艱難梭菌抗菌藥物耐藥機(jī)制研究進(jìn)展

胥騰，黃海輝

綜 述

艱難梭菌抗菌藥物耐藥機(jī)制研究進(jìn)展

胥騰，黃海輝

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

艱難梭菌(，CD)是醫(yī)療機(jī)構(gòu)感染性腹瀉最常見的病原之一，并被美國(guó)疾病控制和預(yù)防中心列為需要緊急和積極應(yīng)對(duì)的耐藥威脅。許多耐藥基因可在醫(yī)療機(jī)構(gòu)、社區(qū)和自然環(huán)境中不同菌種間轉(zhuǎn)移，隨著新耐藥機(jī)制的產(chǎn)生與獲得，CD的抗微生物藥物耐藥性(antimicrobial resistance，AMR)也在不斷演變。CD的耐藥機(jī)制多種多樣，包括化學(xué)修飾造成失效、藥物靶點(diǎn)的修飾以及藥物的主動(dòng)外排等。既往CD對(duì)大環(huán)內(nèi)脂類和喹諾酮類藥物耐藥性及耐藥機(jī)制研究較為充分，但對(duì)甲硝唑、萬古霉素等艱難梭菌感染(infection，CDI)治療藥物的耐藥機(jī)制研究尚處于起步階段。近年來研究發(fā)現(xiàn)，一些既往CD研究中未考慮的機(jī)制如質(zhì)粒介導(dǎo)的耐藥，同樣可能在艱難梭菌AMR中發(fā)揮重要作用。本文主要綜述了CD對(duì)甲硝唑、萬古霉素和非達(dá)霉素等治療用抗菌藥物的耐藥機(jī)制研究進(jìn)展，以期為CDI的防治與新抗菌藥物和新耐藥菌檢測(cè)試劑盒的研發(fā)提供參考。

艱難梭菌；耐藥機(jī)制；甲硝唑；萬古霉素

抗微生物藥物耐藥(antimicirobial resistance，AMR)是全球公共衛(wèi)生面臨的重要威脅[1]。艱難梭菌(，CD)是一種重要的多重耐藥菌，常對(duì)大環(huán)內(nèi)酯類、林可酰胺類、四環(huán)素類、頭孢菌素類和氟喹諾酮類呈多重耐藥[2]。該特征使得CD能夠在應(yīng)用廣譜抗菌藥物導(dǎo)致腸道菌群失調(diào)時(shí)進(jìn)行優(yōu)勢(shì)生長(zhǎng)、產(chǎn)生毒素從而造成艱難梭菌感染(infection，CDI)，同時(shí)其產(chǎn)芽孢的特性可造成CD在醫(yī)療機(jī)構(gòu)內(nèi)的廣泛傳播[3]。從2013年開始，CD就被被美國(guó)疾病控制和預(yù)防中心(Centers for Disease Control and Prevention，CDC)列為最緊迫的公共衛(wèi)生威脅，每年給美國(guó)醫(yī)療保健系統(tǒng)造成 10 億美元的損失[4,5]。至2019年美國(guó)CDC仍將其納入五大抗生素耐藥性緊急威脅之一。

當(dāng)前臨床上推薦治療CDI的抗菌藥物有甲硝唑(metronidazole，MTZ)、萬古霉素(vancomycin)和非達(dá)霉素(fidaxomicin)[6～8]。除此之外，有研究表明替加環(huán)素(tigecycline)可有效治療重度CDI[9]，利福昔明(rifaximin)可能有助于預(yù)防CDI復(fù)發(fā)[10]。然而，近年來發(fā)現(xiàn)CD對(duì)這些藥物的敏感性較前下降。來自美國(guó)休斯頓的一項(xiàng)研究結(jié)果顯示CDI患者糞便分離株中26%對(duì)萬古霉素不敏感，29%對(duì)MTZ不敏感；而在肯尼亞內(nèi)羅畢進(jìn)行的另外一項(xiàng)研究顯示對(duì)二者不敏感的分離株比例可高達(dá)67%和85%[11]。與此同時(shí)，這兩種藥物治療失敗的報(bào)道也并不少見。在國(guó)內(nèi)，艱難梭菌不屬于CARSS和CHINET細(xì)菌耐藥監(jiān)測(cè)網(wǎng)監(jiān)測(cè)病原菌范疇，但某些小規(guī)模流行病學(xué)研究仍然提示耐藥菌株的存在。Meta分析顯示2007～2013年間我國(guó)臨床來源艱難梭菌對(duì)利福平的耐藥率為18.3%，對(duì)四環(huán)素的耐藥率為46.8%，對(duì)甲硝唑和萬古霉素則完全敏感[12]。浙江杭州兩所三甲醫(yī)院2012～2015年間腹瀉患者糞便l來源的411株艱難梭中有15.6%對(duì)甲硝唑耐藥， 36.3%對(duì)四環(huán)素耐藥，所有菌株均對(duì)萬古霉素敏感，且產(chǎn)毒株的多重耐藥率(96.4%)遠(yuǎn)高于非產(chǎn)毒菌株(31.4%)[13]。

