幽門螺旋桿菌對克拉霉素、甲硝唑、左氧氟沙星耐藥的研究進(jìn)展

劉雪芳， 馬嵐青

昆明醫(yī)科大學(xué)第一附屬醫(yī)院消化內(nèi)科，云南 昆明 650032

幽門螺旋桿菌對克拉霉素、甲硝唑、左氧氟沙星耐藥的研究進(jìn)展

劉雪芳， 馬嵐青

昆明醫(yī)科大學(xué)第一附屬醫(yī)院消化內(nèi)科，云南 昆明 650032

幽門螺旋桿菌(Helicobacter pylori，H.pylori)感染可導(dǎo)致慢性胃炎、消化性潰瘍、胃癌和胃黏膜相關(guān)淋巴組織(mucosa-associated lymphoid tissue, MALT)淋巴瘤。而H.pylori根除治療失敗與克拉霉素、甲硝唑及左氧氟沙星高的耐藥率有關(guān)。現(xiàn)已證實這些抗生素的耐藥機制與H.pylori基因突變有關(guān)。提高對這些抗生素耐藥機制的了解對發(fā)展和證實以活檢組織為基礎(chǔ)的耐藥性檢測方法很有必要?？焖贆z測H.pylori耐藥的基因突變有助于H.pylori根除治療方案的選擇。

幽門螺旋桿菌；抗生素耐藥；基因突變；克拉霉素；甲硝唑；左氧氟沙星

幽門螺旋桿菌(Helicobacter pylori，H.pylori)是革蘭氏陰性微需氧細(xì)菌。H.pylori感染普遍出現(xiàn)在各個國家，發(fā)展中國家的感染率超過70%,發(fā)達(dá)國家感染率20%～50%。H.pylori感染可以導(dǎo)致慢性胃炎、消化性潰瘍、胃癌和胃黏膜相關(guān)淋巴組織(mucosa-associated lymphoid tissue, MALT)淋巴瘤。根除H.pylori可以降低胃癌的發(fā)病率。

三聯(lián)療法曾被推薦作為一線治療H.pylori感染的方法，這種治療方法包括質(zhì)子泵抑制劑和兩種抗生素。然而隨著這種治療方法根除H.pylori失敗率的增加，許多研究人員發(fā)現(xiàn)H.pylori對抗生素耐藥是導(dǎo)致這種方法根除失敗的主要原因。目前含有鉍劑的四聯(lián)療法被推薦為根除H.pylori的首選方法?？死顾亍⒓紫踹?、左氧氟沙星作為根除H.pylori最常選用的抗生素，H.pylori對其的耐藥直接影響根除率。所以了解耐藥機制和快速檢測H.pylori在H.pylori感染根除治療中有重要意義。本文就H.pylori對克拉霉素、甲硝唑及左氧氟沙星的耐藥機制和檢測方法作一概述。

1 克拉霉素

1.1 耐藥機制H.pylori對克拉霉素耐藥受多種因素影響，研究表明H.pylori23S rRNA肽基轉(zhuǎn)移酶彎曲部分基因2143和2144腺嘌呤突變?yōu)轼B嘌呤，2142腺嘌呤突變?yōu)榘奏1]。突變A2143G和A2144G是被確認(rèn)為H.pylori耐克拉霉素的原因[2]，韓國科學(xué)家Kim等[3]研究發(fā)現(xiàn)T2182C突變，同時報道了基因突變可以同時出現(xiàn)2～3個。但是Moder等[4]研究T2182C突變與克拉霉素耐藥并無關(guān)聯(lián)。有報道[3]指出A2115G、C2694A也與克拉霉素耐藥有關(guān)，西班牙科學(xué)家Agudo等[5]研究并未發(fā)現(xiàn)以上突變，但是指出H.pylori對克拉霉素的抵抗可能與人種、地區(qū)、年齡、性別有關(guān)。H.pylori對克拉霉素耐藥率存在明顯地域差異：伊朗的耐藥率為45.2%[6]，巴西的耐藥率為8.0%[7]，意大利的耐藥率為9.9%[8]。H.pylori對克拉霉素耐藥率存在年齡及性別的差異：Boyanova等[9]研究發(fā)現(xiàn)65歲以上的耐藥率低于65歲以下，且男性耐藥率高于女性。同時還發(fā)現(xiàn)非消化性潰瘍患者克拉霉素的耐藥率高于消化性潰瘍患者。服用非甾體類抗炎藥的患者對克拉霉素的耐藥率高于未服用患者；H.pylori耐藥率與出生地、是否吸煙、飲食習(xí)慣等有關(guān)。而O’Connor等[10]研究發(fā)現(xiàn)有根除治療經(jīng)驗患者耐藥率高于未進(jìn)行根除治療患者(32.4%vs9.3%)。因此，克拉霉素對H.pylori的耐藥率是受多種因素影響。

