高瓊，黃海輝

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艱難梭菌耐藥性及耐藥機(jī)制研究進(jìn)展

高瓊，黃海輝

復(fù)旦大學(xué)附屬華山醫(yī)院抗生素研究所，衛(wèi)生部抗生素臨床藥理重點(diǎn)實(shí)驗(yàn)室，上海 200040

艱難梭菌()是醫(yī)療保健相關(guān)性腹瀉最主要的病原菌。2002年起歐美地區(qū)艱難梭菌感染發(fā)病率和病死率均明顯增高，耐藥艱難梭菌的出現(xiàn)和傳播更給臨床治療和預(yù)防帶來了挑戰(zhàn)。絕大多數(shù)臨床分離菌對(duì)甲硝唑及萬(wàn)古霉素仍呈高度敏感，但已有異質(zhì)性耐藥或最低抑菌濃度上升的報(bào)道；對(duì)紅霉素和莫西沙星等其他抗菌藥物的耐藥率在不同國(guó)家和地區(qū)則有較大差異。艱難梭菌對(duì)甲硝唑或萬(wàn)古霉素敏感性下降產(chǎn)生的耐藥機(jī)制尚不明確，而對(duì)紅霉素、氟喹諾酮類、四環(huán)素和利福霉素形成的耐藥機(jī)制主要是因?yàn)樽饔冒悬c(diǎn)發(fā)生了改變。文章簡(jiǎn)述了近年來國(guó)際上艱難梭菌耐藥性及耐藥機(jī)制方面的研究進(jìn)展。

艱難梭菌；耐藥性；耐藥機(jī)制

艱難梭菌(Clostridium difficile)為革蘭陽(yáng)性厭氧芽孢桿菌，是醫(yī)院獲得性腹瀉最主要的病原菌，約25%~33%的抗生素相關(guān)性腹瀉以及90%的假膜性腸炎由艱難梭菌所致，統(tǒng)稱為艱難梭菌感染(Clostridium difficile infection, CDI)。2002年起，歐美地區(qū)多次發(fā)生艱難梭菌感染的暴發(fā)與流行，由此導(dǎo)致的發(fā)病率和病死率迅速增高。CDI流行病學(xué)的這一新變化部分與高產(chǎn)毒菌株如核糖體型027的出現(xiàn)相關(guān)[1]。2008年后，在荷蘭等歐洲國(guó)家又出現(xiàn)另一個(gè)檢出率較前明顯增加的高毒力菌株—核糖體型078[2]。同時(shí)CDI人群除住院患者及老年人外，蔓延至既往身體健康未曾住院且未用過抗生素的年輕人甚至孕婦中[3]。中國(guó)近年來對(duì)CDI亦越來越重視。黃海輝等[4]報(bào)道復(fù)旦大學(xué)附屬華山醫(yī)院住院病人中2008年CDI發(fā)病率為17.1例/10000入院病人，與瑞典等國(guó)相仿。陳云波等[5]報(bào)道CDI占醫(yī)院感染性腹瀉30.7%。國(guó)內(nèi)臨床分離株以核糖體型017為多見，直至2014年初廣州南方醫(yī)院才報(bào)道了第一例也是迄今為止唯一的一例027菌株感染[6]。

廣譜抗菌藥物的應(yīng)用導(dǎo)致腸道菌群失調(diào)是CDI最主要的危險(xiǎn)因素。艱難梭菌對(duì)紅霉素、克林霉素和氟喹諾酮類等多種抗菌藥物耐藥也使其更易在醫(yī)院內(nèi)外廣泛傳播[7]，例如歐洲和北美廣泛流行的027菌株即對(duì)莫西沙星呈高水平耐藥[8, 9]。數(shù)十年來甲硝唑和萬(wàn)古霉素一直是治療CDI的首選藥物[10]，但近期亦有報(bào)道艱難梭菌對(duì)這2種藥物的敏感性下降[11]。本文將對(duì)艱難梭菌對(duì)常用抗菌藥物的耐藥性及耐藥機(jī)制作一綜述。