艱難梭菌對(duì)抗菌藥物耐藥機(jī)制是多種多樣的(圖1)。細(xì)菌對(duì)抗菌藥物主要的幾類耐藥機(jī)制在艱難梭菌中均比較常見[14]。艱難梭菌可通過改變藥物結(jié)構(gòu)造成抗菌藥物失效：例如通過轉(zhuǎn)座子Tn4453a和Tn4453b上攜帶的編碼的氯霉素乙酰轉(zhuǎn)移酶，對(duì)氯霉素伯羥基進(jìn)行乙酰化修飾導(dǎo)致藥物失效無法與細(xì)菌50S核糖體亞基結(jié)合；此外某些CD菌株編碼 D類β-內(nèi)酰胺酶可破壞β-內(nèi)酰胺環(huán)使頭孢菌素類藥物失去活性。艱難梭菌還可對(duì)抗菌藥物靶點(diǎn)進(jìn)行修飾：例如操縱子的表達(dá)可造成CD肽聚糖前體末端d-Ser修飾，使該位點(diǎn)與萬古霉素的親和力降低。迄今為止，在CD臨床株RpoB利福平耐藥決定區(qū)(rifampin resistance determining region，RRDR)中也已發(fā)現(xiàn)十余種耐藥突變，可能阻礙利福霉素類藥物與靶點(diǎn)RpoB蛋白的結(jié)合。艱難梭菌編碼不同家族的轉(zhuǎn)運(yùn)蛋白對(duì)抗菌藥物進(jìn)行主動(dòng)外排：例如CD中編碼的MATE家族轉(zhuǎn)運(yùn)蛋白與氟喹諾酮耐藥有關(guān)，而ABC家族轉(zhuǎn)運(yùn)蛋白CprABC介導(dǎo)了CD對(duì)抗微生物肽的耐藥性。生物膜的形成同樣參與了艱難梭菌對(duì)藥物的耐受：亞抑菌濃度的萬古霉素和甲硝唑可增強(qiáng)CD生物膜的形成，同時(shí)CD生物膜又可耐受更高濃度的甲硝唑(10～100 mg/L)和萬古霉素(20 mg/L)。除此之外，一些既往未考慮的機(jī)制如可轉(zhuǎn)移質(zhì)粒介導(dǎo)的耐藥(推測(cè)編碼N-乙酰胞壁酰-L-丙氨酸酰胺酶的萬古霉素耐藥質(zhì)粒pX18-498，甲硝唑耐藥質(zhì)粒pCD-METRO等)，同樣可能在艱難梭菌AMR中發(fā)揮重要作用。本文主要對(duì)常用治療CDI藥物(甲硝唑、萬古霉素、非達(dá)霉素、利福昔明和四環(huán)素類等)的耐藥機(jī)制的研究進(jìn)展展開綜述，以期為臨床治療和新藥研發(fā)提供參考。

圖1 艱難梭菌對(duì)臨床常用CDI治療抗菌藥物的耐藥機(jī)制

艱難梭菌的甲硝唑耐藥機(jī)制包括質(zhì)粒pCD-METRO的水平轉(zhuǎn)移和內(nèi)源基因(如鐵轉(zhuǎn)運(yùn)蛋白編碼基因等)的突變。萬古霉素耐藥則可由質(zhì)粒pX18-498或vanG操縱子介導(dǎo)，當(dāng)vanG操縱子過表達(dá)時(shí)D-Ser可取代D-Ala對(duì)肽聚糖進(jìn)行末端修飾。和的突變以及同源基因的突變可導(dǎo)致艱難梭菌對(duì)非達(dá)霉素敏感性降低。來自抗菌藥物選擇壓力、群體感應(yīng)信號(hào)和基因共同調(diào)節(jié)艱難梭菌生物膜形成，對(duì)甲硝唑和萬古霉素的敏感性降低。外排泵同樣參與了抗菌藥物耐藥，例如630Δ菌株中ATP結(jié)合盒轉(zhuǎn)運(yùn)蛋白CD2068的缺失導(dǎo)致其對(duì)甲硝唑的IC50上升1.4倍。

1 甲硝唑

MTZ是硝基咪唑類抗生素，自20世紀(jì)90年代末以來一直是治療輕中度CDI的首選藥物。然而，自從流行毒株NAP1/027出現(xiàn)后[15]，許多國(guó)家陸續(xù)報(bào)道了對(duì)MTZ低水平耐藥和異質(zhì)性耐藥的菌株，近年來甚至分離到高水平耐藥株。對(duì)MTZ治療響應(yīng)降低的CDI病例報(bào)道也越來越多見。

MTZ是一種前體藥物，攝入菌體后，在胞內(nèi)經(jīng)厭氧菌特有的低氧化還原電位酶促反應(yīng)還原激活，雜環(huán)裂變后形成羥乙基肟酸和乙酰胺[16]。MTZ的殺菌機(jī)制尚不明確，可能為活化反應(yīng)伴隨生成的硝基自由基對(duì)厭氧菌產(chǎn)生細(xì)菌毒性，造成細(xì)菌死亡[16]。

