季銨鹽類消毒劑及大腸桿菌對其耐藥性研究進展

鄒立扣，吳國艷,2，程 琳，何雪梅,2，郭麗娟,2，龍 梅,2

季銨鹽類消毒劑及大腸桿菌對其耐藥性研究進展

鄒立扣1，吳國艷1,2，程 琳1，何雪梅1,2，郭麗娟1,2，龍 梅1,2

（1.四川農業大學 都江堰校區微生物學實驗室，四川 都江堰 611830；2.四川農業大學資源環境學院，四川 成都 611130）

研究表明，食源性細菌消毒劑耐藥嚴重，大腸桿菌是食品污染狀況及耐藥性監測的指示菌，對季銨鹽類消毒劑表現出比革蘭氏陽性菌更強的抗性，且大腸桿菌對消毒劑與抗生素耐藥性可共傳播。鑒于此，本文綜述了季銨鹽類消毒劑的結構與種類、作用機制、大腸桿菌消毒劑耐藥產生、耐藥基因、基因型與表型的關系以及 與抗生素耐藥共傳播機制等的研究進展。食源性大腸桿菌對季銨鹽類消毒劑抗性的耐藥機制研究很少，研究大腸桿菌對季銨鹽類消毒劑耐藥性，可為消毒劑的規范使用以及食源性大腸桿菌的防控提供科學依據及理論基礎。

大腸桿菌；季銨鹽類；消毒劑；耐藥

隨著社會經濟的發展與人民生活水平的提高，食品安全問題已經成為人們關注的焦點，而食品污染中致病微生物引起的食 源性疾病是影響食品安全的最主要因素之一[1-2]。大腸桿菌（Escherichia coli）被很多國家作為各類食品污染狀況的指示菌進行檢測，是保障食品安全的重要指標之一，其可導致腹瀉，感染某些致病性較強的血清型時還可能致命，如O157∶H7引起的出血性腹瀉和溶血性尿毒綜合征（heomlytic uremic syndrome，HUS）等[3]。季銨鹽類化合物（quaternary ammonium compounds，QACs）消毒劑常被用來防止食品工業中致病微生物的擴散及污染[4]，可以有效減少食品中的病原微 生物，然而，抗菌藥物的廣泛或不合理使用可產生篩選壓力，QACs消毒劑的使用會導致細菌的適應和耐藥菌的生長，是消毒劑抗性增加的潛在動力[5]。目前，對革蘭氏陽性細菌消毒劑耐藥及耐藥基因流行已有較多報道和研究，如葡萄球菌（Staphylococcus spp.）及腸球菌（Enterococcus spp.）等[6-8]。相對革蘭氏陽性細菌，革蘭氏陰性細菌對消毒劑表現出更強的抗性[9]，但對革蘭氏陰性細菌，特別是食源性革蘭氏陰性細菌對QACs抗性的發生及耐藥機制研究很少，全面調查大腸桿菌對QACs消毒劑耐藥性已經迫在眉睫[10-11]。鑒于此，本文綜述了季銨鹽類消毒劑的結構與種類、作用機制、大腸桿菌消毒劑耐藥產生、耐藥基因、基因型與表型的關系以及與抗生素耐藥共傳播機制等研究進展。

