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。     

主动外排机制介导鲍曼不动杆菌多重耐药研究进展

鲍曼不动杆菌是医院感染和机会感染的主要致病菌之一,并且其耐药性高,常发生泛耐药或多重耐药。主动外排机制在细菌多重耐药发生中起着重要的作用。与鲍曼不动杆菌多重耐药有关的外排蛋白主要有AdeABC、AdeIJK、Tet（A）、Tet（B）和AheM外排泵。本文结合当前主动外排机制研究进展,对与鲍曼不动杆菌多重耐药有关的主动外排蛋白的分类、组成、基因表达调控以及耐药情况进行综述。通过对鲍曼不动杆菌主动外排机制的深入研究,对新型抗菌药物的开发和治疗方法的改进有着极大的推动作用。     

Multidrug efflux in Pseudomonas aeruginosa: components, mechanisms and clinical significance

Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an intrinsic resistance to multiple antimicrobial agents and the ability to develop high-level (acquired) multidrug resistance during antibiotic therapy. Much of this resistance is promoted by highly homologous three-component efflux systems of broad substrate specificity, of which four have been identified to date. These include MexA-Mexs-OprM and MexX-MexY-OprM, which are expressed constitutively in wild type cells and, thus, provide for intrinsic multidrug resistance, and MexC-MexD-OprJ and MexE-MexF-OprN, whose expression so far has only been seen in acquired multidrug resistant mutant strains. Additional homologues of these efflux systems are identifiable in the recently released genome sequence, though their roles, if any, in antimicrobial efflux are unknown. These tripartite pumps are composed of an integral cytoplasmic membrane drug-proton antiporter of the resistance-nodulation-cell division (RND) family of exporters, a channel-forming outer membrane efflux protein (or outer membrane factor [OMF]) and a periplasmic membrane fusion protein (MFP) that links the other two. In addition to a number of antimicrobials of clinical significance, these pumps also export dyes, detergents, disinfectants, organic solvents and acylated homoserine lactones involved in quorum-sensing. While the natural functional of these pumps remains undefined, the fact that they contribute to antimicrobial resistance in P. aeruginosa makes them reasonable targets for therapeutic intervention.     

细菌多重耐药外排泵抑制剂研究进展

细菌耐药性，尤其是多重耐药性（multi-drug resistance，MDR）已经成为非常严重的医疗问题，而多种类型细菌外排泵（efflux pumps）的存在是细菌多重耐药的重要机制，因此寻找有应用前景的外排泵抑制剂（efflux pump inhibitors，EPI）是十分必要且迫切的。目前已经发现外排泵抑制剂的作用机制分为：（1）干扰外排泵组装；（2）阻断外排泵能量来源；（3）阻碍底物通过外排通道；（4）机制未知。本文按照作用机制对已经发现的细菌多重耐药外排泵抑制剂的特点进行分述。     

铜绿假单胞菌的耐药性及碳青霉烯类抗生素的研究进展

铜绿假单胞菌（Pseudomonas aeruginosa）是一种常见的医院内获得性感染致病菌，其耐药性强，耐药谱广。亚胺培南等碳青霉烯类抗生素是近年来治疗铜绿假单胞菌疗效较好的药物，但随着临床的广泛应用，铜绿假单胞菌对亚胺培南等产生了耐药性。铜绿假单胞菌对碳青霉烯类抗生素的耐药机制有β-内酰胺酶的水解、外膜通透性降低和主动外排系统的排出等，这些耐药机制之间相互协同作用而产生高度耐药。针对这些耐药机制，开发活性更高、安全性更好的碳青霉烯类抗生素显得极为迫切。本文对铜绿假单胞菌的耐药性及碳青霉烯类抗生素的研究进展进行了综述。     

Efflux pumps: their role in antibacterial drug discovery

The emergence of active efflux as a major causative factor in antibiotic resistance has been one of the most significant trends in antiinfective chemotherapy over the last decade. The phenomenon affects virtually all classes of antibiotics and frequently results in multi-drug resistant phenotypes. This review analyzes efflux pumps of clinical significance and examines their impact on different antibiotic classes relative to other mechanisms of resistance. Progress in strategies to combat efflux-mediated resistance by modification of existing antibiotics or identification of efflux pump inhibitors is also reviewed.     

