CN114214238A - 一种多重耐药印第安纳沙门菌及其应用 - Google Patents
一种多重耐药印第安纳沙门菌及其应用 Download PDFInfo
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Abstract
本发明涉及耐药菌株技术领域,尤其涉及一种多重耐药印第安纳沙门菌及其应用。本发明发现一株同时携带五个喹诺酮药物耐药突变位点(gyrAS83F、gyrAD87N、parCT57S、parCS80R、parEA344V)、超广谱β‑内酰胺基因(blaCTX‑M‑65),以及其他多种类型抗菌药物耐药基因的MDR印第安纳沙门菌,该印第安纳沙门菌对氟喹诺酮类药物(环丙沙星、左氧氟沙星和莫西沙星)产生高水平耐药,同时,对第三、四代头孢菌素类药物(头孢噻肟和头孢曲松)产生高水平耐药。鉴于此,本发明的印第安纳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。
Description
技术领域
本发明涉及耐药菌株技术领域,尤其涉及一种多重耐药印第安纳沙门菌及其应用。
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。目前已发现2600多种沙门菌血清型,印第安纳沙门菌(Salmonella entericasubsp.enterica serovar Indiana),最早于1955年分离自美国印第安纳州一个呕吐、腹泻、发热患者。1984年首次报道在广东地区发现了印第安纳沙门菌,但在其后较长一段时间内只有极少数关于印第安纳沙门菌的报道。近年来在病人、动物、食品和环境中均有印第安纳沙门菌的检出,鸡肉是其最主要的宿主,并且印第安纳沙门菌具有广泛的耐药性,耐药范围成逐年上升趋势。
喹诺酮类药物和β-内酰胺类药物(第三、四代广谱头孢菌素)是目前临床上用于治疗沙门菌感染并具有较好治疗效果的重要抗生素。喹诺酮类药物是一类广谱抗菌药,其通过识别菌株靶标蛋白,影响菌株DNA的复制,产生抑菌作用。β-内酰胺类药物是现有抗菌药物中使用最广泛的一类,其通过与细菌内膜蛋白结合,抑制转肽酶的转肽作用,阻止细菌细胞壁和肽聚糖的合成,导致细菌死亡而产生抑菌作用。沙门菌对喹诺酮类药物敏感性的降低与其靶位的改变有关。喹诺酮类药物耐药决定区(quinolone resistance-determiningregions,QRDR)的DNA螺旋酶(编码基因gyrA和gyrB)及拓扑异构酶IV(编码基因parC和parE)发生基因突变,使酶结构发生改变,从而影响喹诺酮类药物的结合。质粒介导喹诺酮耐药基因(plasmid-mediated quinolone resistance,PMQR),包括:qnrA、qnrB、qnrC、qnrD、qnrS、qnrVC、qepA、oqxAB以及aac-(6’)-Ib-cr,也是导致沙门菌对喹诺酮类药物敏感性降低的原因。沙门菌对喹诺酮类药物的耐药程度通常与突变位点的位置和数量有关。单一位点突变可引起低水平耐药,若同时具有2个或多个突变位点,则会导致高水平耐药菌株的产生。
细菌对β-内酰胺类药物最常见、最重要的耐药机制是产生β-内酰胺酶(β-lactamase),该酶能水解药物抗菌活性必需基团(β-内酰胺环),导致药物失去活性。β-内酰胺酶种类繁多,产生超广谱β-内酰胺酶(Extended spectrumβ-lactamases,ESBLs)是导致沙门菌等肠杆菌科细菌对第三、四代广谱头孢菌素耐药的主要因素,并且大部分ESBLs基因由质粒编码,很容易在菌株间转移和传播,使得ESBLs基因快速流行。目前发现的ESBLs主要包括SHV、TEM、OXA、CTX-M和其他型,其中CTX-M型酶是20世纪80年代中期出现的一种新型超广谱β-内酰胺酶。
发明内容
