CN103130881B - 一种肺炎克雷伯菌的耐药新基因 - Google Patents
一种肺炎克雷伯菌的耐药新基因 Download PDFInfo
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Abstract
本发明公开了一种肺炎克雷伯菌的耐药新基因，所述基因是通过从1株对亚胺培南、厄他培南耐药的肺炎克雷伯细菌中提取获得，所述耐药新基因其核苷酸序列为SEQ ID NO.1，氨基酸的序列为SEQ ID NO.2。本发明还通过质粒转导实验和原核表达实验研究了新基因的功能，结果显示新基因对多种药物耐药。所述的新耐药基因对于合理使用抗生素、减少严重感染时产生碳青霉烯类耐药细菌，以及研发新药物抑制细菌耐药性意义重大。
Description
技术领域
本发明涉及一种肺炎克雷伯菌的耐药新基因，具体而言，本发明涉及在病人的血液培养标本中分离出的耐药肺炎克雷伯菌中发现的一种耐药新基因以及该基因编码的蛋白质，并且证实了该基因在细菌耐药中发挥重要作用。
背景技术
克雷伯菌为杆状革兰氏阴性菌，通常都有大量的黏性多糖形成的荚膜包覆，其中肺炎克雷伯菌对人致病性较强，是重要的条件致病菌和医源性感染菌之一。
肺炎克雷伯菌(Klebsiella pneumoniae)，又称肺炎杆菌或Friedlander杆菌，是最早被认识可引起肺炎的革兰阴性杆菌。此菌形态为较短粗的杆菌，大小为0.5～0.8×(1～2)μm，单独、成双或短链状排列。该菌无芽胞，无鞭毛，有较厚的荚膜，多数有菌毛。对营养要求不高，在普通琼脂培养基上可以形成较大的灰白色粘液菌落，以接种环挑之，易拉成丝，有助鉴别；在肠道杆菌选择性培养基上能发酵乳糖，呈现有色菌落。
本菌存在于人体肠道、呼吸道。为人体本身存在菌属，当受到感染或免疫力下降时，便经呼吸道进入肺内而引起大叶或小叶融合性实变，以上叶较为多见。病变中渗出液粘稠而重，致使叶间隙下坠。细菌具有荚膜，在肺泡内生长繁殖时，引起组织坏死、液化、形成单个或多发性脓肿。病变累及胸膜、心包时，可引起渗出性或脓性积液。病灶纤维组织增生活跃，易于机化；纤维素性胸腔积液可早期出现粘连。此菌引起临床症状的特点如下：
①起病急剧，有高热、咳嗽、痰量多和胸痛；
②可有发绀、气急、心悸，约半数患者有畏寒，可早期出现休克；
③临床表现可类似严重的肺炎球菌肺炎，但痰多呈黏稠脓性、量多、带血，灰绿色或红砖色，胶胨状；
④X线检查显示肺叶或小叶实变，有多发性蜂窝状肺脓肿，叶间隙下坠。
在院内感染的败血症中，克雷伯杆菌为重要病原菌，病死率较高。肺炎克雷伯菌也可引起尿道感染、创伤感染及腹泻等，有时导致严重的败血症、脑膜炎、腹膜炎等。
目前对于肺炎克雷伯菌主要采用抗生素治疗，尽早使用有效抗生素是治愈的关键。首选氨基甙类抗生素，如庆大霉素、卡那霉素、妥布霉素、丁胺卡那霉素，可肌注、静滴或管腔内用药。重症宜加用头孢菌素如头孢孟多、头孢西丁、头孢噻肟等。哌拉西林，美洛西林与氨基甙类联用、以及氧氟沙星疗效亦佳。部分病例使用氯霉素、四环素及SMZ-TMP亦有效。重症多有肺组织损伤，慢性病例有时需行肺叶切除。
随着抗生素的频繁使用，细菌的耐药性不断增强，碳青霉烯类抗生素的发现使这一难题有所缓解，此类抗生素如亚胺培南及美罗培南，是产超广谱-内酰胺酶以及高产AmpC酶肠杆菌科细菌感染治疗最有效的药物。随着临床上碳青霉烯类抗菌药物的广泛使用，产生了对亚胺培南或美罗培南耐药的肠杆菌。由于肠杆菌是临床上重要的医院感染菌，其对碳青霉烯类抗菌药物的耐药给临床抗感染治疗带来了极大困难。尤其是耐药基因位于可接合质粒上，通过水平传播使其他细菌获得耐药性，有引起严重医院感染的潜在危险。而且，随着“超级细菌”数量的不断增多，抗生素管道频临枯竭，解决细菌耐药性问题成为当今世界面临的棘手难题，人们试图通过开发新的抗生素来解决耐药问题。开发一种新抗生素需要10年，需花费5-10亿美元，一代耐药菌的产生只要2年时间，开发新的抗生素已经远远赶不上细菌耐药的脚步，因此研究细菌耐药的机制将成为今后一项很重要的课题。
