抗生素耐药性的主动外排机制

细菌对抗生素产生耐药性的机制多种多样，常见的有：细菌产生酶对抗生素的修饰与灭活；药物作用靶点发生改变；膜通透性下降；膜产生主动外排作用；生物被膜的形成。膜的主动外排机制是由各种外排蛋白系统介导的把抗生素等药物从细菌细胞内泵出的主动排出过程，外排系统具有能量依赖性、底物广泛性、系统多样性及功能复杂性的特点；与常用抗生素、合成抗菌药、染料、去污剂及金属离子的多重耐药性密切相关，尤其对四环素、氯霉素及氟喹诺酮类药物等广谱抗菌药的作用更为明显；其生理功能与正常物质转运和代谢有关。以利血平为代表的外排泵抑制剂能有效地对抗主动外排作用的产生，此类抑制剂将是防制细菌多重耐药性的有效措施之一。     

抗生素药性的主动外排机制

细菌对抗生素产生耐药性的机制多种多样，常见的有；细菌产生酶对抗生素的修饰与灭活；药物作用靶点发生改变；膜通透性下降；膜产生主动外排作用；生物被膜的形成，膜的主动外排机制是由各种外排蛋白系统介导的把抗生素等药物从细菌细胞内泵出的主动排出过程，外排系统具有能量依赖性，底物广泛性，系统多样性及功能复杂性的特点；与常用抗生素，合成抗菌药，染料，去污剂及金属离子的多重耐药性密切相关，尤其对四环素，氯霉素及氟喹诺酮类药等广谱抗菌药的作用更为明显；其生理功能与正常物质转运和代谢有关，以利血平为代表的外排泵抑制剂能有效地对抗主动外排作用的产生，此类抑制剂将是防制细菌多重耐药性的有效措施之一。     

细菌外排泵介导耐药性研究进展

随着抗生素的广泛应用,临床上产生一些多重耐药菌株。多重耐药菌株的产生使常规抗生素在临床细菌性感染治疗中失效。细菌主动外排是耐药菌株产生多重耐药的主要机制之一;因此有关细菌主动外排泵的研究也是近年来细菌多重耐药机制研究中,相对较新和活跃的领域。本文结合当前主动外排泵研究的进展,对主动外排蛋白的分类、组成和主要的主动外排系统的基因表达调控以及耐药性情况进行综述。     

鲍曼不动杆菌抗生素主动外排泵转运系统与外排泵抑制剂

鲍曼不动杆菌(Acinetobacter baumannii)是医院内感染的重要病原菌,其耐药率呈现上升的趋势。该菌存在多种耐药机制,其中药物主动外排转运系统或外排泵介导的抗菌药物主动外排与多重耐药(multi-dmg resistance,MDR)密切相关,是近年来研究细菌多重耐药机制的重点。外排泵抑制剂(efflux pump inhibitors,EPIs)的研发有助于克服细菌主动外排机制导致的多重耐药性,为逆转细菌的多重耐药提供了一条新思路。本文就近年来鲍曼不动杆菌的药物主动外排转运系统与外排泵抑制剂的研究进展进行综述。     

铜绿假单胞菌主动外排泵介导的多重抗生素耐药性

铜绿假单胞菌作为一种机会致病菌，对多种临床常用抗生素呈现明显的固有与获得性耐药。这种多重耐药性的形成机制在于该菌具有的多种能量依赖性的药物主动外排泵和低通透性外膜屏障协同作用所致。近10年对铜绿假单胞菌药物外排泵的研究已经颇为深入，本文对有关进展予以讨论。在铜绿假单胞菌已报道了6种属于“耐药-生节-分裂(RND)”类的药物外排泵系统，它们可在天然野生株表达或者由于基因突变而诱导表达，从而介导了对β-内酰胺类(包括β-内酰胺酶抑制剂)、氨基糖苷类、氟喹诺酮类、大环内酯类及四环素类等的耐药性。这些外排泵中尤其以MexAB—OprM系统的作用底物范围最广，在耐药性形成中起主要作用。RND类外排泵系统也普遍存在于其它革兰氏阴性细菌。各外排泵通常由内膜转运体蛋白、内膜融合蛋白及外膜通道蛋白一起形成功能性转运复合体将药物排至胞外。药物外排泵常影响对灭活酶较稳定的抗菌药物。一些新抗菌药物如Linezolid(Oxa—zolidinoes类)、Telithromyein(Ketolides类)及Tigecycline(Glycyclines类)也是MexAB-OprM等RND类外排泵的作用底物，故这些药物抗革兰氏阳性细菌的活性较强。铜绿假单胞菌外排泵表达的调控机制多是在转录水平上受局部阻遏物或激活物的作用。这些局部调节子的基因突变能导致外排泵活性增强。并常见于应用抗生素或其它抗菌消毒制剂后从临床分离的或实验室筛选的多重耐药铜绿假单胞菌。这些耐药菌的形成再次说明合理限制抗菌药物应用的必要性。外排泵系统还与其它耐药机制如细菌的药物作用靶位改变或产生药物灭活酶等一起发挥明显的协同作用，使细菌的耐药程度进一步地增高。现已研制了针对铜绿假单胞菌等细菌的外排泵抑制剂。应用外排泵抑制剂可阻断药物外排泵这一耐药机制，以维护或提高抗菌药物的抗菌活性。如外排泵抑制剂在体外和动物感染模型均被证实明显增强了氟喹诺酮类对铜绿假单胞菌敏感株和耐药株的抗菌活性，并降低了耐药菌的发生率。外排泵抑制剂尚处于临床前的基础实验研制阶段。     

