Advances in Microbiology
Vol.06 No.04(2017), Article ID:22932,10 pages
10.12677/AMB.2017.64013

The Research Progress on Occurrence of Candida Drug Resistance

Wanrui Fu, Lingzhi Li, Xin Zhong, Yinghan Hu, Xiaofeng Zhu, Zhangyong Song*

College of Preclinical Medicine, Southwest Medical University, Luzhou Sichuan

Received: Nov. 17th, 2017; accepted: Nov. 29th, 2017; published: Dec. 6th, 2017

ABSTRACT

In recent years, the morbidity and mortality of candidiasis are increasing rapidly. At the same time, the extensive use of antifungal agents has led to the development of drug-resistant strains. This review summarizes the clinical symptoms, the development of detection methods, the development and resistance mechanism of drug-resistant strains, and the development and usage of antifungal agents so as to provide a basis for diagnosis and treatment of candidiasis.

Keywords:Candidiasis, Multiresistance, Mechanism of Multiple Antibiotic Resistance

临床上念珠菌耐药性发生的研究进展

付婉瑞,李凌志,钟鑫,胡莺菡,朱晓凤,宋章永*

西南医科大学基础医学院,四川 泸州

收稿日期:2017年11月17日;录用日期:2017年11月29日;发布日期:2017年12月6日

摘 要

近年来,临床上以念珠菌为代表的侵袭性真菌感染的发病率和致死率呈急剧上升趋势,且临床菌株的耐药性也随之发生且日益严重。本文综述了念珠菌的临床症状、检验技术的发展、耐药菌株的发生和耐药机理研究现状和抗真菌药物的使用和开发,为诊断和治疗临床上日益严重的念珠菌病提供依据。

关键词 :念珠菌病,多重耐药性,耐药机理

Copyright © 2017 by authors and Hans Publishers Inc.

This work is licensed under the Creative Commons Attribution International License (CC BY).

http://creativecommons.org/licenses/by/4.0/

1. 引言

近年来,临床上以念珠菌为代表的侵袭性真菌病的发病率和致死率呈逐年上升趋势 [1] ,侵袭性真菌感染除发生在免疫力低下者外,也发生在免疫功能正常的人群中 [2] 。耐药性菌株的出现、有效诊断技术的缺乏和新抗真菌药物的缺少是造成目前侵袭性真菌难以防治的根本原因。本文将对念珠菌的临床症状、检验技术的发展、临床上耐药菌株的发生及其机理及抗真菌药物的使用和新方向开发进行概述,旨在为诊断和治疗念珠菌病的发生提供依据。

2. 念珠菌的临床症状

近年来,因为老年病人和慢性病人的增多,或者免疫缺陷病人增多,特别是艾滋病患者的增加,更或是抗生素的滥用、免疫抑制剂使用增多以及各种侵袭性治疗等因素导致念珠菌感染正呈上升趋势。念珠菌(Candida spp.),又称假丝酵母菌,是人体中常见的机会致病真菌,常寄生于人体体表或口腔、肠道、阴道等黏膜系统处。在免疫力低下或正常寄居部位菌群失调的人体内增加念珠菌感染的概率 [1] 。

目前医院内侵袭性真菌感染最常见的是念珠菌感染,主要以白色念珠菌(C. albicans)、光滑念珠菌(C. glabrata)和热带念珠菌(C. tropicalis)为主,极少数的近平滑念珠菌(C. parapsilosis)、假热带念珠菌(C. pseudotropicalis)和季也蒙念珠菌(C. guilliermondi)等 [3] [4] ,其中白色念珠菌所占比例最高。近年来,临床上白色念珠菌的分离率有所下降,非白色念珠菌(如光滑念珠菌)的分离率则有所升高 [4] [5] 。

念珠菌病主要引起以下几种类型疾病:① 皮肤念珠菌病:引起皮肤浸渍发白,去除浸渍表皮其基地潮红、有渗液,微痒或疼痛感的指(趾)间糜烂症状;在腋窝等褶皱部位引起丘疹、疱疹或脓疱的念珠菌性间擦疹等症状;② 粘膜念珠菌病:在口腔可引起鹅口疮,表现为粘膜、咽、舌、牙龈等处出现边界清楚的白色假膜和外围红晕,以及可能引起口角糜烂、皲裂等症状;在生殖器黏膜处可引起生殖器念珠菌病,表现为女性阴道壁上可见灰白色假膜样斑片,有瘙痒或灼热感;男性包皮及龟头潮红,包皮内侧及冠状沟可见覆有假膜斑片;③ 内脏念珠菌病:以肠念珠菌病或肺念珠菌病较常见的深度感染,甚至严重的可涉及全身内脏器官。肺念珠菌病包含单发,新旧病灶并存,或多发粟粒样结节影的肺型和沿支气管血管束分布的片状影的支气管型。目前随着现代诊疗技术的发展,肺部的真菌感染率显著增加,但由于其临床症状不明显且缺乏早期诊断特异性的指标,严重影响着治疗。

