中华预防医学杂志    2018年10期 弯曲菌耐药机制研究进展    PDF     文章点击量:46    
中华预防医学杂志2018年10期
中华医学会主办。
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文章信息

白瑶 叶淑瑶 李凤琴
BaiYao,YeShuyao,LiFengqin
弯曲菌耐药机制研究进展
Overview of resistance mechanisms in Campylobacter
中华预防医学杂志, 2018,52(10)
http://dx.doi.org/10.3760/cma.j.issn.0253-9624.2018.10.021

文章历史

投稿日期: 2017-12-05
上一篇:金黄色葡萄球菌中可移动遗传元件与耐药传播机制研究进展
下一篇:人群队列研究的数据管理与质量控制策略
弯曲菌耐药机制研究进展
白瑶 叶淑瑶 李凤琴     
白瑶 100021 北京,国家食品安全风险评估中心卫生部食品安全风险评估重点实验室
叶淑瑶 西北农林科技大学食品科学与工程学院
李凤琴 100021 北京,国家食品安全风险评估中心卫生部食品安全风险评估重点实验室
摘要: 弯曲菌是全球范围内引发人类急性胃肠炎的主要食源性致病菌之一。虽然人类感染弯曲菌后死亡率较低,但可能导致严重的并发症如格林-巴利综合征、肠易激综合征等。氟喹诺酮类、大环内酯类等抗生素是临床治疗感染性胃肠炎的经验用药,但弯曲菌多重耐药性的出现和耐药程度的加剧给临床用药带来了严峻的挑战。本文结合国内外最新的耐药文献,选择临床治疗弯曲菌感染常用的五类抗生素,综述了弯曲菌对其产生的耐药作用机制和传播规律,以期为临床医疗和畜牧养殖业提供合理的用药方案,并为新型抗生素的药物研发提供理论依据。
关键词 :弯曲杆菌属;抗药性;外排泵;主要外膜蛋白
Overview of resistance mechanisms in Campylobacter
BaiYao,YeShuyao,LiFengqin     
Key Laboratory of Food Safety Risk Assessment of Health, China National Center for Food Safety Risk Assessment, Beijing 100021, China
Corresponding author: Li Fengqin, Email: lifengqin@cfsa.net.cn
Abstract:Campylobacter is a major cause of food-borne gastroenteritis worldwide. While mortality is low when people was infected with Campylobacter, morbidity imparted by post-infectious sequelae such as Guillain-Barré syndrome and irritable bowel syndrome is significantly noteworthy. Although fluoroquinolones and macrolides were the first line drug for the treatment of Campylobacter infections, there is a tough challenge in clinical treatment with high antimicrobial resistant rate and multi antimicrobial resistance arise. Based on the latest literature acquired in this work, we have chosen five classes of antibiotics always used in clinical, and discussed antibiotic resistance mechanisms and transmission of Campylobacter, in order to provide proper therapy both in the veterinary and human populations, and support basis data for the development of new drugs.
Key words :Campylobacter;Drug resistance;Efflux pump;Major outer membrane protein
全文

弯曲菌是目前发达国家和发展中国家普遍存在的主要食源性致病菌,也是一类常见的人兽共患病病原菌,属于微需氧的革兰阴性菌。弯曲菌属包括20多个菌种和多个亚种[1],可通过污染食物或水等多种途径而使人类患病,人类弯曲菌感染约90%由空肠弯曲菌引起,其次是结肠弯曲菌[2]。全世界每年由空肠弯曲菌引起的食源性腹泻病例达4~5亿人次[3]。弯曲菌病典型的临床表现为急性或自限性肠炎,但20%以上的患者会出现病情复发或病程延长现象,严重者可能并发格林-巴利综合征、赖特综合征及反应性关节炎、肠易激综合征等。发达国家如英国、新西兰、丹麦、瑞典等在1980—1998年期间,弯曲菌感染率同比增加了10~50倍[2]。2005年,欧洲国家弯曲菌病发病率首次超过沙门菌,2006—2011年,仅弯曲菌导致的食源性疾病每年有19万多人次,且发病率呈持续上升趋势[4]。美国每年约有200万人次患弯曲菌病,其中15%需住院治疗[5]。本文选择临床治疗弯曲菌感染常用抗生素,综述药物杀菌作用机制及弯曲菌对其产生的耐药机制,为弯曲菌病的治疗和防控提供理论依据。