CD對(duì)MTZ耐藥亦可能涉及多種機(jī)制，其一是藥物還原激活通路的改變。參與藥物氧化還原反應(yīng)的電子傳遞蛋白如丙酮酸黃多辛氧化還原酶PFOR等在該酶促反應(yīng)中發(fā)揮了重要作用，這類蛋白編碼基因的突變參與了耐藥形成。Lynch等[17]對(duì)一株臨床來源的異質(zhì)性耐藥CD分離株CD26A54通過體外誘導(dǎo)獲得MTZ穩(wěn)定耐藥株CD26A54-R (MIC=12～ 256 mg/L)。對(duì)該耐藥株進(jìn)行基因組和蛋白質(zhì)組學(xué)分析表明，編碼的甘油-3-磷酸脫氫酶(Ala229Thr)，以及編碼的PFOR(Gly423Glu)均存在突變[17]。這些突變很可能使電子傳遞中斷，從而改變菌體能量代謝和胞內(nèi)氧化還原電位，進(jìn)而影響MTZ主動(dòng)轉(zhuǎn)運(yùn)和胞內(nèi)還原激活的效率。這些電子傳遞中斷或缺陷的細(xì)菌往往表現(xiàn)為小菌落變異(small colony variant，SCV)，具有菌落較小、生長(zhǎng)緩慢、呼吸減少、菌體分離減弱和對(duì)抗生素耐藥等特點(diǎn)[18,19]。作者通過掃描電子顯微鏡觀察CD26A54-R菌落同樣存在上述特征，符合電子傳遞功能缺陷的表現(xiàn)。另一項(xiàng)體外研究進(jìn)一步證實(shí)了電子傳遞蛋白PFOR對(duì)CD的MTZ耐藥有重要作用，Deshpande等[20]收集了491858，490054和上述CD26A54-R三株存在PFOR突變的MTZ耐藥的CD菌株。其中491858和490054菌株的MTZ MIC均為8～16 mg/L，且PFOR存在Ala1018Val突變。數(shù)據(jù)庫(kù)比對(duì)顯示該氨基酸突變位點(diǎn)緊鄰蛋白的4Fe-4S輔因子接合部。作者對(duì)三株菌分別回補(bǔ)了野生型PFOR編碼基因及其啟動(dòng)子，發(fā)現(xiàn)回補(bǔ)株的MTZ MIC為4～8 mg/L，顯著低于回補(bǔ)前親本株(<0.05)[20]。然而，由于對(duì)CD胞內(nèi)MTZ激活代謝產(chǎn)物(如還原性亞硝酸鹽、NO、乙酰胺)濃度的定量檢測(cè)均未能成功，因此PFOR突變是否通過阻止MTZ胞內(nèi)還原激活而導(dǎo)致耐藥產(chǎn)生尚未確定。

鐵離子作為組成PFOR等電子傳遞蛋白功能性輔基(如含鐵硫簇)的重要元素，在MTZ還原激活途徑中發(fā)揮重要作用，因此鐵代謝/穩(wěn)態(tài)的變化也被認(rèn)為與CD對(duì)MTZ耐藥有關(guān)。Moura等[21]對(duì)一株臨床分離RT10型不產(chǎn)毒MTZ耐藥株(MIC=32 mg/L)進(jìn)行了蛋白組學(xué)分析，發(fā)現(xiàn)在MTZ暴露期間耐藥株中鐵蛋白缺失。Lynch等[17]對(duì)上述CD26A54-R菌株通過蛋白質(zhì)組學(xué)分析發(fā)現(xiàn)，MTZ暴露后亞鐵離子轉(zhuǎn)運(yùn)蛋白FeoB1表達(dá)水平下降2.2倍，鐵化合物ABC轉(zhuǎn)運(yùn)蛋白底物接合蛋白(CDR20291_1548)表達(dá)水平下降1.7倍，提示在耐藥菌株中可能存在鐵攝取減少。同時(shí)這些菌株表現(xiàn)出適應(yīng)性缺陷，需要在培養(yǎng)基中加入鐵以幫助生長(zhǎng)。Deshpande等[20]通過敲除DNA錯(cuò)配修復(fù)基因構(gòu)建了一株可高度突變的CD菌株，以研究其體外MTZ誘導(dǎo)耐藥的基因演變，該研究中菌株對(duì)MTZ的體外耐藥性是逐步發(fā)展的，MIC從2 mg/L增加到16 mg/L。作者發(fā)現(xiàn)截?cái)嗟膩嗚F轉(zhuǎn)運(yùn)蛋白FeoB1是產(chǎn)生低水平抗性的第一步，隨后發(fā)生的是突變，繼而出現(xiàn)(黃嘌呤脫氫酶編碼基因)突變和(一種鐵硫簇調(diào)節(jié)因子)突變，導(dǎo)致更高水平的MTZ抗性(MIC=64 mg/L)。在CD中，F(xiàn)eoB1是主要的鐵轉(zhuǎn)運(yùn)蛋白，作者證實(shí)在ATCC 700057中的突變降低了菌體內(nèi)鐵含量，使細(xì)菌轉(zhuǎn)而利用黃素氧還蛋白(flavodoxin)進(jìn)行能量代謝，黃素氧還蛋白電子傳遞效率遠(yuǎn)低于鐵氧環(huán)蛋白(ferredoxin)因此可能造成MTZ還原激活速率降低。和基因的突變雖然提高了MTZ的MIC，但在沒有發(fā)生突變的情況下不能獨(dú)自介導(dǎo)菌株產(chǎn)生耐藥性，相反僅存在缺失的菌株即可表現(xiàn)出低水平的MTZ耐藥[20]。然而在體內(nèi)該機(jī)制是否同樣有意義仍有爭(zhēng)議，因?yàn)镕eoB1對(duì)CD定植和毒力的產(chǎn)生至關(guān)重要，CDI小鼠中CD的上調(diào)200倍，因而能定植并感染機(jī)體的CD不太可能自發(fā)篩選富集出FeoB1突變[22]。事實(shí)上，在目前已有的臨床分離MTZ耐藥株中，也沒有發(fā)現(xiàn)FeoB1突變的菌株。