3. 念珠菌的检测技术

针对临床上念珠菌病的发生,传统的检测技术有直接镜检、培养、血清生化检查、分子生物学方法、组织病理检查等。通过分离纯化培养菌株,挑取可疑菌落进行KOH溶液处理后,在显微镜下直接观察,可清楚观察到较为清晰的分隔菌丝及厚壁孢子,进而观察其菌落形态可初步进行鉴定。通过对患者血清中白色念珠菌抗体的检测,也可达到检测的目的,但因人体内存在许多甘露聚糖抗体,影响了检测的特异性,使得敏感度较低和假阳性率较高 [6] 。采用免疫胶体金技术进行检验:将特异性抗体固定于硝酸纤维膜的某一区带,当样品中真菌的抗原通过毛细作用,与固体抗体特异结合时,该区域呈现一定的颜色,即为阳性 [7] 。患者血清中1-3-β-葡聚糖、阿拉伯糖醇、烯醇的含量变化也作为侵袭真菌早期感染的检测指标 [8] 。此外,PCR检测技术具有快速、灵敏度高和特异性强的优点,是较为常见的检测方法之一 [6] 。

高通量测序技术的发展和应用使得真菌基因组数据呈爆炸式增长,为PCR、基因探针等分子检测技术提供了有用的数据参考。基于多种方法的联合使用可以有效提高检测敏感度和特异性 [9] 。病理组织观察可观察病理组织的变化和病原体,是真菌感染诊断的金标准,但不同染色方法步骤的差异和组织对试剂的敏感性差异,对镜下观察和鉴定影响较大 [10] 。除上述方法外,临床上为节省检测时间,采用MALDI-TOF (Matrix-assisted laser desorption ionization-time of flight) MS (mass spectrometry)技术进行快速检测 [11] ,但对于目前临床上出现的耐药菌株以及耐药特性,尚不能够快速准确鉴别 [12] 。采用定量PCR结合等位基因特异分子检测技术为耐药性菌株的检测提供了可能 [13] 。

4. 念珠菌的耐药现状

随着HIV、肿瘤化疗和器官移植等发生发展,念珠菌病的发生率在逐年上升,念珠菌主要引起真菌血症、泌尿系统感染和全身性感染。在医院血源感染中位于第四位,病死率居第一位,其中ICU最常发生 [14] [15] 。目前临床上光滑念珠菌的检出率明显增加,其在某些地区医院已成为仅次于白色念珠菌的第2位或第3位的菌 [4] [5] 。

不规范使用和滥用抗真菌药物导致了临床上耐药性念珠菌的不断出现。临床上首选氟康唑等唑类药物对念珠菌进行抗真菌治疗。由于氟康唑的选择压力作用,使得耐氟康唑的白色念珠菌不断增多,另外,也导致非白色念珠菌感染及其耐药性日趋严重 [5] [12] [16] 。念珠菌对三种唑类药物氟康唑、伏立康唑和伊曲康唑的耐药率调查显示:白色念珠菌9.6%、0.0%、16%,近平滑念珠菌19.3%、3.6%、39.8%,热带年念珠菌6.0%、0.0%、31.3%,光滑念珠菌4%、6%、4% [14] 。另一研究机构调查显示:念珠菌对伊曲康唑和伏立康唑均具有较强的耐药性,耐药率分别达19.7%和15.8% [17] 。2010、2011和2012年常见念珠菌对氟康唑、伏立康唑的敏感性显示,非白色念珠菌的耐药明显高于白色念珠菌 [14] 。南京军区总医院对2010~2012年间的非白色念珠菌感染情况进行调查,发现非白色念珠菌对氟康唑、伏立康唑和伊曲康唑的耐药率均逐年上升,分别高达74%、21.7%和26.7%,其中光滑念珠菌尤为突出 [18] 。面对如此严峻的耐药形式,寻找合理的抗真菌方法或药物迫在眉睫。

5. 念珠菌的耐药分子机制

念珠菌的耐药性问题日益严重,与此同时其耐药机理也逐渐研究透彻,主要体现在以下5个方面:1) 增强药物外排泵的表达;2) 药物靶酶过表达或突变;3) 药物靶标合成减少;4) 生物膜形成;5) 其他耐药途径。阐明耐药菌株的耐药性机理将有助于指导临床合理用药以及为新抗真菌药物的开发指明方向。