1.2 檢測方法H.pylori耐克拉霉素檢測方法主要是基于PCR技術(shù)的分子診斷技術(shù)，這些技術(shù)都是建立在與H.pylori耐藥相關(guān)基因突變的基礎(chǔ)上。熒光定量PCR(Light Cycle)可以在1 h內(nèi)檢測出23S rRNA上的點突變[1]。Nasted PCR可以快速、準(zhǔn)確地檢測出H.pylori耐克拉霉素23S rRNA的突變[11]。Schabereiter-Gurtner等[12]研究表明通過Biprones 技術(shù)聯(lián)合Real-time PCR可成為精確的非創(chuàng)傷性檢測H.pylori感染的方法，提高Real-time PCR特異性檢測H.pylori感染及對克拉霉素耐藥的23S rRNA上的點突變，同時雙探針熒光能量轉(zhuǎn)移(FRET)聯(lián)合Real-time PCR技術(shù)，其擴增產(chǎn)物通過溶解曲線進(jìn)行分析，在幾個小時內(nèi)能夠同時檢測H.pylori感染及大環(huán)內(nèi)酯類耐藥性[13]。van Doorn等[14]通過線性探針反向雜交技術(shù)(PCR-LiPA)能夠快速、準(zhǔn)確地檢測出H.pylori耐大環(huán)內(nèi)酯類藥物的23S rRNA的突變。Moder等[4]也證明了焦磷酸測序技術(shù)是一種可靠、快速、高度準(zhǔn)確的檢測H.pylori對克拉霉素耐藥的方法。此外，比色法DNA芯片能夠檢測野生株和任何位置單個點突變，對檢測H.pylori耐藥基因有高度特異性，且不要求昂貴的設(shè)備，花費低、操作簡單，具有高流通量和在臨床上應(yīng)用的技術(shù)可行性[15]，尤其適用于發(fā)展中國家的小型和中等規(guī)模的醫(yī)院。因此，比色法DNA芯片也是一種可靠、快速、準(zhǔn)確地檢測有關(guān)H.pylori克拉霉素耐藥的方法。

2 甲硝唑

2.1 耐藥機制H.pylori對甲硝唑耐藥最根本的原因是H.pylori基因突變。早在1998年Goodwin等[16]報道對氧敏感的NADPH硝基還原酶活性與H.pylori對甲硝唑敏感性有關(guān)。突變的H.pylori的rdxA基因可以鈍化對氧敏感的NADPH硝基還原酶基因的編碼和表達(dá)。大量研究調(diào)查顯示H.pylori耐甲硝唑與H.pylori的基因rdxA或frxA基因突變有關(guān)[17-19]。而H.pylori對甲硝唑的高耐藥率與rdxA基因低突變率[20]，提示H.pylori對甲硝唑耐藥還存在其他機制。Kaakoush等[21]認(rèn)為H.pylori耐甲硝唑不僅涉及rdxA或frxA基因突變，更多的是涉及復(fù)雜新陳代謝的改變。Tsugawa等[22]研究發(fā)現(xiàn)不是所有對甲硝唑耐藥的H.pylori都具有硝基還原酶活性，只有少部分菌株具有此酶活性。并指出多數(shù)對甲硝唑耐藥的H.pylori是由于鐵吸收蛋白調(diào)節(jié)器(fur)上的兩個氨基酸類突變(C78Y，P114S)。同時Choi等[23]報道fur基因產(chǎn)物可能與其他細(xì)胞成分、新陳代謝線路相互作用，在胃小凹內(nèi)形成有利于H.pylori生存的特殊環(huán)境。此外，RND流出泵過度表達(dá)增加H.pylori對甲硝唑的耐藥率[24]，不含cagA基因的H.pylori菌株相對于含cagA基因的H.pylori更易對甲硝唑產(chǎn)生耐藥[25]。愛爾蘭O’Connor 等[10]報道H.pylori對甲硝唑與性別有關(guān)，女性耐藥率為35.4%，男性耐藥率為28.5%。

2.2 檢測方法H.pylori耐甲硝唑是基于多種因素，檢測甲硝唑耐藥菌株方法也涉及多方面。Singh等[26]重復(fù)共有基因序列(repetitive intergenic consensus sequences，ERIC)結(jié)合隨機擴增基因多態(tài)性PCR技術(shù)(random amplified polymorphic DNA-PCR，RAPD-PCR)識別耐甲硝唑菌株。Jenks等[17]、Tankovic等[18]采用PCR技術(shù)特意性擴增H.pylorirdxA基因片段，然后對該片段進(jìn)行核苷酸序列測定，以檢測出耐藥菌株。Mehrabadi等[24]通過RT-PCR方法和同源分析法檢測出RND流出泵過度表達(dá)，從而鑒別H.pylori耐甲硝唑菌株。Morimoto等[27]采用重復(fù)序列PCR(rep-PCR)聯(lián)合高級微生物基因分型系統(tǒng)(DiversiLab Microbial Typing System DL)快速、高敏感地識別耐甲硝唑、克拉霉素菌株及克拉霉素同時耐藥的菌株。高分辨率溶解曲線分析技術(shù)(high resolution melting analysis，HRM)利用溶解溫度和變性程度之間關(guān)系進(jìn)行基因片段分析，完全基于核酸物理性質(zhì)進(jìn)行分析，無需序列特異性探針，不受突變堿基類型和位點局限，在PCR結(jié)束后直接運行HRM既可以對未知突變進(jìn)行篩查、掃描，又可以對已知突變進(jìn)行分析，也可用于短片段重復(fù)序列的分析[28]。

