幽门螺旋杆菌对克拉霉素、甲硝唑、左氧氟沙星耐药的研究进展

刘雪芳， 马岚青

昆明医科大学第一附属医院消化内科，云南 昆明 650032

幽门螺旋杆菌对克拉霉素、甲硝唑、左氧氟沙星耐药的研究进展

刘雪芳， 马岚青

昆明医科大学第一附属医院消化内科，云南 昆明 650032

幽门螺旋杆菌(Helicobacter pylori，H.pylori)感染可导致慢性胃炎、消化性溃疡、胃癌和胃黏膜相关淋巴组织(mucosa-associated lymphoid tissue, MALT)淋巴瘤。而H.pylori根除治疗失败与克拉霉素、甲硝唑及左氧氟沙星高的耐药率有关。现已证实这些抗生素的耐药机制与H.pylori基因突变有关。提高对这些抗生素耐药机制的了解对发展和证实以活检组织为基础的耐药性检测方法很有必要。快速检测H.pylori耐药的基因突变有助于H.pylori根除治疗方案的选择。

幽门螺旋杆菌；抗生素耐药；基因突变；克拉霉素；甲硝唑；左氧氟沙星

幽门螺旋杆菌(Helicobacter pylori，H.pylori)是革兰氏阴性微需氧细菌。H.pylori感染普遍出现在各个国家，发展中国家的感染率超过70%,发达国家感染率20%～50%。H.pylori感染可以导致慢性胃炎、消化性溃疡、胃癌和胃黏膜相关淋巴组织(mucosa-associated lymphoid tissue, MALT)淋巴瘤。根除H.pylori可以降低胃癌的发病率。

三联疗法曾被推荐作为一线治疗H.pylori感染的方法，这种治疗方法包括质子泵抑制剂和两种抗生素。然而随着这种治疗方法根除H.pylori失败率的增加，许多研究人员发现H.pylori对抗生素耐药是导致这种方法根除失败的主要原因。目前含有铋剂的四联疗法被推荐为根除H.pylori的首选方法。克拉霉素、甲硝唑、左氧氟沙星作为根除H.pylori最常选用的抗生素，H.pylori对其的耐药直接影响根除率。所以了解耐药机制和快速检测H.pylori在H.pylori感染根除治疗中有重要意义。本文就H.pylori对克拉霉素、甲硝唑及左氧氟沙星的耐药机制和检测方法作一概述。

1 克拉霉素

1.1 耐药机制H.pylori对克拉霉素耐药受多种因素影响，研究表明H.pylori23S rRNA肽基转移酶弯曲部分基因2143和2144腺嘌呤突变为鸟嘌呤，2142腺嘌呤突变为胞嘧啶[1]。突变A2143G和A2144G是被确认为H.pylori耐克拉霉素的原因[2]，韩国科学家Kim等[3]研究发现T2182C突变，同时报道了基因突变可以同时出现2～3个。但是Moder等[4]研究T2182C突变与克拉霉素耐药并无关联。有报道[3]指出A2115G、C2694A也与克拉霉素耐药有关，西班牙科学家Agudo等[5]研究并未发现以上突变，但是指出H.pylori对克拉霉素的抵抗可能与人种、地区、年龄、性别有关。H.pylori对克拉霉素耐药率存在明显地域差异：伊朗的耐药率为45.2%[6]，巴西的耐药率为8.0%[7]，意大利的耐药率为9.9%[8]。H.pylori对克拉霉素耐药率存在年龄及性别的差异：Boyanova等[9]研究发现65岁以上的耐药率低于65岁以下，且男性耐药率高于女性。同时还发现非消化性溃疡患者克拉霉素的耐药率高于消化性溃疡患者。服用非甾体类抗炎药的患者对克拉霉素的耐药率高于未服用患者；H.pylori耐药率与出生地、是否吸烟、饮食习惯等有关。而O’Connor等[10]研究发现有根除治疗经验患者耐药率高于未进行根除治疗患者(32.4%vs9.3%)。因此，克拉霉素对H.pylori的耐药率是受多种因素影响。

