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展

劉雪芳， 馬嵐青

昆明醫科大學第一附屬醫院消化內科，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展

劉雪芳， 馬嵐青

昆明醫科大學第一附屬醫院消化內科，云南 昆明 650032

幽門螺旋桿菌(Helicobacter pylori，H.pylori)感染可導致慢性胃炎、消化性潰瘍、胃癌和胃黏膜相關淋巴組織(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?，F已證實這些抗生素的耐藥機制與H.pylori基因突變有關。提高對這些抗生素耐藥機制的了解對發展和證實以活檢組織為基礎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菌。H.pylori感染普遍出現在各個國家，發展中國家的感染率超過70%,發達國家感染率20%～50%。H.pylori感染可以導致慢性胃炎、消化性潰瘍、胃癌和胃黏膜相關淋巴組織(mucosa-associated lymphoid tissue, MALT)淋巴瘤。根除H.pylori可以降低胃癌的發病率。

三聯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，許多研究人員發現H.pylori對抗生素耐藥是導致這種方法根除失敗的主要原因。目前含有鉍劑的四聯療法被推薦為根除H.pylori的首選方法?？死顾?、甲硝唑、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肽基轉移酶彎曲部分基因2143和2144腺嘌呤突變為鳥嘌呤，2142腺嘌呤突變為胞嘧啶[1]。突變A2143G和A2144G是被確認為H.pylori耐克拉霉素的原因[2]，韓國科學家Kim等[3]研究發現T2182C突變，同時報道了基因突變可以同時出現2～3個。但是Moder等[4]研究T2182C突變與克拉霉素耐藥并無關聯。有報道[3]指出A2115G、C2694A也與克拉霉素耐藥有關，西班牙科學家Agudo等[5]研究并未發現以上突變，但是指出H.pylori對克拉霉素的抵抗可能與人種、地區、年齡、性別有關。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]研究發現65歲以上的耐藥率低于65歲以下，且男性耐藥率高于女性。同時還發現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、飲食習慣等有關。而O’Connor等[10]研究發現有根除治療經驗患者耐藥率高于未進行根除治療患者(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技術的分子診斷技術，這些技術都是建立在與H.pylori耐藥相關基因突變的基礎上。熒光定量PCR(Light Cycle)可以在1 h內檢測出23S rRNA上的點突變[1]。Nasted PCR可以快速、準確地檢測出H.pylori耐克拉霉素23S rRNA的突變[11]。Schabereiter-Gurtner等[12]研究表明通過Biprones 技術聯合Real-time PCR可成為精確的非創傷性檢測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移(FRET)聯合Real-time PCR技術，其擴增產物通過溶解曲線進行分析，在幾個小時內能夠同時檢測H.pylori感染及大環內酯類耐藥性[13]。van Doorn等[14]通過線性探針反向雜交技術(PCR-LiPA)能夠快速、準確地檢測出H.pylori耐大環內酯類藥物的23S rRNA的突變。Moder等[4]也證明了焦磷酸測序技術是一種可靠、快速、高度準確的檢測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，且不要求昂貴的設備，花費低、操作簡單，具有高流通量和在臨床上應用的技術可行性[15]，尤其適用于發展中國家的小型和中等規模的醫院。因此，比色法DNA芯片也是一種可靠、快速、準確地檢測有關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。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。大量研究調查顯示H.pylori耐甲硝唑與H.pylori的基因rdxA或frxA基因突變有關[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，更多的是涉及復雜新陳代謝的改變。Tsugawa等[22]研究發現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。并指出多數對甲硝唑耐藥的H.pylori是由于鐵吸收蛋白調節器(fur)上的兩個氨基酸類突變(C78Y，P114S)。同時Choi等[23]報道fur基因產物可能與其他細胞成分、新陳代謝線路相互作用，在胃小凹內形成有利于H.pylori生存的特殊環境。此外，RND流出泵過度表達增加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更易對甲硝唑產生耐藥[25]。愛爾蘭O’Connor 等[10]報道H.pylori對甲硝唑與性別有關，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]重復共有基因序列(repetitive intergenic consensus sequences，ERIC)結合隨機擴增基因多態性PCR技術(random amplified polymorphic DNA-PCR，RAPD-PCR)識別耐甲硝唑菌株。Jenks等[17]、Tankovic等[18]采用PCR技術特意性擴增H.pylorirdxA基因片段，然后對該片段進行核苷酸序列測定，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，從而鑒別H.pylori耐甲硝唑菌株。Morimoto等[27]采用重復序列PCR(rep-PCR)聯合高級微生物基因分型系統(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(high resolution melting analysis，HRM)利用溶解溫度和變性程度之間關系進行基因片段分析，完全基于核酸物理性質進行分析，無需序列特異性探針，不受突變堿基類型和位點局限，在PCR結束后直接運行HRM既可以對未知突變進行篩查、掃描，又可以對已知突變進行分析，也可用于短片段重復序列的分析[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(quinolone resistance determining region，QRDR)突變導致[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^對抗生素的使用劑量與適應證制定標準不同，H.pylori對左氧氟沙星耐藥出現地域、年齡差異。Megraud等[33]研究調查中顯示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]發現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技術的分子診斷技術。大多檢測H.pylori耐左氧氟沙星都是采用PCR技術特意性擴增H.pylorigyrA基因的QRDR片段，然后對該片段進行核苷酸序列測定[29-31]。德國學者Glocker等[36]利用Real-time PCR結合熒光共振能量轉換技術(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]構建等位基因特異性PCR(Allele-specific PCR，AS-PCR)，在3～5 h內檢測出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用，耐藥菌株也不斷出現，H.pylori根除治療面臨著嚴峻的考驗。因此，設計一種快速、準確、高敏感性及操作簡便的檢測對抗生素耐藥菌株的方法，對指導臨床用藥有著深遠的意義。近年來，基因芯片技術逐漸趨于成熟，HRM因其快速、低成本、高敏感性、特異性好、重復性好及操作簡便，在國外興起一種用于突變掃描和基因分型的最新遺傳學分析方法，成為國外新興的遺傳學、方法學研究和應用熱點。二者均具有較高的臨床應用價值及推廣意義。

