張智健 逄宇 趙雁林 劉長(zhǎng)庭
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·綜述·
膿腫分枝桿菌復(fù)合群的研究進(jìn)展
張智健 逄宇 趙雁林 劉長(zhǎng)庭
膿腫分枝桿菌復(fù)合群(Mycobacteriumabscessuscomplex,MABC)是可引起人體致病的重要非結(jié)核分枝桿菌(non-tuberculous Mycobacteria, NTM),它由膿腫分枝桿菌、馬賽分枝桿菌和Mycobacteriumbolletii3個(gè)菌種組成。多靶位基因測(cè)序?yàn)镸ABC準(zhǔn)確可靠的菌種鑒定方法??死顾厥荕ABC感染所致疾病治療的基石,近年來,MABC研究較大的進(jìn)展就是紅霉素核糖體甲基化酶(41)[erythromycin ribosome methytransferase,erm(41)]基因的發(fā)現(xiàn),erm(41)與克拉霉素誘導(dǎo)耐藥相關(guān)。膿腫分枝桿菌和M.bolletii均攜帶完整的erm(41)基因,而馬賽分枝桿菌erm(41)基因有2個(gè)片段缺失。膿腫分枝桿菌28位堿基具有多態(tài)性,T28序列型可誘導(dǎo)耐藥,C28序列型不誘導(dǎo)耐藥。為檢測(cè)膿腫分枝桿菌是否對(duì)克拉霉素誘導(dǎo)耐藥,建議培養(yǎng)時(shí)間由3 d 延長(zhǎng)至14 d。另外,已有證據(jù)表明MABC可能在人與人之間傳染,有必要研究MABC的基因分型技術(shù)。
分枝桿菌屬; 非結(jié)核分枝桿菌; 抗藥性, 細(xì)菌; 基因型
膿腫分枝桿菌復(fù)合群(Mycobacteriumabscessuscomplex,MABC)是由膿腫分枝桿菌、馬賽分枝桿菌和Mycobacteriumbolletii3個(gè)菌種組成的復(fù)合群,是可引起人體致病的重要非結(jié)核分枝桿菌(non-tuberculous Mycobacteria, NTM)。在所有NTM中,MABC分離率占第二位,僅次于鳥分枝桿菌復(fù)合群[1]。在快生型NTM中分離率占第一位,65%~80%的快生型NTM肺病是由MABC引起[2]。MABC是致病性最高,耐藥性最強(qiáng)的快生型NTM。
近年來,NTM感染患者明顯增多,其中以MABC感染為多見[3]。MABC主要侵犯人體肺臟,引起MABC肺病,還可以侵犯肺外組織器官如皮膚和軟組織、淋巴結(jié)、骨骼、關(guān)節(jié)等,甚至引起角膜炎、心內(nèi)膜炎等,重癥患者可引起全身播散性疾病、MABC菌血癥[4-7]。MABC感染所致的疾病診斷困難,治療棘手,所以引起各國(guó)學(xué)者的廣泛關(guān)注。可以說,MABC是目前國(guó)際上NTM研究的重點(diǎn)和熱點(diǎn)。關(guān)于MABC的研究成果和進(jìn)展,也是如今NTM研究領(lǐng)域的一大亮點(diǎn)。
MABC的命名經(jīng)歷了較大的歷史變化。最早并不是一個(gè)復(fù)合群,僅有膿腫分枝桿菌單一菌種,并且是歸屬于龜分枝桿菌的一個(gè)亞種,稱為龜分枝桿菌。1953年Moore和Frerichs[8]報(bào)道1例由該菌引起膝關(guān)節(jié)膿腫樣感染的患者,“膿腫”由此得名。隨后將其命名為龜分枝桿菌膿腫亞種(Mycobacteriumchelonaesubsp.abscessus)。一直到1992年,美國(guó)胸科學(xué)會(huì)(American Thoracic Society, ATS)根據(jù)該菌的藥物敏感性試驗(yàn)(簡(jiǎn)稱“藥敏試驗(yàn)”)及核酸序列分析將膿腫亞種從龜分枝桿菌中獨(dú)立出來,成為一個(gè)單獨(dú)的菌種,稱為膿腫分枝桿菌[9]。
