肺炎克雷伯菌对黏菌素的耐药机制研究与监测
夏薇，博士、二级教授、博士生导师，北华大学医学技术学院院长。教育部医学技术类教学指导委员会委员，国家级一流专业建设点负责人、全国高等院校医学检验业校际协作理事会理事长、享受国务院特殊津贴专家、吉林省B类国家级领军人才、吉林省教学名师，吉林省检验医学学会检验教育分会主任委员、吉林省医学会检验分会副主任委员等。从事医学检验专业教学34年。主要研究方向为疾病的诊断指标及方法。
刘晓妤，临床检验诊断学在读硕士研究生，师从夏薇教授、徐英春教授、杨启文教授。研究方向：中国多中心粘菌素耐药肺炎克雷伯菌分子耐药机制及毒力差异研究，《医学参考报微生物与感染频道》编辑，《检验医学教程-临床微生物分册》编写秘书。
肺炎克雷伯菌（Klebsiella pneumoniae）是一种常见的革兰阴性杆菌，根据2014年世界卫生组织发布的全球抗菌药物耐药性监测报告显示，肺炎克雷伯菌被认为是引起人类感染的前三大物种之一，包括尿路感染、肺炎、脑膜炎、外科伤口感染和败血症[1]。黏菌素是一种由多粘芽孢杆菌产生的环聚七肽阳离子抗菌剂[2]。在结构上，粘菌素是带阳离子的一个疏水七肽环，由四个二氨基丁酸、两个亮氨酸和一个苏氨酸残基组成，带有三个带正电的氨基和一个带有两个不同区域的尾部，折叠呈一个复杂的三维结构[3]。一般通过与脂多糖（LPS）上的脂质A上游离的带阴离子磷酸基团结合产生作用，因其具有比带正电荷的二价阳离子如Ca2+和Mg2+更高的亲和力从而破坏脂多糖的稳定性，导致外膜完整性降低[4]。进而导致外膜渗透性增加，黏菌素通过“自促摄取”吸收进入周质，并可能将分子插入内膜。目前黏菌素的杀菌机制尚不清楚，但其插入可能会导致内外膜的融合，导致细菌膜的整体破坏[5]。黏菌素常被视作碳青霉烯耐药的多重耐药的肺炎克雷伯菌的“最后一线”用药[6]，随着其应用增多，黏菌素耐药的肺炎克雷伯菌被频繁报道于世界各地[7, 8]。本文将概述肺炎克雷伯菌的黏菌素的耐药机制的研究进展，以期对黏菌素耐药的肺炎克雷伯菌的治疗提供依据。
1. 质粒介导的耐药机制：质粒介导的mcr基因在肺炎克雷伯菌中流行率较低[9-11]，编码的MCR蛋白磷酸乙醇胺转移酶家族的一员，它是通过将磷酸乙醇胺（PEtN）添加至脂质A从而降低黏菌素与结合位点的亲和力导致菌株耐药。mcr-1-mcr-10都有被报道，但在肺炎克雷伯菌中存在mcr-1[8]，mcr-7[12]和mcr-8[13, 14]，其中mcr-1是目前报道最多的基因[15]。但研究者们发现mcr基因相对独立，并不会影响到细菌的染色质介导的耐药机制[16]。
2. 染色质介导的耐药机制：肺炎克雷伯菌的外部阳离子环境刺激，例如暴露于阳离子抗菌肽、低pH值、低Mg2+浓度、高Fe3+和Al3+浓度，以及巨噬细胞的吞噬作用，都可以导致肺炎克雷伯菌通过双组分系统（TCS）及其相关调控基因产生特定突变或者异常表达使阳离子化合物包括PEtN和L-4-氨基-阿拉伯糖（L-Ara4N）修饰带负电的LPS[17]。目前pmrAB和phoPQ系统上的单氨基酸突变可以引起黏菌素MIC上升4-1000倍，例如pmrB上的T157P[18]和D313N[19]，phoP上的D191Y[20]和phoQ上的L26P[21]。mgrB是调节phoPQ系统的负调节基因，phoP上调后mgrB表达升高，MgrB蛋白反过来抑制phoQ基因的表达，由于单氨基酸突变或插入序列插入可导致mgrB失活[22-24]是目前报道最为广泛的肺炎克雷伯菌黏菌素耐药机制，插入序列导致耐药的机制也在异质性耐药中被认为可以通过质粒打破。crrAB系统的生理机制目前尚不清楚，并不是所有的肺炎克雷伯菌都存在这一系统，它可能来自水平传播[25]，有研究表明crrAB系统通过调控pmrA或者crrC对黏菌素耐药发挥作用[26]，但crrB的特定单氨基酸突变可以导致肺炎克雷伯菌的耐药，crrC基因的存在对耐药存在贡献[27]，有研究表明crrB的错义突变可以导致肺炎克雷伯菌拥有比mgrB失活后更高的黏菌素MIC[28]，并且可以通过上调外排泵表达导致肺炎克雷伯菌对黏菌素耐药[29]。
3. 其他耐药机制：其他耐药机制研究较少，目前有研究者提出荚膜多糖可能与黏菌素耐药相关，通过包裹细菌减少与黏菌素接触使肺炎克雷伯菌耐药[30, 31]，但也有研究提出荚膜产生与黏菌素MIC之间无显著关系[32]。外排泵表达上调可以导致耐药[29]，几项研究表明，外排泵可参与降低对黏菌素的耐药中[33]，包括AcrAB-TolC外排泵的过表达[34]和其调节因子ramR的过表达[35]和广谱抗菌药物外排泵KpnEF[36]。
4. 结语：肺炎克雷伯菌黏菌素耐药的报道日益增多，并在全球范围内广泛传播，黏菌素耐药的肺炎克雷伯菌常伴随多重耐药，泛耐药和全耐药也时有报道，这使得对其耐药机制的研究显得尤为重要。其耐药机制以染色质介导的耐药机制为主，双组分系统及其上下游调控因子的异常表达与耐药直接相关，其根本原因可能为调控基因上的特定点突变或者基因结构变异。目前也有报道黏菌素耐药会导致肺炎克雷伯菌毒力上升[37]，导致较为严重的医院感染，所以目前亟需对肺炎克雷伯菌的黏菌素耐药机制建立准确的研究和定期的监测。
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