本发明的目的在于克服现有技术的不足,提供一种多重耐药印第安纳沙门菌及其应用。
本发明发现一株同时携带五个喹诺酮药物耐药突变位点(gyrAS83F、gyrAD87N、parCT57S、parCS80R、parEA344V)、超广谱β-内酰胺基因(blaCTX-M-65),以及其他多种类型抗菌药物耐药基因的MDR印第安纳沙门菌,该菌株的gyrA基因发生双位点突变:Ser83→Phe(丝氨酸突变为苯丙氨酸,简写为S83F)、Asp87→Asn(天冬氨酸突变为天冬酰胺,简写为D87N),同时parC基因也发生双位点突变:Thr57→Ser(苏氨酸突变为丝氨酸,简写为T57S)、Ser80→Arg(丝氨酸突变为精氨酸,简写为S80R),以及parE基因发生单位点突变(Ala344→Val)(丙氨酸突变为缬氨酸,简写为A344V),并且该菌株携带新型超广谱β-内酰胺基因——blaCTX-M-65。这是首次发现一株同时携带五个喹诺酮药物耐药突变位点(gyrAS83F、gyrAD87N、parCT57S、parCS80R、parEA344V)、超广谱β-内酰胺基因(blaCTX-M-65),以及其他多种类型抗菌药物耐药基因的MDR印第安纳沙门菌,该菌株在国内外均未见报道。其中,parEA344V是第一次报道的本发明中发现的新的突变位点。这些耐药基因不仅导致沙门菌77-5菌株对多种传统药物表现出耐药,而且对目前临床治疗沙门菌感染的一线药物——氟喹诺酮类和第三、四代广谱头孢菌素类药物也表现出高水平耐药。
为实现上述目的,本发明采取的技术方案为:提供一种印第安纳沙门菌,所述印第安纳沙门菌同时携带五个喹诺酮药物耐药突变位点,所述突变位点分别为:
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天冬酰胺;
parC基因编码产物的第57位的苏氨酸突变为丝氨酸;
parC基因编码产物的第80位的丝氨酸突变为精氨酸;
parE基因编码产物的第344位的丙氨酸突变为缬氨酸。
作为本发明所述印第安纳沙门菌的优选实施方式,所述印第安纳沙门菌还携带超广谱β-内酰胺基因blaCTX-M-65。
作为本发明所述印第安纳沙门菌的优选实施方式,所述印第安纳沙门菌包括以下耐药基因:
(1)QRDR基因和PMQR基因:
gyrA基因编码产物的第83位的丝氨酸突变为苯丙氨酸,第87位的天冬氨酸突变为天冬酰胺;parC基因编码产物的第57位的苏氨酸突变为丝氨酸,第80位的丝氨酸突变为精氨酸;parE基因编码产物的第344位的丙氨酸突变为缬氨酸;aac(6')-Ib-cr;
(2)β-内酰胺类药物耐药基因:blaCTX-M-65、blaOXA-1、blaTEM-1B;
(3)氨基糖苷类药物耐药基因:aac(3)-IV、aac(6')-Iaa、aph(3”)-Ib、aph(3')-IIa、aph(4)-Ia、aph(6)-Id、aadA5、armA;
(4)磺胺类药物耐药基因:sul1、sul2;
(5)甲氧苄氨嘧啶类药物耐药基因:dfrA17;
(6)四环素类药物耐药基因:tetA;
(7)苯丙醇类药物耐药基因:catB3、floR。
本发明的多重耐药(multidrug-resistant,MDR)印第安纳沙门菌,血清型为印第安纳,该菌株携带多种类型抗菌药物耐药基因,包括:喹诺酮类药物耐药决定区(quinoloneresistance-determining regions,QRDR)突变位点gyrAS83F、gyrAD87N、parCT57S、parCS80R、parEA344V,质粒介导喹诺酮类药物耐药(plasmid-mediated quinolone resistance,PMQR)基因aac(6')-Ib-cr、β-内酰胺类药物耐药基因(blaCTX-M-65、blaOXA-1和blaTEM-1B)、氨基糖苷类药物耐药基因(aac(3)-IV、aac(6')-Iaa、aph(3”)-Ib、aph(3')-IIa、aph(4)-Ia、aph(6)-Id、aadA5和armA)、磺胺类药物耐药基因(sul1、sul2)、甲氧苄氨嘧啶类药物耐药基因(dfrA17)、四环素类药物耐药基因(tetA)以及苯丙醇类药物耐药基因(catB3、floR)。