3 左氧氟沙星

3.1 耐藥機制H.pylori對氟喹諾酮類藥物耐藥99%是由于H.pylorigyrA基因的喹諾酮類藥物耐藥決定區(qū)(quinolone resistance determining region，QRDR)突變導(dǎo)致[29]。已有13種突變被檢測出，它們位于基因gyrA的86、87、88和91位點[30-32]。有研究表明83.8%的H.pylori對左氧氟沙星耐藥是由于H.pylori上的基因gyrA在Asn87或是Asp91位點上點突變[31]。且H.pylori的基因gyrA在Asn87位點上的突變對左氧氟沙星耐藥率高于H.pylori的基因gyrA在Asp91位點上點突變。而gyrB基因在463位點上點突變是H.pylori對氟喹諾酮類藥物耐藥的新機制?？赡苡捎诓煌貐^(qū)對抗生素的使用劑量與適應(yīng)證制定標(biāo)準(zhǔn)不同，H.pylori對左氧氟沙星耐藥出現(xiàn)地域、年齡差異。Megraud等[33]研究調(diào)查中顯示H.pylori對左氧氟沙星耐藥率在成人與兒童中是不同的：成人耐藥率為14.1%，兒童為8.0%；不同地區(qū)對左氧氟沙星的耐藥率也是不同的：北歐為7.7%，南/中歐為18.6%，西歐為13.1%，馬來西亞為0[34]；O’Connor等[35]發(fā)現(xiàn)H.pylori耐左氧氟沙星存在年齡差異：45歲以下耐藥率為2.8%，45歲以上耐藥率為19.1%。

3.2 檢測方法H.pylori耐左氧氟沙星檢測主要是檢測突變的基因，這些方法主要是基于PCR技術(shù)的分子診斷技術(shù)。大多檢測H.pylori耐左氧氟沙星都是采用PCR技術(shù)特意性擴增H.pylorigyrA基因的QRDR片段，然后對該片段進(jìn)行核苷酸序列測定[29-31]。德國學(xué)者Glocker等[36]利用Real-time PCR結(jié)合熒光共振能量轉(zhuǎn)換技術(shù)(fluorescence resonance energy transfer-based real-time PCR)檢測出不同突變類型基因突變菌株的熔解溫度，通過熔解溫度鑒別耐喹諾酮類藥物的H.pylori。Nishizawa等[37]構(gòu)建等位基因特異性PCR(Allele-specific PCR，AS-PCR)，在3～5 h內(nèi)檢測出H.pylorigyrA基因上的突變，是一種快速、可靠地識別耐氟喹諾酮類藥物的菌株方法。Rajper等[38]利用反向雜交PCR線性探針測定快速測定耐左氧氟沙星的H.pylorigyrA基因上的突變。此外，GenoType HelicoDR測試是依賴DNA條帶方法學(xué)檢測出耐左氧氟沙星的H.pylorigyrA基因上的突變及耐克拉霉素H.pylori的23S rRNA的突變，也可檢測出對克拉霉素和左氧氟沙星同時耐藥的菌株[39]，這種方法操作簡單便于在臨床使用。

隨著抗生素在根除H.pylori感染治療中的廣泛應(yīng)用，耐藥菌株也不斷出現(xiàn)，H.pylori根除治療面臨著嚴(yán)峻的考驗。因此，設(shè)計一種快速、準(zhǔn)確、高敏感性及操作簡便的檢測對抗生素耐藥菌株的方法，對指導(dǎo)臨床用藥有著深遠(yuǎn)的意義。近年來，基因芯片技術(shù)逐漸趨于成熟，HRM因其快速、低成本、高敏感性、特異性好、重復(fù)性好及操作簡便，在國外興起一種用于突變掃描和基因分型的最新遺傳學(xué)分析方法，成為國外新興的遺傳學(xué)、方法學(xué)研究和應(yīng)用熱點。二者均具有較高的臨床應(yīng)用價值及推廣意義。

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(責(zé)任編輯：王豪勛)

堪誤：本刊2015年第12期第1494頁第一作者王銳的文章《危險因素對胃黏膜癌前病變中血管內(nèi)皮生長因子表達(dá)的影響》左下角基金項目：全軍“十二五”課題面上項目(CWS11J005),標(biāo)注有誤，本文無基金項目，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

云南省聯(lián)合專項基金(2012FB027)

劉雪芳，碩士在讀，研究方向：消化系統(tǒng)疾病。E-mail：532180042@qq.com

馬嵐青，博士，研究生導(dǎo)師，副主任醫(yī)師，研究方向：消化系統(tǒng)疾病。E-mail：malanqing@aliyun

R37

A

1006-5709(2016)01-0101-04

2014-12-25