隨著細(xì)菌分離培養(yǎng)及分子鑒定技術(shù)的發(fā)展,相繼有新的與膿腫分枝桿菌親緣關(guān)系密切的菌種被發(fā)現(xiàn)。2004年,法國(guó)馬賽l例50歲女性肺炎患者痰液中分離出一株NTM,表型和膿腫分枝桿菌相似,16SrRNA測(cè)序顯示和膿腫分枝桿菌標(biāo)準(zhǔn)株相似度達(dá)100%,但rpoB測(cè)序相似度僅為96%,系統(tǒng)進(jìn)化分析顯示其來源于膿腫分枝桿菌,因其發(fā)現(xiàn)于馬賽故將其命名為“馬賽分枝桿菌”[10]。隨后馬賽分枝桿菌相繼在美國(guó)等其他國(guó)家被發(fā)現(xiàn)和報(bào)道[11-13]。2006年Adékambi等[14]又發(fā)現(xiàn)一株與膿腫分枝桿菌親緣關(guān)系密切的菌種,并以其已故同事,一位叫Bollet的微生物家命名,稱為“Mycobacteriumbolletii”。這樣一來,廣義的膿腫分枝桿菌不再是單一菌種,而是由3個(gè)菌種形成的一個(gè)復(fù)合群。目前多數(shù)文獻(xiàn)認(rèn)同,廣義的膿腫分枝桿菌改稱為膿腫分枝桿菌復(fù)合群(MABC),它由膿腫分枝桿菌、馬賽分枝桿菌和M.bolletii3個(gè)菌種組成[15]。少數(shù)文獻(xiàn)不改變膿腫分枝桿菌的稱謂,將其所屬3個(gè)菌種稱為其亞種:分別為:膿腫分枝桿菌膿腫亞種(Mycobacteriumabscessussubsp.abscessus)、膿腫分枝桿菌馬賽亞種(Mycobacteriumabscessussubsp.massiliense) 和膿腫分枝桿菌bolletii亞種(Mycobacteriumabscessussubsp.bolletii)。
也有學(xué)者對(duì)上述三分類法持異議,他們根據(jù)多靶位基因的系統(tǒng)進(jìn)化樹中馬賽分枝桿菌和M.bolletii位置接近,認(rèn)為兩者應(yīng)該合二為一,統(tǒng)稱為膿腫分枝桿菌bolletii亞種[16]。這樣,膿腫分枝桿菌就只包含2個(gè)亞種:膿腫分枝桿菌膿腫亞種和膿腫分枝桿菌bolletii亞種。
所以,到底是三分類還是二分類,目前爭(zhēng)議較大,各方觀點(diǎn)見仁見智,多數(shù)人傾向于三分類??紤]到馬賽分枝桿菌在對(duì)克拉霉素敏感性上優(yōu)于M.bolletii,我們認(rèn)同三分類法。最新有研究支持我們的觀點(diǎn),該研究根據(jù)全基因組單核苷酸多態(tài)性位點(diǎn)建立的進(jìn)化樹顯示:MABC明確包含3個(gè)進(jìn)化支,這3個(gè)進(jìn)化支分別為膿腫分枝桿菌、馬賽分枝桿菌和M.bolletii;有意思的是,M.bolletii位置和膿腫分枝桿菌還更為鄰近,而不是和馬賽分枝桿菌更為鄰近[17]。
現(xiàn)有研究資料統(tǒng)計(jì)發(fā)現(xiàn),MABC的菌種構(gòu)成中不同國(guó)家地區(qū)有不同的分布。膿腫分枝桿菌占71%~43%;馬賽分枝桿菌占21%~56%;M.bolletii較為少見,占1%~18%[18-25]。
我國(guó)對(duì)MABC的研究較少,大多還停留在“龜-膿腫分枝桿菌復(fù)合群”或者“膿腫分枝桿菌” 的概念,文獻(xiàn)中的稱謂比較混亂,對(duì)國(guó)際上的研究熱點(diǎn)馬賽分枝桿菌,僅有個(gè)別文獻(xiàn)的個(gè)案報(bào)道,M.bolletii在我國(guó)更是還未見報(bào)道[26]。
準(zhǔn)確的菌種鑒定是臨床正確診斷、有效治療的前提。并且,現(xiàn)有研究發(fā)現(xiàn),膿腫分枝桿菌、馬賽分枝桿菌及M.bolletii對(duì)克拉霉素具有不同的藥敏特性,膿腫分枝桿菌及M.bolletii大多對(duì)克拉霉素耐藥,而馬賽分枝桿菌大多對(duì)克拉霉素敏感[27]。所以臨床上觀察到,同樣的治療藥物,對(duì)MABC有不同的治療效果。對(duì)于MABC這一異質(zhì)性種群來說,僅局限于菌群鑒定遠(yuǎn)遠(yuǎn)不能滿足臨床的需要,準(zhǔn)確的菌種鑒定顯得尤為重要。