细菌RND外排泵的结构与作用机制研究进展

细菌RND(resistance-nodulation-cell division)外排泵是一类由内膜转运蛋白、周质融合蛋白、外膜外排蛋白组成的蛋白复合体,能够将抗菌药物和多种小分子化合物如毒素、染料、洗涤剂、脂质、群感信号分子等排出细菌细胞外。通过影响RND外排泵的组装和功能,可逆转多重耐药性的发生与防止耐药性的发展。细菌具有多种外排泵,本文以RND外排泵为代表,介绍与其结构以及外排药物的转运和调控机制的最新进展。     

Study on antibiotics susceptibility and mechanism of carbapenem-resistance in clinical isolates of Pseudomonas aeruginosa]

The susceptibility of 260 strains of Pseudomonas aeruginosa to several antibiotics and the mechanism of resistance to carbapenems were investigated. The number of strains of P. aeruginosa moderately resistant or resistant to ofloxacin, ceftazidime and imipenem (IPM) were 76 (29.2%), 31 (11.9%) and 30 (11.5%), respectively. There was no clear relationship between the drug resistance of P. aeruginosa and serum type. Fourteen strains (46.6%) out of 30 IPM-resistant strains were susceptible to meropenem (MEPM). Twenty seven (90.0%) IPM-resistant strains showed cross resistant to panipenem (PAPM), and 12 strains (44.4%) out of the 27 strains showed high susceptibility to MEPM. P. aeruginosa becomes resistant to IPM and PAPM only by the decrease in the outer membrane permeability of these carbapenems. In contrast, P. aeruginosa becomes equally resistant to MEPM by concurrent occurrence of the increase in the efflux of the antibiotics and the decrease in the outer membrane permeability of the antibiotics. The possibility that both mechanisms are taken place concurrently in P. aeruginosa is considered to be low, and it was also supported by the results of the present study.     

泵抑制剂对环丙沙星在铜绿假单胞菌内聚积的影响

目的探讨耐药铜绿假单胞菌（PA）中是否存在主动外排系统,以揭示PA对氟喹诺酮类药物的耐药机制。方法检测碳酰氰间氯苯腙（CCCP）对16株PA药物M IC的影响;荧光法检测PA耐药菌和敏感菌对环丙沙星（CIP）的摄取,以及加入CCCP后对CIP累积量的变化情况。结果外排泵阳性株为4株,CCCP可以使PA 16菌体内环丙沙星的累积量显著上升,而其它菌株中加入CCCP后,环丙沙星的累积量变化不大。结论本地区PA中可能存在氟喹诺酮药物的主动外排系统。     

Bacterial Efflux Pumps Involved in Multidrug Resistance and their Inhibitors: Rejuvinating the Antimicrobial Chemotherapy

Active efflux of antibiotics is one of the major mechanisms of drug resistance in bacteria. The efflux process is mediated by membrane transporters with a large variety of unrelated compounds as their substrates. Though these pumps are responsible for the low intrinsic resistance of a bacterium to a drug, their overexpression, accumulation of mutations in these proteins and their synergy with other drug resistance mechanisms hampers effective antimicrobial treatment. As efflux pumps have been reported to play vital roles in mediating multidrug resistance in clinical isolates from varied geographic locations and varied populations, the inhibition of efflux pumps appears to be an attractive approach to combat the problem of drug resistance. Efflux pump inhibitors can be utilized for increasing the antibiotic concentration inside a pathogenic cell making these drugs more effective, reduce the accumulation of other resistance mechanisms in a cell and for diagnostic purposes to evaluate the presence and contribution of the efflux mechanism in a pathogen. A large number of inhibitors have been discovered and patented in last two decades but the process of discovery, testing and commercialization is rather slow. Some of the important inhibitors include the energy decouplers, phenothiazines, analogs of popular antibiotics, inhibitors of serotonin re-uptake, to name a few, that have been used as adjuvants in the antimicrobial chemotherapy to potentiate the activity of some important antimicrobials in deadly pathogens that have worried the mankind since long. This review describes the role of efflux pumps in governing the resistance phenotype of a pathogen, efflux pumps found in bacteria and the efflux pump inhibitors that have been studied and patented so far.     