肺炎克雷伯菌碳霉烯酶(Klebsiella pneumoniae carbapenemase，KPC酶)最早在一株亚胺培南耐药的肺炎克雷伯菌中被发现，是能够明显水解亚胺培南或美罗培南的一类β2内酰胺酶，包括Ambler分子分类为A、B、D3类酶。A类为丝氨酸酶，其活性部位具有丝氨酸结构，属于Bush分群中的第2f亚组。A类碳青霉烯酶少见，这类酶都是青霉素酶。D类为丝氨酸酶，属于Bush分群中的第2d亚组，其活性部位具有丝氨酸结构，仅见于不动杆菌。B类是金属酶，属于Bush分类3，是一种需金属离子发挥活性的β2内酰胺酶，可被EDTA所抑制，由染色体介导或质粒介导，存在于多种不同革兰阳性和革兰阴性细菌中。金属酶均可明显水解亚胺培南，能水解除单环类抗菌药物以外的绝大多数β2内酰胺类抗菌药物。临床使用亚胺培南等碳青霉烯类抗菌药物大大增加，导致金属β2内酰胺酶产生率有不断上升的趋势。目前尚未开发出有效的金属酶抑制剂。
除了表达水解碳青霉烯类β2内酰胺酶外，肺炎克雷伯菌对碳青酶烯类抗菌药物的耐药机制还可能涉及如下几个方面：(1)高产AmpC酶伴膜孔蛋白Omp缺失。革兰阴性菌的细胞壁外膜的蛋白通道是药物进入细菌的重要途径，膜孔蛋白对β2内酰胺类抗菌药物的导入作用有其特异性，当某种膜孔蛋白表达减少或缺失时，相应的抗菌药物就不能进入细菌发挥作用；(2)青霉素结合蛋白改变。β2内酰胺类抗菌药物通过膜孔蛋白进入周质间隙，然后与青霉素结合蛋白结合而发挥抗菌作用，此蛋白发生结构变化，β2内酰胺类抗菌药物与之亲和力下降或不与其结合，则不能有效干扰细菌细胞壁的合成而产生耐药；(3)主动外排系统的活跃。细菌细胞内膜存在能量依赖性蛋白外排泵，通过主动外排作用将药物从菌体排出，使达到作用靶位的药量明显减少，不足以发挥杀菌或抑菌作用。
半个世纪以前，革兰阴性杆菌肺炎(gram-negative bacillarypneumonia，GNBP)曾被认为是一种非常少见的疾病，很少受到临床关注。除克雷伯杆菌外，几乎没有关于革兰阴性杆菌(gram-negativebacterium，GNB)引起肺炎的报道。近二、三十年来，随着易感人群的改变、抗菌药物广泛应用与耐药菌的变迁以及各种微生物检测技术的提高与普及，GNBP已成为进入抗生素时代的现代医学的一种重要疾病。GNB在肺炎病原所占比例已从原先的0.5％～5.0％上升到现在社区获得性肺炎的9％～37％和医院内获得性肺炎(nosocomialpneumonia，NP)的70％以上。迄今对医院内、外获得的GNBP的临床和流行病学特征、易感因素、病原学诊断、抗感染治疗药物和方法等领域已进行了大量研究，但是病死率仍居高不下。研究和总结GNBP发病机制和诊治经验，仍是今后一段时间肺部感染性疾病领域的重要课题。
及时发现细菌中出现的耐药新基因，对人类的健康具有重要的意义，人类可以通过最新耐药基因的研究，开发新的抗菌药物。当超级菌导致人类患病时有相应的抗菌药物进行治疗。
发明内容
碳霉烯酶自2000年以后陆续在美国的新英格兰和亚特兰大地区被发现，主要在克雷伯菌属中，也在其他菌株中被发现。目前产碳霉烯酶的细菌已经在包括中国在内的全球范围内广泛传播，并对碳青霉烯类、青霉素类、头孢菌素类和氨曲南在内的几乎所有β2内酰胺类抗菌药物耐药，而且产碳霉烯酶的菌株种类正在增加，随时可能引起医院感染的爆发和流行。因此产碳霉烯酶菌的感染将是我们面临的严重问题，应引起微生物学家和临床医师的高度重视。对产碳霉烯酶菌的治疗目前尚无特别有效的药物。因此，新耐药基因的研究对临床、微生物实验室及新药研制均有十分重要的意义。