[1]Ribeiro ML, Vitiello L, Miranda MC, et al. Mutations in the 23S rRNA gene are associated with clarithromycin resistance in Helicobacter pylori isolates in Brazil [J]. Ann Clin Microbiol Antimicrob, 2003, 2: 11.

[2]Momynaliev KT, Selezneva OV, Kozlova AA, et al. A2144G is the main mutation in the 23S rRNA gene of Helicobacter pylori associated with clarithromycin resistance [J]. Genetika, 2005, 41(10): 1338-1344.

[3]Kim JM, Kim JS, Kim N, et al. Gene mutations of 23S rRNA associated with clarithromycin resistance in Helicobacter pylori strains isolated from Korean patients [J]. J Microbiol Biotechnol, 2008, 18(9): 1584-1589.

[4]Moder KA, Layer F, Konig W, et al. Rapid screening of clarithromycin resistance in Helicobacter pylori by pyrosequencing [J]. J Med Microbiol, 2007, 56(Pt 10): 1370-1376.

[5]Agudo S, Perez-Perez G, Alarcon T, et al. High prevalence of clarithromycin-resistant Helicobacter pylori strains and risk factors associated with resistance in Madrid, Spain [J]. J Clin Microbiol, 2010, 48(10): 3703-3707.

[6]Abadi AT, Taghvaei T, Mobarez AM, et al. Frequency of antibiotic resistance in Helicobacter pylori strains isolated from the northern population of Iran [J]. J Microbiol, 2011, 49(6): 987-993.

[7]Eisig JN, Silva FM, Barbuti RC, et al. Helicobacter pylori antibiotic resistance in Brazil: clarithromycin is still a good option [J]. Arq Gastroenterol, 2011, 48(4): 261-264.

[8]De Francesco V, Giorgio F, Ierardi E, et al. Primary clarithromycin resistance in Helicobacter pylori: the Multicentric Italian Clarithromycin Resistance Observational (MICRO) study [J]. J Gastrointestin Liver Dis, 2011, 20(3): 235-239.