细菌体内药物蓄积浓度减少与氟喹诺酮耐药性的研究进展

对氟喹诺酮耐药性细菌体内药物蓄积浓度减少机制--膜通透屏障和主动外排泵激活的研究进展进行了讨论,该机制涉及细菌染色体非特异性基因位点的突变,通过基因间的相互作用,激活多重抗生素耐药操纵子marRAB,导致细菌外膜孔道蛋白异常和主动外排泵功能增强.该机制可由其它药物诱发,可单独或与药物作用靶位改变共同引起细菌发生氟喹诺酮耐药性.     

细菌耐药性的产生及防控对策

细菌对抗菌药物的耐药性已成为全球关注的医学与社会问题,给临床感染性疾病的治疗带来极大的困难。细菌耐药基因可由染色体突变或质粒、转座子、整合子等转移并传播,介导产生抗生素灭活酶或钝化酶,通过改变药物作用靶位、外膜通透性、抗生素的主动外排泵系统和代谢途径及形成生物被膜来发挥对药物的抗药性。细菌耐药性的产生受到自然因素和社会因素的影响,尤其是抗菌药物的滥用和环境中抗生素的污染是促进耐药菌产生的主要因素。通过合理控制抗菌药物的使用,研制新型抗菌药物,开发治疗感染性疾病的新疗法等多种有效措施来控制或减少细菌耐药性的发生与传播,需要不断地探索,仍然是人类所面临的长期挑战。     

细胞体内药物蓄积浓度减少与氟喹诺酮耐药性的研究进展

对氟喹诺酮耐药性细菌体内药物蓄积浓度养活机制－膜通透屏障和主动外排腺激活的研究进展进行了探讨,该机制涉及细菌染色体非特异性基因位点的突变,通过基因间的相互作用,激活多重抗生素耐药操纵子marRAB,导致细菌外膜孔道蛋白异常和主动外排泵功能增强。该机制可由其它药物诱发,可单独或药物作用靶位改变共同引起细菌发生氟喹诺酮耐药性。     

微生物药物外排泵及其抑制剂研究

细菌自身的不断进化及抗生素的不合理使用导致了耐药性细菌的出现和蔓延，微生物的主动外排是导致细菌产生多重耐药的重要原因。微生物外排泵的研究是目前的热点之一，也被认为是开发新抗生素的理想靶标。本文从基因组和比较基因组水平对外排泵的类型、作用机制、表达调控及研究方法等方面概述，并结合正在开展的分枝杆菌的药物外排泵进行了比较基因组分析。这些工作有利于开发新的抗生素或抗生素增效剂。     

感染致病菌耐药与临床治疗对策

细菌对常用抗菌药物的耐药成为人类健康事业面临的严重问题之一。细菌可通过产生灭活酶或钝化酶，改变抗茵药物作用靶位，改变细菌细胞壁的通透性、主动外排作用以及形成细菌生物被膜而对抗菌药物耐药。本文介绍了临床常见致病菌对各类抗菌药物的主要耐药机制及耐药基因，并总结了针对常见细菌耐药的合理用药及相关防治对策，以期为临床常见致病菌耐药提供解决方案。     

