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文章信息
- 王于莉, 姜昕汝, 郑月娟
- WANG Yu-Li, JIANG Xin-Ru, ZHENG Yue-Juan
- 外排泵介导肺炎克雷伯菌耐药的研究进展
- The progress on drug resistance of Klebsiella pneumoniae caused by efflux pump
- 微生物学通报, 2016, 43(6): 1351-1357
- Microbiology China, 2016, 43(6): 1351-1357
- DOI: 10.13344/j.microbiol.china.150982
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文章历史
- 收稿日期: 2015-11-28
- 接受日期: 2016-03-01
- 优先数字出版日期(www.cnki.net): 2016-03-10
肺炎克雷伯菌是导致医院内感染的重要致病菌[1]。2012 中国CHINET 耐药监测显示以肺炎克雷伯菌为主的克雷伯菌属耐药菌占革兰阴性耐药菌的18.49%[2]。该菌一般感染免疫系统受损的病人,继而导致严重的感染,如伤口感染、尿路感染、 肺炎、败血症和脑膜炎等[3-4]。近年来,随着激素、 免疫抑制剂和广谱抗生素的广泛使用,肺炎克雷伯菌的耐药情况更加严峻[5]。肺炎克雷伯菌的耐药机制包括:产生灭活抗菌药物的酶(如碳青霉烯酶)、 改变抗菌药物作用的靶标、阻碍抗菌药物的渗透、 促进外排泵系统的表达等[6]。其中,外排泵系统的表达是该菌产生耐药性的重要机制[7]。细菌的外排泵系统包括: 耐药结节化细胞分化家族 (Resistance-nodulation-division family,RND)、ATP 结合盒家族(ATP-binding cassette family,ABC)、主要易化子超家族(Major facilitator superfamily ,MFS)、小多重耐药家族(Small multidrug resistance family ,SMR) 和多药及毒性化合物外排家族 (Multidrug and toxic compound extrusion family,MATE)[8]。本文将对外排泵介导肺炎克雷伯菌的耐药现状、外排泵的分子结构和基因水平的调节及主要外排泵抑制剂的研究进展做一简要综述。
1 外排泵介导肺炎克雷伯菌耐药的现状同一株细菌可存在多种外排泵,外排不同种类的抗生素如内酰胺类、喹诺酮类和大环内酯类等,不同外排泵的外排强度不同导致细菌对多种抗生素产生耐药。王亮等[9]发现在肺炎克雷伯菌中 AcrAB-TolC外排泵对喹诺酮类抗生素环丙沙星的外排强度最大,由此导致的耐药率达33.8%。Pakzad 等[10]在烧伤病人身上分离出52株肺炎克雷伯菌,由于外排泵AcrAB的作用,导致40株肺炎克雷伯菌对环丙沙星耐药,耐药率达76.9%。Yuan等[11]研究发现肺炎克雷伯菌对环丙沙星和左氧氟沙星的耐药率分别为66.9%和50.7%,且所有耐药肺炎克雷伯菌株均携带外排泵基因。刘军等[12]的研究则表明肺炎克雷伯菌对阿奇霉素、四环素和氯霉素等产生耐药性与携带AcrAB-TolC外排泵密切相关,其中对内酰胺类抗生素头孢他啶的耐药率最高,达76.82%,对其他抗生素如阿奇霉素、四环素和氯霉素的耐药率均超过50%。蒋燕群等[13]也发现对头孢他啶耐药的肺炎克雷伯菌株中,外排泵基因转录水平增高的菌株占样本总量的51.9%,说明因外排作用导致的耐药所占比重很大。