5.1. 增强药物外排泵的表达

念珠菌减少胞内药物浓度的机制之一是增强药物外排泵的表达,此机制主要体现在耐唑类药物方面。念珠菌细胞膜上有两种重要的耐药蛋白:ABC转运蛋白超家族(ATP binding cassette transporters, ABCT)和主要易化扩散载体超家族(major facilitator super-family, MFS),两者都具有药物外排功能。念珠菌基因组上的ABCT家族能编码相应蛋白:Cdr1、Cdr2 (白色念珠菌),CgCDR1,CgCDR2,CgSNQ2 (光滑念珠菌),CpCdr1 (近平滑念珠菌),CkAbc1 (克柔念珠菌),CdCDR1 (都柏林念珠菌),与氟康唑耐药有关 [19] [20] 。研究发现白色念株菌的Cdr1对氟康唑耐药性的影响比Cdr2p更大 [21] [22] 。而且Cdr1和Cdr2的表达是受锌簇转录因子Tac1 (Transcriptional activator of CDR)基因调控,目前已发现Tac1基因中有19种获得性功能(Gain-of-function, GOF)突变与氟康唑耐药性有关 [23] [24] 。

白色念珠菌、近平滑念珠菌与都柏林念珠菌的MFS中,Mdr1是与耐唑类药物有关的蛋白 [25] [26] [27] 。其受一种锌簇转录因子Mrr1的调控。研究表明Mrr1蛋白失活后会导致白色念珠菌对氟康唑耐药性的丧失 [28] 。在没有额外刺激的情况下,Tac1与Mrr1的GOF突变就能使转运蛋白编码基因处于活跃状态和固有的高水平表达 [29] [30] 。在光滑念珠菌中,Tpo3p转运蛋白与Pdr1转录因子与唑类耐药有关 [31] [32] ,但在其他的念珠菌中,尚未发现明显与唑类耐药相关的MFS转运蛋白。

5.2. 药物靶酶过表达或突变

由ERG11基因编码的羊毛甾醇14α-去甲基化酶(14DM)在真菌细胞膜成分-麦角甾醇合成途径中发挥关键作用。唑类药物通过与14DM结合发挥作用。ERG11基因的过表达能使14DM产生增多,从而保证在唑类药物作用下仍有足够14DM参与麦角甾醇合成,进而导致唑类药物耐药性的产生。锌簇转录因子Upc2调控ERG11基因的表达,其GOF突变是导致白色念珠菌ERG11基因上调表达的常见原因 [33] 。热带念珠菌耐唑类药物菌株中存在过表达ERG11基因,但光滑念珠菌与克柔念珠菌中,ERG11基因的过表达对耐药性的产生影响不大 [20] 。

ERG11基因突变是念珠菌耐药性产生的另一重要机制。在白色念珠菌ERG11基因中已发现超过140种大多在105~165,266~287和405~488位点的与唑类耐药有关的氨基酸置换 [34] [35] 。氨基酸的置换可通过另外两种途径引起唑类药物的耐药:1) 氨基酸置换位点位于与唑类药物结合的14DM位点,突变导致药物与靶酶的亲和力降低,如:V437I、S453F、N490K,Y132F,Y132H [36] [37] ;2) 14DM的空间结构改变,药物不易与靶酶结合,如:V51G,C75W,Y79D,V94G,T123I [37] [38] 。ERG11基因突变(Y132F位点)也在非白色念珠菌(如近平滑念珠菌与热带念珠菌)的耐唑类药物菌株中被发现 [26] [39] [40] 。

5.3. 药物靶标合成减少

两性霉素B作用于麦角甾醇的合成,两性霉素B压力筛选下,念珠菌通过降低麦角甾醇合成途径中的基因表达使麦角甾醇合成量减少,增强菌株自身的两性霉素B的耐药性。白色念珠菌中,干扰麦角甾醇合成中的相关基因(ERG2、ERG3、ERG6、和ERG11基因)则能减少麦角甾醇合成,ERG2,ERG3,ERG5和ERG11基因的缺失或同时突变会导致念珠菌对唑类和两性霉素B交叉耐药的产生 [35] [41] 。光滑念珠菌的CgERG6基因突变也与耐多烯类药物有关 [42] 。近平滑念珠菌的ERG3基因R135I错义突变与耐唑类药物相关 [43] 。