基于以上原因，本发明人等进行了反复悉心的研究，结果发现了一种新的耐药基因KPC，并命名KPC-15，所述耐药基因具体是通过以下方法获得：
1、在台州市立医院心胸外科病人血液培养阳性的标本中分离出的1株对亚胺培南、厄他培南耐药的肺炎克雷伯细菌。
2、然后将分离的单克隆菌株按操作程序用VITEK2和药敏平板琼脂扩散方法(补充药敏纸片)进行细菌鉴定及药敏试验，经鉴定此菌株为肺炎克雷伯菌。同时，试验证实此菌株对多种抗生素有显著抗性。
3、将上述筛选得到的耐药菌株培养扩增，用质粒提取试剂盒提取所述耐药菌株的质粒，并以此质粒为模板，设计KPC基因的引物进行PCR扩增，利用设计的测序引物对PCR产物进行测序，得到该耐药基因的编码区核苷酸序列，其序列如序列表SEQ ID No.1所示，其编码的蛋白质序列，如序列表SEQ ID No.2所示。
4、通过比对发现该KPC-15耐药基因为新的KPC变异基因，主要变异是其氨基酸序列的第119位突变成L(亮氨酸)和第146位突变成K(赖氨酸)。
本发明通过细菌质粒转导实验检测KPC-15基因的功能，将包含KPC-15基因的质粒耐药菌株与E.coli J53AzR(对叠氮化钠耐药)菌株共培养，通过含叠氮化钠(300mg/L)和环丙沙星(0.03mg/L)平板筛选结合子，将结合子做药敏试验，受体菌拥有供体菌的抗性。
为进一步证实该受体菌获得的抗性来源于KPC-15基因，本发明将该基因克隆入pET32a载体中，并将其转化入野生型E.coli JM109中，通过含有Amp的平板选阳性克隆(含有pET32a-KPC-15质粒的细菌)。将阳性克隆做药敏试验，结果显示KPC-15基因可以使细菌具有对以下药物耐药：氨曲南、环丙沙星、头孢替坦、妥布霉素、丁胺卡拉、头孢唑林、头孢吡肟、头孢他啶、头孢曲松、庆大霉素、亚胺培南、左氧氟沙星、哌拉西林/他唑巴坦、呋喃妥因、复方新诺明、头孢哌酮/舒巴坦(舒普深)、米诺霉素。
附图说明
图1表示目前14种KPC家族蛋白的序列比对结果。
具体实施方式
实施例1新抗性基因KPC-15的发现
一材料和方法
1、材料
法国bioMerieux的VITEK2；细菌鉴定卡；VITEK2GN Test kit；细菌药敏卡；法国bioMerieux的AST-GN13；英国Oxoid Ltd的补充药敏纸片(75μg/30μg头孢哌酮/舒巴坦、30μg米诺环素)；TakaRa公司的miniBEST plasmid purification Kit质粒小量抽提试剂盒；高保真聚合酶，其他试剂均为市购。
2、方法
2.1肺炎克雷伯菌KP1241菌株的分离鉴定
将台州市立医院心胸外科病人血液培养阳性的菌株转种到血平板上分离培养，在35℃含5％CO2孵育箱中培养16-18h，然后将分离的单克隆菌株按操作程序用bioMerieux的VITEK2细菌鉴定及药敏分析系统和药敏平板琼脂扩散方法(补充药敏纸片)进行细菌鉴定及药敏试验。按CLSI2012标准鉴定药敏结果。
2.2质粒提取
(1)肺炎克雷伯菌的培养：从平板中挑取单菌落接种至2ml含有抗生素(亚胺培南含量1mg/L)的液体培养基中，37℃过夜培养。
(2)过夜培养菌液，12,000rpm，离心2min，弃上清液。
(3)用250μl的Solution I(含RNaseA I)充分悬浮细菌沉淀。
(4)加入250μl的Solution II轻轻上下翻转混合5-6次，使菌体充分裂解，形成透明溶液。
(5)加入400μl的4℃预冷的Solution III，轻轻上下翻转混合5-6次，直至形成紧实凝集块，然后室温静置2min.