1 食源性大腸桿菌簡介

大腸桿菌是人和動物重要的共生腸道微生物，絕大部分大腸桿菌屬于腸道正常菌群，但仍有部分菌株可導致人和動物感染、發病，這些致病性大腸桿菌可引起人類的腸胃炎、尿道感染、新生兒敗血癥或腦膜炎等。根據所含毒力因子的種類，致病性大腸桿菌可分為5 類，腸致病性大腸桿菌（enteropathogenic E. coli，EPEC）、腸產毒性大腸桿菌（enterotoxigenic E. coli，ETEC）、腸侵襲性大腸桿菌（enteroinvasive E. coli，EIEC）、腸黏附性大腸桿菌（enteroaggregative E. coli，EAEC）及腸出血性大腸桿菌（enterohemorrhagic E. coli，EHEC）等，這些大腸桿菌均可通過食物鏈進入人體并對人類的健康構成威脅[3]。食品中污染大腸桿菌主要發生在食品生產、處理、分配及零售等過程，20世紀以來，由于奶制品、水果、蔬菜、甜點、肉類及水產品等食品污染，引發了大腸桿菌的全球暴發，而暴發的場所有飯店、快餐店、自助餐廳、野餐、養老院及社區等[12]。除了具有較強的致病力，大腸桿菌可通過自身基因突變或捕獲外源耐藥基因產生耐藥性，也可將耐藥基因通過質粒及整合子等傳遞到在腸道或環境中與其共存的其他細菌，因此，被認為是耐藥性監測中良好的指示菌，目前發現，眾多食品特別是肉類食品中分離出的大腸桿菌抗生素耐藥嚴重[13-14]，是抗生素耐藥大腸桿菌重要的“貯存庫”[15-16]，這些耐藥菌可通過被污染的食品使人感染，對人類的健康構成威脅[17]。

2 季銨鹽類消毒劑結構與種類

消毒劑是食品生產清洗、消毒過程中使用的主要化合物，可保證食品產品的微生物安全。為了避免在食品和普通消費者市場中微生物污染及感染的風險，消毒劑在共同衛生中使用正呈現增長的趨勢，消毒劑中有眾多的化學活性物質，主要用于消毒與保存。常用的消毒防腐劑有季胺鹽類化合物、酚類化合物、雙胍類、碘及其復合物、醛類、過氧化物和銀化合物等，這些消毒劑有些已經使用了近百年，相對于抗生素，消毒劑可表現出更寬的廣譜活性，且具有多個靶位點，而抗生素可能只有一個特異的胞內位點[18]。

在食品工業中，常規清潔和控制食品、環境中可產生嚴重疾病的病原微生物水平十分重要，清潔步驟中消毒劑的使用，可以減少表面微生物的生存[19-20]。QACs消毒劑由于具有無腐蝕、無刺激性、較穩定且毒性低等優點而被廣泛地用于食品和環境的消毒[21-22]。QACs消毒劑亦被作為獸藥控制動物疾病[23]，使用QACs消毒劑可降低禽類養殖中細菌污染[24-25]。QACs包含一個4價氮， 基本化學結構為N+R1R2R3R4X-，其中R代表一個氫原子、一個烷基或烷基被替代的其他功能基團，X代表一個陰離子，比如氯或溴等[26]（圖1）。

圖1 季銨鹽類消毒劑結構通式[18]Fig.1 General chemical structur e of QACs[18]

美國國家環境保護局（U.S. Environmental Protection Agency，USEPA）把QACs分為四大類[27]，而根據功能基團的不同，可分為三大類，具體見表1。

表1 QACs消毒劑的分類Table 1 Classification of QAC disinfectants

目前，用于衛生消毒的QACs種類主要有N-烷基二甲基芐基氯化銨（N-alkyl dimethyl benzyl ammonium chloride，A D BAC）、苯扎氯銨（benzalkonium chloride，BC）、溴化十六烷基三甲銨（cetyltrimethylammonium bromide，CTAB）、溴化溴棕三甲銨（cetrimide bromi de，CB）、氯化十六烷基吡啶（cetylpyridinium chloride，CTPC）、雙十烷基二甲基氯化銨（N,N-didecyl-N,N-dimethylammonium chloride，DDAC）、司拉氯銨（stearalkonium chloride）及芐索氯銨（benzethonium chloride）等，其中BC、CTAB及ADBAC等已被廣泛應用于食品工業，其中BC、CB等已使用超過40年[26]，幾種主要的QACs結構見圖2。

a. ADBAC[28]/BC[29]；b. CB[28]；c. CTPC[28]；d. CTAB[30]；e. DDAC[27]。

圖3 QACs反應機理示意圖[26]Fig.3 C artoon showing the mechanism of action of QACs[26]