傳統(tǒng)表型鑒定方法,如生化法及對(duì)硝基苯甲酸和(或)噻吩-2-羧酸肼鑒別培養(yǎng)基,由于耗時(shí)費(fèi)力,鑒定結(jié)果可靠性差[28-29]。最主要的問題是不能準(zhǔn)確鑒定到種。所以,在MABC的鑒定中已沒有地位。
以靶位基因PCR為基礎(chǔ)的分子鑒定為目前分枝桿菌主流的菌種鑒定方法。具體包括核酸探針、基因芯片、PCR-限制性片段長(zhǎng)度多態(tài)性(restriction fragment length polymorphism,RFLP)、PCR-直接測(cè)序法等。對(duì)于MABC來說,由于膿腫分枝桿菌、馬賽分枝桿菌及M.bolletii親緣關(guān)系密切,以16s rRNA為靶位基因的任何鑒定方法都無法對(duì)它們鑒別。
于是,核酸探針、基因芯片等多以16s rRNA為靶位基因的商品化的鑒定試劑盒對(duì)于MABC的鑒定就顯得無能為力。PCR-RFLP(PRA)作為較為成熟的NTM鑒定方法,已得到了廣泛應(yīng)用。最常用的靶位基因?yàn)閞poB、hsp65。但具體對(duì)于MABC來說, PRA-hsp65有兩種酶切圖譜[30],文獻(xiàn)最初稱為hsp65 Ⅰ型和hsp65 Ⅱ型,后來研究發(fā)現(xiàn),hsp65 Ⅰ型為膿腫分枝桿菌,hsp65 Ⅱ型為馬賽分枝桿菌和M.bolletii[31]。所以PRA-hsp65只能鑒定出膿腫分枝桿菌,不能鑒定馬賽分枝桿菌和M.bolletii。PRA-rpoB對(duì)MABC更是只有一種酶切圖譜,只能鑒定到菌群,不能準(zhǔn)確鑒定到菌種[32]。PRA在MABC的鑒定中也受到限制。
所以,PCR-直接測(cè)序法為目前MABC鑒定的主要方法, 靶基因包括間隔區(qū)序列(internal transcribed spacer,ITS)、rpoB、hsp65、sodA、recA等。但研究發(fā)現(xiàn),單靶位基因測(cè)序鑒定MABC有時(shí)可能得到錯(cuò)誤的結(jié)果[33]。所以,目前多采用多靶位基因測(cè)序,測(cè)序結(jié)果相互驗(yàn)證[33-34]。對(duì)于多靶位基因測(cè)序鑒定結(jié)果不一致的現(xiàn)象,文獻(xiàn)稱之為“菌種間復(fù)合模式”, 處理原則為取多數(shù)靶位基因鑒定一致的結(jié)果[31]。多靶位基因聯(lián)合測(cè)序鑒定快速,結(jié)果可靠,值得在MABC的菌種鑒定中推廣應(yīng)用;缺點(diǎn)是費(fèi)用成本較高。
以克拉霉素和阿奇霉素為代表的大環(huán)內(nèi)酯類抗生素是MABC感染所致疾病治療的基石??死顾氐牡匚挥绕渫怀?,它在所有的大環(huán)內(nèi)酯類抗生素中抗分枝桿菌的活力最強(qiáng)[35]。 MABC對(duì)克拉霉素的耐藥機(jī)制成為了近年來人們關(guān)注的焦點(diǎn)。
克拉霉素作用靶位為細(xì)菌核糖體50S大亞基中23S rRNA 結(jié)構(gòu)域Ⅴ區(qū),該區(qū)具有肽酰轉(zhuǎn)移酶活性,克拉霉素與其結(jié)合后阻礙肽鏈延伸,阻止蛋白的翻譯合成[36]。MABC對(duì)克拉霉素的耐藥機(jī)制主要有兩種情況:一種是獲得性耐藥,為編碼 23S rRNA 的rrl基因2058或者2059位點(diǎn)A堿基的點(diǎn)突變,導(dǎo)致克拉霉素失去作用靶位而耐藥[37]。rrl的突變是由于長(zhǎng)期的克拉霉素治療引起,細(xì)菌在抗生素的選擇壓力下發(fā)生的自發(fā)點(diǎn)突變。rrl突變率早期較低, 1996年Wallace等[38]報(bào)道,在800例MABC肺病及全身性感染的患者中,僅有18例,占2.3%。近年來有增高趨勢(shì),韓國(guó)報(bào)道突變率在7%~23%[25,39]。
