环境放线菌中的达托霉素抗性机制

[目的]研究环境放线菌中的达托霉素抗性机制,为临床耐药机制的出现提供预警.[方法]通过测定土壤放线菌(49株)和药用植物内生放线菌(10株)的达托霉素耐受谱,筛选达托霉素抗性菌株;通过达托霉素灭活实验,确定抗性菌株的灭活能力;通过形态观察和16S rRNA序列分析分类鉴定达托霉素降解菌.通过PCR扩增检测达托霉素去酰基化酶基因在降解菌株中的分布情况.[结果]本研究中所有的环境放线菌均耐受达托霉素.在土壤放线菌中和药物植物内生放线菌中,分别有24株(49.0％)和4株(40％)能够灭活达托霉素,25 (51.0％)株和6株(60％)通过其他机制耐受达托霉素.序列测定表明,链霉菌属(Streptomyces)、小单孢菌属(Mcromonospora)和诺卡氏菌属(Nocardia)的部分菌株有灭活达托霉素的能力.PCR扩增表明,5株(17.9％)放线菌含有编码达托霉素去酰基酶的基因.[结论]环境放线菌具有超高的达托霉素抗性频率,灭活达托霉素是主要抗性机制之一.     

应用响应面法优化发酵培养基提高达托霉素产量

[背景]达托霉素来自玫瑰孢链霉菌NRRL 11379的发酵产物,是重要的临床用抗生素.其原始产生菌发酵周期长,影响达托霉素的生产效率.本实验室前期在天蓝色链霉菌中重构了达托霉素的生物合成途径,有效地缩短了发酵周期,但重组菌株K10中达托霉素发酵产量很低,制约了后续的研究和开发.[目的]利用响应面法优化产达托霉素的重组菌...     

院内感染复数菌败血症的临床和耐药分析

目的 探讨院内感染复数菌败血症的临床特征、危险因素、致病菌分布及耐药情况。方法 回顾性分析1970～2004年经血培养和临床资料证实的126例院内感染的复数菌败血症。结果 院内感染复数菌败血症均发生在较严重的基础疾病上．创伤性手术和操作、广谱抗菌药物和免疫抑制剂的应用、放疗与化疗为该病的危险因素。院内感染复数菌败血症感染病死率达52％,致病菌以G^-菌占多数,合并真菌感染者死亡率高。致病菌多为耐药菌株。结论 院内感染复数菌败血症病情危重,病死率高,多系耐药菌株感染。应尽早治疗原发基础疾病,避免危险因素,严格掌握侵入性诊疗手段的运用指征,合理应用抗菌药物。并重视院内环境卫生,严格做好洗手、消毒等是防止耐药菌播散的主要措施。     

嗜麦芽窄食单胞菌耐药机制研究进展

嗜麦芽窄食单胞菌是条件致病菌,主要感染医院内免疫力低下患者,所引起的医院感染日益增多。该菌属于多重耐药菌,对多种抗菌药物及一些消毒剂表现出抗药性。其耐药机制复杂,包括产生水解酶、外膜低渗透性、外排泵系统以及整合子、质粒、转座子等介导的可转移性耐药机制等。     

不动杆菌耐药机制的研究现状

不动杆菌（Acinetobacter）是一种毒力较低的条件致病菌,广泛存在于自然界、人体皮肤及黏膜上,以往该菌所引起的感染很少见.近年来随着抗菌药物的临床广泛应用,诱导了大量该菌属耐药株的产生,该菌的临床分离率越来越高,并形成了对单一抗菌药物耐药到多重耐药（multidrug resistant）,由低耐药率到高耐药率的发展,主要导致肺炎、泌尿系感染、脑膜炎等.因此对这些常用抗菌药物的耐药机制的研究以及如何减少耐药株的产生已成为当前该领域较为关注的主题.     

达托霉素生物合成研究进展

达托霉素是由玫瑰孢链霉菌(Streptomyces roseosporus)生产的一种环脂肽类抗生素, 具有强大的抗革兰氏阳性致病细菌的作用, 是继“抗生素最后一道防线”万古霉素后的新型抗生素。本文主要对达托霉素的结构、作用机制、合成基因簇及合成机制等当前的研究成果进行综述, 并且总结了利用组合生物学对达托霉素进行结构改造的策略, 以此来研究结构与活性之间的关系, 并寻找更广谱高效的抗生素。最后, 本文总结了提高达托霉素产量的策略, 为工业上降低达托霉素生产成本提供理论参考。     