3 季銨鹽類消毒劑作用機制及殘留檢測

3.1 作用機制

細菌細胞表面攜帶負電荷，常通過陽性離子維持細胞膜的穩定性。QACs是陽離子型表面活性劑和抗菌劑，可通過正電荷與細胞膜相互作用，其抗菌活性是N-烷基的功能。N-烷基賦予QACs親脂性特征，通過陽性氮基團與細菌細胞膜上酸性磷脂的結合，疏水端整合入細菌疏水膜的核心，在高濃度時，QACs通過形成混合膠束聚集來溶解疏水細胞膜成分[26]（圖3）。總體來說，QACs發揮抗菌活性主要依靠破壞和變性蛋白和酶、破壞細胞膜整體性和使細胞內含物泄漏等[26,31]。對不同微生物的抗菌活性取決于烷基鏈的長度：鏈長12～14烷基的QACs對革蘭氏陽性細菌和酵母表現最適活性；14～16烷基的QACs對革蘭氏陰性細菌表現最適活性；鏈長小于4或大于18的QACs幾乎無活性[26]。除對細菌具有抗菌活性，QACs消毒劑對一些病毒、真菌、酵母和原生動物也具有活性[32]。

3.2 殘留檢測

研究表明QACs殘留可危害動物健康[33]，食品中殘留的季銨鹽類消毒劑對人鼻上皮細胞有害，可引發、加重鼻炎[34]。同時，殘留消毒劑可導致細菌耐藥性，歐盟規定水果和蔬菜中QACs殘留不得超過0.01 mg/kg，因此對季銨鹽類消毒劑殘留檢測凸顯重要。在眾多方法中，高效液相色譜（high performance liquid chromatography，HPLC）、液相質譜（liquid chromatograph-mass spectrometer，LC-MS）方法被成功用于檢測QACs[35]。Shen等[29]利用HPLC系統反相模式檢測了阿奇霉素眼藥水中的BC，選用Venusil- XBP（L）-C18（150 mm×4.6 mm，5 μm）柱，柱溫50 ℃，流動相甲醇-磷酸鉀 （16∶5，V/V），樣品前處理中使用去蛋白步驟，結果表明HPLC是監測阿奇霉素眼藥水中的BC含量的有效方法。 Ford等[36]建立了在HPLC結合電噴霧電離質譜方法基礎上的對QACs定性及定量檢測方法，對BC的檢測限達到了3 ng/mL，且可同時檢測苯基、雙烷基二甲基氨鹽化合物，此方法已成功應用于分析衛生采樣裝置、產品及游泳池水。MALDI-TOFMS方法亦被用于分析商業產品漱口水中QACs的組成，研究發現除了產品中標明的CPC、四烷基氨基氨鹽化合物及三烷基氨鹽化合物外，還包含一個復雜的氨鹽類混合物[28]。以ABDAC、四烷基氨基氨鹽化合物為檢測對象，研究表明毛細血管電泳（capillary electrophoresis，CE）法要比電噴霧質譜分離效率高，檢測限要高于兩個數量級[37]。

4 大腸桿菌對季銨鹽類消毒劑耐藥性

4.1 大腸桿菌對消毒劑耐藥性產生基礎

QACs的使用是消毒劑抗性增加的潛在的重要動力，食品工業中QACs的廣泛使用會導致細菌的適應和耐藥菌的生長[5]。QACs可以有效減少食品中病原微生物，為了能快速殺死病原菌，在屠宰、肉類處理，特別是那些小且難接觸到的區域，消毒劑的使用濃度要遠高于它們對微生物的最小抑菌濃度[38-39]，這種濃度可以達到千倍的最小抑菌濃度（minimal inhibitory concentration，MIC）值，而細菌要戰勝快速、猛烈的消毒劑攻擊并產生耐藥生幾乎是不可能的。大部分QACs在應用后不需要用水沖洗或沖洗不及時等，因此細菌與QACs的接觸時間可以延長，長時間暴露于低濃度的QACs，可以使微生物處于亞抑制濃度中，如此，會使那些只對QACs高濃度MIC敏感的細菌才會生存下來[40]，細菌對消毒劑的耐藥性逐漸增大，最終導致消毒劑在食品行業中使用失敗，并出現影響人類健康等嚴重的問題。