β-1,3-D葡聚糖是真菌细胞壁的组成成分,是棘白菌素的作用位点。其主要催化亚基由FKS1基因编码,该基因的点突变会导致棘白菌素耐药性的产生,FSK1基因的点突变主要位于两个不同的热点突变区域:HS1和HS2 [44] 。临床上分离得到白色念珠菌的FSK1突变主要发生在相应氨基酸区域640~650 (HS1)及1345~1365 (HS2)区域 [45] 。并且,发生在HS1的点突变S645P,S645Y,以及S645F能显著的降低白色念珠菌对棘白菌素的敏感性 [46] 。而近平滑念珠菌对棘白菌素耐药很可能是由于FSK1基因突变热点区域的自然多态性(P660A,P647A) [47] [48] 。耐棘白菌素药物的光滑念珠菌、热带念珠菌、克柔念珠菌中,FSK1基因也发生了点突变 [44] [49] 。

5.4. 生物被膜的形成

生物被膜是一种由真菌形成的富含多糖细胞外基质的微生物群落,能有效阻止药物进入细胞。形成生物被膜中的白色念珠菌对唑类药物的抗性比浮游菌高1000倍,抗棘白菌素类高2~20多倍 [50] [51] 。研究表明,白色念珠菌形成的生物被膜能对多种药物产生耐药 [52] 。生物被膜耐药的机制与以下几个原因相关:1) 细胞外基质形成物理屏障,防止抗菌药物进入细胞群落 [53] [54] 。2) 生物被膜各组分参与和维持生物被膜功能,如ABC与MFS转运蛋白参与生物膜的形成 [55] ,HSP90调节生物被膜的耐药功能 [56] [57] ,eDNA维持生物膜结构完整性和稳定性 [58] ,耐药株细胞补充被破坏的生物被膜 [59] 等。

5.5. 其他耐药途径

由ERG3基因编码的甾醇Δ5,6-去饱和酶在麦角甾醇生物合成后期发挥作用。唑类药物作用该酶位点,使得甾醇Δ5,6-去饱和酶会将无毒的14-甲基甾醇中间产物转化成细胞毒性甾醇14-甲基-3,6-二醇,并在细胞中积累,最终导致细胞死亡。因此,ERG3基因失活或缺失会阻止毒性物质积累,同时甾醇中间产物会转化为14-去甲基粪甾醇,该物质能部分替代麦角甾醇的功能,从而导致念珠菌对所有唑类药物的交叉耐药性的产生 [49] [60] 。同时,ERG3基因缺失后会导致细胞膜麦角甾醇的缺失,也表现出对两性霉素B的交叉耐药 [61] 。白色念珠菌中,已发现超过18种与耐药有关的ERG3基因位点突变,其中,W332R、H243N、T330A、A351V、K97E、V237A、D147G、K97E、L193P、A353T、D19E氨基酸置换菌株均对氟康唑、伊曲康唑、伏立康唑等药物产生耐药,并发现在氟康唑或伊曲康唑的压力下,耐药菌株中14-甲基粪甾醇含量明显增多 [62] [63] 。

氟胞嘧啶在真菌细胞内发挥药效需要胞嘧啶脱氨酶(cytosine deaminase, CD)和尿嘧啶磷酸核糖转移酶(uracil phosphoribosyl transferase, UPRT)的转化。真菌对氟胞嘧啶的耐药产生多是编码UPRT的FUR1基因突变以及编码CD的FCA1基因(白色念珠菌)或FCY1基因突变造成的 [64] [65] 。

6. 念珠菌的治疗药物

目前临床常用药物包括两性霉素B、制霉菌素等多烯类、氟康唑、伏立康唑和伊曲康唑等唑类和卡泊芬净、米卡芬净等棘白菌素类药物进行治疗念珠菌病。不同类型药物通过作用真菌不同靶点来实现杀菌作用,但由于这些临床药物的长期使用导致临床上耐药菌株的不断涌现,使得这些药物的使用面临着挑战。

6.1. 常用药物

以两性霉素B为代表的多烯类药物,可全身给药。该类药物可通过与真菌细胞膜的麦角甾醇结合,损伤细胞膜,影响其通透性,从而影响真菌正常的新陈代谢进而抑制生长。该类药物常用于治疗系统性真菌感染,是治疗侵袭性曲霉病和侵袭性念珠菌病的主要药物,也是治疗由杜氏利什曼原虫引起的内脏和皮肤黑热病的二线药物 [66] 。但由于多烯类药物也能与哺乳动物细胞膜上的胆固醇结合,产生较强的中毒性肾损害,副作用较大。