1 艱難梭菌對(duì)常用抗菌藥物的耐藥性

1.1 不同國(guó)家和地區(qū)艱難梭菌對(duì)抗菌藥物的耐藥率

調(diào)查研究發(fā)現(xiàn)艱難梭菌對(duì)抗菌藥物的耐藥率在不同國(guó)家和地區(qū)的差異較大(表1)。絕大多數(shù)的艱難梭菌臨床分離株對(duì)甲硝唑和萬(wàn)古霉素仍呈敏感，但已有對(duì)甲硝唑敏感性下降、異質(zhì)性耐藥甚至高度耐藥的報(bào)道[13,21]，同時(shí)對(duì)萬(wàn)古霉素敏感性下降的報(bào)道亦非少見[8,16]。艱難梭菌對(duì)紅霉素、克林霉素等其他抗菌藥物的耐藥率則在不同國(guó)家地區(qū)間有較大差異。Tickler IA等[8]報(bào)道美國(guó)32家醫(yī)院508株艱難梭菌對(duì)克林霉素的耐藥率為36.8%，瑞典[12]報(bào)道艱難梭菌對(duì)克林霉素的耐藥率高達(dá)65%，但同為歐洲國(guó)家的波蘭的一項(xiàng)研究[9]顯示克林霉素耐藥率則僅為27.7%，亞洲中國(guó)臺(tái)灣[16]和韓國(guó)[17]報(bào)道其臨床分離株對(duì)克林霉素的耐藥率分別為73.5%與67.9%。艱難梭菌對(duì)莫西沙星耐藥率在不同的國(guó)家和地區(qū)差異更大：新西蘭[14]的一項(xiàng)研究結(jié)果顯示其對(duì)莫西沙星的耐藥率僅為2%，而波蘭[9]報(bào)道的耐藥率可高達(dá)83.1%。

1.2 流行菌株對(duì)常用抗菌藥物的耐藥率較非流行菌株為高。

Tickler IA等[8]報(bào)道美國(guó)508株艱難梭菌中核糖體型027最為多見，占28.1%(143/508)，其對(duì)克林霉素和莫西沙星的耐藥率分別為50.3%和92.3%，且39.1%的027菌株對(duì)萬(wàn)古霉素敏感性降低(MIC90為4mg/L)；非流行菌株核糖體型106和002對(duì)克林霉素和莫西沙星的耐藥率則較低，分別為3.2%和16.7%，以及9.7%和5.6%。Waslawski S等[22]和Lachowicz D等[9]的研究結(jié)果也都證實(shí)流行株027對(duì)克林霉素、莫西沙星、紅霉素等臨床常用藥物的耐藥率高于非027菌株。在亞洲核糖體型027菌株并不多見，但Kim J等[17]報(bào)道韓國(guó)流行株核糖體型018和017其對(duì)克林霉素和莫西沙星的耐藥率亦明顯高于非流行菌株。

表1 不同國(guó)家/地區(qū)艱難梭菌對(duì)常見臨床抗菌藥物的MICs和耐藥率

注：MIC(minimum inhibitory concentration)表示最小抑菌濃度；MIC90表示在一批實(shí)驗(yàn)中能抑制90％受試菌所需MIC；a試驗(yàn)藥物為美羅培南。