一、弯曲菌的耐药机制  弯曲菌的耐药性的产生主要由基因水平转移和耐药基因获得引起,其耐药机制的遗传作用元件位于染色体上或由质粒介导,呈现内源性耐药或获得性耐药。弯曲菌常见的耐药机制包括4类:(1)抗生素靶点的改变或表达,如合成DNA酶的位点突变;(2)阻碍抗生素与靶点的结合,主要外膜蛋白(major outer membrane protein,MOMP)发挥作用等;(3)多种外排泵发挥作用,如CmeABC外排泵等;(4)对抗生素的修饰或使其失活,如β内酰胺酶的产生等。弯曲菌对临床常用五大类抗生素的耐药机制及抗生素作用机制见表1

表1弯曲菌对临床常用抗生素的耐药机制

(一)大环内酯类  大环内酯类化合物是一类具有14~16元大环内酯环的广谱抗菌药,在医疗卫生和养殖业中广泛应用[6]。大环内酯类药物可透过细胞膜进入细菌体内,与核糖体50S大亚基的P位点可逆性结合。14元大环内酯类药物的主要作用基团是德糖胺,在靠近多肽链合成的肽酰基转移酶中心,通过2-羟基与A2058、A2059位点以氢键形式结合,同时,内酯环的6-羟基或6-甲氧基、11-羟基和12-羟基各自键合A2062、U2609、U2609的位点,通过这些结合位点,阻断核糖核酸和多肽链的转移、促进转录阶段肽的早熟性解离而影响核糖体蛋白的移位、阻碍肽链延伸、抑制细菌蛋白质的合成[7]。虽然14或15元大环内酯类药物能抑制细菌核糖体蛋白移位和肽链延长,但细菌仍有一定合成短肽链的能力,而16元大环内酯类药物由于C5位上存在双糖侧链,能完全抑制肽酰基的转移,从而完全抑制核糖体合成肽链[8]。此外,大环内酯类药物还可通过抑制细菌50S大亚基的组装而抑制细菌蛋白质的合成,从而发挥抑菌作用[9]
        弯曲菌对大环内酯类药物的耐药机制主要包括核糖体靶点突变、核糖体蛋白变构、细胞膜上外排泵改变、ermB基因及相关耐药岛的存在以及核糖体RNA甲基化等[10,11,12],鲜有质粒介导倾向的报道[3]
        弯曲菌核糖体23S rRNA基因的2074和2075位点突变是导致其对红霉素产生高水平耐药的主要原因[13,14,15,16,17]。突变位点导致红霉素与弯曲菌核糖体23S rRNA亚基V区的结合部位结构发生改变,影响药物与靶点的结合,使弯曲菌对红霉素产生耐药[11, 18]。虽然弯曲菌对红霉素的耐药程度与23S rRNA基因上突变位点的拷贝数量没有直接关系[10,19],但在大多数对红霉素耐药的弯曲菌菌株中,与耐药有关的突变位点在靶基因中有二三个拷贝才能引起耐药[20],其中突变位点A2075G在高水平大环内酯类耐药株中比较常见;而位点A2074G和A2074C突变会导致核糖体结构发生轻微改变,使弯曲菌生长不佳,因此这两个位点突变导致的耐药株产生率较低[11, 21]。对红霉素耐药的弯曲菌菌株也易对阿奇霉素和克拉霉素等其他大环内酯类药物产生一定的耐药性[22],但23S rRNA基因上A2075G位点的突变对第三代大环内酯类抗生素(如泰利霉素)影响较小。
        核糖体蛋白L4上74位点(G74D)突变和L22(86或98位点有插入序列)核糖体蛋白结构的改变可导致弯曲菌对大环内酯类药物产生中低水平的耐药[10, 13,14, 21]。核糖体蛋白L4和L22结合于23S rRNA的Ⅰ区,参与维持23S rRNA的主体构象,与新生肽链的移位过程密切相关。核糖体蛋白L4及L22的突变均可使细菌多肽链通道发生改变,其中L4突变可使入口通道变窄而不能与药物结合,从而降低药物与靶点的结合能力;而L22突变则导致入口通道扩大而增大与药物结合的无效途径。核糖体蛋白变构亦可改变23S rRNA Ⅱ、Ⅲ、Ⅳ区的构象,进而影响与Ⅴ区肽转移酶中心结合的药物的抗菌活性。
        CmeABC外排泵是弯曲菌对大环内酯类产生耐药性至关重要的因素,合成CmeABC外排泵的基因发生突变可引起CmeABC蛋白过度表达,导致弯曲菌对大环内酯类药物高度耐药[13,23]。CmeABC外排泵活性的改变与弯曲菌对红霉素产生低水平耐药有关,CmeABC失活或被抑制时,可使弯曲菌对红霉素的耐药性降低2~4倍[13,23,24],并可降低对红霉素耐药的突变菌株传递率[25]。此外,CmeABC外排泵也可与修饰后的核糖体蛋白L22和L4协同作用,介导弯曲菌对大环内酯类药物高度耐药[14,25,26]
        甲基化酶ermB)基因与细菌核糖体RNA的甲基化密切相关,可介导结肠弯曲菌对大环内酯类药物高度耐药。ermB)基因可存在于细菌染色体上多重耐药基因决定区,此耐药决定区可能从革兰阳性菌水平转移而来,并可在空肠弯曲菌与结肠弯曲菌间通过自然转导方式水平传递,还可介导弯曲菌对林可胺类和链阳菌素B类耐药[12,27,28]