由于携带这些耐药基因,导致沙门菌77-5对以上这些抗菌药物均表现出耐药性,萘啶酮酸、环丙沙星、左氧氟沙星、莫西沙星、头孢噻肟、头孢曲松、氨苄西林、阿莫西林/克拉维酸、链霉素、阿米卡星、庆大霉素、复方磺胺、四环素、氯霉素、氟苯尼考对该菌株最小抑菌浓度分别为4096μg/mL、16μg/mL、8μg/mL、8μg/mL、>8μg/mL、>16μg/mL、>128μg/mL、>128/64μg/mL、>64μg/mL、>64μg/mL、>32μg/mL、>16/304μg/mL、>64μg/mL、128μg/mL、>128μg/mL。
作为本发明所述印第安纳沙门菌的优选实施方式,所述印第安纳沙门菌是印第安纳沙门菌(Salmonella enterica subsp.enterica serovar Indiana)77-5,其保藏编号为GDMCC No:62103。
本发明的沙门菌77-5,为革兰氏阴性、两端钝圆的短杆状,质谱鉴定结果为沙门菌,得分值为2.27。其在沙门菌属显色平板上为紫色、圆形、光滑、边缘规则的菌落形态,在营养琼脂平板上为灰白色、圆润、稍凸起的菌落形态,具有沙门菌典型的菌落形态特征。血清抗原式鉴定为1,4,12:z,1,7,是典型的印第安纳沙门菌血清型。本发明的沙门菌77-5可培养于LB、BHI和NA培养基中。
本发明还提供一种微生物菌剂,所述微生物菌剂包含所述的印第安纳沙门菌。
本发明还提供所述的印第安纳沙门菌在筛选沙门菌新型抗菌药物中的应用。
作为本发明所述应用的优选实施方式,所述应用为将所述印第安纳沙门菌作为筛选沙门菌新型抗菌药物的模式菌株。
作为本发明所述应用的优选实施方式,所述筛选具体为:将待测药物配制成溶液,测定其对所述印第安纳沙门菌的最低抑菌浓度和最低杀菌浓度。
本发明的有益效果:
本发明的沙门菌77-5同时携带五个QRDR突变位点gyrAS83F、gyrAD87N、parCT57S、parCS80R、parEA344V以及PMQR基因aac(6')-Ib-cr,导致菌株对氟喹诺酮类药物(环丙沙星、左氧氟沙星和莫西沙星)产生高水平耐药。同时,菌株77-5还携带超广谱β-内酰胺酶(Extended spectrumβ-lactamases,ESBLs)基因——blaCTX-M-65,导致菌株对第三、四代头孢菌素类药物(头孢噻肟和头孢曲松)产生高水平耐药。鉴于此,沙门菌77-5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。
其中,印第安纳沙门菌(Salmonella enterica subsp.enterica serovarIndiana)77-5,于2021年12月9日保藏于广东省微生物菌种保藏中心,地址:广州市先烈中路100号大院59号楼5楼,保藏编号为GDMCC No:62103。
附图说明
图1为沙门菌77-5菌株在沙门菌属显色平板上的菌落形态图。
图2为沙门菌77-5菌株在营养平板上的菌落形态图。
图3为沙门菌77-5菌株的镜检观察形态图。
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。