Understanding efflux in Gram-negative bacteria: opportunities for drug discovery

INTRODUCTION: Bacteria evolved an arsenal of mechanisms to deal with toxic compounds and metabolic stresses, including antimicrobial agents. Efflux pumps are major players in the multidrug resistance of Gram-negative bacteria and pose major hurdles in the drug discovery process. However, recent advances in our understanding of efflux in these bacteria provide opportunities and assets for drug discovery. AREAS COVERED: This review provides an overview of drug efflux in Gram-negative bacteria and its role in antimicrobial resistance, stress responses and other biological processes such as biofilm formation, and virulence. The discussion includes comments on the significance of synergy between a low-permeability outer membrane and efflux, notably the role of porins and lipopolysaccharide. The author then summarizes efforts aimed at inhibiting efflux pumps as a means to extend the utility of clinically useful antibiotics. This includes highlights of identification and characterization of small molecule efflux pump inhibitors (EPIs) from natural and synthetic sources, as well as novel strategies such as gene silencing and inhibitory antibodies. EXPERT OPINION: Options for treating infections caused by multidrug-resistant bacteria are limited. Given the attractiveness of the therapeutic potential of efflux pump inhibition, further studies exploring novel strategies to interfere with efflux pump expression and function are warranted. This includes rational EPI design facilitated by pump structure information, exploitation of genetically defined efflux-proficient and efflux-compromised strain panels and non-traditional approaches such as pump inhibition by gene silencing, antibodies and perhaps even phage.     

多黏菌素异质性耐药的研究进展及临床意义

Due to the extensive use of antibiotics, bacterial resistance is constantly emerging. Nowadays, polymyxins have become the last-resort to defend against multidrug-resistant bacteria, especially carbapenem-resistant Gram-negative bacteria. However, in recent years, there are more and more reports about polymyxin resistance, and the heteroresistance of polymyxins has been found to be a severe situation. Heteroresistance is the intermediate process from sensitivity to drug resistance. Routine clinical tests cannot effectively detect the heteroresistance, which poses a huge threat to the clinical treatment of drugs, resulting in repeated infections and drug failure. However, there are few researches on its mechanism. At present, it is mainly related to the mutations of LPS related genes on chromosomes, the unstable amplification of drug-resistant genes, and the efflux pump system. In this paper, we summarize the related research of bacterial heteroresistance, especially heteroresistance of polymyxins and its clinical     

多黏菌素异质性耐药的研究进展及临床意义

由于抗菌药物的大量使用,细菌的耐药性不断产生。如今,多黏菌素已成为抗多重耐药菌,尤其是碳青霉烯类耐药革兰阴性菌感染的最后一道防线药物。然而近几年来关于多黏菌素耐药的报道不断增加,并且已有研究发现多黏菌素的异质性耐药现象。异质性耐药是细菌由敏感到耐药的中间过程,常规的临床检验无法有效检测出异质性耐药,这对临床治疗用药造成了巨大的威胁,造成患者的反复感染和用药失败。但对其机制的研究较少,目前发现的主要与染色体上脂多糖(LPS)相关基因的突变,耐药基因的不稳定扩增和外排泵系统有关。本文总结了细菌异质性耐药,尤其是多黏菌素异质性耐药的研究及其临床意义。     

Practical applications and feasibility of efflux pump inhibitors in the clinic--a vision for applied use

The world of antibiotic drug discovery and development is driven by the necessity to overcome antibiotic resistance in common Gram-positive and Gram-negative pathogens. However, the lack of Gram-negative activity among both recently approved antibiotics and compounds in the developmental pipeline is a general trend despite the fact that the plethora of covered drug targets are well-conserved across the bacterial kingdom. Such intrinsic resistance in Gram-negative bacteria is largely attributed to the activity of multidrug resistance (MDR) efflux pumps. Moreover, these pumps also play a significant role in acquired clinical resistance. Together, these considerations make efflux pumps attractive targets for inhibition in that the resultant efflux pump inhibitor (EPI)/antibiotic combination drug should exhibit increased potency, enhanced spectrum of activity and reduced propensity for acquired resistance. To date, at least one class of broad-spectrum EPI has been extensively characterized. While these efforts indicated a significant potential for developing small molecule inhibitors against efflux pumps, they did not result in a clinically useful compound. Stemming from the continued clinical pressure for novel approaches to combat drug resistant bacterial infections, second-generation programs have been initiated and show early promise to significantly improve the clinical usefulness of currently available and future antibiotics against otherwise recalcitrant Gram-negative infections. It is also apparent that some changes in regulatory decision-making regarding resistance would be very helpful in order to facilitate approval of agents aiming to reverse resistance and prevent its further development.     

MC-207110 Daiichi Seiyaku/Microcide Pharmaceuticals

Microcide and Daiichi are collaborating on the discovery of new bacterial efflux pump inhibitorsfor the treatment of drug-resistant Pseudomonas aeruginosa and other bacterial infections. The bacterial efflux pump technology program focuses on Gram-negative bacteria that have developed antibiotic resistance by means of the efflux pump [192681], [341408]. MC-207110 was the initial screening hitfrom Microcide's library and is a low molecular weight dipeptide amide, which shows minimal antibacterial activity but potentiated the activity of levofloxacin by 8-fold at 10 microg/ml. This lead is being optimized to improve on its biological and pharmacological profile [341183]. Optimization studies have been conducted on MC-207110, yielding a class of broad-spectrum efflux pump inhibitors for P aeruginosa. A member of this class has demonstrated in vivo activity against P aeruginosa in a murine neutropenic thigh model [351686].     

