影响多粘菌素B对绿脓杆菌杀菌效力的主要环境因素的研究

本文通过改变温度,水活度,气体条件和营养含量等影响绿脓杆菌生长的主要环境因素,测定多粘菌素B对绿脓杆菌的最小杀菌浓度(MBC)。结果表明环境因素导致或显著影响绿脓杆菌对抗生素的生态耐受性。实验表明多粘菌素B对绿脓杆菌的杀菌效力,除药物对细菌特有的药理学作用外,还取决于细菌的生长环境。结合冷休克率试验表明,环境影响细菌群体处于分裂状态的菌数。若分裂状态菌数下降表明生长速度减慢。提示了多粘菌素B对绿脓杆菌的效力指数,定量分析可以作为其综合效力作用的表现。以同步培养法确定在单个细胞周期中的抗生素敏感阶段。同时以冷休克率试验资料证明细菌处于分裂状态和幼龄期是其敏感阶段。初步阐述了生长速度缓慢与药物的生态耐受性密切相关。     

群体感应与微生物耐药性

微生物耐药性已成为全球关注的严重问题,其演化机制和调控机理也已成为研究热点。近年来的研究发现,一些微生物耐药性机制受到群体感应系统的调控。群体感应是一种在微生物界广泛存在并与菌体密度关联的细胞-细胞间的通讯系统。高密度的菌落群体能够产生足够数量的小分子信号,激活下游包括致病毒力和耐药性机制在内的多种细胞进程,耐受抗生素并且危害寄主。本文结合国内外最新的研究进展,对微生物群体感应系统的研究现状进行了概括性介绍,重点阐述了群体感应系统对微生物耐药性机制的调控作用,如微生物生物被膜形成和药物外排泵调控等方面的作用,并探讨了利用群体淬灭控制微生物耐药性的新策略。     

综述与专论：细菌的信号转导系统及其在耐药中的作用

耐药菌的日益增多给临床治疗带来巨大的困难,揭示耐药机制成为遏制耐药菌的基本环节。细菌的信号系统是菌体之间信息交流的主要渠道,在调控细菌耐药性方面发挥重要的作用。本文梳理了细菌双组分系统、群体感应系统、第二信使、吲哚等细菌信号系统(分子)与细菌耐药性的关系,总结了各信号系统调控细菌耐药性的机制和途径,包括调控生物膜的形成、调节药物外排泵的活性、激活抗生素灭活酶、提高耐药基因表达水平、促进耐药基因转移、修饰细胞壁结构等,涉及到细菌耐药的多个环节。各信号系统不仅可以独立调控耐药,还可以互相作用,形成调控网络,从多个层面调节细菌耐药性。因此,靶向细菌信号系统,阻断菌体之间的信号联络,有望成为遏制细菌耐药性日益严重的新策略。     

RND家族外排泵及其与微生物群体感应系统的相互关系

抗生素耐药性一直是细菌病害防治的难题,药物外排泵过量表达是细菌耐药性形成的重要机制之一。在革兰氏阴性细菌中,RND(Resistance-nodulation-cell division)家族外排泵在耐药性中发挥着重要作用,近年来的研究表明,依赖于小分子信号物质进行调控的群体感应系统与RND外排泵家族之间存在紧密的相互作用关系。本文在介绍RND家族外排泵的结构、转运机理和群体感应系统的类型及调控方式的基础上,剖析了群体感应系统对RND外排泵的调控机理以及RND外排泵对群体感应系统信号分子转运的影响。深入研究RND家族外排泵与群体感应系统之间的相互依赖、相互制约关系有利于阐明RND家族外排泵的调控机理,并有可能为克服微生物耐药性问题提供新的思路。     

群体感应系统介导细菌生物膜形成的研究进展

群体感应(QS)是微生物之间的通讯机制,通过信号分子调控基因表达,这种交流可使细菌表达不同的生理行为,包括病原微生物的毒性、对抗生素的形成、生物膜的形成与生长等。生物膜的形成对微生物的代谢、毒力因子的表达等密切相关。群体感应现象与生物膜的形成相互依赖,生物膜提供菌体聚集场所,避免群体感应信号分子的扩散,聚集菌体的群体感应现象为生物膜的形成提供基础。群体感应系统不仅可直接介导细菌生物膜的形成,还可调节胞内第二信使分子水平,间接调控生物膜的生成。本文中,笔者从直接和协同其他信号分子两方面对细菌生物膜形成机制研究进展进行综述,为在工业应用中降低细菌耐药性、指导食品生产安全、提高功能性生物膜产量等方面提供理论依据。     