包括咪唑类和三唑类衍生物的唑类药物,是最常用的抗真菌药,通过抑制真菌细胞色素P450依赖酶- 14α-羊毛脂醇脱甲基酶来阻止真菌细胞膜的麦角甾醇合成,使麦角甾醇合成途径中的中间代谢产物经旁路代谢合成毒性甾醇,在细胞内积累,影响细胞膜的流动性和通透性,达到抑菌的效果。由于其在临床上的大量使用,目前临床上分离的对唑类药物耐药性菌株越来越多 [5] [12] [67] 。但研究发现唑类药物在治疗血管瘤和肿瘤方面能够发挥一定的疗效 [68] [69] ,因此拓展了抗真菌药物的使用范围。

抑制真菌葡聚糖合酶的脂肽大分子——棘白菌素类药物是通过破坏真菌13-(1,3)-D-葡聚糖的合成,造成细胞壁的缺失,使细胞渗透压失衡,最终导致真菌细胞溶解死亡 [70] 。棘白菌素类药物在临床上的应用为抢救性治疗侵袭性曲霉病,被推荐为侵袭性念珠菌病治疗的主要药物,60%的念珠菌血症患者使用棘白菌素类药物。但口服药效率很低,必须静脉注射。白色念珠菌的Fks1蛋白突变将导致棘白菌素类药物耐药性的产生 [48] 。

5-氟胞嘧啶也为常用的经典抗菌药物,其抑菌机制为借助一个或多个嘌呤–胞嘧啶通透酶,进入真菌细胞被胞嘧啶脱氨酶转变为5-氟尿嘧啶,5-氟尿嘧啶被尿嘧啶磷酸核糖转移酶磷酸化后整合到RNA中,进而干扰蛋白质的合成、抑制DNA合成和核分裂。当念珠菌的胞嘧啶透酶发生突变时可导致5-氟尿嘧啶的耐药性快速产生 [71] 。

6.2. 新型药物的开发

针对侵袭性真菌的感染,治疗常采取预防用药或经验性用药或长周期用药的方式,这些治疗方式导致侵袭性真菌耐药现象日渐严重,因此,新型抗真菌药物的研发迫在眉睫。

研发方向着重于以下几个方面:1) 作用真菌新靶点的挖掘:如抑制钙调磷酸酶或作用于真菌线粒体等药物研发 [39] 。也可借助新的研究成果来挖掘,如大多数真菌感染与生物膜形成有关,真菌生物膜的发现与抑制剂的使用为治疗与生物膜相关的耐药性的机会性感染提供了可能。到目前为止,许多生物膜抑制剂大部分都是销售药物、已知的生物活性分子和自然物质。值得注意的是,生物膜电位器的协同作用则能加强现有的抗真菌药物在治疗与生物膜相关的耐药感染的效果 [72] 。2) 抗真菌的疫苗开发:加深认识理解真菌感染的致病机制,能够更好地开发高效、耐用、安全的疫苗来预防真菌病的发生 [73] 。3) 联合用药:现存药物的联合使用可以发现抑制真菌的新型研究思路 [74] 。4) 其他方面药物的新功能发现:研究显示曲马多能通过靶向调节白色念珠菌G蛋白耦联受体来抑制白色念珠菌双型形态转变,并对白色念珠菌的生长和生物被膜形成具有抑制作用 [75] ,使其成为潜在的抗念珠菌局部给药的候选药物。5) 新结构基团的次生代谢产物的挖掘:如紫檀芪是天然植物来源的二苯乙烯衍生的植物抗毒素物质,其具有显著的体外抑制生物被膜的形成和破坏已成形的生物被膜功能,但对浮游的白色念珠菌无抑菌效果 [76] 。

7. 结语

目前临床上耐药性菌株日益严重,并且超级念珠菌的出现 [77] ,使得形势更为严峻,因此建立快速的耐药性菌株检测技术和基于耐药机理开发出新的抗真菌药物是当前的重要任务。治疗真菌感染的当务之急是开发新的工具来治疗病人。除开发新的抗真菌药物外,疫苗是一个重要的选择,联合使用抗真菌药物也是一种有益的治疗方法 [78] 。

基金项目

大学生创新创业训练项目(No. 2017035和No. 201710632005)。

文章引用

付婉瑞,李凌志,钟 鑫,胡莺菡,朱晓凤,宋章永. 临床上念珠菌耐药性发生的研究进展
The Research Progress on Occurrence of Candida Drug Resistance[J]. 微生物前沿, 2017, 06(04): 98-107. http://dx.doi.org/10.12677/AMB.2017.64013

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  79. NOTES

    *通讯作者。

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