2 常见的外排泵系统及其结构目前,在肺炎克雷伯菌中已经发现的外排泵有 RND 家族的AcrAB-TolC 和KexD,MFS 家族的 KmrA、KpnGH 和KdeA,以及SMR 家族的KpnEF。
2.1 耐药结节化细胞分化家族(Resistancenodulation- division family,RND)RND 外排泵是以细胞膜两侧的质子差作为驱动势,将药物排出体外。它由3 个蛋白结构域构成,分别是内膜蛋白、膜融合蛋白和外膜蛋白。外排泵 AcrAB-TolC 的检出率最高,其中,AcrA 是膜融合蛋白,横跨细胞膜,连接内膜蛋白AcrB 和外膜蛋白TolC,使之形成一个贯穿细胞膜的密封通道,将药物由胞内运输到胞外[14]。内膜蛋白AcrB 的主要作用是摄取并转运特异的底物,外排多种抗生素如喹诺酮类、大环内酯类和内酰胺类等,还能为整个 AcrAB-TolC 泵提供能量[15]。外膜蛋白TolC 的序列相对保守,可与不同的膜融合蛋白结合,形成多种外排泵的外膜通道[16]。当TolC 基因缺失时,外排作用减弱,细菌对药物的敏感性也随之显著增加[17]。 有研究表明,移除肺炎克雷伯菌的AcrAB-TolC 泵后,细菌的耐药性、适应性以及毒力都受到影响[18]。 肺炎克雷伯菌对喹诺酮类药物高度耐药和对碳青霉烯类药物的敏感性下降都与外排泵AcrAB-TolC 有关[19]。Ogawa 等[20]发现外排泵KexD 的氨基酸序列同AcrAB-TolC 外排泵家族成员的内膜蛋白AcrB 高度相似,也可以连接膜融合蛋白AcrA,并利用外膜蛋白TolC 将各种抗生素(如青霉素、罗他霉素等)泵出菌体。
2.2 主要易化子超家族(Major facilitator superfamily,MFS)MFS外排泵家族是由编码跨膜结构的基因重复表达而形成的多次跨膜转运蛋白复合物(通常为 12或14次跨膜)。质子交换为MFS家族转运底物提供能量,外排底物包括多种抗生素,如四环素类、氯霉素类和氨基糖苷类。kmrA编码KmrA外排泵,Ogawa等将该基因在肺炎克雷伯菌株MKG78578中高水平表达,工程菌株可外排红霉素和四环素等多种抗生素,但该基因并不因特异性底物的存在而增加表达量[21]。kpnG和kpnH编码KpnGH外排泵,Srinivasan等发现插入失活的kpnGH基因片段,可以增加肺炎克雷伯菌对抗生素(如阿奇霉素、头孢他啶、环丙沙星、厄他培南、多粘菌素B、链霉素等) 的敏感性[22]。耐药肺炎克雷伯菌株MGH78578的特定基因kdeA编码KdeA外排泵,该工程菌株可外排氯霉素和卡那霉素等多种抗生素,但KdeA外排泵在肺炎克雷伯菌株ATCC10031中的外排作用不明显[23]。
2.3 小多重耐药家族(Small multidrug resistance family,SMR)SMR 家族成员通常由110 个左右的氨基酸残基组成,一般由4 个跨膜结构域和一个α螺旋组成,是分子量最小的一类外排泵。SMR 发挥外排功能需要质子交换和构象改变,即氨基酸序列较保守的跨膜结构域与底物结合区协同作用,共同运输药物等小分子。Srinivasan 等用特定外排基因kpnE 和kpnF 编码外排泵KpnEF ,其在肺炎克雷伯菌株 NTUH-K2044 中能识别多种底物,如头孢曲松、粘菌素、红霉素、四环素和链霉素等。当kpnEF 基因被敲除时,KpnEF 泵对上述抗生素的敏感性降低。 在同样的抗生素环境中,当使用外排泵抑制剂氰氯苯腙(CCCP)后,细菌萎缩及死亡情况明显增加,该现象在临床分离株中也得到验证[24]。
3 主要外排泵的基因水平调节及外排泵抑制剂 3.1 主要外排泵的基因水平调节关于外排泵基因水平调节的研究开展得不多,并且主要集中在AcrAB-TolC泵。AcrAB-TolC由基因acrA和acrB编码构成,它们上游具有共同的操纵子序列marRAB,marRAB由启动子marO、阻遏基因marR、和转录激活基因marA、marB组成 。