Olaitan等[23]發(fā)現(xiàn)大多數(shù)甲硝唑耐藥艱難梭菌的耐藥表型較為獨(dú)特，耐藥菌僅在含血紅素瓊脂板上才表現(xiàn)出甲硝唑耐藥，而在普通瓊脂板上耐藥表型消失。研究發(fā)現(xiàn)，這些艱難梭菌菌株在基因的啟動(dòng)子中存在T-to-G突變(作者稱之為PnimBG)，從而導(dǎo)致的組成型轉(zhuǎn)錄。沉默或敲除后，這些MTZ耐藥株的耐藥表型消失。NimB是一種血紅素依賴的黃素酶，可降解硝基咪唑類抗菌藥物使其喪失抗菌活性。此外，作者發(fā)現(xiàn)PnimBG突變的發(fā)生似乎與DNA聚合酶的Thr82Ile突變有關(guān)，而后者已被證實(shí)可介導(dǎo)艱難梭菌對(duì)氟喹諾酮類抗菌藥耐藥。該研究結(jié)果表明，對(duì)氟喹諾酮耐藥的艱難梭菌菌株中可能也有部分菌株同時(shí)對(duì)甲硝唑的敏感性降低。

Boekhoud等[24]發(fā)現(xiàn)質(zhì)粒也可介導(dǎo)MTZ耐藥，他們從MTZ治療失敗的CDI患者糞便標(biāo)本CD臨床分離株中(RT 020)發(fā)現(xiàn)了一種約7 kb大小的高拷貝質(zhì)粒(pCD METRO)，攜帶該質(zhì)粒使CD對(duì)MTZ產(chǎn)生耐藥性，將pCD METRO質(zhì)粒轉(zhuǎn)化敏感株后MIC增加可超過24倍。流調(diào)顯示該質(zhì)粒僅存在于MTZ耐藥的菌株的中，該研究在585株CD中發(fā)現(xiàn)3.8%的菌株攜帶pCD METRO，攜帶質(zhì)粒的菌株來自不同歐洲國(guó)家，并屬于不同核糖體分型(RT027、010和020)。然而，該質(zhì)粒介導(dǎo)MTZ抗性的確切機(jī)制尚不明確。該質(zhì)粒含有一個(gè)與脆弱擬桿菌基因同源的小假基因(small pseudogene)，但該基因缺乏編碼催化結(jié)構(gòu)域，在實(shí)驗(yàn)室菌株中誘導(dǎo)該基因高表達(dá)也不會(huì)介導(dǎo)耐藥。攜帶該質(zhì)粒不會(huì)造成菌株的生長(zhǎng)速率降低，且耐藥菌株在非選擇性培養(yǎng)基上重復(fù)傳代也不會(huì)丟失該質(zhì)粒。該質(zhì)粒的鳥嘌呤胞嘧啶含量(GC%)為41.6%，與染色質(zhì)的標(biāo)準(zhǔn)值(約28%～30%)不匹配，表明該質(zhì)粒是艱難梭菌通過水平轉(zhuǎn)移從另一種未知生物體處獲得[24]。

除上述已被驗(yàn)證的耐藥機(jī)制之外，在暴露于MTZ的耐藥菌株中還檢測(cè)到DNA修復(fù)蛋白R(shí)ecA的差異表達(dá)[21,25]。在其他菌種中，具有DNA修復(fù)缺陷的突變株對(duì)MTZ更敏感[17]。在耐藥菌株中也發(fā)現(xiàn)了氧化應(yīng)激相關(guān)蛋白的差異表達(dá)[23]，硫胺素合成酶()和甘油-3-氧化還原酶()基因的突變也與CD的耐藥性相關(guān)。另一株RT010耐藥臨床分離株中存在372位突變?cè)斐删幋a蛋白的碳端結(jié)構(gòu)缺失[26]。但是這些基因突變或表達(dá)水平變化在MTZ耐藥中發(fā)揮的作用尚未經(jīng)實(shí)驗(yàn)室研究確認(rèn)。

艱難梭菌對(duì)甲硝唑耐藥可能與甲硝唑的臨床療效下降有關(guān)。近期研究表明，艱難梭菌對(duì)MTZ MIC≥1 μg/mL是基于MTZ的初始治療方案臨床治療失敗的獨(dú)立預(yù)測(cè)因素[27]。來自美國(guó)與歐洲的兩個(gè)多中心隊(duì)列RCT研顯示甲硝唑標(biāo)準(zhǔn)方案下CDI初始治療失敗率接近30%，且后續(xù)出現(xiàn)CDI復(fù)發(fā)的概率總體達(dá)到23%，兩項(xiàng)指標(biāo)均劣于萬古霉素。因此近5年歐洲和美國(guó)指南中MTZ地位有所下降，僅推薦用于治療不能耐受或無法獲得萬古霉素/非達(dá)霉素的輕中度CDI初次發(fā)作患者。

