17241菌株的API 20E生化鉴定示意图。
图4:本发明沙门菌Sal17241中gyrA基因发生突变的具***点;gyrA(324C→T,Ser83→Phe;336A→G,Asp87→Gly)。
图5:本发明沙门菌Sal17241中parC基因发生突变的具***点;parC(253A→C,Ser80→Arg)。
图6:本发明沙门菌Sal17241中parE基因发生突变的具***点;parE(1372T→C,Ser458→Pro)。
具体实施方式
为更清楚地表述本发明的技术方案,下面结合具体实施例进一步说明,但不能用于限制本发明,此仅是本发明的部分实施例。
实施例1菌株的分离
沙门菌Sal17241分离自中国南宁市某超市的鲜猪肉食品中,对采集的样品进行检测,检测方法参照《食品安全国家标准食品微生物学检验沙门氏菌》GB 4789.4-2010。取样25g(mL)加入225mL缓冲蛋白胨水(BPW)的无菌均质袋中均质摇匀,于37℃培养8h~18h。轻轻摇动培养过的样品混合物,从中取1mL转种于10mL四硫磺酸钠煌绿(TTB)增菌液内,于42℃培养18h~24h,同时,另取1mL,转种于10mL***盐胱氨酸(SC)增菌液,于37℃培养18h~24h。分别用接种环取增菌液1环,划线接种于沙门菌属显色培养基平板,于37℃分别培养18h~24h,观察平板上生长的菌落。其典型的沙门菌菌落在显色平板上为紫色、圆型、湿润、边缘平整(图1)。将目标菌落从营养琼脂(NA)平板上转接到脑心浸液营养肉汤(BHI)中,于37℃过夜培养。在无菌条件下将菌液加入终浓度为50%甘油管中,保存于-40℃冰箱,并进行冻干管保存,由此得到菌株Sal17241。
实施例2菌株的鉴定和培养
将纯化后的菌株Sal17241进行形态特征、生理生化、血清型等方面的鉴定。
(1)染色镜检:将菌落涂片,进行革兰氏染色,镜检观察形态。沙门菌为革兰氏阴性、短杆状(图2)。
(2)API 20E鉴定:从NA平板上刮取单个菌落,用生理盐水制备成浊度适当的细胞悬浮液,使用API 20E生化鉴定试剂条鉴定(图3)。
(3)血清型鉴定:采用玻片凝集法对沙门菌分离菌株进行血清分型。首先检查沙门菌的菌体抗原(O抗原),然后依次确定菌株的第Ⅰ相和第Ⅱ相鞭毛抗原(H抗原),最后参照沙门菌属诊断抗原表,做出血清型诊断。
菌株Sal17241为革兰氏阴性、短杆菌,API 20E鉴定为沙门菌,符合率为89.4%。其生化特征如下:精氨酸双水解酶、赖氨酸脱羧酶、鸟氨酸脱羧酶阳性、分解柠檬酸盐、产生硫化氢,发酵葡萄糖、甘露醇、山梨醇、鼠李糖、密二糖、***糖,尿素酶、苯丙氨酸脱氨酶、氧化酶、ONPG试验、吲哚试验、VP试验均为阴性,不能液化明胶,不发酵肌醇、蔗糖、苦杏仁苷,具沙门菌典型生理生化特征。血清抗原式鉴定为1,4,5,12:i:1,2,为典型的鼠伤寒沙门菌血清型。
综合判断菌株Sal17241的外观、形态、革兰氏染色、生化反应及血清学反应可鉴定菌株Sal17241为沙门菌属肠道沙门菌肠道亚种鼠伤寒血清型(Salmonella entericasubsp.enterica serovar Typhimurium),命名为沙门菌Sal17241。
沙门菌Sal17241于2020年9月30日保藏于广东省微生物菌种保藏中心(GDMCC),保藏地址:广州市先烈中路100号大院59号楼5楼,保藏号:GDMCC No:61226。
实施例3沙门菌Sal17241药敏特征分析