正向调控AcrAB-TolC外排泵的基因有soxS、 marA、ramA和rob等。soxS可以编码操纵子marRAB,促进AcrAB和TolC的表达,从而提高对抗生素的外排水平。marA则能特异性结合启动子marO,增强 RNA聚合酶与启动子的亲合力,提高转录起始的速率,增加AcrA和AcrB的表达[25]。ramA结合位点位于AcrAB基因的上游,促进AcrAB的表达从而提高肺炎克雷伯菌的外排抗生素的能力[26-27],如喹诺酮类[28]、β内酰胺类[29]以及甘氨酰四环素类(替加环素)[30]。rob的过表达会促进marRAB等8个目的基因表达,从而激活内膜蛋白AcrB,导致AcrAB-TolC 的外排作用亢进[31]。
负向调控AcrAB-TolC外排泵的基因有acrR和 marR。其中,AcrR由阻遏基因acrR编码,与AcrAB 启动子上的反向重复序列(24个碱基对组成)结合,抑制AcrAB的表达。marR是操纵子marRAB的转录阻遏基因,与上游启动子marO结合后,负向调节 marRAB的转录,从而抑制AcrAB的表达。
另外,de Majumdar 等发现基因rarA,rarA 也可以调节acrAB,进而提高AcrAB外排泵的表达[32]。
3.2 外排泵抑制剂外排泵过度表达是细菌产生耐药的重要原因和方式,因此,外排泵被认为是治疗耐药菌的潜在作用靶点。国内外研究已经证实有效并展开深入研究的外排泵抑制剂有邻氨基苯甲酸的衍生物 (Tariquidar ,XR9576) ,三酸盐(Zosuquidar ,LY335979),维拉帕米和氰氯苯腙(CCCP)等。虽然某些抑制剂已经进入临床试验阶段,但是大部分抑制剂仍停留在临床前基础研究阶段(药理学研究和毒理学研究)。除了具有抑制剂功能的临床药物如维拉帕米、利血平等,目前还没有成功应用于临床的外排泵抑制剂。究其原因,主要是因为这些抑制剂的使用剂量较大,副作用大,对机体存在毒性伤害。 目前,已发现多种植物(传统中药占有相当大的比重) 来源的成分有抑制细菌产生耐药和消除耐药性等作用,现在对植物来源的外排泵抑制剂的相关研究已经逐步开展起来。下面根据不同的作用机制来介绍几种外排泵抑制剂。
3.2.1 干扰外排泵组装的抑制剂:格罗泊霉素 (Globomycin)是来自链霉菌的一种环肽抗生素,能够抑制膜融合蛋白AcrA的信号肽酶LspA,而LspA 是AcrA的一个剪切位点,该位点被抑制后会影响 AcrA的成熟,干扰AcrAB-TolC外排泵的组装,减少药物的外排。格罗泊霉素仅仅在RND家族的外排泵成员中发挥抑制作用,这限制了它的使用范围[33]。
3.2.2阻断外排泵能量来源的抑制剂:聚乙二醇 1000维生素E琥珀酸酯(TPGSs1000)通过降低ATP 的水解能力,减少外排泵的能量来源,发挥对P-gp 泵(ATP结合盒家族,ATP-binding cassette family,ABC家族的重要成员)的抑制作用,进而减少药物的外排,目前仅停留在临床前基础研究阶段[34];而邻氨基苯甲酸的衍生物(Tariquidar,XR9576)是特异性、非竞争性的结合在P-gp的ATP结合位点上,通过抑制ATP酶活性而减少ATP的水解[35-37]。从 Tariquidar的临床试验效果来看,它可持续发挥对外排泵的抑制作用,而且机体对其耐受性较强,已经通过一期临床安全性评价[38-39]。外排泵抑制剂可以与抗生素发挥协同作用,增加耐药菌对抗生素的敏感性[40]。三酸盐(Zosuquidar,LY335979)是喹啉类衍生物,兼具高效性和高选择性,在治疗老年急性髓性白血病临床试验中具有减少药物外排,延长生存期的作用[41],且其与阿霉素联合治疗肿瘤的一期临床试验已经完成[42]。另外,一些外排泵抑制剂也可以通过抑制ATP 盒的活性[43] 或者直接消耗 ATP[44],来减少P-gp泵的能量来源。