氨基糖苷类抗生素的耐药机制研究进展

氨基糖苷类抗生素(AG)是高效的广谱抗生素，用于治疗许多革兰阴性菌和一些革兰阳性菌感染，随着临床的广泛应用，细菌的耐药性日趋严重。本文主要从核糖体修饰作用、氨基糖苷类抗生素的修饰酶的作用、药物的外排泵系统等方面对 AGs的耐药机制进行阐述，为能合理使用AGs、控制细菌耐药性蔓延以及新型AG药物的开发提供参考。     

鲍曼不动杆菌8种RND外排泵介导替加环素耐药表型的研究

目的 探讨鲍曼不动杆菌中8种RND外排泵基因的分布与替加环素耐药表型间的关系。方法 微量肉汤稀释法检测120株鲍曼不动杆菌对替加环素的耐药性,PCR扩增8种外排泵及调控基因。琼脂二倍稀释法测定替加环素耐药菌株对18种抗菌药物的最低抑菌浓度(minimal inhibitory concentration, MIC),利用5种外排泵抑制判定外排泵表型。采用实时荧光定量PCR检测外排泵基因的表达水平。结果 筛选出替加环素耐药菌8株,对18种抗菌药物耐药情况严重,其中6株为广泛耐药菌;3种外排泵抑制剂CCCP、PAβN及维拉帕米的表型阳性率分别为50%、37.5%和12.5%。耐替加环素菌株除ACICU_02904和ACICU_03412基因外均为阳性;替加环素敏感菌中外排泵(adeABC-adeRS、adeFGH-adeL和adeIJK-adeN)基因检测率为38.4%,新近发现外排泵(ACICU_00143、ACICU_03066和ACICU_03646)基因检测率为50.9%,然而,ACICU_02904和ACICU_03412基因检出率为0.9%。在转录水平上,替加环素耐药组的adeB相对表达量是敏感组的23.5倍。结论 鲍曼不动杆菌中adeB相对表达量伴随菌株对替加环素MIC值增加而升高,主动外排泵表达活性增加是替加环素耐药的一个重要机制。     

Membrane permeability and regulation of drug "influx and efflux" in enterobacterial pathogens

In Enterobacteriaceae, membrane permeability is a key in the level of susceptibility to antibiotics. Modification of the bacterial envelope by decreasing the porin production or increasing the expression of efflux pump systems has been reported. These phenomena are frequently associated with other resistance mechanisms such as alteration of antibiotics or modification of the drug targets, in various clinical isolates showing a Multi Drug Resistant phenotype (MDR). In Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae and Salmonella enterica several genes and external factors are involved in the emergence of MDR isolates. These bacterial isolates exhibit a noticeable reduction of functional porins per cell due to a decrease, a complete shutdown of synthesis, or the expression of an altered porin and a high expression of efflux systems (e.g. overexpression of the pump). The combined action of these mechanisms during an infection confers a significant decrease in bacterial sensitivity to antibiotherapy ensuring dissemination and colonization of the patient and favours the acquisition of additional mechanisms of resistance. MarA and ramA are involved in a complex regulation cascade controlling membrane permeability and actively participate in the triggering of the MDR phenotype. Mutations in regulator genes have been shown to induce the overproduction of efflux and the down-regulation of porin synthesis. In addition, various compounds such as salicylate, imipenem or chloramphenicol are able to activate the MDR response. This phenomenon has been observed both in vitro during culture of bacteria in the presence of drugs and in vivo during antibiotic treatment of infected patients. These effectors activate the expression of specific global regulators, marA, ramA, or target other genes located downstream in the regulation cascade.     

51株鲍曼不动杆菌耐药表型及外排蛋白基因表达研究

目的:探讨鲍曼不动杆菌耐药表型以及adeB外排泵基因的表达情况。方法:收集2004年2月～2005年2月北京协和医院51株非重复鲍曼不动杆菌,应用聚合酶链反应法测定鲍曼不动杆菌主动外排系统adeB结构基因及序列分析。结果:受试菌株对常用广谱抗生素均不敏感;多重耐药鲍曼不动杆菌携带adeB主动外排泵基因。结论:本研究中鲍曼不动杆菌多重耐药性与产生β-内酰胺酶无关,另有其它耐药机制存在,主动外排作用可能是鲍曼不动杆菌多重耐药的重要机制之一。