ppGpp介导的抗生素胁迫应答机制研究进展

抗生素是由微生物在生长发育后期产生的次级代谢产物,具有杀死或抑制细菌生长的能力,因此被广泛应用于细菌感染的临床治疗。在长期的进化过程中,细菌采取多种方式应对环境中抗生素的威胁。除了广为人知的抗生素耐药性(resistance)之外,细菌还能对抗生素产生耐受性(tolerance)和持留性(persistence),严重影响抗生素的临床疗效。鸟苷四磷酸(guanosine tetraphosphate, ppGpp)和鸟苷五磷酸(guanosine pentaphosphate, pppGpp) (本文统称ppGpp)是细菌应对营养饥饿等不利环境时产生的"报警"信号分子,其能够在全局水平调控基因的表达,使细菌适应不利的环境。越来越多的研究表明,ppGpp与细菌应对抗生素胁迫密切相关。基于此,本文综述了细菌中ppGpp的合成与水解及其作用机制,并重点阐述了ppGpp介导抗生素胁迫应答的分子机制,以期为新型抗生素的开发提供新思路。     

细菌的信号转导系统及其在耐药中的作用

耐药菌的日益增多给临床治疗带来巨大的困难,揭示耐药机制成为遏制耐药菌的基本环节.细菌的信号系统是菌体之间信息交流的主要渠道,在调控细菌耐药性方面发挥重要的作用.本文梳理了细菌双组分系统、群体感应系统、第二信使、吲哚等细菌信号系统(分子)与细菌耐药性的关系,总结了各信号系统调控细菌耐药性的机制和途径,包括调控生物膜的形成...     

下一代底盘微生物Vibrio sp. FA2的抗生素抗性

【背景】细菌耐药是当前人类面临的重大挑战之一,抗生素耐药性研究是解决耐药问题的重要途径。Vibrio sp. FA2是环境中分离的一株快速生长菌,其生长速度快于目前报道的快速生长的需钠弧菌(Vibrio natriegens) 14048,并且具有多样的碳源利用能力,是具有较大潜力的下一代微生物底盘,可用于开发化合物高效生产菌株。【目的】前期对FA2菌株的常见抗生素耐药性测试发现该菌具有多重抗生素耐受性,不利于基因工程操作,并且在工业应用中会带来生态安全风险,因此有必要研究其抗生素抗性,并敲除其耐药性基因。【方法】采用抗生素药敏试验测试菌株耐药性,并且利用基因组注释分析筛选目标基因,通过基因敲除构建了关于目标基因的敲除突变株,并比较了FA2野生株和敲除株的抗生素敏感性和生长情况。【结果】系统分析了FA2菌株对以氨苄西林为代表的β-内酰胺类及氨基糖苷类等多种抗生素的耐受性,分析筛选出一系列与FA2菌株耐药性相关的基因。其中,FA2菌株对几种β-内酰胺类抗生素都有较强的耐受性,并且注释到carB6、ampC2和ampC1这3个可能的氨苄西林耐受性基因。研究了氨苄西林耐受性,对3个基因进行了...     

细菌中的群体感应

群体感应(quorum sensing)是细菌根据细胞密度变化进行基因表达调控的一种生理行为。具有群体感应的细菌能产生并释放一种被称为自体诱导物(autoinducer)的信号分子,它随着细胞密度增加而同步增加。当自体诱导物积累到一定浓度时会改变细菌特定基因的表达。革兰氏阳性及阴性细菌通过群体感应与周围环境进行信息交流,从而改变细菌的一系列生理活性,这些细菌的生理特性包括共生、细菌毒性、竞争、接合、抗生素的产生、运动性、孢子及生物膜的形成。这种信号传递方式可能对低等的细胞进一步进化,并形成高等的生物体有重要作用。细菌中群体感应系统的进化可能是多细胞体形成的早期阶段。     

Exploring the role of the immune response in preventing antibiotic resistance

For many bacterial infections, drug resistant mutants are likely present by the time antibiotic treatment starts. Nevertheless, such infections are often successfully cleared. It is commonly assumed that this is due to the combined action of drug and immune response, the latter facilitating clearance of the resistant population. However, most studies of drug resistance emergence during antibiotic treatment focus almost exclusively on the dynamics of bacteria and the drug and neglect the contribution of immune defenses. Here, we develop and analyze several mathematical models that explicitly include an immune response. We consider different types of immune responses and investigate how each impacts the emergence of resistance. We show that an immune response that retains its strength despite a strong drug-induced decline of bacteria numbers considerably reduces the emergence of resistance, narrows the mutant selection window, and mitigates the effects of non-adherence to treatment. Additionally, we show that compared to an immune response that kills bacteria at a constant rate, one that trades reduced killing at high bacterial load for increased killing at low bacterial load is sometimes preferable. We discuss the predictions and hypotheses derived from this study and how they can be tested experimentally.     