按照美国临床实验室标准化委员会(Clinical and Laboratory Standardsinstitute,CLSI)2018版的方法和结果判断标准,采用肉汤稀释法考察沙门菌Sal17241菌株对喹诺酮类药物耐药水平的高低,所测试的药物包括萘啶酮酸、环丙沙星、左氧氟沙星和莫西沙星。于装有4mL MH肉汤的试管中接种沙门菌Sal17241菌株,孵育至对数期,用0.5麦氏比浊管进行比浊,调制菌液浓度为1×107cfu/mL左右。取上述菌液,用新鲜MH肉汤按1:200稀释,混匀。选择无菌96孔平底微量培养板,在每排第1孔中加入MH肉汤培养基100μL,然后于每排第1孔加入待测药物100μL,混合均匀,并取出100μL移至第2孔中,以此类推直至第12孔混匀后吸取100μL弃去。最后于各孔中加入混匀后的本发明沙门菌Sal17241(1×105cfu/mL)的菌悬液100μL。选择合适孔位加入200μL MH肉汤培养基作为阴性对照;加入100μL沙门菌Sal17241菌液和100μL MH肉汤培养基作为阳性对照。每组平行操作3次。将培养板置于37℃恒温箱中培养18-20h观察结果,测定其MIC值。自培养箱取出培养板,用酶标仪读取OD值,获得药敏结果及分析报告。或肉眼判读各个孔的阴阳性结果,混浊为阳性,清晰为阴性。
结果表明,萘啶酮酸、环丙沙星、左氧氟沙星和莫西沙星对沙门菌Sal17241菌株最小抑菌浓度(Minimal inhibitory concentration,MIC)分别为8192μg/mL,32μg/mL,16μg/mL,8μg/mL。沙门菌Sal17241菌株的具体药敏结果如表1所示。
表1沙门菌Sal17241菌株对喹诺酮类药物的耐药情况
实施例4沙门菌Sal17241菌株喹诺酮类药物耐药基因的检测
(1)全基因组二代测序及Blast序列分析目标耐药基因
对沙门菌Sal17241菌株进行全基因组二代测序。利用Covaris M200超声波破碎仪将基因组DNA打断为400bp的片段,并利用Ion Plus Fragment Library Kit进行文库构建工作。全基因组测序采用Ion Torrent S5测序仪进行。基因组de novo组装由SPAdesv3.6.2进行。同时,利用prokka对拼接后的基因组序列进行编码基因、tRNA、rRNA等基因组成分预测,并作编码基因功能注释。
采用本地Blast序列分析技术检测喹诺酮类药物耐药基因。本地Blast分析法:采用操作命令“makeblastdb-in 17241.ffn-dbtype nucl-parse_seqids-out 17241db.nt”以沙门菌Sal17241菌株的全基因组测序序列信息文档构建本地数据库db.nt(17241.ffn为菌株全基因组测序序列信息文档名,17241db.nt为构建的数据库名),再以目标位点(QRDR基因:gyrA、gyrB、parC、parE;PMQR基因:q nrA、qnrB、qnrC、qnrD、qnrS、qnrVC、aac(6')-Ib-cr、qepA、oxqAB)的序列信息文档为目标(以分析gyrA基因为例,gyrA.txt为目标基因的序列信息文档),以操作命令“blastn–db 17241db.nt-evalue 1e-5-outfmt 0-num_description s 10-num_threads 64-query gyrA.txt-outgyrAresult”对本地数据库17241db.nt进行gyrA基因的Blast分析(outgyrAresult为输出结果的命名)。最后查看输出文件,进行结果判定。
(2)PCR扩增目标耐药基因及Sanger法一代测序分析