1.2 检测方法H.pylori耐克拉霉素检测方法主要是基于PCR技术的分子诊断技术，这些技术都是建立在与H.pylori耐药相关基因突变的基础上。荧光定量PCR(Light Cycle)可以在1 h内检测出23S rRNA上的点突变[1]。Nasted PCR可以快速、准确地检测出H.pylori耐克拉霉素23S rRNA的突变[11]。Schabereiter-Gurtner等[12]研究表明通过Biprones 技术联合Real-time PCR可成为精确的非创伤性检测H.pylori感染的方法，提高Real-time PCR特异性检测H.pylori感染及对克拉霉素耐药的23S rRNA上的点突变，同时双探针荧光能量转移(FRET)联合Real-time PCR技术，其扩增产物通过溶解曲线进行分析，在几个小时内能够同时检测H.pylori感染及大环内酯类耐药性[13]。van Doorn等[14]通过线性探针反向杂交技术(PCR-LiPA)能够快速、准确地检测出H.pylori耐大环内酯类药物的23S rRNA的突变。Moder等[4]也证明了焦磷酸测序技术是一种可靠、快速、高度准确的检测H.pylori对克拉霉素耐药的方法。此外，比色法DNA芯片能够检测野生株和任何位置单个点突变，对检测H.pylori耐药基因有高度特异性，且不要求昂贵的设备，花费低、操作简单，具有高流通量和在临床上应用的技术可行性[15]，尤其适用于发展中国家的小型和中等规模的医院。因此，比色法DNA芯片也是一种可靠、快速、准确地检测有关H.pylori克拉霉素耐药的方法。

2 甲硝唑

2.1 耐药机制H.pylori对甲硝唑耐药最根本的原因是H.pylori基因突变。早在1998年Goodwin等[16]报道对氧敏感的NADPH硝基还原酶活性与H.pylori对甲硝唑敏感性有关。突变的H.pylori的rdxA基因可以钝化对氧敏感的NADPH硝基还原酶基因的编码和表达。大量研究调查显示H.pylori耐甲硝唑与H.pylori的基因rdxA或frxA基因突变有关[17-19]。而H.pylori对甲硝唑的高耐药率与rdxA基因低突变率[20]，提示H.pylori对甲硝唑耐药还存在其他机制。Kaakoush等[21]认为H.pylori耐甲硝唑不仅涉及rdxA或frxA基因突变，更多的是涉及复杂新陈代谢的改变。Tsugawa等[22]研究发现不是所有对甲硝唑耐药的H.pylori都具有硝基还原酶活性，只有少部分菌株具有此酶活性。并指出多数对甲硝唑耐药的H.pylori是由于铁吸收蛋白调节器(fur)上的两个氨基酸类突变(C78Y，P114S)。同时Choi等[23]报道fur基因产物可能与其他细胞成分、新陈代谢线路相互作用，在胃小凹内形成有利于H.pylori生存的特殊环境。此外，RND流出泵过度表达增加H.pylori对甲硝唑的耐药率[24]，不含cagA基因的H.pylori菌株相对于含cagA基因的H.pylori更易对甲硝唑产生耐药[25]。爱尔兰O’Connor 等[10]报道H.pylori对甲硝唑与性别有关，女性耐药率为35.4%，男性耐药率为28.5%。

2.2 检测方法H.pylori耐甲硝唑是基于多种因素，检测甲硝唑耐药菌株方法也涉及多方面。Singh等[26]重复共有基因序列(repetitive intergenic consensus sequences，ERIC)结合随机扩增基因多态性PCR技术(random amplified polymorphic DNA-PCR，RAPD-PCR)识别耐甲硝唑菌株。Jenks等[17]、Tankovic等[18]采用PCR技术特意性扩增H.pylorirdxA基因片段，然后对该片段进行核苷酸序列测定，以检测出耐药菌株。Mehrabadi等[24]通过RT-PCR方法和同源分析法检测出RND流出泵过度表达，从而鉴别H.pylori耐甲硝唑菌株。Morimoto等[27]采用重复序列PCR(rep-PCR)联合高级微生物基因分型系统(DiversiLab Microbial Typing System DL)快速、高敏感地识别耐甲硝唑、克拉霉素菌株及克拉霉素同时耐药的菌株。高分辨率溶解曲线分析技术(high resolution melting analysis，HRM)利用溶解温度和变性程度之间关系进行基因片段分析，完全基于核酸物理性质进行分析，无需序列特异性探针，不受突变碱基类型和位点局限，在PCR结束后直接运行HRM既可以对未知突变进行筛查、扫描，又可以对已知突变进行分析，也可用于短片段重复序列的分析[28]。