如今，對消毒劑耐藥性研究的對象主要為革蘭氏陽性菌中的葡萄球菌（Staphylococcus spp.），由于消毒劑的過量使用，導致葡萄球菌，特別是耐甲氧西林金黃色葡萄球菌（methicillin-resistant Staphylococcus aureus，MRSA）對消毒劑的耐藥。相對革蘭氏陽性細菌，革蘭氏陰性細菌對消毒劑表現出更強的抗性[9]。大腸桿菌對QACs消毒劑的MICs遠高于葡萄球菌，如大腸桿菌對BC的MIC為50 mg/L，遠高于葡萄球菌的0.5 mg/L[18]，假單胞菌（Pseudomonas sp.）對BC的MIC達到了200 mg/L，遠高于葡萄球菌的4～11 mg/L[41]，革蘭氏陰性細菌對QACs的高抗性源自于所攜帶的特異性耐藥基因[42]。

4.2 大腸桿菌對季銨鹽類耐藥基因型

迄今為止，有7 種不同質粒介導的QAC特異的抗性基因在革蘭氏陰性細菌大腸桿菌中被發現，包括qacE、qacEΔ1、qacF、qacG、qacH、qacI及sugE[43-45]。這些基因編碼外排泵蛋白賦予對QACs的抗性，屬于小多重抗性（small multiple resistance，SMR）蛋白家族[42,45]，SMR家族基因可由質粒或整合子介導，對QACs高濃度MICs的菌株經常通過獲得可移動基因元件，如質粒、1型整合子等獲得這些消毒劑基因[46]，而1型整合子大多存在于可接合的質粒上[47-48]，因此，qac、sugE（p）基因可在革蘭氏陰性細菌中水平及垂直傳播[49-50]。由于質粒型消毒劑基因的可傳播性，以及具有與抗生素耐藥基因的共傳播特性，相對于染色體編碼QACs特異性耐藥基因，qac、sugE（p）基因在消毒劑耐藥中扮演著重要的角色。qacE、qacEΔ1基因發現存在于革蘭氏陰性菌質粒上1型整合子3端[51-52]，qacEΔ1基因是qacE基因的突變缺陷體[43]。QacEΔ1蛋白對季銨鹽類消毒劑和染料的耐藥水平低于QacE，這些差異是由于QacEΔ1蛋白失去了第四個跨膜片段和羧基末端的高度保守殘基所造成的[53]。qacG基因報道由1型整合子介導[45]，qacF、qacH、qacI基因與qacE的同源率達到67.8%，亦常發現于革蘭氏陰性細菌質粒介導的整合子[54-55]、質粒pB8及IncP-1βpR751上[56]。與qac基因類似，sugE基因被發現存在于質粒，首先發現于肺炎克雷伯氏菌（Klebsiella pneumoniae）β-內酰胺藥物抗性質粒pTKH11上[57]，之后，從大腸桿菌、沙門氏菌分離的質粒上也檢測出sugE（p）基因[39,58-60]。對于sugE基因，Chung等[61]認為大腸桿菌（E. coli）中的sugE的超量表達，表現出對部分QACs耐藥；Son等[62]認為sugE僅僅可通過一個氨基酸突變即可表現出對QAC抗性。最新研究表明，qac、sugE（p）基因共存于多重耐藥（multi drug resistance，MDR）質粒：InA/C、pSN254上[60]，可介導高水平消毒劑耐藥。

除以上基因之外，某些非特異性外排基因，如大腸桿菌[63]以及大腸桿菌O157∶H7[64]中質粒介導TehA基因[65]，耐藥節結化細胞分化（resistance nodulation cell division，RND）家族外排泵AcrAB-TolC也表現出對QACs的非特異性的耐藥，但其表達與調控基因marOR、soxS的表達密切相關[10]。另有5 種染色體編碼基因sugE（c）（染色體型sugE）、emrE、ydgE/ydgF及mdfA等也特異抗性地賦予對QACs的抗性，但不具傳播性[66-67]。綜上，質粒介導的QACs消毒劑耐藥基因不僅在革蘭陽性菌中已經流行，在革蘭氏陰性菌中也已經存在，質粒介導消毒劑基因傳播危害性較大，應引起重視。