Targeting bioenergetics is key to counteracting the drug-tolerant state of biofilm-grown bacteria

Embedded in an extracellular matrix, biofilm-residing bacteria are protected from diverse physicochemical insults. In accordance, in the human host the general recalcitrance of biofilm-grown bacteria hinders successful eradication of chronic, biofilm-associated infections. In this study, we demonstrate that upon addition of promethazine, an FDA approved drug, antibiotic tolerance of in vitro biofilm-grown bacteria can be abolished. We show that following the addition of promethazine, diverse antibiotics are capable of efficiently killing biofilm-residing cells at minimal inhibitory concentrations. Synergistic effects could also be observed in a murine in vivo model system. PMZ was shown to increase membrane potential and interfere with bacterial respiration. Of note, antibiotic killing activity was elevated when PMZ was added to cells grown under environmental conditions that induce low intracellular proton levels. Our results imply that biofilm-grown bacteria avoid antibiotic killing and become tolerant by counteracting intracellular alkalization through the adaptation of metabolic and transport functions. Abrogation of antibiotic tolerance by interfering with the cell’s bioenergetics promises to pave the way for successful eradication of biofilm-associated infections. Repurposing promethazine as a biofilm-sensitizing drug has the potential to accelerate the introduction of new treatments for recalcitrant, biofilm-associated infections into the clinic.     

Bioinformatics approaches for new drug discovery: a review

Prolonged antibiotic therapy for the bacterial infections has resulted in high levels of antibiotic resistance. Initially, bacteria are susceptible to the antibiotics, but can gradually develop resistance. Treating such drug-resistant bacteria remains difficult or even impossible. Hence, there is a need to develop effective drugs against bacterial pathogens. The drug discovery process is time-consuming, expensive and laborious. The traditionally available drug discovery process initiates with the identification of target as well as the most promising drug molecule, followed by the optimization of this, in-vitro, in-vivo and in pre-clinical studies to decide whether the compound has the potential to be developed as a drug molecule. Drug discovery, drug development and commercialization are complicated processes. To overcome some of these problems, there are many computational tools available for new drug discovery, which could be cost effective and less time-consuming. In-silico approaches can reduce the number of potential compounds from hundreds of thousands to the tens of thousands which could be studied for drug discovery and this results in savings of time, money and human resources. Our review is on the various computational methods employed in new drug discovery processes.     

Persisters and beyond: Mechanisms of phenotypic drug resistance and drug tolerance in bacteria

Abstract

One of the challenges in clinical infectious diseases is the problem of chronic infections, which can require long durations of antibiotic treatment and often recur. An emerging explanation for the refractoriness of some infections to treatment is the existence of subpopulations of drug tolerant cells. While typically discussed as “persister” cells, it is becoming increasingly clear that there is significant heterogeneity in drug responses within a bacterial population and that multiple mechanisms underlie the emergence of drug tolerant and drug-resistant subpopulations. Many of these parallel mechanisms have been shown to affect drug susceptibility at the level of a whole population. Here we review mechanisms of phenotypic drug tolerance and resistance in bacteria with the goal of providing a framework for understanding the similarities and differences in these cells.     

Transposon mutagenesis identifies genes which control antimicrobial drug tolerance in stationary-phase Escherichia coli

Tolerance to antimicrobial agents is a universal phenomenon in bacteria which are no longer multiplying or whose growth rate slows. Since slowly multiplying bacteria occur in clinical infections, extended periods of antimicrobial chemotherapy are needed to eradicate these organisms and to achieve cure. In this study, the molecular basis of antibiotic tolerance was investigated using transposon mutagenesis. We screened 5000 Escherichia coli Tn10Cam mutants for reduction of kanamycin tolerance in late stationary phase and found that 4935 mutants were able to grow to late stationary phase. Reduced tolerance was observed in nine mutants which became sensitive to killing by kanamycin. The mutant KS639 was the most sensitive one to kanamycin, and its genome was disrupted in an intergenic region which lies between aldB and yiaW open reading frames. This mutant showed increased sensitivity not only to kanamycin but also to gentamicin, ciprofloxacin and rifampicin. Reduced tolerance of KS639 to kanamycin was also observed in a murine thigh infection model. P1 transduction to the wild type strains confirmed that the intergenic region was responsible for the tolerance of the bacterium to antibiotics. Using PCR-directed one-step gene replacement, we inactivated the genes aldB, yiaW and yiaV. We also deleted the intergenic region. There was no difference in kanamycin tolerance between each mutant (DeltaaldB, DeltayiaW and DeltayiaV) and the parental strain. But the mutant lacking the intergenic region showed reduced tolerance to kanamycin. These data suggest that the intergenic region between aldB and yiaW genes may be involved in tolerance to antimicrobial agents in E. coli. Furthermore, they show that it is important in murine infection during antibiotic treatment and lead to a faster kill of the mutant bacteria.     