对菌株全基因组二代测序及Blast序列分析目标耐药基因的检测结果进一步采用PCR扩增及Sanger法一代测序分析的方法进行验证。根据已发表基因序列设计目标耐药基因PCR扩增引物,引物序列见表1。采用单重PCR的方法,PCR扩增反应体系(25μL):12.5μL 2×Premix Taq,上、下游引物各120nmol/L,模板2μL,超纯水补齐;PCR扩增反应条件:95℃预变性5min;95℃变性45s、55-60℃退火45s、72℃延伸45s,共进行30个循环;72℃延伸10min。将扩增片段进行电泳检测(120V,30min)、纯化和一代测序分析,获得扩增目标基因的序列信息。对测序结果进行Blast分析和结果判定。研究中所设计的各目标基因PCR扩增引物如下:
表2沙门菌gyrA、parC、parE引物及扩增片段大小
(3)沙门菌Sal17241菌株喹诺酮类药物耐药基因的检测结果
与鼠伤寒沙门菌标准菌株LT2基因序列进行比对,沙门菌Sal17241菌株QRDR基因中gyrA、parC、parE检出突变;gyrA基因编码产物发生双突变:Ser83→Phe(丝氨酸突变为苯丙氨酸,简写为S83F)、Asp87→Gly(天冬氨酸突变为甘氨酸,简写为D87G),同时parC基因编码产物发生单位点突变(Ser80→Arg)(丝氨酸突变为精氨酸,简写为S80R),以及parE基因编码产物发生单位点突变(Ser458→Pro)(丝氨酸突变为脯氨酸,简写为S458P)。而PMQR基因(qnrA、qnrB、qnrC、qnrD、qnrS、qnrVC、aac(6')-Ib-cr、qepA、oxqAB)则全部没有检出。进一步采用PCR扩增及Sanger法一代测序进行验证,两种方法检测结果一致。
氨基酸符号表示的意义为:丝氨酸(Serine,缩写Ser或S)、苯丙氨酸(Phenylalanine,缩写Phe或F)、天冬氨酸(Aspartic,缩写Asp或D)、甘氨酸(Glycine,缩写Gly或G)、精氨酸(Arginine,缩写Arg或R)、脯氨酸(Proline,缩写Pro或P)。
沙门菌Sal17241菌株QRDR基因中gyrA、parC、parE检出突变详细情况分别如图4-6所示。
实施例5耐喹诺酮类药物沙门菌Sal17241菌株的应用
沙门菌Sal17241的具体应用方法主要体现在两个方面:
(1)沙门菌Sal17241同时携带四个喹诺酮类药物耐药突变位点gyrAS83F、gyrAD87G、parCS80R和parES458P,对四种喹诺酮类抗菌药物(萘啶酮酸、环丙沙星、左氧氟沙星和莫西沙星)产生高水平耐药,特别是环丙沙星,被认为是治疗由沙门菌所引起的感染的良药,近年来效果一直较为显著。喹诺酮耐药决定区基因的多个突变在同一菌种中的出现,对于探讨喹诺酮类药物的耐药发生机制,对治疗沙门菌感染的新策略的研究具有重要指导意义,可作为寻求细菌耐药机制的重要材料。
(2)沙门菌Sal17241同时携带四个喹诺酮类药物耐药突变位点gyrAS83F、gyrAD87G、parCS80R和parES458P,对四种喹诺酮类抗菌药物(萘啶酮酸、环丙沙星、左氧氟沙星和莫西沙星)产生高水平耐药,还可作为筛选新型抗菌药物的重要模式菌株。
抗菌药物的筛选方法如下:
精密称取一定量的待测药物,用合适溶媒溶解,配制成30mg/mL的溶液待用。再用合适溶媒配制成浓度为2000μg/mL的溶液,用来测定其最低抑菌浓度(minimum inhibitoryconcentration,MIC)和最低杀菌浓度(minimum bactericidal concentration,MBC),置于冰箱中-40℃保存备用。实验前一天,取出于冰箱-40℃保存的本发明沙门菌Sal17241,待放至室温,用接种环分别挑取少量菌落,分别接种于MH琼脂培养基上,在37℃恒温箱中培养18-24h。于新长出菌落的MH琼脂培养基上,用接种环挑取少量活化菌落于无菌生理盐水稀释配制成1×107cfu/mL的菌悬液(标准比浊管对照),备用。