除非特别指明,否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法(例如,分子生物学和核酸化学实验方法)。参见例如,Sambrook等人,Molecular Cloning:A Laboratory Manual,第2版,Cold SpringHarbor Laboratory Press,Cold Spring Harbor,N.Y.(1989);和Ausubel等人,CurrentProtocols in Molecular Biology,Greene Publishing Associates(1992),其全部通过引用合并入本文。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
在本发明中,除非另有说明,否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且,本文中所用的分子遗传学、核酸化学和分子生物学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时,为了更好地理解本发明,下面提供相关术语的定义和解释。
如本文中所使用的,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(Asparticacid,缩写Asp或D)、天冬酰胺(Asparagine,缩写Asn或N)、苏氨酸(Threonine,缩写Thr或T)、精氨酸(Arginine,缩写Arg或R)、丙氨酸(Alanine,缩写Ala或A)、缬氨酸(Valine,缩写Val或V)。
如本文中所使用的,术语“突变”,当用于描述基因或DNA时,是指基因序列或DNA序列中一个或多个(例如,几个)碱基的添加、缺失和/或置换;当用于描述蛋白质时,是指蛋白质氨基酸序列中一个或多个(例如,几个)氨基酸残基的添加、缺失和/或置换。
实施例1菌株的分离、纯化
沙门菌77-5分离自中国石家庄市中储海鲜城的鸡肉样品中,参照《食品安全国家标准食品微生物学检验沙门氏菌检验》GB 4789.4-2016中沙门菌的检测方法,进行样品检测。无菌操作称取25g(mL)样品置于225mL缓冲蛋白胨水(BPW)的无菌容器中均质混匀,于37℃培养8h~18h。轻轻摇动培养后的样品混合物,用无菌吸头移取1mL混合物溶液,转种于10mL四硫磺酸钠煌绿(TTB)增菌液,于42℃培养18h~24h,同时,另取1mL,转种于10mL亚硒酸盐胱氨酸(SC)增菌液,于37℃培养18h~24h。用接种环取1环增菌液,划线接种于沙门菌属显色培养基平板,于37℃培养18h~24h,观察平板上菌落的形态特征。典型的沙门菌菌落在沙门菌属显色培养基平板上为紫色、圆形、光滑、边缘规则的菌落。将目标菌落划线接种至营养琼脂(NA)平板纯化培养后,挑取单菌落转接到脑心浸出液(BHI)营养肉汤中,于37℃过夜培养。无菌操作将菌液加入50%甘油小管中,于-40℃保存,并进行冻干管保存,由此获得菌株77-5。
实施例2菌株的鉴定
将纯化后的菌株77-5进行菌落形态特征、质谱鉴定、血清分型等方面的鉴定。
(1)染色镜检:挑取单菌落菌体涂片,革兰氏染色进行制片,镜检观察形态。沙门菌为革兰氏阴性、两端钝圆的短杆状(图3)。
(2)质谱鉴定:挑取新鲜NA平板上单菌落菌体,涂抹到MALDI靶板上,自然干燥后,在单菌落涂层上加入1μL 70%甲酸水溶液,自然干燥后,再加入1μL基质溶液,自然干燥后,放入基质辅助激光解析电离-飞行时间质谱仪进行检测(表1)。
表1沙门菌77-5菌株的质谱鉴定结果
(3)血清型鉴定:采用玻片凝集法对沙门菌分离株进行血清分型,依次检查沙门菌的菌体抗原(O抗原)、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77-5为革兰氏阴性、两端钝圆的短杆状,质谱检测鉴定为沙门菌,得分值为2.27。在沙门菌属显色培养基平板上为沙门菌典型的紫色、圆形、光滑、边缘规则的菌落,在营养琼脂平板上为灰白色、圆润、稍凸起的菌落。血清抗原式鉴定为1,4,12:z,1,7,为典型的印第安纳沙门菌血清型。