Shooting yourself in the foot: How immune cells induce antibiotic tolerance in microbial pathogens

Antibiotic treatment failure of infection is common and frequently occurs in the absence of genetically encoded antibiotic resistance mechanisms. In such scenarios, the ability of bacteria to enter a phenotypic state that renders them tolerant to the killing activity of multiple antibiotic classes is thought to contribute to antibiotic failure. Phagocytic cells, which specialize in engulfing and destroying invading pathogens, may paradoxically contribute to antibiotic tolerance and treatment failure. Macrophages act as reservoirs for some pathogens and impede penetration of certain classes of antibiotics. In addition, increasing evidence suggests that subpopulations of bacteria can survive inside these cells and are coerced into an antibiotic-tolerant state by host cell activity. Uncovering the mechanisms that drive immune-mediated antibiotic tolerance may present novel strategies to improving antibiotic therapy.     

Failure of antibiotic treatment in microbial populations

The tolerance of bacterial populations to biocidal or antibiotic treatment has been well documented in both biofilm and planktonic settings. However, there is still very little known about the mechanisms that produce this tolerance. Evidence that small, non-mutant subpopulations of bacteria are not affected by an antibiotic challenge has been accumulating and provides an attractive explanation for the failure of typical dosing protocols. Although a dosing challenge can kill the susceptible bacteria, the remaining persister cells can serve as a source of population regrowth. We give a condition for the failure of a periodic dosing protocol for a general chemostat model, which supports the simulations of an earlier, more specialized batch model. Our condition implies that the treatment protocol fails globally, in the sense that a mixed bacterial population will ultimately persist above a level that is independent of the initial composition of the population. We also give a sufficient condition for treatment success, at least for initial population compositions near the steady state of interest, corresponding to bacterial washout. Finally, we investigate how the speed at which the bacteria are wiped out depends on the duration of administration of the antibiotic. We find that this dependence is not necessarily monotone, implying that optimal dosing does not necessarily correspond to continuous administration of the antibiotic. Thus, genuine periodic protocols can be more advantageous in treating a wide variety of bacterial infections.      

Directly observing therapy: a new view of drug tolerance in tuberculosis

Drug tolerance in bacteria is widely believed to be due to metabolic changes that accompany growth arrest. A study in this issue of Cell reveals a drug tolerance mechanism in replicating mycobacteria that is induced by residence in macrophages and depends on drug efflux.     

Influence of antibiotic treatment and Wolbachia curing on sexual isolation among Drosophila melanogaster cage populations

Speciation depends on the establishment of reproductive isolation between populations of the same species. Whether assortative mating evolves as a by-product of adaptation is a major question relevant to the origin of species by reproductive isolation. The long-term selection populations used here were originally established 30 years ago from a single cage population (originating from a maternal one) and subsequently subjected to divergent selection for tolerance of toxins in food (heavy metals versus ethanol) to investigate this question. Those populations now differ in sexual isolation and Wolbachia infection status. Wolbachia are common and widespread bacteria infecting arthropods and nematodes. Attention has recently focused on their potential role in insect speciation, due to post-mating sperm-egg incompatibilities induced by the bacteria. In this paper we examine the potential effect of Wolbachia on the level of sexual isolation. By antibiotic curing, we show that removal of Wolbachia decreases levels of mate discrimination (sexual isolation index) between populations by about 50%. Backcrossing experiments confirm that this effect is due to infection status rather than to genetic changes in the populations resulting from antibiotic treatment. Antibiotic treatment has no effect on mate discrimination level between uninfected populations. Our findings suggest that the presence of Wolbachia (or another undetected bacterial associate) act as an additive factor contributing to the level of pre-mating isolation between these Drosophila melanogaster populations. Given the ubiquity of bacterial associates of insects, such effects could be relevant to some speciation events.