筛选对耐药菌株敏感的待测药物,根据美国临床实验室标准化委员会(CLSI)推荐的纸片扩散法来测定菌株对不同药物的敏感性。用无菌棉签蘸取已制备好的标准细菌浓度为1×107cfu/mL的菌悬液,用涂布棒均匀涂布至相应的MH琼脂培养基。将灭菌的牛津杯放置在涂布后的MH琼脂培养基上,用微量移液器向牛津杯内加入50μL配好的药液(30mg/mL);置37℃恒温箱中培养18-24h,测量抑菌圈大小。取等体积MH肉汤培养基加入溶媒作为空白对照。
筛选对耐药菌株敏感的待测药物,根据美国临床实验室标准化委员会(CLSI)推荐的肉汤稀释法来测定菌株对不同药物的MIC值,每组平行操作3次,得出浓度平均值。测定MIC值时,用无菌生理盐水将1×107cfu/mL的菌悬液再次稀释,使之浓度为1×105cfu/mL。选择无菌96孔平底微量培养板,在每排第1孔中加入MH肉汤培养基100μL,然后于每排第1孔加入待测药物100μL,混合均匀,并取出100μL移至第2孔中,以此类推直至第12孔混匀后吸取100μL弃去。最后于各孔中加入混匀后的本发明沙门菌Sal17241(1×105cfu/mL)的菌悬液100μL。选择合适孔位加入200μL MH肉汤培养基作为阴性对照;加入100μL沙门菌Sal17241菌液和100μL MH肉汤培养基作为阳性对照。将培养板置于37℃恒温箱中培养18-20h观察结果,测定其MIC值。自培养箱取出培养板,用酶标仪读取OD值,获得药敏结果及分析报告。或肉眼判读各个孔的阴阳性结果,混浊为阳性,清晰为阴性。当确定MIC值后,取MIC值前3-5个孔的细菌和MH肉汤培养基混合物,转种在MH琼脂培养基上,置37℃的培养箱中,培养22h后取出观察,平均数小于5个的最低药物浓度即确定为该化合物的MBC。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
SEQUENCE LISTING
<110> 广东省微生物研究所(广东省微生物分析检测中心)
<120> 一种同时携带四个喹诺酮类药物耐药突变位点的鼠伤寒沙门菌及其应用
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Claims (3)
1.一种鼠伤寒沙门菌,其特征在于,所述鼠伤寒沙门菌同时携带四个喹诺酮类药物耐药突变位点,所述四个喹诺酮类药物耐药突变位点分别为:
gyrA基因编码产物的第83位的丝氨酸突变为苯丙氨酸;
gyrA基因编码产物的第87位的天冬氨酸突变为甘氨酸;
parC基因编码产物的第80位丝氨酸突变为精氨酸;
parE基因编码产物的第458位丝氨酸突变为脯氨酸;
所述鼠伤寒沙门菌是鼠伤寒沙门菌(Salmonella enterica subsp. enterica serovar Typhimurium)Sal17241,其分类命名为鼠伤寒沙门菌(Salmonella enterica subsp. enterica serovar Typhimurium),已于2020年9月30日保藏于广东省微生物菌种保藏中心,保藏地址:广州市先烈中路100号大院59号楼5楼,保藏编号:GDMCC No:61226。
2.一种微生物菌剂,其特征在于,所述微生物菌剂包含权利要求1所述的鼠伤寒沙门菌。
3.权利要求1所述的鼠伤寒沙门菌在作为筛选新型抗菌药物的模式菌株中的应用。
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