2 萬古霉素

最常見的萬古霉素耐藥機(jī)制是基因介導(dǎo)的藥物靶點(diǎn)修飾，造成萬古霉素與細(xì)胞壁親和力降低[28]。系列基因主要介導(dǎo)d-Lac或d-Ser兩種末端修飾。其中，d-Lac修飾(d-Ala-d-Lac)由和基因簇編碼，可造成高水平萬古霉素抗性；d-Ser修飾(d-Ala-d-Ser)由、和基因簇編碼，可造成低水平抗性。目前已在CD中鑒定出多個(gè)同源基因，包括和，并且與萬古霉素MIC的升高相關(guān)[29～31]。這些基因的表達(dá)由雙組分調(diào)節(jié)系統(tǒng)控制，該系統(tǒng)包含組氨酸激酶VanS和反應(yīng)調(diào)節(jié)子VanR[32]。VanS通常識(shí)別萬古霉素的存在，導(dǎo)致自身磷酸化并將其磷酸基團(tuán)轉(zhuǎn)移到VanR，隨后，磷酸化的VanR結(jié)合到啟動(dòng)子區(qū)域以誘導(dǎo)的轉(zhuǎn)錄。有研究顯示約85%的CD攜帶基因[31]，但與萬古霉素耐藥的關(guān)系仍不明確。Ramírez-Vargas等[30]分析了哥斯達(dá)黎加當(dāng)?shù)蒯t(yī)院流行的(NAPCR1型) 38個(gè)分離株，發(fā)現(xiàn)所有分離株都具有樣序列，但其中只有四個(gè)分離株萬古霉素耐藥(MIC= 4 mg/L)。有學(xué)者對(duì)此做出解釋，認(rèn)為存在其他調(diào)控機(jī)制使得在敏感株中保持沈默[30,33,34]。有研究在萬古霉素耐藥CD臨床菌株(MIC=4～8 mg/L)以及實(shí)驗(yàn)室誘導(dǎo)突變株(MIC=8～ 16 mg/L)中發(fā)現(xiàn)，原先沉默的vanG發(fā)生組成性表達(dá)，原因是這些菌株攜帶的調(diào)控vanG的雙組分系統(tǒng)發(fā)生了兩處突變[31]。該研究對(duì)參考菌株R20291進(jìn)行連續(xù)傳代構(gòu)建萬古霉素耐藥株(MIC=8～16 mg/L)，這些耐藥株的都發(fā)生了突變。然后作者又分析了11株萬古霉素MIC升高(4～ 8 mg/L)的臨床株，發(fā)現(xiàn)它們存在相似的突變，導(dǎo)致組成型高表達(dá)[31]。其一是調(diào)節(jié)蛋白VanR中的Thr115Ala突變使得VanR持續(xù)處于DNA結(jié)合構(gòu)象，從而更易誘導(dǎo)vanG的轉(zhuǎn)錄。近期研究顯示該位點(diǎn)突變與2016年美國(guó)佛羅里達(dá)州艱難梭菌臨床分離株對(duì)萬古霉素的MIC增加有關(guān)[35]。其二是組氨酸激酶VanS保守區(qū)域的突變（Arg314Leu），該區(qū)域影響了VanS的磷酸酶活性，因此可能增加VanR的磷酸化水平。

除基因外，萬古霉素耐藥CD中也發(fā)現(xiàn)了、、和等同源基因的存在，但這些基因與耐藥關(guān)系仍不明確。Saldanha等[32]對(duì)巴西 7 株萬古霉素耐藥臨床分離CD進(jìn)行全基因組測(cè)序，發(fā)現(xiàn)有五株存在至少一個(gè)基因。然而，對(duì)萬古霉素敏感的兩個(gè)分離株也含有和基因，這表明單獨(dú)存在基因與萬古霉素耐藥性無關(guān)[32]。未來需要對(duì)基因的表達(dá)水平以及誘導(dǎo)其表達(dá)的上游調(diào)控機(jī)制進(jìn)行深入研究，以解釋這些基因與萬古霉素耐藥性的關(guān)聯(lián)。

多藥外排泵是存在于細(xì)菌細(xì)胞膜中的主動(dòng)轉(zhuǎn)運(yùn)蛋白，其中一個(gè)主要家族是ATP結(jié)合盒(ATP- binding cassette，ABC)轉(zhuǎn)運(yùn)蛋白，水解ATP供能，對(duì)簡(jiǎn)單離子或大分子的溶質(zhì)如抗菌藥物進(jìn)行轉(zhuǎn)運(yùn)外排[36]。已有研究在幾種梭狀芽孢桿菌中證實(shí)轉(zhuǎn)運(yùn)蛋白是造成多藥耐藥的主要原因[37,38]。在CD中，陽(yáng)離子抗菌肽(cathelicidin antimicrobial peptide，CAMP)可誘導(dǎo)ABC轉(zhuǎn)運(yùn)蛋白操縱子高表達(dá)，從而降低各種 CAMP的有效性[39]。Ngernsombat等[40]發(fā)現(xiàn)并驗(yàn)證了多藥外排泵ABC轉(zhuǎn)運(yùn)蛋白CD2068。作者參考 CD630菌株的基因組分析確定CD2068與和[37,38]中的其他兩個(gè)已知 ABC轉(zhuǎn)運(yùn)蛋白具有高度同源性。在暴露于萬古霉素 (0.25 mg/L)后基因表達(dá)水平顯著增加。在大腸埃希菌中過表達(dá)CD2068使萬古霉素對(duì)大腸埃希菌的半數(shù)最大抑制濃度(half maximal inhibitory concentration，IC50)升高2.6倍。然而，在敏感模式株CD630Δ中敲除、回補(bǔ)，未觀察到萬古霉素IC50的顯著差異。CD2068造成CD對(duì)多藥耐藥性能力減弱的原因尚不清楚，但作者提出了幾種假設(shè)，如CD敏感株中CD2068的表達(dá)水平較低；其他ABC轉(zhuǎn)運(yùn)蛋白的代償；和/或其他機(jī)制介導(dǎo)某些抗生素耐藥。CD630基因組中共有243處基因經(jīng)預(yù)測(cè)編碼ABC轉(zhuǎn)運(yùn)蛋白[41]，因此需要進(jìn)行更多的研究來確定CD2068或其他轉(zhuǎn)運(yùn)蛋白在CD對(duì)萬古霉素耐藥中的作用及機(jī)制。