综合判断菌株77-5的菌落形态、外观、革兰氏染色、质谱鉴定及血清凝集反应的结果,可鉴定菌株77-5为沙门菌属肠道沙门菌肠道亚种印第安纳血清型(Salmonellaenterica subsp.enterica serovar Indiana),命名为沙门菌77-5。
实施例3菌株药敏特征分析
依据美国临床实验室标准化委员会(Clinical and Laboratory Standardsinstitute,CLSI)2018版的实验方法和结果判断标准,采用微量肉汤稀释法考察沙门菌77-5菌株对喹诺酮类、β-内酰胺类、氨基糖苷类、磺胺类、甲氧苄氨嘧啶类、四环素类和苯丙醇类抗菌药物耐药水平的高低。
将甘油管中菌株77-5划线转种于NA平板活化培养,挑取单菌落菌体至含4mL水解酪蛋白(MH)肉汤的试管中,37℃,200rpm,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1×108cfu/mL左右。无菌操作移取上述菌液,以新鲜MH肉汤按1:200进行稀释混匀。于无菌96孔平底微量培养板A1孔中加入MH肉汤100μL,同一孔中加入待测抗生素溶液100μL,用无菌微量移液器轻柔吹打混匀,移取100μL至A2孔中,依次稀释至A12孔,并从A12孔的匀液中吸弃100μL。最后于各孔中加入混匀后的本发明沙门菌77-5(1×105cfu/mL)的菌悬液100μL,并选择合适孔位加入200μL MH肉汤培养基作为阴性对照,另一个孔中加入100μL沙门菌77-5稀释菌悬液和100μL MH肉汤培养基作为阳性对照。将该培养板置于37℃恒温培养箱中培养18h~20h,观察每个孔位菌液的混浊情况(浑浊为阳性,澄清为阴性),用酶标仪测定OD600值,判读其MIC值。经实验测定,菌株77-5对喹诺酮类、β-内酰胺类、氨基糖苷类、磺胺类、甲氧苄氨嘧啶类、四环素类和苯丙醇类抗菌药物均表现出耐药性。沙门菌77-5菌株的药敏测定结果如表2所示。
表2沙门菌77-5菌株对15种抗生素的耐药情况
实施例4沙门菌77-5菌株抗菌药物耐药基因的检测
(1)菌株全基因组二代测序及Blast分析目标耐药基因
通过菌株DNA提取、质检、构建文库、上机测序、数据处理等对沙门菌77-5菌株进行全基因组二代测序。采用美基生物D3146-02细菌DNA试剂盒提取菌株DNA,测定DNA浓度并稀释至合适浓度,转至自动构库仪专用96孔板中,进行文库构建。构建文库后,对文库进行质检,逐步稀释至合适浓度,变性并上机测序。程序结束后,将测序数据拷贝至本地服务器,拆分数据后依次操作“gunzip*.gz”、“/data/database/command/pre0”、“/data/database/command/ill0”、“/data/database/command/ill00”等命令对测序数据进行组装拼接。同时,利用prokka对拼接后的基因序列进行编码基因、rRNA、tRNA等基因组成分的预测,并做编码基因的功能注释。
采用本地Blast序列分析技术检测沙门菌77-5菌株的喹诺酮类、β-内酰胺类、氨基糖苷类、磺胺类、甲氧苄氨嘧啶类、四环素类和苯丙醇类抗菌药物耐药基因。本地Blast分析过程:
1)以沙门菌77-5菌株的全基因组测序序列信息文件构建本地数据库(77-5assembly.fasta为菌株全基因组测序序列信息文件名,77-5db为构建的本地数据库名),运行命令“makeblastdb-in 77-5assembly.fasta-dbtypenucl-parse_seqids-out 77-5db”;