[9]Boyanova L, Ilieva J, Gergova G, et al. Numerous risk factors for Helicobacter pylori antibiotic resistance revealed by extended anamnesis: a Bulgarian study [J]. J Med Microbiol, 2012, 61(Pt 1): 85-93.

[10]O’Connor A, Taneike I, Nami A, et al. Helicobacter pylori resistance to metronidazole and clarithromycin in Ireland [J]. Eur J Gastroenterol Hepatol, 2010, 22(9): 1123-1127.

[11]Noguchi N, Rimbara E, Kato A, et al. Detection of mixed clarithromycin-resistant and -susceptible Helicobacter pylori using nested PCR and direct sequencing of DNA extracted from faeces [J]. J Med Microbiol, 2007, 56(Pt 9): 1174-1180.

[12]Schabereiter-Gurtner C, Hirschl AM, Dragosics B, et al. Novel real-time PCR assay for detection of Helicobacter pylori infection and simultaneous clarithromycin susceptibility testing of stool and biopsy specimens [J]. J Clin Microbiol, 2004, 42(10): 4512-4518.

[13]Regnath T, Enninger A, Schalasta G. Real-time PCR assay for rapid detection of clarithromycin-resistant helicobacter pylori in gastric biopsy specimens [J]. Z Gastroenterol, 2004, 42(12): 1371-1375.

[14]van Doorn LJ, Glupczynski Y, Kusters JG, et al. Accurate prediction of macrolide resistance in Helicobacter pylori by a PCR line probe assay for detection of mutations in the 23S rRNA gene: multicenter validation study [J]. Antimicrob Agents Chemother, 2001, 45(5): 1500-1504.

[15]Xuan SH, Zhou YG, Shao B, et al. Enzymic colorimetry-based DNA chip: a rapid and accurate assay for detecting mutations for clarithromycin resistance in the 23S rRNA gene of Helicobacter pylori [J]. J Med Microbiol, 2009, 58(Pt 11): 1443-1448.

[16]Goodwin A, Kersulyte D, Sisson G, et al. Metronidazole resistance in Helicobacter pylori is due to null mutations in a gene (rdxA) that encodes an oxygen-insensitive NADPH nitroreductase [J]. Mol Microbiol, 1998, 28(2): 383-393.

[17]Jenks PJ, Ferrero RL, Labigne A. The role of the rdxA gene in the evolution of metronidazole resistance in Helicobacter pylori [J]. J Antimicrob Chemother, 1999, 43(6): 753-758.

[18]Tankovic J, Lamarque D, Delchier JC, et al. Frequent association between alteration of the rdxA gene and metronidazole resistance in French and North African isolates of Helicobacter pylori [J]. Antimicrob Agents Chemother, 2000, 44(3): 608-613.

[19]Mirzaei N, Poursina F, Moghim S, et al. The mutation of the rdxA gene in metronidazole-resistant Helicobacter pylori clinical isolates [J]. Adv Biomed Res, 2014, 3: 90.

[20]Kargar M, Baghernejad M, Doosti A. Role of NADPH-insensitive nitroreductase gene to metronidazole resistance of Helicobacter pylori strains [J]. Daru, 2010, 18(2): 137-140.

[21]Kaakoush NO, Asencio C, Megraud F, et al. A redox basis for metronidazole resistance in Helicobacter pylori [J]. Antimicrob Agents Chemother, 2009, 53(5): 1884-1891.

[22]Tsugawa H, Suzuki H, Satoh K, et al. Two amino acids mutation of ferric uptake regulator determines Helicobacter pylori resistance to metronidazole [J]. Antioxid Redox Signal, 2011, 14(1): 15-23.

[23]Choi SS, Chivers PT, Berg DE. Point mutations in Helicobacter pylori’s fur regulatory gene that alter resistance to metronidazole, a prodrug activated by chemical reduction [J]. PLoS One, 2011, 6(3): e18236.

[24]Mehrabadi JF, Sirous M, Daryani NE, et al. Assessing the role of the RND efflux pump in metronidazole resistance of Helicobacter pylori by RT-PCR assay [J]. J Infect Dev Ctries, 2011, 5(2): 88-93.