3.2.3 阻碍底物通过外排通道的抑制剂:维拉帕米是罂粟碱的衍生物,作为钙离子通道阻滞剂,临床作为冠脉扩张剂,用于治疗高血压、心绞痛等疾病。 研究显示其可用于肺炎克雷伯菌耐药的逆转,当红霉素中加入维拉帕米后,其最小抑菌浓度减小到原来的1/8[45],但在作为外排泵抑制剂使用时所需剂量较高,容易发生心血管系统的不良反应,从而限制了其临床应用。Cholkar 等研究发现消退素E1 衍生物(RX-10045)可以作为P-gp 的底物,同抗菌药物竞争性结合底物结合位点而抑制药物的外排,目前处在临床前基础研究阶段[46]。Fadli 等从摩洛哥百里香(Thymus maroccanus)中提取了一种精油,能逆转包括肺炎克雷伯菌在内的部分革兰氏阴性菌的耐药性,并提示该精油能够阻塞菌体的外排泵通道,减少药物的外排[47]。最近有研究发现其具有抗真菌作用[48],该精油临床应用的价值较高,尚待进一步的研究。
4 传统中药开辟应对外排泵引起的细菌耐药的新途径传统中药汉防己具有解热、镇痛和抗炎等多种功能,其有效成分粉防己碱可以竞争性结合或者遮掩外排泵的底物结合位点,从而抑制AcrAB-TolC 的外排作用[48]。连翘脂苷B也可以抑制肺炎克雷伯菌外排泵的活性[49]。另外,由于肺炎克雷伯菌和大肠埃希菌同属肠杆菌科细菌,且高度同源,治疗外排泵引发的耐药性大肠埃希菌的中药及有效成分也可为治疗外排泵引发的肺炎克雷伯菌耐药提供参考,比如:青蒿琥酯(Artesunate,AS)在浓度为 512 mg/L时可降低大肠埃希菌外排泵相关基因 acrA、acrB的表达水平,且与氨苄西林联用时可明显减少AcrAB-TolC上游正向调控基因soxS和marA 的表达量进而抑制外排泵AcrAB-TolC的表达[50]。中药黄连在亚抑菌浓度下可改变外排基因acrA的部分碱基,从而影响acrA基因mRNA的表达、减少大肠埃希菌对四环素类、氯霉素类和喹诺酮类抗生素的耐药[51]。
传统中药的作用不仅仅在于杀菌、抑菌,还具有“调节整体、改善局部”的特色与优势,通过调节神经内分泌免疫三大调节系统中的激素、细胞因子来发挥整体的免疫效应[52-54]。在细菌感染过程中,从调控机体免疫反应角度入手,绕开一味地用抗生素给病原体施加筛选压力,最终达到抑制过度炎症反应、提高机体免疫力、促进免疫细胞对病原体的吞噬、抗原提呈,并发挥后续的适应性免疫反应,以达到清除病原体的目的。比如,麻黄附子细辛汤联合抗生素在抗肺炎克雷伯菌体外实验中效果明显[55];扶正颗粒在正常小鼠及免疫抑制小鼠的肺炎克雷伯菌感染中均具有良好的防治作用,这一现象与其提高机体免疫能力有关[56]。该治疗策略既可以减缓临床用药对耐药菌的筛选压力,又能为广谱耐药菌感染的患者提供新的治疗方案和思路。
5 展望(1) 外排泵引起肺炎克雷伯菌的耐药现状日趋严重,同时外排泵抑制剂并未真正在临床展开应用。针对性地开发结构稳定、无明显细胞毒性的外排泵抑制剂,有利于恢复或增强抗生素的活性,有望联合抗生素治疗外排泵引发的肺炎克雷伯菌感染[57-58]。(2) 从纳米粒子、纳米胶束和脂质体等层面研发抗生素新剂型,可以规避药物外排作用、提高药物的敏感性、减少给药量和降低不良反应等。(3) 加大对调节机体免疫反应的传统中药的研发,发挥其增加机体免疫或抗炎等作用,绕开病原体的耐药性问题,为治疗外排泵引起的耐药菌提供可替代方案。
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