多重耐药性金黄色葡萄球菌(MRSA)的临床药物治疗及耐药机制研究

近年来,由多重耐药性金黄色葡萄球菌引起的院内感染不仅是临床医师必须经常面临的棘手问题,而且已对全人类的健康构成极大威胁。一直以来,万古霉素都是临床治疗革兰阳性菌感染的首选药物,曾被称为临床抗感染的"最后底线"。但目前,万古霉素耐药性金黄色葡萄球菌已经在许多国家分离出来,这就对开发新型抗感染治疗药物提出了迫切要求。就万古霉素及新型替代药物利奈唑胺和达托霉素的作用机制、临床应用情况及耐药状况做一综述。     

外排泵介导肺炎克雷伯菌耐药的研究进展

肺炎克雷伯菌(Klebsiella pneumoniae)是重要的条件致病菌,近年来肺炎克雷伯菌感染在医院内感染中所占的比率持续上升,耐药率也不断攀升,这给临床治疗带来极大的困难。肺炎克雷伯菌发生耐药的重要机制之一就是其细胞膜上存在的外排泵系统,它们将渗入细菌体内的药物不断泵出,导致菌体内的药物浓度过低,不足以发挥抗菌作用。本文主要针对外排泵介导肺炎克雷伯菌的耐药现状,外排泵的分子结构和基因调节,外排泵抑制剂以及传统中药在耐药菌治疗方面的应用等做系统性梳理,以期为临床治疗耐药肺炎克雷伯菌提供一些新思路。     

铜绿假单胞菌对喹诺酮类药物耐药机制的研究进展

铜绿假单胞菌是一种重要的医院内感染条件致病菌,喹诺酮类药物是一类主要用于革兰阴性菌感染的抗菌药物。随着该类药物的广泛使用,该菌的耐药率也逐年上升。大量的研究表明,该菌对喹诺酮类药物的耐药机制主要包括：①编码喹诺酮类药物作用靶位的DNA旋转酶和拓扑异构酶Ⅳ的基因突变,改变了酶的结构,使药物不能与酶－DNA复合物稳定结合;②外排系统调节基因的变异而导致细胞内药物浓度降低。     

食源性致病菌耐药机制研究进展

食源性致病菌是危害食品安全与人体健康的关键因素,而细菌耐药性的不断出现与传播,更加剧了食源性致病菌的潜在风险,成为全球普遍关注的公共卫生焦点问题。本文中,笔者首先综述4种主要的细菌耐药机制:降低细胞膜通透性机制、外排泵机制、药物靶标位点突变机制以及酶解作用机制。在此基础之上,系统回顾了常见食源性致病菌耐药机制的研究进展,并对食源性致病菌耐药机制进一步的研究方向进行了展望,以期为食源性致病菌耐药机制的深入研究提供基础资料,为食源性致病菌耐药性风险的控制提供科学依据。     

Daptomycin,a Bacterial Lipopeptide Synthesized by a Nonribosomal Machinery

Daptomycin (Cubicin®) is a branched cyclic lipopeptide antibiotic of nonribosomal origin and the prototype of the acidic lipopeptide family. It was approved in 2003 for the nontopical treatment of skin structure infections caused by Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), and in 2006 for the treatment of bacteremia. Understanding the ribosome-independent biosynthesis of daptomycin assembly will provide opportunities for the generation of daptomycin derivatives with an altered pharmaceutical spectrum to address upcoming daptomycin-resistant pathogens. Herein, the structural properties of daptomycin, its biosynthesis, recent efforts for the generation of structural diversity, and its proposed mode of action are discussed.     

Regulation mechanisms underlying the biosynthesis of daptomycin and related lipopeptides

Daptomycin is a lipopeptide antibiotics used to treat Gram-positive pathogens infections, including drug-resistant strains. In-depth exploration of its biosynthesis and regulation is crucial for metabolic engineering improvement of this ever-increasing important antibiotic. The past years have witnessed the significant progresses in the understanding of the molecular mechanisms underlying the biosynthesis and regulation of daptomycin. This information was updated in our review, with special focus on the regulatory network integrating a wide variety of physiological and environmental inputs. This should provide novel insight into the regulatory mechanism of biosynthesis of daptomycin and nodes for strain improvement to increase the yields of daptomycin.     