(二)氟喹诺酮类  氟喹诺酮类药物是一类对革兰阴性菌和阳性菌普遍有效的浓度依赖型广谱抗菌药物,如氧氟沙星、环丙沙星等是临床治疗肠道微生物感染的经验用药,其对革兰阴性菌的杀菌机制是通过作用于细菌靶点拓扑异酶Ⅱ(DNA促旋酶,由gyrAgyrB基因分别编码其两个亚单位A和B)和拓扑异构酶Ⅳ(由parCparE基因分别编码其亚单位),干扰细菌DNA的复制。在菌体内,氟喹诺酮类药物与靶位酶形成稳定的复合物,使得靶位酶与DNA分离,阻碍DNA双链的延伸,抑制细菌DNA的合成,从而发挥抑菌和杀菌的作用[29]
        弯曲菌对氟喹诺酮类药物耐药机制可分为两类,一是药物作用靶点的改变,由菌体DNA促旋酶gyrA及拓扑异构酶Ⅳ的parC基因突变引起拓扑异构酶的变化;二是菌体对药物的外排作用,通过细菌外排泵和外膜孔道蛋白改变引起[30,31,32]
        弯曲菌拓扑异构酶的改变,将会降低氟喹诺酮类药物对其的亲和力,从而降低弯曲菌对药物的敏感性。弯曲菌DNA促旋酶内gyrA基因位点突变导致的氨基酸取代包括:Thr-86-Ile,Ala-70-Thr,Asp-90-Ala和Asp-90-Asn等。与沙门菌或大肠埃希菌耐药机制不同的是,沙门菌等对氟喹诺酮类高度耐药需要gyrAparC基因突变的逐步积累,而以上基因单一位点突变即可介导弯曲菌对氟喹诺酮类药物不同水平耐药,其中Thr-86-Ile突变最为常见,可介导弯曲菌对萘啶酸和氟喹诺酮类药物高度耐药,而Thr-86-Ala突变较少见,且仅介导弯曲菌对萘啶酸耐药,并不介导对氟喹诺酮类耐药[33]gyrA基因中Asp-90-Asn和Ala-70-Thr突变可介导弯曲菌对氟喹诺酮类药物中度水平耐药[34,35]。此外,弯曲菌还存在gyrA基因Thr-86-Ile和拓扑异构酶Ⅳ的parC基因Arg-139-Gln协同突变,可介导对氟喹诺酮类药物的高度耐药[36]。弯曲菌gyrB基因突变也有报道,但与氟喹诺酮耐药无关[37,38,39]。另有研究发现,有些空肠弯曲菌和结肠弯曲菌缺乏parCparE基因,菌体内缺乏氟喹诺酮类药物的作用靶点,因此不会对其产生耐药[37,38,40,41]
        CmeABC外排泵的作用可降低菌体内氟喹诺酮类和其他多种药物的浓度,在弯曲菌的固有耐药和获得性耐药中都发挥了重要的作用[42]。弯曲菌CmeABC外排泵和DNA促旋酶的协同作用,可介导对氟喹诺酮类药物高度耐药[30,31,32]。在低水平耐药的弯曲菌中,未见CmeABC外排泵和gyrA基因位点突变同时存在的现象[31]。CmeABC外排泵的存在有助于gyrA基因位点Thr-86-Ile突变在短时间内发生,且能在无抗菌药物条件下稳定存在,比敏感菌更具有生存优势[43]。与其他革兰阴性菌中CmeABC外排泵过表达才导致耐药不同的是,弯曲菌中CmeABC外排泵只要正常表达,即可对氟喹诺酮类药物产生中度耐药。CmeABC的敲除可降低弯曲菌对氟喹诺酮类药物的耐药水平,其过表达则增强对氟喹诺酮类药物的耐药程度[31]
        研究推测CmeG外排泵也可导致弯曲菌对氟喹诺酮类耐药,插入突变序列的CmeG外排泵可导致弯曲菌对环丙沙星耐药性降低4倍,而CmeG过表达则可导致菌株的耐药性增加8~32倍[44]