2)以目标基因位点(QRDR基因:gyrA、gyrB、parC、parE;PMQR基因:qnrA、qnrB、qnrC、qnrD、qnrS、qnrVC、qepA、oqxAB、aac-(6’)-Ib-cr;β-内酰胺类药物耐药基因:blaOXA-1、blaTEM-1B、blaTEM-135、blaCTX-M-14、blaCTX-M-15、blaCTX-M-55、blaCTX-M-65、blaCTX-M-123;氨基糖苷类药物耐药基因:aac(3)-IV、aac(6')-Iaa、aph(3”)-Ib、aph(3')-IIa、aph(4)-Ia、aph(6)-Id、aadA、aadB、armA、rmtB;磺胺类药物耐药基因:sul1、sul2、sul3;甲氧苄氨嘧啶类药物耐药基因:dfrA、dfrB;四环素类药物耐药基因:tetA、tetB、tetC、tetD;苯丙醇类药物耐药基因:catA、catB、floR、cmlA)的序列信息文件为参照,(以parE基因为例,parE.txt为目标基因位点的序列信息文件,77-5_parE_out为输出的结果文件名)运行命令“blastn-db 77-5db-evalue 1e-5-outfmt 0-num_descriptions 10-num_threads 64-query parE.txt-out77-5_parE_out”,对本地数据库77-5db进行parE基因的Blast分析;
3)查看输出的结果文件,进行Blast结果判定。
(2)PCR扩增目标耐药基因及一代测序分析
进一步采用PCR扩增及一代测序分析的方法,对沙门菌77-5菌株的全基因组二代测序及Blast序列分析目标耐药基因的判定结果进行验证。根据已发表基因序列设计目标耐药基因的PCR扩增引物(引物序列见表3)。采用单重PCR的方法,25μL PCR扩增反应体系(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。将扩增片段产物进行琼脂糖凝胶电泳检测(120V,30min)、纯化和一代测序分析,获得扩增目标基因的序列信息,并对测序结果进行Blast分析,以进行结果判定。
表3沙门菌gyrA、parC、parE引物序列及片段大小
(3)沙门菌77-5菌株抗菌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77-5菌株QRDR基因中gyrA、parC、parE检出突变,而PMQR基因中只有aac(6')-Ib-cr基因检出;其中,gyrA基因发生双位点突变:Ser83→Phe(丝氨酸突变为苯丙氨酸,简写为S83F)、Asp87→Asn(天冬氨酸突变为天冬酰胺,简写为D87N),同时parC基因也发生双位点突变:Thr57→Ser(苏氨酸突变为丝氨酸,简写为T57S)、Ser80→Arg(丝氨酸突变为精氨酸,简写为S80R),以及parE基因发生单位点突变(Ala344→Val)(丙氨酸突变为缬氨酸,简写为A344V)。
β-内酰胺类药物耐药基因中检出blaCTX-M-65、blaOXA-1和blaTEM-1B基因,氨基糖苷类药物耐药基因中检出aac(3)-IV、aac(6')-Iaa、aph(3”)-Ib、aph(3')-IIa、aph(4)-Ia、aph(6)-Id、aadA5和armA基因,磺胺类药物耐药基因中检出sul1、sul2基因,甲氧苄氨嘧啶类药物耐药基因中检出dfrA17基因;四环素类药物耐药基因中检出tetA基因,以及苯丙醇类药物耐药基因中检出catB3和floR基因。采用PCR扩增及一代测序分析的方法进一步验证,两种方法检测结果一致。
沙门菌77-5菌株中抗菌药物耐药基因检出情况如下:
1)QRDR基因和PMQR基因:
①gyrA(324C→T,Ser83→Phe;335G→A,Asp87→Asn)
②parC(170C→G,Thr57→Ser;240C→A,Ser80→Arg)
③parE(1031C→T,Ala344→Val)
④aac(6')-Ib-cr
2)β-内酰胺类药物耐药基因:blaCTX-M-65、blaOXA-1、blaTEM-1B;