MABC對(duì)克拉霉素的另外一種耐藥機(jī)制為誘導(dǎo)耐藥。MABC的誘導(dǎo)耐藥為近年來研究的熱點(diǎn)。最早于1992年,Brown等[35]觀察到,在MABC的體外藥敏試驗(yàn)中,隨著培養(yǎng)時(shí)間的延長(zhǎng),最低抑菌濃度(minimum inhibitory concentration,MIC)有逐漸增高的現(xiàn)象,這種情況一時(shí)找不到合理的解釋。后來,人們?cè)诮Y(jié)核分枝桿菌[40]、恥垢分枝桿菌[41]、偶發(fā)分枝桿菌[42]中相繼發(fā)現(xiàn)紅霉素核糖體甲基化酶(erythromycin ribosome methytransferase,erm)基因,分別命名為erm(37)、erm(38)、erm(39),這種基因編碼的erm能使克拉霉素的作用位點(diǎn)2058或2059位點(diǎn)腺嘌呤的甲基化,導(dǎo)致克拉霉素失去作用靶位而失活。2009年,Nash等[43]從上述分枝桿菌中存在erm基因得到啟示,是否MABC中也存在erm基因?通過基因重組及轉(zhuǎn)化,最終證實(shí)erm基因在MABC中的存在,將其命名為erm(41)。
erm(41) 可被克拉霉素誘導(dǎo)表達(dá),有活性的erm(41)能催化23S rRNA 2058或2059位腺嘌呤甲基化,導(dǎo)致常規(guī)培養(yǎng)3 d藥敏試驗(yàn)結(jié)果為敏感的MABC,在延長(zhǎng)培養(yǎng)至14 d后,對(duì)克拉霉素表現(xiàn)出耐藥。但不是所有MABC都會(huì)產(chǎn)生誘導(dǎo)耐藥。不少文獻(xiàn)詳細(xì)研究并總結(jié)了erm(41)基因在膿腫分枝桿菌、馬賽分枝桿菌及M.bolletii之間的結(jié)構(gòu)差異和它們相應(yīng)的在誘導(dǎo)耐藥方面的特點(diǎn),結(jié)果發(fā)現(xiàn):結(jié)構(gòu)上,膿腫分枝桿菌和M.bolletii均攜帶完整的erm(41)基因,而馬賽分枝桿菌erm(41)基因有2個(gè)片段缺失,分別為276 bp長(zhǎng)片段和2 bp短片段缺失[43-45]。另外,基因序列第28位堿基存在多態(tài)性,膿腫分枝桿菌有T28序列型或C28序列型,馬賽分枝桿菌和M.bolletii只有T28序列型。誘導(dǎo)耐藥表型上,只有T28序列型的膿腫分枝桿菌和M.bolletii可表現(xiàn)為誘導(dǎo)耐藥。C28序列型的膿腫分枝桿菌和馬賽分枝桿菌沒有誘導(dǎo)耐藥。
erm(41)的發(fā)現(xiàn)解釋了臨床困擾已久的MABC對(duì)克拉霉素治療反應(yīng)的異質(zhì)性問題?,F(xiàn)在明白,這種情況是由于不同的菌種,或者是膿腫分枝桿菌不同的28位堿基序列型引起。治療反應(yīng)好的菌株為沒有誘導(dǎo)耐藥表型的馬賽分枝桿菌或C28序列型的膿腫分枝桿菌;相應(yīng)地,治療反應(yīng)差的為有誘導(dǎo)耐藥表型的M.bolletii或者T28序列型的膿腫分枝桿菌。
對(duì)于克拉霉素的耐藥表型和基因型的對(duì)應(yīng)關(guān)系,不同的研究的有不同的結(jié)論。多數(shù)研究認(rèn)為,培養(yǎng)3 d的耐藥菌株對(duì)應(yīng)的改變?yōu)閞rl基因突變,培養(yǎng)14 d的誘導(dǎo)耐藥株對(duì)應(yīng)的erm(41)的基因型為T28序列型[38]。也有研究持不同看法,有研究發(fā)現(xiàn)培養(yǎng)3 d的耐藥菌株并不都存在rrl基因突變,可能還有其他機(jī)制[27]。還有研究發(fā)現(xiàn),并不是所有的T28序列型膿腫分支桿菌都能夠誘導(dǎo)耐藥[46]。另外,erm(41)和rrl一般認(rèn)為屬于兩個(gè)獨(dú)立的機(jī)制,兩者可以在一株菌中共存;但有的研究認(rèn)為,erm(41)和rrl兩種機(jī)制只存在二選一的問題,不可能共存[47]。上述爭(zhēng)議還有待于進(jìn)一步深入地進(jìn)行研究。