2 艱難梭菌對(duì)常用抗菌藥物的耐藥機(jī)制

2.1 甲硝唑

當(dāng)前絕大多數(shù)臨床分離株體外對(duì)甲硝唑仍呈高度敏感。但自1994年P(guān)eláez T等[23]首次檢測(cè)到耐甲硝唑的艱難梭菌后，已有越來越多對(duì)甲硝唑敏感性下降甚至耐藥的報(bào)道。2002年P(guān)eláez T等[24]發(fā)現(xiàn)在臨床分離的415株艱難梭菌中6.3%對(duì)甲硝唑耐藥，2008年[25]再次報(bào)道12%的臨床分離株對(duì)甲硝唑呈異質(zhì)性耐藥，且與臨床治療失敗相關(guān)。Baines SD等[26]在一項(xiàng)研究中發(fā)現(xiàn)24.4%的001型菌株對(duì)甲硝唑敏感性降低(MIC為4~8 mg/L)。黃海輝等[13]報(bào)道分離自上海腹瀉患者的188株艱難梭菌中18株對(duì)甲硝唑呈異質(zhì)性耐藥。當(dāng)前大多數(shù)國(guó)家的微生物體外藥敏試驗(yàn)判斷標(biāo)準(zhǔn)均參照美國(guó)臨床和實(shí)驗(yàn)室標(biāo)準(zhǔn)協(xié)會(huì)(CLSI)的標(biāo)準(zhǔn),直至2015年該標(biāo)準(zhǔn)中艱難梭菌對(duì)甲硝唑的耐藥折點(diǎn)仍為≥32 mg/L，但是該折點(diǎn)是基于血清治療濃度而非腸腔內(nèi)治療濃度[27]。歐洲臨床微生物與感染協(xié)會(huì)(ESCMID)按照艱難梭菌感染主要在腸腔，甲硝唑主要在腸腔內(nèi)作用的原則，自2012年起更改折點(diǎn)為>2 mg/L為耐藥[28]。因此如果按照歐洲抗菌藥物敏感性試驗(yàn)委員會(huì)(EUCAST)標(biāo)準(zhǔn)，耐甲硝唑艱難梭菌的檢出率將比現(xiàn)在的文獻(xiàn)報(bào)道明顯為高。

甲硝唑的殺菌機(jī)制尚未完全闡明，可能為被還原后的甲硝唑代謝物非特異性地與細(xì)菌DNA結(jié)合，抑制細(xì)菌DNA合成，并使DNA鏈斷裂從而導(dǎo)致細(xì)菌死亡。同時(shí)艱難梭菌等厭氧菌對(duì)甲硝唑敏感性下降/耐藥的機(jī)制也不明確。研究者推測(cè)可能與甲硝唑進(jìn)入菌體減少而泵出增加、甲硝唑的還原活化過程被阻斷、細(xì)菌DNA修復(fù)能力增強(qiáng)有關(guān)[29]。Lynch T等[30]對(duì)甲硝唑耐藥株進(jìn)行全基因組測(cè)序，發(fā)現(xiàn)在單核苷酸多態(tài)性(Single nucleotide polymorphism,SNP)，SNP水平有一些參與核心代謝通路的基因，如鐵利用、電子傳遞和能量產(chǎn)生的基因發(fā)生了變異；Chong PM等[31]隨后又采用2D-LC-MS/MS方法對(duì)該耐藥株進(jìn)行蛋白組學(xué)分析，結(jié)果顯示艱難梭菌對(duì)甲硝唑耐藥可能有多種機(jī)制參與，包括鐵代謝和DNA修復(fù)能力的改變等。但具體哪些基因真正參與了艱難梭菌對(duì)甲硝唑耐藥及其機(jī)制目前尚未見報(bào)道。