(三)β-内酰胺类  β-内酰胺类药物包括临床最常用的青霉素类、头孢菌素类、碳青霉烯类和单环β-内酰胺类等,具有杀菌活性强、毒性低、适应证广及临床疗效好的优点,其杀菌机制是通过结合在细菌的肽聚糖转肽酶上,破坏细胞壁结构的完整性,导致细胞渗透膨胀、菌体裂解死亡。
        弯曲菌对β-内酰胺类药物的耐药机制可分为3类:一是细菌染色体编码的β-内酰胺酶失活,二是外膜孔蛋白结构的改变,三是外排泵的作用。弯曲菌中青霉素酶类的β-内酰胺酶表达,可介导对青霉素、阿莫西林、替卡西林等抗生素耐药,但可被克拉维酸、舒巴坦等β-内酰胺酶抑制剂类药物抑制[45]。青霉素酶类不影响弯曲菌对碳青霉烯类和头孢菌素类的敏感性[45]
        与弯曲菌对大环内酯类的耐药机制相似的是,弯曲菌中阳离子选择型的MOMP可以排出多数分子量大于360 KDa的阳离子型β-内酰胺酶或者阴离子型β-内酰胺酶。分子结构发生局部改变的药物和小分子量的亚胺培南(299 KDa)和氨苄西林(333 KDa)等可通过MOMP,因此弯曲菌对这些抗生素敏感,而阿莫西林(365 KDa)则可能部分通过MOMP,或通过非MOMP依赖性的机制介导其进入菌体内[46]
        CmeABC是对β-内酰胺类药物最主要的外排泵,CmeB外排泵的作用可使弯曲菌对β-内酰胺类药物产生耐药。空肠弯曲菌中CmeB突变,可使得菌株对氨苄西林的敏感性增加4~32倍,而CmeB过表达则使得菌株耐药性增加4倍[42,47]。插入突变的CmeE导致弯曲菌CmeDEF外排泵失活,则对氨苄西林和头孢噻肟耐药性增加2倍[48],但CmeG的失活不影响弯曲菌NCTC11168对头孢噻肟的耐药性[44]

(四)四环素类  四环素类药物是一类重要的广谱活性抗生素,对多数革兰阴性菌和阳性菌广泛有效。此类药物是亲脂性蛋白合成抑制剂,通过疏水性途径进入菌体内,与细菌核糖体30S亚基发生可逆性结合,阻碍氨酰-tRNA和核糖体A位点结合,导致肽链延伸终止,抑制新生多肽链的合成,从而发挥抑菌和杀菌的作用。
        弯曲菌对四环素类药物的耐药机制可分为核糖体靶位点靶点的改变和外排泵作用两大类。弯曲菌中普遍存在tetO基因,可存在于质粒上或染色体上[17, 49],其序列与革兰阳性菌中该基因同源性高达99%以上,且G-C含量高于弯曲菌,推测该基因可能由革兰阳性菌水平转移而来[50]tetO基因编码核糖体保护蛋白TetO,识别被四环素结合的核糖体A位点,并与未占位的A位点结合,发挥保护核糖体的作用,使得四环素类药物缺少与核糖体结合的靶点,导致弯曲菌对四环素类耐药[51,52]
        细胞膜上的外排泵与tetO基因的协同作用,增加了对四环素的耐药程度。CmeG外排泵突变的弯曲菌对四环素敏感程度增加四倍[44]。CmeB基因突变导致CmeABC外排泵失活的弯曲菌对四环素敏感程度增加8~64倍[42,53,54]