體外藥敏試驗(yàn)是幫助克服盲目經(jīng)驗(yàn)性用藥、促進(jìn)治療有的放矢的有力工具和最佳途徑。為此,國(guó)內(nèi)外指南對(duì)NTM的體外藥敏試驗(yàn)作了綱領(lǐng)性規(guī)定。2007版ATS的NTM診治指南及我國(guó)2012版《非結(jié)核分枝桿菌病診斷與治療專家共識(shí)》都建議,有條件應(yīng)盡可能做體外藥敏試驗(yàn),然后根據(jù)藥敏試驗(yàn)結(jié)果選擇相應(yīng)敏感的治療藥物[2,48]。并且,ATS指南推薦,對(duì)于快生型NTM,常規(guī)藥敏試驗(yàn)應(yīng)包括九種藥物:克拉霉素、阿米卡星、頭孢西汀、亞胺培南、氟喹諾酮類、多西環(huán)素、復(fù)方新諾明、妥布霉素、利奈唑胺。指南還明確規(guī)定了NTM體外藥敏試驗(yàn)的方法:肉湯微量稀釋法測(cè)定MIC。美國(guó)臨床與實(shí)驗(yàn)室標(biāo)準(zhǔn)委員會(huì)(Clinical and Laboratory Stan-dards Institute, CLSI)也更新了NTM體外藥敏試驗(yàn)指南(CLSI 2011版M24-A2)[49],為各種類型NTM的肉湯微量稀釋法提供了標(biāo)準(zhǔn)化的操作方法步驟。這些指南有力地推動(dòng)和規(guī)范了NTM體外藥敏試驗(yàn)的開展。
對(duì)MABC的體外藥敏試驗(yàn),進(jìn)展最大的是:由于MABC對(duì)克拉霉素可能產(chǎn)生誘導(dǎo)耐藥,所以培養(yǎng)時(shí)間由常規(guī)培養(yǎng)3 d,要求進(jìn)一步延長(zhǎng)至14 d,以檢測(cè)誘導(dǎo)耐藥的現(xiàn)象。以前僅僅培養(yǎng)3 d的體外藥敏試驗(yàn)結(jié)果只反映MABC的獲得性耐藥情況,沒有體現(xiàn)出誘導(dǎo)耐藥的情況。
相對(duì)于結(jié)核分枝桿菌的基因分型技術(shù)的成熟,NTM的基因分型剛剛起步。以前人們一直樂觀預(yù)期NTM不會(huì)在人與人之間傳染,但最新的多個(gè)研究表明NTM可能在人與人之間傳染[50-52]。較多文獻(xiàn)報(bào)道MABC在醫(yī)院內(nèi)暴發(fā)和流行[6, 53-54],所以對(duì)于MABC的基因分型,目前已提上研究日程。
隨著各種NTM標(biāo)準(zhǔn)株全基因測(cè)序的完成,可變數(shù)量串聯(lián)重復(fù)序列(variable number of tandem repeats,VNTR)分型技術(shù)在NTM檢測(cè)中逐漸興起,先后有胞內(nèi)分枝桿菌[55]、鳥分枝桿菌[56]、MABC[57]根據(jù)自身標(biāo)準(zhǔn)株的全基因序列建立了相應(yīng)的VNTR分型技術(shù)。這些分型技術(shù)有力地推動(dòng)了NTM院內(nèi)暴發(fā)感染傳播機(jī)制的研究。不僅如此,有文獻(xiàn)還發(fā)現(xiàn)了VNTR分型的基因型和體外藥敏試驗(yàn)結(jié)果,以及臨床表型和臨床預(yù)后之間有一定內(nèi)在聯(lián)系[58]。這無疑拓寬了VNTR的應(yīng)用范圍,為預(yù)測(cè)NTM患者的藥物治療反應(yīng)及最終預(yù)后提供了新的視角。
總之,近年來有關(guān)MBAC的研究成果頗為豐富。即便如此,我國(guó)MABC感染所致疾病的防治形勢(shì)依然嚴(yán)峻。要攻克和解決MABC感染所致疾病的診治困境和難題,我們還任重道遠(yuǎn)。
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(本文編輯:薛愛華)