4.3 大腸桿菌對季銨鹽類耐藥基因型與表型關系

不同的消毒劑基因介導對QACs的不同程度的耐藥，目前，已知部分消毒劑基因與其表型之間的關系[18,66]，但尚有基因與表型之間的關系未建立。鑒于相關流行病學調查不夠系統、全面，關于qac基因型與表型之間的關系，有兩種觀點。一種觀點主要基于對qacEΔ1基因的研究，此觀點認為，qac基因介導低水平QACs抗性，不同qac基因攜帶株對QACs的MICs無明顯差異[43]。在綠膿假單胞菌（Pseudomonas aeruginosa）中，qacEΔ1基因并沒有對QACs成員之一的苯扎氯銨表現高抗性[68]。其中的一個解釋是基于qac基因介導對眾多陽離子化合物表現抗性，從而可能導致對QACs底物表現非高水平的特異性[66,69]。qac基因介導對QACs的抗性，同時可對30多種親脂性陽離子化合物表現抗性，這些化合物至少隸屬于12個不同的化學家族，包括單價陽離子化合物，如吖啶黃、結晶紫及絕大部分QACs等，雙價陽離子化合物包括雙胍類、聯脒及部分QACs等[70]。qacEΔ1、qacE啟動子的類型與表達水平也可能導致對QACs的低水平耐藥[42]。然而另一觀點認為，qac基因和對不同陽離子化合物抗性之間存在緊密的聯系，由于qac基因的表達，細菌對消毒劑的抗性逐漸增加[71]。Smith等[72]研究發現qac基因陰性與陽性菌株間QACs的MICs有顯著差異，攜帶qac基因菌株MIC值可為不攜帶該基因菌株的2 倍。質粒介導的qacG基因可使菌株對消毒劑的MIC值高于敏感菌株5 倍，且暴露在20 mg/L QAC中的存活時間高于敏感株1萬 倍[73]。pNVH01質粒上攜帶的qacJ基因陽性菌株對BC和CATB消毒劑的MIC值分別高于陰性菌株的4.5～5.5 倍[46]。

除此之外，目前尚缺乏對革蘭氏陰性細菌中qacF、qacH、qacI及sugE（p）等基因與消毒劑耐藥表型關系的研究，因此，需系統調查質粒介導消毒基因的耐藥表型，在此基礎上，確立消毒劑基因與對QACs的MICs之間的對應關系。