3)氨基糖苷类药物耐药基因:aac(3)-IV、aac(6')-Iaa、aph(3”)-Ib、aph(3')-IIa、aph(4)-Ia、aph(6)-Id、aadA5、armA;
4)磺胺类药物耐药基因:sul1、sul2;
5)甲氧苄氨嘧啶类药物耐药基因:dfrA17;
6)四环素类药物耐药基因:tetA;
7)苯丙醇类药物耐药基因:catB3、floR。
实施例5 MDR沙门菌77-5菌株的应用
沙门菌77-5的具体应用方法主要体现在三方面:
(1)沙门菌77-5同时携带五个喹诺酮药物耐药突变位点(gyrAS83F、gyrAD87N、parCT57S、parCS80R、parEA344V)、超广谱β-内酰胺类药物耐药基因(blaCTX-M-65),以及多种类型抗菌药物耐药基因,对喹诺酮类、β-内酰胺类、氨基糖苷类、磺胺类、甲氧苄氨嘧啶类、四环素类和苯丙醇类等15种抗菌药物均表现出耐药性,特别是氟喹诺酮类和第三、四代头孢菌素类药物是目前沙门菌感染临床治疗的重要药物。沙门菌77-5中同时出现了多种类型抗菌药物耐药基因,对于探讨菌株对氟喹诺酮类、β-内酰胺类等药物耐药性发生机制、沙门菌感染防治新方案的形成具有重要的指导意义,其可作为探究细菌耐药机制的重要材料。
(2)菌株致病性是沙门菌感染宿主后是否导致宿主患病的重要条件之一。沙门菌77-5同时携带五个喹诺酮药物耐药突变位点(gyrAS83F、gyrAD87N、parCT57S、parCS80R、parEA344V)、超广谱β-内酰胺类基因(blaCTX-M-65),以及多种类型抗菌药物耐药基因,对喹诺酮类、β-内酰胺类、氨基糖苷类、磺胺类、甲氧苄氨嘧啶类、四环素类和苯丙醇类等15种抗菌药物均表现出耐药性,可作为探究沙门菌耐药性与其致病性关系的重要材料。
(3)沙门菌77-5同时携带五个喹诺酮药物耐药突变位点(gyrAS83F、gyrAD87N、parCT57S、parCS80R、parEA344V)、超广谱β-内酰胺类基因(blaCTX-M-65),以及多种类型抗菌药物耐药基因,对喹诺酮类、β-内酰胺类、氨基糖苷类、磺胺类、甲氧苄氨嘧啶类、四环素类和苯丙醇类等15种抗菌药物均表现出耐药性,可作为筛选沙门菌新型抗菌药物的重要模式菌株。筛选对待测菌株具有抑制作用的药物,筛选方法如下:
精密称取一定量待测试药物,以合适溶媒溶解混合均匀,配制成浓度为30mg/mL的母液,再以该溶媒配制成浓度为2000μg/mL的使用溶液,用以测定其对沙门菌的最低抑菌浓度(minimum inhibitory concentration,MIC)和最低杀菌浓度(minimum bactericidalconcentration,MBC),保存于-40℃冰箱。药敏实验前一天,于-40℃冰箱中取出本发明沙门菌77-5,放至室温后,用接种环将菌液划线转种于MH琼脂培养基上,37℃恒温培养箱中培养18h~24h。用接种环挑取MH琼脂培养基上活化的少量菌落,置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),混合均匀备用。
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。用无菌棉棒蘸取制备好的浓度为1×107cfu/mL的标准细菌菌悬液,均匀涂布至相应的MH琼脂培养基。将无菌牛津杯置于涂布后的MH琼脂培养基上,用微量移液器取50μL药液(30mg/mL)于牛津杯中(以等体积MH肉汤培养基加入溶媒作为空白对照),37℃培养18h~24h,测量抑菌圈大小。