Efflux systems in bacterial pathogens: an opportunity for therapeutic intervention? An industry view

The efflux systems of bacteria protect cells from antibiotics and biocides by actively transporting compounds out of the cytoplasm and/or periplasm and thereby limit their steady-state accumulation at their site(s) of action. The impact of efflux systems on the efficacy of antibiotics used in human medicine and animal husbandry is becoming increasingly apparent from the characterization of drug-resistant strains with altered drug efflux properties. In most instances, efflux-mediated antibiotic resistance arises from mutational events that result in their elevated expression and, in the case of efflux pumps with broad substrate specificity, can confer multi-drug resistance (MDR) to structurally unrelated antibiotics. Knowledge of the role of efflux systems in conferring antibiotic resistance has now been successfully exploited in the pharmaceutical industry and contributed, in part, to the development of new members of the macrolide and tetracycline classes of antibiotics that circumvent the efflux-based resistance mechanisms that have limited the clinical utility of their progenitors. The therapeutic utility of compounds that inhibit bacterial drug efflux pumps and therein potentiate the activity of a co-administered antibiotic agent remains to be validated in the clinical setting, but the approach holds promise for the future in improving the efficacy and/or extending the clinical utility of existing antibiotics. This review discusses the potential of further exploiting the knowledge of efflux-mediated antibiotic resistance in bacteria toward the discovery and development of new chemotherapeutic agents.     

Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and impact on treatment

Pseudomonas aeruginosa continues to be a major cause of infections in Western society, in part because of its high intrinsic resistance to antibiotics. It has been demonstrated that this intrinsic resistance arises from the combination of unusually restricted outer-membrane permeability and secondary resistance mechanisms such as energy-dependent multidrug efflux and chromosomally encoded periplasmic β-lactamase. Given this high level of natural resistance, mutational resistance to most classes of antibiotics can readily arise. In this review we summarize new insights into the mechanisms of resistance, and describe therapeutic approaches that can be used in the face of this continuing resistance threat, as well as new approaches that are being developed to combat resistance.     

The role of multidrug efflux pumps in the antibiotic resistance of Pseudomonas aeruginosa and other gram-negative bacteria. Insights from the Society of Infectious Diseases Pharmacists

Gram-negative bacteria remain clinically important pathogens in both hospital and community settings. Recent research indicates that efflux pumps play a prominent role in the multidrug resistance of Pseudomonas aeruginosa and many other gram-negative bacteria. Four multidrug efflux pump systems have been well characterized in P. aeruginosa: MexA-MexB-OprM, MexC-MexD-OprJ, MexE-MexF-OprN, and MexX-MexY-OprM. These efflux pumps have different substrate specificities, and their production and activity can be increased by many factors commonly present in infections (e.g., high inocula of bacteria, low pH, and stationary-phase growth). Moreover, fluoroquinolone antibiotics can commonly select mutants that constitutively overproduce Mex-Opr efflux pump systems. Based on most recent studies, the prevalence of efflux pump overproduction in clinical strains of P. aeruginosa may range from 14-75%. The best treatment for infections caused by bacteria that overproduce efflux pumps is unknown, but pharmacodynamic optimization of antibiotics and the use of antibiotic combinations that are substrates for different pump systems may represent reasonable strategies until more data are available.     

Redefining penems

The antimicrobial class of penems has the potential to address most of the relevant resistance issues associated with beta-lactam antibiotics because of their exceptionally broad spectrum of antibacterial activity and their intrinsic stability against hydrolytic attack by many beta-lactamases including ESBL and AmpC enzymes. The subclass of carbapenems covers the spectrum of hospital pathogens whereas the subclass of penems covers community pathogens. The only currently available penem, faropenem, has a low propensity for resistance development, beta-lactamase induction and selection of carbapenem-resistant Pseudomonas aeruginosa. This makes it attractive for the treatment of community-acquired infections and for step-down or sequential therapy following carbapenem treatment without jeopardizing the activity of carbapenems or the entire beta-lactam class in the hospital environment.     

Inhibition of bacterial efflux pumps: a new strategy to combat increasing antimicrobial agent resistance

Resistance to multiple drugs in medically important bacteria results in therapeutic challenges for the clinician. The mechanisms by which bacteria evade the effects of antimicrobial agents are many, but in recent years it has become apparent that efflux is a significant means of resistance and probably explains the intrinsic resistance to numerous drugs observed in species such as Pseudomonas aeruginosa. Drug efflux is mediated by membrane-based hydrophobic proteins belonging to several distinct families, the members of which are related by structural characteristics, mechanism of action and energy source for the transport process. The multi-drug efflux transporters are particularly problematic as they are capable of extruding numerous structurally dissimilar drugs. Inhibition of these pumps, and even those with more limited substrate specificity, has been shown to decrease intrinsic resistance, reverse acquired resistance and reduce the emergence of mutants with higher-level target-based mutational resistance. Combining broad spectrum efflux pump inhibitors with current drugs that are pump substrates can recover clinically relevant activity of those compounds and thus may reduce the need for the discovery and development of new antimicrobial agents that are not pump substrates. Additional effort toward the identification, characterisation and determination of the clinical utility of efflux pump inhibitors is warranted.     