2.2 萬(wàn)古霉素

迄今為止，僅波蘭于1991 年曾報(bào)道用紙片擴(kuò)散法檢測(cè)到3株對(duì)萬(wàn)古霉素耐藥的艱難梭菌[32]。但自1996年起陸續(xù)有瓊脂稀釋法或E試驗(yàn)法檢測(cè)到萬(wàn)古霉素敏感性下降菌株的報(bào)道。2013年Dong D等[33]分離到2株萬(wàn)古霉素敏感性下降株(MIC為8 mg/L)，Tickler IA等[8]于2014年報(bào)道13.2%(40/302)的菌株對(duì)萬(wàn)古霉素敏感性降低(MIC范圍為2~4 mg/L)。已知萬(wàn)古霉素的主要作用機(jī)制為與細(xì)菌肽聚糖前體末端的D-丙氨酰-D-丙氨酸結(jié)合，抑制細(xì)胞壁肽聚糖的合成。腸球菌對(duì)萬(wàn)古霉素的主要耐藥機(jī)制即為產(chǎn)生一組功能相似的連接酶，導(dǎo)致合成D-丙氨酰-D-乳酸取代正常的細(xì)胞壁肽聚糖末端的D-丙氨酰-D-丙氨酸，使萬(wàn)古霉素不能與其靶點(diǎn)結(jié)合(VanA等)；或者合成D-丙氨酰-D-絲氨酸取代正常細(xì)胞壁的結(jié)構(gòu)(VanE等)。但是，當(dāng)前艱難梭菌對(duì)于萬(wàn)古霉素敏感性降低的機(jī)制仍不明確。Leeds等[34]誘導(dǎo)獲得2株萬(wàn)古霉素耐藥株(MIC為16 mg/L)，測(cè)序發(fā)現(xiàn)其中一株耐藥株中參與肽聚糖合成的基因發(fā)生突變(P108L)，另一株耐藥菌RNA聚合酶β’亞基發(fā)生突變(D244Y)，但這些突變與萬(wàn)古霉素耐藥的關(guān)系仍需進(jìn)一步研究。

2.3 非達(dá)霉素

非達(dá)霉素2011年在美國(guó)上市，其作用機(jī)制為抑制細(xì)菌轉(zhuǎn)錄過程中的RNA聚合酶。迄今僅從1例CDI復(fù)發(fā)患者中分離到1株非達(dá)霉素耐藥株(MIC為16 mg/L),該患者之前曾接受過非達(dá)霉素治療并治愈。PCR擴(kuò)增測(cè)序顯示該耐藥菌基因發(fā)生突變[35]。Seddon等[36]對(duì)實(shí)驗(yàn)室誘導(dǎo)的非達(dá)霉素敏感性下降菌株(MIC為1~4 mg/L)測(cè)序，結(jié)果亦顯示這些耐藥株的(K1073H，Q1074K, Q1074H, V1143G和/或V1143D)或者(I10R，R89G，Q781R和/或D1127E)出現(xiàn)突變。因與分別為RNA聚合酶β亞基和β’亞基的編碼基因，因此上述研究結(jié)果初步證實(shí)RNA聚合酶為艱難梭菌對(duì)非達(dá)霉素的耐藥靶點(diǎn)。除此之外，Leeds等[34]發(fā)現(xiàn)一株敏感性降低的實(shí)驗(yàn)菌株中，與轉(zhuǎn)錄調(diào)節(jié)因子MarR家族同源的一個(gè)基因發(fā)生了移碼突變，但該突變的意義還有待于進(jìn)一步研究。

2.4 紅霉素和克林霉素

紅霉素和克林霉素是蛋白合成抑制劑大環(huán)內(nèi)酯類-林可霉素類-鏈陽(yáng)霉素B類(Macrolide-lincosa-mide-streptogramin B, MLSB)家族的成員，對(duì)于這一類藥物目前各國(guó)報(bào)道的耐藥率均較高。該類藥物作用于細(xì)菌核糖體50S亞單位，通過抑制肽鏈延長(zhǎng)而影響細(xì)菌蛋白質(zhì)的合成?，F(xiàn)階段研究發(fā)現(xiàn)，臨床致病菌對(duì)紅霉素和克林霉素的耐藥主要通過核糖體靶點(diǎn)改變或主動(dòng)外排，同時(shí)滅活酶的產(chǎn)生也可引起細(xì)菌對(duì)其耐藥。由基因編碼的核糖體甲基化酶可對(duì)核糖體23S rRNA進(jìn)行修飾，導(dǎo)致細(xì)菌對(duì)這類藥物呈現(xiàn)高水平耐藥[37]?；蚋鶕?jù)其序列相似性分為不同的類別，其亞型目前已超20種，其中最常見的為基因[38]。編碼核糖體23S rRNA甲基化酶引起的耐藥表型為MLSB,即對(duì)大環(huán)內(nèi)酯類、林可酰胺類和鏈陽(yáng)菌素B交叉耐藥[39]。