Daptomycin treatment impacts resistance in off-target populations of vancomycin-resistant Enterococcus faecium

The antimicrobial resistance crisis has persisted despite broad attempts at intervention. It has been proposed that an important driver of resistance is selection imposed on bacterial populations that are not the intended target of antimicrobial therapy. But to date, there has been limited quantitative measure of the mean and variance of resistance following antibiotic exposure. Here we focus on the important nosocomial pathogen Enterococcus faecium in a hospital system where resistance to daptomycin is evolving despite standard interventions. We hypothesized that the intravenous use of daptomycin generates off-target selection for resistance in transmissible gastrointestinal (carriage) populations of E. faecium. We performed a cohort study in which the daptomycin resistance of E. faecium isolated from rectal swabs from daptomycin-exposed patients was compared to a control group of patients exposed to linezolid, a drug with similar indications. In the daptomycin-exposed group, daptomycin resistance of E. faecium from the off-target population was on average 50% higher than resistance in the control group (n = 428 clones from 22 patients). There was also greater phenotypic diversity in daptomycin resistance within daptomycin-exposed patients. In patients where multiple samples over time were available, a wide variability in temporal dynamics were observed, from long-term maintenance of resistance to rapid return to sensitivity after daptomycin treatment stopped. Sequencing of isolates from a subset of patients supports the argument that selection occurs within patients. Our results demonstrate that off-target gastrointestinal populations rapidly respond to intravenous antibiotic exposure. Focusing on the off-target evolutionary dynamics may offer novel avenues to slow the spread of antibiotic resistance.

This study shows that systemic daptomycin use causes selection for daptomycin resistance in gastrointestinal populations of vancomycin-resistant Enterococcus faecium, highlighting the importance of off-target selection on the evolution of antimicrobial resistance.     

Manipulation of kynurenine pathway for enhanced daptomycin production in Streptomyces roseosporus

Daptomycin is a cyclic lipopeptide natural product produced by Stretptomyces roseosporus, displaying good bactericidal activity against a wide range of gram‐positive pathogens. Daptomycin contains a 13 amino acid and kynurenine (Kyn) is essential for optimal activity of daptomycin. In this study, we characterized the Kyn pathway in S. roseosporus and investigated its role in supplying precursor for daptomycin biosynthesis. Two genes (dptJ and tdo) coding for tryptophan‐2,3‐dioxgenase existed in the chromosome. dptJ is located in the daptomycin biosynthetic gene cluster, while tdo is in other locus. Disruption of dptJ or tdo resulted in reduced yield by ～50%. The introduction of an additional copy of dptJ but not tdo led to enhanced production of daptomycin by 110%. Furthermore, disruption of kyn encoding kynureninase showed improved daptomycin productivity by 30%. Our results demonstrated that the enhancement of Kyn supply through metabolic engineering approach is an efficient way to increase daptomycin production. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:847–852, 2013     

Antibiotic resistance is prevalent in an isolated cave microbiome

Antibiotic resistance is a global challenge that impacts all pharmaceutically used antibiotics. The origin of the genes associated with this resistance is of significant importance to our understanding of the evolution and dissemination of antibiotic resistance in pathogens. A growing body of evidence implicates environmental organisms as reservoirs of these resistance genes; however, the role of anthropogenic use of antibiotics in the emergence of these genes is controversial. We report a screen of a sample of the culturable microbiome of Lechuguilla Cave, New Mexico, in a region of the cave that has been isolated for over 4 million years. We report that, like surface microbes, these bacteria were highly resistant to antibiotics; some strains were resistant to 14 different commercially available antibiotics. Resistance was detected to a wide range of structurally different antibiotics including daptomycin, an antibiotic of last resort in the treatment of drug resistant Gram-positive pathogens. Enzyme-mediated mechanisms of resistance were also discovered for natural and semi-synthetic macrolide antibiotics via glycosylation and through a kinase-mediated phosphorylation mechanism. Sequencing of the genome of one of the resistant bacteria identified a macrolide kinase encoding gene and characterization of its product revealed it to be related to a known family of kinases circulating in modern drug resistant pathogens. The implications of this study are significant to our understanding of the prevalence of resistance, even in microbiomes isolated from human use of antibiotics. This supports a growing understanding that antibiotic resistance is natural, ancient, and hard wired in the microbial pangenome.     

The action mechanism of daptomycin

Daptomycin is a lipopeptide antibiotic produced by the soil bacterium Streptomyces roseosporus that is clinically used to treat severe infections with Gram-positive bacteria. In this review, we discuss the mode of action of this important antibiotic. Although daptomycin is structurally related to amphomycin and similar lipopeptides that inhibit peptidoglycan biosynthesis, experimental studies have not produced clear evidence that daptomycin shares their action mechanism. Instead, the best characterized effect of daptomycin is the permeabilization and depolarization of the bacterial cell membrane. This activity, which can account for daptomycin’s bactericidal effect, correlates with the level of phosphatidylglycerol (PG) in the membrane. Accordingly, reduced synthesis of PG or its increased conversion to lysyl-PG promotes bacterial resistance to daptomycin. While other resistance mechanisms suggest that daptomycin may indeed directly interfere with cell wall synthesis or cell division, such effects still await direct experimental confirmation. Daptomycin’s complex structure and biosynthesis have hampered the analysis of its structure activity relationships. Novel methods of total synthesis, including a recent one that is carried out entirely on a solid phase, will enable a more thorough and systematic exploration of the sequence space.     