(6)室温12,000rpm，离心10min，取上清液。
(7)将试剂盒中的Spin Column安置于Collection Tube上。
(8)将(6)的上清液转移至Spin Column中，12,000rpm，离心1min，弃滤液。
(9)将500μl的Rinse A加入Spin Column中，12,000rpm，离心30sec，弃滤液。
(10)将700μl的Rinse B加入Spin Column中，12,000rpm，离心30sec，弃滤液。
(11)重复操作步骤(10)。
(12)将Spin Column安置于Collection Tube上，12,000rpm，离心1min。
(13)将Spin Column安置于新的1.5ml的离心管上，在Spin Column膜的中央处加入60μl的灭菌蒸馏水或Elution Buffer，室温静置1min。12000rpm，离心1min洗脱DNA。取25μl的DNA加入PCR反应体系。
2.3PCR扩增耐药新基因
根据KPC的基因序列设计扩增引物，引物序列如下：
F：5′-CGAGCAACTATGGATGAACG-3′
R：5′-GTATCTGTGAGGGCGAAGG-3′
以肺炎克雷伯菌KP1241菌株提取的质粒为模板，PCR反应体系如下：
PCR循环参数为：94℃预变性3分钟，94℃变性30秒，55℃退火30秒，72℃延伸1分钟，共35个循环，最后72℃延伸10分钟，PCR产物用1％琼脂糖凝胶电泳检测。
2.4测序及结果分析
根据KPC的基因序列设计该基因的测序引物，如下：
F：5′-CGGAACCATTCGCTAAACTCG-3′
R：5′-CGCCAACTCCTTCAGCAACA-3′
将PCR产物送华大测序。
3、结果：
3.1)耐药菌株的鉴定及对药物敏感性分析：
生化特性：中等大小的革兰氏阴性杆菌，KIA产酸/产酸，葡萄糖与乳糖产酸产气，氧化酶阴性，VP阳性，吲哚阴性，枸橼酸盐阳性，动力阴性，不产H2S，符合肺炎克雷伯菌的特性，经VITEK2微生物分析仪鉴定为肺炎克雷伯菌，将其编号为KP1241，鉴定概率为99％。
药敏实验结果如表1所示：
表1肺炎克雷伯临床耐药菌KP1241的药敏检测结果
注：MIC指最低抑菌所需药物浓度，其单位为(ug/ml)，R为耐药，*纸片扩散法。
MIC值越大代表该菌株对相应抗菌药物越不敏感，即该菌株对抗菌药越耐药，从上述实验结果显示：肺炎克雷伯菌KP1241对氨苄西林/舒巴坦、氨曲南、环丙沙星、头孢替坦、妥布霉素、丁胺卡拉、氨苄西林、头孢唑林、头孢吡肟、头孢他啶、头孢曲松、庆大霉素、亚胺培南、左氧氟沙星、哌拉西林/他唑巴坦、呋喃妥因、复方新诺明、头孢哌酮/舒巴坦(舒普深)、米诺霉素都具有耐药性，其中对哌拉西林/他唑巴坦和呋喃妥因耐药性最强。
3.2)KPC-15新基因的确定：
测序并经序列比对分析，发现KP1241菌株的质粒具有新KPC基因，其核苷酸序列为SEQ ID NO.1，其所编码的蛋白质序列为SEQID NO.2。通过比对发现该KPC-15耐药基因为新的KPC变异基因，主要变异是其氨基酸序列的第119位突变成L(亮氨酸)和第146位突变成K(赖氨酸)，比对结果见图1。
实施例2细菌质粒转导实验及耐药性检测
一、材料和方法
1、材料
肺炎克雷伯菌(编号KP1241)；野生型E.coli J53。