艱難梭菌對(duì)紅霉素耐藥主要由位于Tn5398轉(zhuǎn)座子的基因所介導(dǎo)[40]，也有研究者報(bào)道基因位于其他位點(diǎn)[41]。ErmB決定區(qū)具有多態(tài)性，在歐洲ErmB決定區(qū)基因結(jié)構(gòu)以E4和E15最為常見，而Dong D等[33]報(bào)道中國(guó)臨床分離株中Erj2更為重要?；蜿幮缘钠D難梭菌亦可對(duì)紅霉素呈高水平耐藥，機(jī)制不明，其中部分菌株23S rDNA 656位堿基發(fā)生突變(C→T)[42]。

2.5 四環(huán)素

四環(huán)素類為快速抑菌劑，其與細(xì)菌核糖體30S亞單位A位特異性結(jié)合，阻止氨基酰-tRNA與核糖體聯(lián)結(jié)，從而抑制肽鏈延長(zhǎng)和蛋白質(zhì)合成。臨床致病菌對(duì)四環(huán)素的耐藥機(jī)制主要為核糖體靶點(diǎn)的保護(hù)()和主動(dòng)外排系統(tǒng)(等)。艱難梭菌主要通過產(chǎn)生核糖體保護(hù)蛋白(TetM)而對(duì)四環(huán)素耐藥。研究發(fā)現(xiàn)，核糖型012和046四環(huán)素耐藥株基因多由Tn5397轉(zhuǎn)座子攜帶，而在核糖型017和078中基因多位于Tn916樣轉(zhuǎn)座子上[43,44]。此外，Spigaglia P[45]等在一株陰性的耐藥株中檢測(cè)到另一個(gè)核糖體保護(hù)蛋白編碼基因。主動(dòng)外排介導(dǎo)的四環(huán)素耐藥在艱難梭菌中目前尚無(wú)相關(guān)報(bào)道。

2.6 氟喹諾酮類

除新西蘭、瑞典和中國(guó)臺(tái)灣以外，大多數(shù)國(guó)家和地區(qū)艱難梭菌對(duì)莫西沙星的耐藥率均已超過20%，部分耐藥率甚至超過80%。高產(chǎn)毒株027的廣泛流行即可能與其對(duì)莫西沙星耐藥相關(guān)。氟喹諾酮類作用于細(xì)菌DNA旋轉(zhuǎn)酶和(或)拓?fù)洚悩?gòu)酶Ⅳ，導(dǎo)致酶-DNA復(fù)合物的斷裂，抑制細(xì)菌DNA合成而殺菌。細(xì)菌對(duì)氟喹諾酮類藥物的耐藥機(jī)制主要分為3類：(1)藥物靶點(diǎn)改變；(2)靶點(diǎn)保護(hù)蛋白的產(chǎn)生；(3)主動(dòng)外排作用[46]。前期研究發(fā)現(xiàn)艱難梭菌對(duì)喹諾酮類耐藥機(jī)制主要為第一種，即DNA旋轉(zhuǎn)酶亞單位GyrA和/或GyrB發(fā)生改變。突變位點(diǎn)多發(fā)生于喹諾酮耐藥決定區(qū)(Quinolone-resistance determining region,QRDR)[47]，其中GyrA變異以T82I最為常見，此外還檢測(cè)到T82V、D71V、D81N、A83V、A118T以及A118V，GyrB最常見的變異位點(diǎn)為426，包括D426N、D426V，以及R447K、R447L、E466K、S416A、S366A、D501E、A503S[33,48,49]。