3 左氧氟沙星

3.1 耐药机制H.pylori对氟喹诺酮类药物耐药99%是由于H.pylorigyrA基因的喹诺酮类药物耐药决定区(quinolone resistance determining region，QRDR)突变导致[29]。已有13种突变被检测出，它们位于基因gyrA的86、87、88和91位点[30-32]。有研究表明83.8%的H.pylori对左氧氟沙星耐药是由于H.pylori上的基因gyrA在Asn87或是Asp91位点上点突变[31]。且H.pylori的基因gyrA在Asn87位点上的突变对左氧氟沙星耐药率高于H.pylori的基因gyrA在Asp91位点上点突变。而gyrB基因在463位点上点突变是H.pylori对氟喹诺酮类药物耐药的新机制。可能由于不同地区对抗生素的使用剂量与适应证制定标准不同，H.pylori对左氧氟沙星耐药出现地域、年龄差异。Megraud等[33]研究调查中显示H.pylori对左氧氟沙星耐药率在成人与儿童中是不同的：成人耐药率为14.1%，儿童为8.0%；不同地区对左氧氟沙星的耐药率也是不同的：北欧为7.7%，南/中欧为18.6%，西欧为13.1%，马来西亚为0[34]；O’Connor等[35]发现H.pylori耐左氧氟沙星存在年龄差异：45岁以下耐药率为2.8%，45岁以上耐药率为19.1%。

3.2 检测方法H.pylori耐左氧氟沙星检测主要是检测突变的基因，这些方法主要是基于PCR技术的分子诊断技术。大多检测H.pylori耐左氧氟沙星都是采用PCR技术特意性扩增H.pylorigyrA基因的QRDR片段，然后对该片段进行核苷酸序列测定[29-31]。德国学者Glocker等[36]利用Real-time PCR结合荧光共振能量转换技术(fluorescence resonance energy transfer-based real-time PCR)检测出不同突变类型基因突变菌株的熔解温度，通过熔解温度鉴别耐喹诺酮类药物的H.pylori。Nishizawa等[37]构建等位基因特异性PCR(Allele-specific PCR，AS-PCR)，在3～5 h内检测出H.pylorigyrA基因上的突变，是一种快速、可靠地识别耐氟喹诺酮类药物的菌株方法。Rajper等[38]利用反向杂交PCR线性探针测定快速测定耐左氧氟沙星的H.pylorigyrA基因上的突变。此外，GenoType HelicoDR测试是依赖DNA条带方法学检测出耐左氧氟沙星的H.pylorigyrA基因上的突变及耐克拉霉素H.pylori的23S rRNA的突变，也可检测出对克拉霉素和左氧氟沙星同时耐药的菌株[39]，这种方法操作简单便于在临床使用。

随着抗生素在根除H.pylori感染治疗中的广泛应用，耐药菌株也不断出现，H.pylori根除治疗面临着严峻的考验。因此，设计一种快速、准确、高敏感性及操作简便的检测对抗生素耐药菌株的方法，对指导临床用药有着深远的意义。近年来，基因芯片技术逐渐趋于成熟，HRM因其快速、低成本、高敏感性、特异性好、重复性好及操作简便，在国外兴起一种用于突变扫描和基因分型的最新遗传学分析方法，成为国外新兴的遗传学、方法学研究和应用热点。二者均具有较高的临床应用价值及推广意义。

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堪误：本刊2015年第12期第1494页第一作者王锐的文章《危险因素对胃黏膜癌前病变中血管内皮生长因子表达的影响》左下角基金项目：全军“十二五”课题面上项目(CWS11J005),标注有误，本文无基金项目，特此更正。

Advances in research of Helicobacter pylori resistance to Clarithromycin, Metronidazole, Levofloxacin

LIU Xuefang, MA Lanqing

Department of Gastroenterology, the First Affilicated Hospital of Kunming Medical University, Kunming 650032, China

Infection with Helicobacter pylori (H.pylori) is associated with chronic gastritis and peptic ulceration and the bacterium. It is also considered as a risk factor for the development of gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. High resistance rates of Clarithromycin, Metronidazole, and Levofloxacin are associated withH.pylorieradication failure. It is widely accepted that the resistance to these antimicrobial is related with mutations ofH.pylorigene. Improving the understanding of the mechanisms of antimicrobial resistance inH.pyloriis essential for the development and validation of biopsy-based tests for detection of resistance. Rapid detection of mutations gene ofH.pyloricontributes to the selection of suitable eradication therapies for patients.

Helicobacter pylori; Antimicrobial resistance; Gene mutations; Clarithromycin; Metronidazole; Levofloxacin

10.3969/j.issn.1006-5709.2016.01.028

云南省联合专项基金(2012FB027)

刘雪芳，硕士在读，研究方向：消化系统疾病。E-mail：532180042@qq.com

马岚青，博士，研究生导师，副主任医师，研究方向：消化系统疾病。E-mail：malanqing@aliyun

R37

A

1006-5709(2016)01-0101-04

2014-12-25