Research progress ofMycobacteriumabscessuscomplex
ZHANGZhi-jian*,PANGYu,ZHAOYan-lin,LIUChang-ting.
*RespiratoryDiseasesDepartmentofNanlou,ChinesePeople’sLiberationArmyGeneralHospital,Beijing100853,China
s:LIUChang-ting,Email:liuchangting301@163.com;ZHAOYan-lin,Email:zhaoyanlin@chinatb.org
Mycobacteriumabscessuscomplex (MABC), an important non-tuberculous mycobacteria (NTM) that can cause human diseases, consistes ofMycobacteriumabscessus,MycobacteriummassilienseandMycobacteriumbolletii. Multi target gene sequencing is deemed to be the most accurate and reliable method for species identification of MABC. Clarithromycin plays a role of cornerstone in the treatment of MABC diseases. Recently, one of the greatest progress of the discovery of erythromycin ribosome methylase (erm(41)) which is associated with inducing resistance to clarithromycin.M.abscessusandM.bolletiiboth have an intacterm(41) gene whileM.massiliensehas two deletions inerm(41) gene.M.abscessusstrains have a T/C polymorphism at the 28th nucleotide: T28 strains demonstrates inducible clarithromycin resistance, while C28 strains are susceptible. For the purpose of detecting induced resistance to clarithromycin ofMycobacteriumabscessus, it’s advised to prolong the incubation from 3 days to 14 days. In addition, recent evidences had raised the possibility that person-to-person transmission could occur among highly susceptible individuals, so it is necessary to study the genotyping of MABC.
Mycobacterium; Nontuberculous mycobacteria; Drug resistance, bacterial; Genotype
10.3969/j.issn.1000-6621.2015.06.017
100853 北京,解放軍總醫(yī)院南樓呼吸科(張智健、劉長(zhǎng)庭);中國(guó)疾病預(yù)防控制中心 國(guó)家結(jié)核病參比實(shí)驗(yàn)室(逄宇、趙雁林)
劉長(zhǎng)庭,Email:liuchangting301@163.com;趙雁林,Email:zhaoyanlin@chinatb.org
2015-04-16)