[25]Khan A, Farooqui A, Manzoor H, et al. Antibiotic resistance and cagA gene correlation: a looming crisis of Helicobacter pylori [J]. World J Gastroenterol, 2012, 18(18): 2245-2252.

[26]Singh V, Mishra S, Maurya P, et al. Drug resistance pattern and clonality inH.pyloristrains [J]. J Infect Dev Ctries, 2009, 3(2): 130-136.

[27]Morimoto N, Takeuchi H, Nishida Y, et al. Clinical application of the diversiLab microbial typing system using repetitive sequence-based PCR for characterization of Helicobacter pylori in Japan [J]. J Clin Lab Anal, 2015, 29(3): 250-253.

[28]Tucker EJ, Huynh BL. Genotyping by high-resolution melting analysis [J]. Methods Mol Biol, 2014, 1145: 59-66.

[29]Garcia M, Raymond J, Garnier M, et al. Distribution of spontaneous gyrA mutations in 97 fluoroquinolone-resistant Helicobacter pylori isolates collected in France [J]. Antimicrob Agents Chemother, 2012, 56(1): 550-551.

[30]Rimbara E, Noguchi N, Kawai T, et al. Fluoroquinolone resistance in Helicobacter pylori: role of mutations at position 87 and 91 of GyrA on the level of resistance and identification of a resistance conferring mutation in GyrB [J]. Helicobacter, 2012, 17(1): 36-42.

[31]Miyachi H, Miki I, Aoyama N, et al. Primary levofloxacin resistance and gyrA/B mutations among Helicobacter pylori in Japan [J]. Helicobacter, 2006, 11(4): 243-249.

[32]Seck A, Burucoa C, Dia D, et al. Primary antibiotic resistance and associated mechanisms in Helicobacter pylori isolates from Senegalese patients [J]. Ann Clin Microbiol Antimicrob, 2013, 12: 3.

[33]Megraud F, Coenen S, Versporten A, et al. Helicobacter pylori resistance to antibiotics in Europe and its relationship to antibiotic consumption [J]. Gut, 2013, 62(1): 34-42.

[34]Goh KL, Navaratnam P. High Helicobacter pylori resistance to metronidazole but zero or low resistance to clarithromycin, levofloxacin, and other antibiotics in Malaysia [J]. Helicobacter, 2011, 16(3): 241-245.

[35]O’Connor A, Taneike I, Nami A, et al. Helicobacter pylori resistance rates for levofloxacin, tetracycline and rifabutin among Irish isolates at a reference centre [J]. Ir J Med Sci, 2013, 182(4): 693-695.

[36]Glocker E, Kist M. Rapid detection of point mutations in the gyrA gene of Helicobacter pylori conferring resistance to ciprofloxacin by a fluorescence resonance energy transfer-based real-time PCR approach [J]. J Clin Microbiol, 2004, 42(5): 2241-2246.

[37]Nishizawa T, Suzuki H, Umezawa A, et al. Rapid detection of point mutations conferring resistance to fluoroquinolone in gyrA of Helicobacter pylori by allele-specific PCR [J]. J Clin Microbiol, 2007, 45(2): 303-305.

[38]Rajper S, Khan E, Ahmad Z, et al. Macrolide and fluoroquinolone resistance in Helicobacter pylori isolates: an experience at a tertiary care centre in Pakistan [J]. J Pak Med Assoc, 2012, 62(11): 1140-1144.

[39]Lee JW, Kim N, Nam RH, et al. GenoType HelicoDR test in the determination of antimicrobial resistance of Helicobacter pylori in Korea [J]. Scand J Gastroenterol, 2014, 49(9): 1058-1067.

(責任編輯：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皮生長因子表達的影響》左下角基金項目：全軍“十二五”課題面上項目(CWS11J005),標注有誤，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

云南省聯合專項基金(2012FB027)

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

馬嵐青，博士，研究生導師，副主任醫師，研究方向：消化系統疾病。E-mail：malanqing@aliyun

R37

A

1006-5709(2016)01-0101-04

2014-12-25