細(xì)胞壁蛋白66()基因編碼艱難梭菌的細(xì)胞表面抗原，既往研究顯示Cwp66在細(xì)胞粘附中起重要作用。Zhou等[42]發(fā)現(xiàn)Cwp66編碼基因缺失的艱難梭菌菌株與野生株相比，對(duì)克林霉素、氨芐青霉素和紅霉素更加敏感，但對(duì)萬古霉素和甲硝唑的敏感性降低。但突變?cè)斐擅舾行越档偷木唧w機(jī)制尚不明確。

生物膜可作為物理屏障抑制宿主免疫反應(yīng)并阻止足夠濃度的抗菌藥物到達(dá)感染部位，與多種病原菌的耐藥性、耐受性及反復(fù)感染有關(guān)[43～45]。目前有兩項(xiàng)研究表明CD生物膜的形成與萬古霉素敏感性降低有關(guān)[46,47]。Dapa等[46]測(cè)定了兩株CD (CD630和R20291)的生物膜生長(zhǎng)，發(fā)現(xiàn)在暴露于20 mg/L萬古霉素(100倍MIC)后，與浮游CD相比，一日和三日齡生物膜中的CD菌株存活率分別升高5倍和12倍。Tijerina-Rodriguez 等[47]研究發(fā)現(xiàn)與浮游細(xì)菌相比，生物膜中CD的萬古霉素 MIC 升高100倍。盡管生物膜已被證明與萬古霉素MIC的增加和CDI復(fù)發(fā)有關(guān)，但CD生物膜的形成是多因素的，涉及細(xì)胞壁表面因子、運(yùn)動(dòng)纖毛、芽孢生成和群體感應(yīng)等多種機(jī)制，目前的研究還不能明確地將任何一種機(jī)制與萬古霉素耐藥聯(lián)系起來。

與MTZ耐藥相似，質(zhì)粒介導(dǎo)的水平轉(zhuǎn)移同樣可造成CD的萬古霉素耐藥。最近有研究報(bào)道來自萬古霉素治療無效患者的分離株中存在由質(zhì)粒介導(dǎo)的萬古霉素敏感性降低(MIC=2 mg/L)[48]。該質(zhì)粒pX18-498是一個(gè)具有51個(gè)ORFs的大型質(zhì)粒，包括一個(gè)推測(cè)編碼N-乙酰胞壁酰-l-丙氨酸氨基酶(一種肽聚糖重塑酶)的基因。該酶對(duì)于以細(xì)胞壁為靶點(diǎn)的抗菌藥物耐藥性的產(chǎn)生至關(guān)重要，將帶有該酰胺酶編碼基因的pX18-498質(zhì)粒轉(zhuǎn)化入CD可導(dǎo)致細(xì)菌的滲透脆性降低。此外，感染攜帶pX18-498的CD菌株的小鼠比感染缺乏該質(zhì)粒的同基因背景菌株的小鼠疾病程度更加嚴(yán)重。作者認(rèn)為pX18-498對(duì)耐藥機(jī)制的影響，以及該質(zhì)粒與細(xì)菌染色質(zhì)基因組之間是否存在相互作用仍需要進(jìn)一步研究。

考慮到萬古霉素耐藥質(zhì)粒pX18-498在非產(chǎn)毒株中也有攜帶，且艱難梭菌可在人體腸道內(nèi)定植，在1歲以下幼兒腸道中定植概率達(dá)70%，而在成人腸道中概率在2%～15%，因此盡早識(shí)別可能的帶菌者，通過PCR等手段篩查是否攜帶pX18-498耐藥質(zhì)粒，或許能夠?qū)θf古霉素耐藥的艱難梭菌及其造成的感染進(jìn)行早識(shí)別、早治療。

3 非達(dá)霉素

窄譜抗菌藥物非達(dá)霉素與細(xì)菌RNA聚合酶(RNA polymerase，RNAP)的夾型結(jié)構(gòu)域結(jié)合，抑制DNA轉(zhuǎn)錄的起始步驟[49]。自2011年起，美國(guó)食品藥品監(jiān)督管理局批準(zhǔn)非達(dá)霉素用于治療CDI。非達(dá)霉素耐藥的CD (MIC=16 μg/mL)是從一名接受非達(dá)霉素治療的 rCDI 患者的糞便標(biāo)本中分離得到的[50]。非達(dá)霉素耐藥性源于RNAP結(jié)合位點(diǎn)的突變，包括RpoB (Gln1074Lys，Val1143Asp、Gly、Phe)和RpoC (Gln781Arg，Asp1127Glu，Asp237Tyr)等多種突變[51,52]。其中，Val1143Asp (MIC>64 mg/L)和Val1143Gly (MIC=16 mg/L)也存在于非達(dá)霉素耐藥的臨床分離CD菌株中[52,53]。RpoB的Val1143Asp突變會(huì)影響CD的適應(yīng)性和毒力[53]。Val1143Asp、Val1143Gly突變的實(shí)驗(yàn)室菌株與其親本菌株R20291相比，整體生長(zhǎng)變緩、競(jìng)爭(zhēng)適應(yīng)性下降且毒素A和B的產(chǎn)生均減少，在CDI 金黃地鼠模型中其毒力也降低。研究人員對(duì)這些 RNAP存在突變的耐藥株的臨床意義進(jìn)行了分析，認(rèn)為目前尚不清楚這批耐藥株是否能在治療濃度的非達(dá)霉素中存活(據(jù)報(bào)道非達(dá)霉素的糞便治療濃度可達(dá)1396 ± 1019 μg/g)[54]。此外，由于這類耐藥突變伴隨著適應(yīng)性降低，且非達(dá)霉素的窄譜活性對(duì)腸道微生物群影響較小[55]，因此如果在治療過程中出現(xiàn)耐藥突變，共生的多種腸道微生物群可能有助于減輕突變帶來的影響。體外誘導(dǎo)的非達(dá)霉素耐藥突變株(MIC=16 mg/L)的基因存在移碼突變，是(多重抗生素抗性調(diào)節(jié)因子)的同源基因，但要確定該突變?cè)诜沁_(dá)霉素抗性中所起的作用仍需要實(shí)驗(yàn)室研究驗(yàn)證[56]。