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。测定MIC值时,每组作3个平行,以平行浓度的均值为最终结果。用无菌生理盐水将1×107cfu/mL的菌悬液稀释至1×105cfu/mL,选择无菌96孔平底微量培养板,于每排第1孔中加入MH肉汤100μL,在同一列孔中加入待测抗生素使用溶液100μL,用无菌微量移液器轻柔吹打混匀,移取100μL至第2孔中,依次稀释至第12孔,并从第12孔的匀液中吸弃100μL。最后取本发明沙门菌77-5匀液(1×105cfu/mL)100μL置于各孔中。并选择合适孔位加入200μL MH肉汤培养基作为阴性对照,将本发明沙门菌77-5匀液100μL(1×105cfu/mL)和100μLMH肉汤培养基加入同一孔作为阳性对照。将培养板置于37℃恒温培养箱中培养18h~20h,观察每个孔位菌液的混浊情况(混浊为阳性,澄清为阴性),用酶标仪测定OD600值,判读其MIC值。确定MIC值后,取MIC值前3~5个孔的细菌肉汤混合物,转种于MH琼脂培养基上,37℃培养22h后观察平板上菌落生长情况,平均数小于5个对应的最低药物浓度即确定为该化合物的MBC值。
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> 广东省科学院微生物研究所(广东省微生物分析检测中心)
<120> 一种多重耐药印第安纳沙门菌及其应用
<130> 2021.12.21
<160> 6
<170> PatentIn version 3.3
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Claims (8)
1.一种印第安纳沙门菌,其特征在于,所述印第安纳沙门菌同时携带五个喹诺酮药物耐药突变位点,所述突变位点分别为:
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天冬酰胺;
parC基因编码产物的第57位的苏氨酸突变为丝氨酸;
parC基因编码产物的第80位的丝氨酸突变为精氨酸;
parE基因编码产物的第344位的丙氨酸突变为缬氨酸。
2.根据权利要求1所述的印第安纳沙门菌,其特征在于,所述印第安纳沙门菌还携带超广谱β-内酰胺基因blaCTX-M-65。
3.根据权利要求1所述的印第安纳沙门菌,其特征在于,所述印第安纳沙门菌包括以下耐药基因:
(1)QRDR基因和PMQR基因:
gyrA基因编码产物的第83位的丝氨酸突变为苯丙氨酸,第87位的天冬氨酸突变为天冬酰胺;parC基因编码产物的第57位的苏氨酸突变为丝氨酸,第80位的丝氨酸突变为精氨酸;parE基因编码产物的第344位的丙氨酸突变为缬氨酸;aac(6')-Ib-cr;
(2)β-内酰胺类药物耐药基因:blaCTX-M-65、blaOXA-1、blaTEM-1B;
(3)氨基糖苷类药物耐药基因:aac(3)-IV、aac(6')-Iaa、aph(3”)-Ib、aph(3')-IIa、aph(4)-Ia、aph(6)-Id、aadA5、armA;
(4)磺胺类药物耐药基因:sul1、sul2;
(5)甲氧苄氨嘧啶类药物耐药基因:dfrA17;
(6)四环素类药物耐药基因:tetA;
(7)苯丙醇类药物耐药基因:catB3、floR。
4.根据权利要求1所述的印第安纳沙门菌,其特征在于,所述印第安纳沙门菌是印第安纳沙门菌(Salmonella enterica subsp.enterica serovar Indiana)77-5,其保藏编号为GDMCC No:62103。
5.一种微生物菌剂,其特征在于,所述微生物菌剂包含权利要求1~4任一所述的印第安纳沙门菌。
6.权利要求1~4任一所述的印第安纳沙门菌在筛选沙门菌新型抗菌药物中的应用。
7.根据权利要求6所述的应用,其特征在于,所述应用为将所述印第安纳沙门菌作为筛选沙门菌新型抗菌药物的模式菌株。
8.根据权利要求7所述的应用,其特征在于,所述筛选具体为:将待测药物配制成溶液,测定其对所述印第安纳沙门菌的最低抑菌浓度和最低杀菌浓度。
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