4.4 季銨鹽類消毒劑與抗生素耐藥基因共傳播

QACs被廣泛使用，然而QACs對抗生素抗性的潛在的篩選壓力卻很少引起關注。Soumet等[74]評估了大腸桿菌菌株反復暴露于不同QACs后，對QACs和抗生素敏感性變化的影響，研究發現，菌株同時表現對QACs、抗生素的耐藥，QACs在亞抑制濃度下的過度使用可導致對抗生素耐藥菌株的篩選，并帶來公共安全風險。QACs類消毒劑的暴露使用，發揮著篩選壓力的 作用，并可以產生共耐藥的基因，編碼對消毒劑和抗生素的共同抗性[75]。從臨床樣品分離的高水平QAC的MICs值的大腸桿菌菌株同時與抗生素抗性密切相關[76]，BC耐藥菌株對大環內酯類、苯唑西林的耐藥率明顯高于非耐藥菌[6]。細菌對消毒劑和抗生素抗性之間存在關聯，消毒劑和抗生素可以共篩選（co-selection）同時具有對消毒劑和抗生素抗性的細菌。為了生存，共篩選的微生物必須獲得對2 種以上不同抗菌物質的抗性。共篩選可以通過兩種機制藥發生，一種機制為交叉耐藥（cross-resistance），指不同的藥物對同一靶位作用或使用同一作用途徑；一般由單個外排泵介導，同時可以泵出QACs和其他抗菌物質[77-78]。如葡萄球菌中qacC基因可賦予宿主對β-內酰胺藥物的抗性[79]。另一種機制為共同耐藥（coresistance），指賦予抗性表型的基因存在于同一個移動基因元件上，比如質粒或整合子。這些基因元件包括兩個或更多的抗性基因或基因單位[80-81]。QACs和抗生素的共耐藥被認為與醫療保健、食品設備中使用QAC有關[50]。不管是交叉耐藥還是共同耐藥，最終結果是一致的：即對一種藥物抗性的發展同時伴隨對另一種藥物抗性的出現。在革蘭氏陰性細菌中，qac基因經常存在于質粒介導的1型整合子上，這些整合子同時攜帶不同的抗生素抗性基因，因此，qac基因和抗生素耐藥基因可同時表達，從而表現出共同耐藥[82]。qacEΔ1基因常發現于1型整合子上，同時包括sul I磺胺類藥物抗性基因[43,83]，qacEΔ1亦被發現與新型金屬β-內酰胺酶基因blaNDM-1同時存在于1型整合子上[84]。qacG基因與其他抗性基因blaIMP-4、aacA4、qnrB4等一起共存在沙門氏菌質粒I型整合子上[49]。sugE（p）基因發現與抗生素抗性基因blaCMY-2、sulI、aadA及tet RA等共存于大腸桿菌或沙門氏菌多重耐藥質粒IncA/C、pSN254上[39,58-60]。在可移動的基因元件上攜帶QAC耐藥基因可保證抗性通過水平基因轉移傳播于菌群中[50]。消毒劑抗性和抗生素抗性可以“共定植”，因此對其中一個篩選可導致另一個抗性的篩選[85]。

5 結 語

目前，國內外對食源性大腸桿菌耐藥基因流行特征及耐藥機制等研究尚不深入，調查食源性大腸桿菌消毒劑耐藥基因流行特征及傳播機制已經迫在眉睫，開展大腸桿菌對消毒劑耐藥流行病學及機制研究，對防控消毒劑耐與抗生素藥性與基因傳播有著重要作用，為QACs的規范使用提供依據和理論基礎，并為食品工業中食源性大腸桿菌及革蘭氏陰性菌的防控提供科學依據。

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Progress in Research on the Resistance of Escherichia coli to Quaternary Ammonium Compounds (QACs)

ZOU Li-kou1, WU Guo-yan1,2, CHENG Lin1, HE Xue-mei1,2, GUO Li-juan1,2, LONG Mei1,2
(1. Laboratory of Microbiology, Dujiangyan Campus of Sichuan Agricultural University, Dujiangyan 611830, China; 2. College of Resources and Environment, Sichuan Agricultural University, Chengdu 611130, China)

Current studies have demonstrated the serious disinfectant resistance in food-borne bacteria. Escherichia coli as an indicator bacterium for food contamination and drug resistance reveals much higher resistance to quaternary ammonium compounds (QACs) than do Gram-positive bacteria. In addition, the resistance to QACs and antibiotics can be disseminated and co-transmitted, thus resulting in co-selected disinfectant and antibiotics-resistant bacteria. Therefore, the chemical structure, species and mechanism of action of QACs, the genetic mechanism as well as genotype and phenotype underlying QACs resistance of Escherichia coli, and the mechanism of co-transmission with antibiotic resistance are summarized in this article. To date, little is known about the mechanism of disinfectant resistance in food-borne E. coli. Thus, further research on the disinfectant resistance is needed. Understanding the mechanism of QACs resistance of E. coli can provide a theoretical and scientific basis for regulating the use of disinfectants and preventing food-borne E. coli infections.

Escherichia coli; quaternary ammonium compounds; disinfectant; antibiotic resistance

Q93；TS207.4

A

1002-6630（2014）17-0338-08

10.7506/spkx1002-6630-201417063

2013-08-07

四川省教育廳重點基金項目（10ZA055）；四川省教育廳青年基金項目（13ZB0282）；教育部“長江學者和創新團隊發展計劃”項目（IRT13083）

鄒立扣（1979—），男，教授，博士，研究方向為微生物分子生物學、食品安全。E-mail：zoulkcn@hotmail.com