4 利福昔明和四環(huán)素類

CDI的替代療法包括利福霉素類的利福昔明以及四環(huán)素類的替加環(huán)素。利福昔明抑制細(xì)菌RNA 聚合酶，并可作為萬古霉素治療rCDI后的序貫治療。細(xì)菌RNA聚合酶β亞基RpoB突變是產(chǎn)生利福霉素抗性的主要機(jī)制[2]。這些突變會(huì)破壞利福霉素和RpoB的直接相互作用或改變RpoB上的利福霉素結(jié)合口袋(rifamycin-binding pocket)結(jié)構(gòu)。已發(fā)現(xiàn)CD中存在多處可造成耐藥的RpoB突變，包括Ser488Tyr，Asp492Tyr，His502Asn/Tyr，Arg505Lys，Ser550Phe/Tyr[2]。與其他菌株不同(例如腦膜炎奈瑟球菌和結(jié)核分枝桿菌)，RpoB突變導(dǎo)致CD對(duì)利福霉素產(chǎn)生耐藥性的同時(shí)不會(huì)產(chǎn)生體外和體內(nèi)適應(yīng)性代價(jià)[53]。CD對(duì)利福霉素的耐藥性發(fā)展迅速，有研究顯示甚至在利福昔明治療CDI期間就可能出現(xiàn)耐藥，導(dǎo)致臨床治療失敗。在一個(gè)病例中，CD菌株(RT056)在利福昔明治療3天內(nèi)產(chǎn)生了耐藥性，MIC從0.002 mg/L增加到32 mg/L以上[57]。此外，對(duì)利福昔明耐藥的C D在醫(yī)療機(jī)構(gòu)內(nèi)也很常見(耐藥率為29.1%～48.9%)，這可能會(huì)增加該藥物治療CDI失敗的風(fēng)險(xiǎn)[58]。由于持續(xù)使用利福霉素較易誘導(dǎo)艱難梭菌產(chǎn)生耐藥突變的特性，目前無論國(guó)內(nèi)還是歐美地區(qū)指南均不推薦該類藥物用作初發(fā)CDI的一、二線治療，僅用于在接近治療終點(diǎn)時(shí)短時(shí)間內(nèi)給藥以減少CDI多次復(fù)發(fā)的可能[59,60]。

四環(huán)素類是靶向細(xì)菌30S核糖體的廣譜抗菌藥物，可阻止氨基酰-tRNA與mRNA結(jié)合從而抑制蛋白質(zhì)翻譯[61]。與利福昔明相比，CD對(duì)替加環(huán)素的耐藥率較低。最近的一項(xiàng)Meta分析表明，20%的分離自人類標(biāo)本的CD菌株對(duì)四環(huán)素具耐藥性[62]。CD通過轉(zhuǎn)座子(例如Tn916、Tn5397和Tn4453)攜帶的各種基因(例如和)編碼的核糖體保護(hù)蛋白即延伸因子(elongation-factor)[63]，介導(dǎo)四環(huán)素類藥物耐藥性的產(chǎn)生。研究發(fā)現(xiàn)在歐洲和北美地區(qū)，是CD中最常見的四環(huán)素耐藥決定基因[64]。但由于替加環(huán)素對(duì)核糖體的親和力高于傳統(tǒng)四環(huán)素藥物，因此它對(duì)攜帶的菌株仍具有活性。對(duì)替加環(huán)素的高水平耐藥性由四環(huán)素類破壞酶基因即編碼，它可以通過酶促反應(yīng)滅活替加環(huán)素[63]。最近發(fā)現(xiàn)的的同源基因出現(xiàn)在家畜和人類標(biāo)本CD分離株中的可移動(dòng)元件上，這也提示替加環(huán)素耐藥性可在環(huán)境、社區(qū)與人群間相互傳播[63]。

5 艱難梭菌抗菌新藥的研發(fā)