2、方法及结果
分别以肺炎克雷伯菌KP1241和野生型E.coli J53AzR(对叠氮化钠耐药)为供体菌和受体菌，将对数生长期的两者各取0.5ml加入4ml新鲜的LB肉汤中静置过夜培养。接合子以含叠氮化钠(300mg/L)和环丙沙星(0.03mg/L)的胰大豆琼脂(TSA)平板筛选，置35℃孵育18-24小时。将筛选到的接合子用bioMerieux的VITEK2细菌鉴定及药敏分析系统和药敏平板琼脂扩散方法(补充药敏纸片)分析其对药物的敏感性，结果如表2所示：
表2E.coliJ53AzR转化菌的药敏检测结果
注：MIC指最低抑菌所需药物浓度，其单位为(ug/ml)，R为耐药，*纸片扩散法。
以上结果显示含有KPC-15质粒的受体菌拥有与肺炎克雷伯菌KP1241相司的耐药种类。
实施例3组成型KPC-15基因表达菌的构建
一、材料和方法
1、材料
E.coli JM109；TaKaRa公司的pMD18T载体；pBR322载体；TaKaRa公司的限制性核酸内切酶Pst I和EcoR I；天根生化科技(北京)有限公司的琼脂糖凝胶回收试剂盒；TaKaRa公司的T4DNA连接酶。
2、方法
2.1组成型KPC-15基因表达菌E.coli JM109(pET32a-KPC-15)构建
将实施例1中的PCR产物与pMD18T载体连接，连接产物加入100μl JM109的感受态中转化，在含有IPTG、x-gal、Amp的平板上培养，测序验证基因序列及其基因插入方向正确。然后将KPC-15基因克隆到pET32a载体中，得到含KPC-15基因的pET32a-KPC-15质粒。将重组质粒转化到感受态E.coli JM109细胞中，在含有Amp的平板上培养，筛选获得重组菌E.coli JM109(pET32a-KPC-15)。
2.2对构建成功的克隆菌进行耐药性检测
采用法国bioMerieux的VITEK2细菌鉴定及药敏分析系统和药敏平板琼脂扩散方法(补充药敏纸片)对重组菌E.coli JM109(pET32a-KPC-15)进行耐药性检测，结果见下表：
表3E.coli JM109(pET32a-KPC-15)的药敏检测结果
注：MIC指最低抑菌所需药物浓度，其单位为(ug/ml)，R为耐药，*纸片扩散法。
结果显示重组菌对氨苄西林/舒巴坦、氨曲南、环丙沙星、头孢替坦、妥布霉素、丁胺卡拉、头孢唑林、头孢吡肟、头孢他啶、头孢曲松、庆大霉素、亚胺培南、左氧氟沙星、哌拉西林/他唑巴坦、呋喃妥因、复方新诺明、头孢哌酮/舒巴坦(舒普深)、米诺霉素耐药，即KPC-15基因的功能是，可以使细菌具有对以下药物耐药：氨曲南、环丙沙星、头孢替坦、妥布霉素、丁胺卡拉、头孢唑林、头孢吡肟、头孢他啶、头孢曲松、庆大霉素、亚胺培南、左氧氟沙星、哌拉西林/他唑巴坦、呋喃妥因、复方新诺明、头孢哌酮/舒巴坦(舒普深)、米诺霉素。
Claims (4)
1.一种KPC-15蛋白，其特征在于，所述KPC-15蛋白的氨基酸序列如序列表SEQ ID NO：2所示。
2.编码权利要求1所述蛋白的核酸。
3.根据权利要求2所述的核酸，其特征在于：其序列如SEQ ID NO：1所示。
4.权利要求2所述的核酸在制备耐药菌株中的应用。
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