(五)氨基糖苷类  氨基糖苷类药物是治疗需氧革兰阴性杆菌严重感染的重要药物[55],其发挥作用的机制分为两类:一是与细菌核糖体结合,作用于肽链合成初期,阻碍肽链从A位点到P位点的移位,导致肽链合成提前终止,从而抑制细菌细胞膜蛋白质的合成,改变细胞膜的完整性。二是干扰肽链合成后期的校对功能,导致错误的氨基酸合并,核糖体蛋白错误表达,形成结构功能异常的蛋白[56]
        弯曲菌对氨基糖苷类的耐药通常由质粒介导,主要通过氨基糖苷磷酸转移酶(Aminoglycoside phosphate transferase, APH)和氨基糖苷乙酰转移酶(Aminoglycoside acetyltransferase, AAD)等氨基糖苷类修饰酶的作用,对抗生素进行修饰,使其减少与核糖体的结合能力。目前在弯曲菌中已经发现四种亚型的APH,分别为APH Ⅰ、Ⅲ、Ⅳ、Ⅶ亚型。其中APH第Ⅲ亚型首次发现于结肠弯曲菌的质粒上,在对链霉素和链丝菌素耐药的菌株中也存在此亚型,是弯曲菌对氨基糖苷类耐药的主要因素[57]。与肠球菌中存在相似的耐药决定簇证明,弯曲菌可能通过水平转移获得此类耐药基因[57]。弯曲菌菌株的质粒上还有许多和tetO基因一起转移来的氨基糖苷类的耐药基因,分别源自革兰阴性菌(如幽门螺杆菌、大肠埃希菌和沙门菌)和革兰阳性菌(如肠球菌)[57,58,59,60,61,67]。对卡那霉素耐药的弯曲菌质粒上存在APH Ⅰ和Ⅶ亚型[57,62,63]。与APHⅠ和Ⅲ型通过水平转移方式而获得性耐药不同,APH Ⅶ亚型被认为是弯曲菌固有存在的,因为它与弯曲菌染色体的基因组G-C含量相似[64]
        弯曲菌染色体上点突变引起的对链霉素耐药仅在结肠弯曲菌中发现,并且这种突变可以通过自然结合的方式转移给其他菌株[54,65],空肠弯曲菌中未见相似突变。
        外排泵在弯曲菌对氨基糖苷类抗生素耐药的作用中尚不明确。外排泵CmeG中插入突变序列,可使得CmeG突变的庆大霉素耐药株的最小抑菌浓度(Minimum inhibitory concentration, MIC)值降低16倍,但对链霉素的耐药性却没有影响,且CmeG过表达并未导致弯曲菌对氨基糖苷类药物耐药性增加[44]

(六)天然耐药性  天然耐药性又称为固有耐药性,是某种细菌固有的特点,可能由于此类细菌具有天然屏障,药物无法进入细菌体内或由于细菌缺少对药物敏感的靶位所致。弯曲菌对新生霉素、头孢哌酮、多粘菌素、利福平、万古霉素等抗生素天然耐药。

二、结语  综上所述,弯曲菌对临床常用药物的耐药机制主要包括染色体突变、核糖体结合蛋白改变、外排泵作用、对药物进行修饰等,阻碍药物进入细菌体内或者阻碍药物与靶点结合而发挥作用。近年来,随着养殖业和医疗中抗生素的过度使用[66,67,68,69],细菌在药物选择压力下可能产生多重耐药性突变,并可能通过水平转移产生各种复杂的耐药机制[70,71,72]。目前全基因组测序技术预测弯曲菌中可能潜在14种外排泵,但仅有CmeABC和CmeG的功能被研究,其他外排泵在耐药机制中的作用有待进一步发现。阐明弯曲菌的耐药机制,对弯曲菌病的防控和新型药物的研发具有重要意义。

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