新型抗菌藥物的研發(fā)是應(yīng)對(duì)耐藥菌感染的重要手段。目前有五種治療CDI的抗菌新藥物已進(jìn)入臨床研究階段。利地利唑(ridinilazole)是新型的苯并咪唑(bis-benzimidazole)類抗菌藥，作用機(jī)制并未未完全闡明，可抑制艱難梭菌二分裂和毒素A、B的產(chǎn)生[65]。II期臨床試驗(yàn)結(jié)果顯示利地利唑有效性非劣效于萬古霉素，且使用利地利唑治療CDI組患者的腸道菌群α多樣性變化更小(<0.0001)，微生物群組成也能更快恢復(fù)到治療前的水平[66]。目前該藥正處于III期臨床研究階段。MGB-BP-3是一種基于奎寧- 遠(yuǎn)霉素(quinoline–distamycin-based)的人工合成抗菌新藥，作用機(jī)制并未完全闡明，可與艱難梭菌DNA小溝結(jié)合抑制基因轉(zhuǎn)錄，對(duì)NAP1/027株具強(qiáng)效殺菌作用，體外藥敏MIC為0.25μg/mL[67]。目前該藥正處于II期臨床試驗(yàn)階段。Ibezapolstat是一種二氯芐基嘌呤衍生物(dichlorobenzyl purine derivative)，為細(xì)菌DNA聚合酶III PolC抑制劑，體外藥效學(xué)研究顯示其對(duì)艱難梭菌的MIC在1～ 8 μg/mL之間[68]。CRS3123是一種苯并吡喃衍生物，作為甲硫氨酰- tRNA合酶抑制劑。臨床前研究顯示CRS3123能夠抑制多種CD臨床分離株的毒素A、B產(chǎn)生，快速緩解CDI癥狀，抑制芽孢形成[69]。該藥物目前已進(jìn)入II期臨床試驗(yàn)。DNV3837是一種喹諾酮類與惡唑烷酮雜化后的化合物，體外藥效學(xué)研究顯示該藥 MIC為0.25 μg/mL[70]。與上述其他新藥不同，DNV3837為靜脈注射給藥，給藥后血酯酶可使 DNV3837去磷酸化為活性形式 DNV3681，并在腸道組織中富集。因此DNV3837可能為不能耐受口服藥物的CDI患者提供一種替代選擇[71]。該藥物目前亦正處于II期臨床試驗(yàn)階段。

6 結(jié)語與展望

艱難梭菌感染仍然是醫(yī)療衛(wèi)生系統(tǒng)的嚴(yán)重威脅和負(fù)擔(dān)，MDR菌株普遍存在，臨床常用治療藥物如甲硝唑和萬古霉素對(duì)CDI的療效下降，替代選擇如利福霉素類藥物更易導(dǎo)致CD耐藥性的快速產(chǎn)生。除傳統(tǒng)機(jī)制如藥物轉(zhuǎn)化、靶位改變、主動(dòng)外排、生物膜形成等可共同參與耐藥形成外，耐藥質(zhì)粒亦可介導(dǎo)艱難梭菌的甲硝唑或萬古霉素耐藥。由于艱難梭菌為產(chǎn)芽孢的厭氧革蘭陽(yáng)性菌，以往用于需氧菌耐藥機(jī)制研究的分子生物學(xué)技術(shù)很多不適用于艱難梭菌耐藥研究，但是近幾年隨著CRISPR-Cas技術(shù)的發(fā)展應(yīng)用，推出了多種基因編輯質(zhì)粒并可商業(yè)獲得，有助于對(duì)艱難梭菌耐藥機(jī)制特別是對(duì)甲硝唑和萬古霉素等治療用藥物的耐藥機(jī)制進(jìn)行深入研究。隨著對(duì)耐藥性變遷和耐藥機(jī)制的更好了解，將為抗菌藥物合理應(yīng)用，遏制耐藥菌的產(chǎn)生和播散、新抗菌藥以及新耐藥菌快速檢測(cè)試劑盒的研發(fā)提供理論基礎(chǔ)。

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Progress on mechanisms of antibiotic resistance in

Teng Xu, Haihui Huang

(CD) is one of the most common pathogens causing health-care-associated infectious diarrhea and is listed by the U.S. Centers for Disease Control and Prevention as an urgent antibiotic resistance (AR) threat. Many resistance genes can be transferred between different CD strains present in the clinical setting, community, and environment. The antimicrobial resistance (AMR) of CD continues to evolve with the emergence and acquisition of new drug resistance mechanisms.CD has developed diverse drug resistance mechanisms, such as drug alteration, modification of the target site, and extrusion of drugs via efflux pumps. Researches have provided comprehensive knowledge about resistance mechanisms of macrolides and quinolonesin CD. However, the mechanisms of resistance for metronidazole, vancomycin, and other therapeutic antibiotics againstinfection (CDI) are only beginning to be elucidated. Some previously unfound mechanisms, such as plasmid-mediated drug resistance in CD, may also play an important role. In this review, we summarize the research progress on drug resistance mechanisms of CD with antimicrobial drugs already used clinically, such as metronidazole, vancomycin, and fidaxomicin, thereby providing the references for the clinical treatment and prevention of CDI, as well as the development of new antibacterial drugs and detection kits for drug resistant bacteria.

clostridioides difficile; resistance mechanism; metronidazole; vancomycin; fidaxomicin

2023-08-18;

2023-10-27;

2023-11-03

上海市自然科學(xué)基金(編號(hào)：21ZR1410800) [Supported by the Natural Science Foundation of Shanghai (No. 21ZR1410800)]

胥騰，博士，住院醫(yī)師，研究方向：細(xì)菌耐藥性與耐藥機(jī)制研究。E-mail: txu20@fudan.edu.cn

黃海輝，博士，主任醫(yī)師，研究方向：感染性疾病的診治與新藥研發(fā)，厭氧菌耐藥性與耐藥機(jī)制研究。E-mail: huanghaihui@fudan. edu.cn

10.16288/j.yczz.23-193

(責(zé)任編委: 謝建平)