Broad-spectrum antimicrobial peptide resistance by MprF-mediated aminoacylation and flipping of phospholipids

Bacteria are frequently exposed to cationic antimicrobial peptides (CAMPs) from eukaryotic hosts (host defence peptides) or from prokaryotic competitors (bacteriocins). However, many bacteria, among them most of the major human pathogens, achieve CAMP resistance by MprF, a unique enzyme that modifies anionic phospholipids with l-lysine or l-alanine thereby introducing positive charges into the membrane surface and reducing the affinity for CAMPs. The lysyl or alanyl groups are derived from aminoacyl tRNAs and are usually transferred to phosphatidylglycerol (PG). Recent studies with MprF from Staphylococcus aureus demonstrated that production of Lys-PG only leads to CAMP resistance when an additional flippase domain of MprF is present that translocates Lys-PG and exposes it at the outer leaflet of the membrane. Thus, MprF exerts two specific functions that have hardly been found in other bacterial proteins. MprF proteins are crucial virulence factors of many human pathogens, which recommends them as targets for new anti-virulence drugs. Intriguingly, specific point mutations in mprF cause resistance to the CAMP-like antibiotic daptomycin in a yet unclear way that may involve altered Lys-PG synthesis and/or Lys-PG flipping capacities. Thus, a thorough characterization of MprF domains and functions will help to unravel how bacteria maintain and protect their cytoplasmic membranes.     

Cardiolipin Prevents Membrane Translocation and Permeabilization by Daptomycin

Daptomycin is an acidic lipopeptide antibiotic that, in the presence of calcium, forms oligomeric pores on membranes containing phosphatidylglycerol. It is clinically used against various Gram-positive bacteria such as Staphylococcus aureus and Enterococcus species. Genetic studies have indicated that an increased content of cardiolipin in the bacterial membrane may contribute to bacterial resistance against the drug. Here, we used a liposome model to demonstrate that cardiolipin directly inhibits membrane permeabilization by daptomycin. When cardiolipin is added at molar fractions of 10 or 20% to membranes containing phosphatidylglycerol, daptomycin no longer forms pores or translocates to the inner membrane leaflet. Under the same conditions, daptomycin continues to form oligomers; however, these oligomers contain only close to four subunits, which is approximately half as many as observed on membranes without cardiolipin. The collective findings lead us to propose that a daptomycin pore consists of two aligned tetramers in opposite leaflets and that cardiolipin prevents the translocation of tetramers to the inner leaflet, thereby forestalling the formation of complete, octameric pores. Our findings suggest a possible mechanism by which cardiolipin may mediate resistance to daptomycin, and they provide new insights into the action mode of this important antibiotic.     

Breeding of High Daptomycin-Producing Strain by Streptomycin Resistance Superposition

Daptomycin is a cyclolipopeptide antibiotic produced by Streptomyces roseosporus. It is widely used to treat drug-resistant bacterial infections; however, daptomycin yield in wild strains is very low. To improve the daptomycin production by the strain BNCC 342432, a modified method of ribosome engineering with superposition of streptomycin resistance was adopted in this study. The highest-yield mutant strain SR-2620 was obtained by increasing streptomycin resistance of BNCC 342432, and achieved daptomycin production of 38.5 mg/l in shake-flask fermentation, 1.79-fold higher than the parent strain and its heredity stability was stable. The morphological characteristics of the two strains were significantly different, and the 440^th base G of the rpsL gene in the mutant strain was deleted, which resulted in a frameshift mutation. Our results demonstrate that gradually increasing strain resistance to streptomycin was an effective breeding method to improve daptomycin yield in S. roseosporus. Open in a separate window     

Structure-activity relationship of daptomycin analogues with substitution at (2S, 3R) 3-methyl glutamic acid position

Daptomycin is a highly effective lipopeptide antibiotic against Gram-positive pathogens. The presence of (2S, 3R) 3-methyl glutamic acid (mGlu) in daptomycin has been found to be important to the antibacterial activity. However the role of (2S, 3R) mGlu is yet to be revealed. Herein, we reported the syntheses of three daptomycin analogues with (2S, 3R) mGlu substituted by (2S, 3R) methyl glutamine (mGln), dimethyl glutamic acid and (2S, 3R) ethyl glutamic acid (eGlu), respectively, and their antibacterial activities. The detailed synthesis of dimethyl glutamic acid was also reported.