2.7 利福霉素類

臨床用于治療CDI的利福霉素類藥物主要為利福平和利福昔明。Tenover FC等報(bào)道北美316 株艱難梭菌對(duì)利福平耐藥率為7.9%[15]。黃海輝等報(bào)道分離的188株艱難梭菌對(duì)利福平的耐藥率為19.8%，且對(duì)利福平高度耐藥者(MIC≥32 mg/L)同時(shí)也對(duì)利福昔明呈耐藥[13]。利福平作用機(jī)制為特異地與依賴于DNA的RNA聚合酶β亞單位(RpoB)結(jié)合，形成穩(wěn)定的復(fù)合物，從而抑制多聚酶活性，抑制DNA 轉(zhuǎn)錄。艱難梭菌等革蘭陽(yáng)性菌對(duì)利福平耐藥亦主要與RNA多聚酶的改變有關(guān)。到目前為止，在艱難梭菌中已報(bào)道八種RpoB變異，所有這些變異都發(fā)生在第488位和第548位氨基酸之間。R505K最為常見，通常導(dǎo)致高水平耐藥(MIC≥32 mg/L)；H502N引起利福平MIC輕度上升(MIC為<32 mg/L)。雙重變異亦有報(bào)道，如R505K合并H502N、R505K合并I548M等[50,51]。

3 結(jié) 語(yǔ)

不同國(guó)家和地區(qū)艱難梭菌對(duì)常用抗菌藥物的耐藥率有很大差異，但絕大多數(shù)菌株對(duì)甲硝唑、萬(wàn)古霉素以及非達(dá)霉素仍呈敏感。通常流行株對(duì)紅霉素、克林霉素和莫西沙星的耐藥率較非流行菌株更高。艱難梭菌對(duì)甲硝唑和萬(wàn)古霉素的耐藥機(jī)制仍不明確，對(duì)其他常用抗菌藥物耐藥主要為作用靶點(diǎn)改變所致。預(yù)期對(duì)艱難梭菌對(duì)甲硝唑等藥物耐藥機(jī)制的進(jìn)一步研究將會(huì)為抗菌藥物的研發(fā)提供新靶點(diǎn)和新思路。同時(shí)在臨床上加強(qiáng)對(duì)艱難梭菌耐藥性的監(jiān)測(cè)，避免盲目使用廣譜抗菌藥物對(duì)于降低耐藥艱難梭菌感染的發(fā)生和傳播也極為重要。

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(責(zé)任編委: 謝建平)

Update on antimicrobial resistance in

Qiong Gao, Haihui Huang

is the leading cause of healthcare-associated diarrhea. Since 2002, the morbidity and mortality rates ofinfection have increased dramatically in Europe and North America. The emergence ofstrains that are resistant to multiple antimicrobial agents can complicate prevention programs and potential treatment. Although most clinical isolates are still susceptible to metronidazole and vancomycin, heteroresistance to metronidazole and increasing vancomycin MICs (minimum inhibitory concentrations) have been reported. The prevalence of resistance to other antimicrobial agents, including erythromycin and moxifloxacin, is highly variable in different countries and regions. The exact mechanism of reduced susceptibility to metronidazole or vancomycin is still not clear. The principal mechanism of erythromycin, fluoroquinolones and rifamycins resistance inis determined by target alterations. This review will focus primarily on the antimicrobial susceptibility patterns and resistance mechanisms ofin order to provide an up-to-date review on the topic.

clostridium difficile; antimicrobial resistance; resistance mechanism

2015-03-26;

2015-04-13

國(guó)家自然科學(xué)基金項(xiàng)目(編號(hào)：30973594, 81101292)

高瓊, 碩士研究生, 研究方向：艱難梭菌耐藥機(jī)制。Tel: 021-52888193; E-mail: 14211220003@fudan.edu.cn

黃海輝, 主任醫(yī)師, 博士生導(dǎo)師, 研究方向：感染性疾病的診治，厭氧菌耐藥性及耐藥機(jī)制。Tel: 021-52888195; E-mail: huanghaihui@fudan.edu.cn

10.16288/j.yczz.15-131

2015-4-29 10:55:15

http://www.cnki.net/kcms/detail/11.1913.R.20150429.1055.001.html