铜绿假单胞菌耐药机制的研究进展

铜绿假单胞菌是耐药性较强的病原菌,β-内酰胺酶的生成和外排泵的表达是产生固有耐药和获得性耐药的重要机制。除了已经熟知的机制外,最新的研究运用多种技术手段如微阵列技术和基因突变频率分析等对铜绿假单胞菌的耐药机制进行了更进一步的研究;同时铜绿假单胞菌的适应性耐药的研究也取得了很大进展。综述近年来发表的有关铜绿假单胞菌耐药机制的研究文献,并对其耐药机制的研究进展作了分析。     

Inhibition of bacterial efflux pumps: a new strategy to combat increasing antimicrobial agent resistance

Resistance to multiple drugs in medically important bacteria results in therapeutic challenges for the clinician. The mechanisms by which bacteria evade the effects of antimicrobial agents are many, but in recent years it has become apparent that efflux is a significant means of resistance and probably explains the intrinsic resistance to numerous drugs observed in species such as Pseudomonas aeruginosa. Drug efflux is mediated by membrane-based hydrophobic proteins belonging to several distinct families, the members of which are related by structural characteristics, mechanism of action and energy source for the transport process. The multi-drug efflux transporters are particularly problematic as they are capable of extruding numerous structurally dissimilar drugs. Inhibition of these pumps, and even those with more limited substrate specificity, has been shown to decrease intrinsic resistance, reverse acquired resistance and reduce the emergence of mutants with higher-level target-based mutational resistance. Combining broad spectrum efflux pump inhibitors with current drugs that are pump substrates can recover clinically relevant activity of those compounds and thus may reduce the need for the discovery and development of new antimicrobial agents that are not pump substrates. Additional effort toward the identification, characterisation and determination of the clinical utility of efflux pump inhibitors is warranted.     

Metallo-beta-lactamase VIM-2 in Pseudomonas aeruginosa isolates from a cystic fibrosis patient

The increased incidence of Pseudomonas aeruginosa isolated from patients with cystic fibrosis (CF) along with an increase in its multidrug resistance makes therapeutic management very problematic. Careful identification and accurate studies of susceptibility to antibiotics are critical for improving therapeutic measures and for facilitating our understanding of the epidemiology of this pathogen. Fifteen P. aeruginosa isolates obtained from five CF children in the Paediatric Hospital of Coimbra were studied. Isolates from a female patient were resistant to all agents tested except colistin. A VIM-2 enzyme inserted in integron In58 was detected, and this isolate presented a unique random amplified polymorphic DNA (RAPD) type. Others isolates were susceptible to beta-lactams, and each isolate had a different RAPD type. VIM-2 confers resistance to the majority of beta-lactams and is associated with other gene cassettes coding for enzymes that inactivate aminoglycosides. Person-to-person transmission of these isolates is not well understood, therefore it is important to design infection control policies to avoid acquisition and dissemination of multiresistant strains.     

Role of efflux pumps and metabolising enzymes in drug delivery

The impact of efflux pumps and metabolic enzymes on the therapeutic activity of various drugs has been well established. The presence of efflux pumps on various tissues and tumours has been shown to regulate the intracellular concentration needed to achieve therapeutic activity. The notable members of efflux proteins include P-glycoprotein, multi-drug resistance protein and breast cancer resistance protein. These efflux pumps play a pivotal role not only in extruding xenobiotics but also in maintaining the body’s homeostasis by their ubiquitous presence and ability to coordinate among themselves. In this review, the role of efflux pumps in drug delivery and the importance of their tissue distribution is discussed in detail. To improve pharmacokinetic parameters of substrates, various strategies that modulate the activity of efflux proteins are also described. Drug metabolising enzymes mainly include the cytochrome P450 family of enzymes. Extensive drug metabolism due to the this family of enzymes is the leading cause of therapeutic inactivity. Therefore, the role of metabolising enzymes in drug delivery and disposition is extensively discussed in this review. The synergistic relationship between metabolising enzymes and efflux proteins is also described in detail. In summary, this review emphasises the urgent need to make changes in drug discovery and drug delivery as efflux pumps and metabolising enzymes play an important role in drug delivery and disposition.