Efflux pumps

外排泵
  • 文章类型: Journal Article
    铜绿假单胞菌是导致严重多部位感染的重要病原体,具有高发病率和死亡率。本研究分析了山东省某三甲医院耐碳青霉烯类铜绿假单胞菌(CRPA),中国,使用全基因组测序(WGS)。目的是探讨碳青霉烯耐药的机制和分子特征。对2022年1月至2023年3月的91株分离株进行了回顾性分析,其中包括菌株鉴定和抗菌药物敏感性测试。WGS用于确定这些CRPA菌株的基因组序列,使用平均核苷酸鉴定(ANI)精确鉴定了该物种,进一步分析了多位点序列分型和菌株相关性。发现一些菌株携带ampD和oprD基因,而只有少数携带碳青霉烯酶基因或相关基因。值得注意的是,所有菌株都拥有mexA,mexE,和mexX基因.鉴定的主要谱系是ST244,其次是ST235。该研究揭示了医院分离株中不同的碳青霉烯耐药机制,与中国大陆以前的研究不同。它强调碳青霉烯抗性不是由于单一机制,而是由于酶介导的抗性的组合。AmpC过表达,OprD功能障碍,和外排泵过度表达。这项研究为该地区CRPA抗性的进化机制和分子特征提供了有价值的见解,协助国家预防和控制CRPA,并为靶向和开发新药提供参考。
    Pseudomonas aeruginosa is a significant pathogen responsible for severe multisite infections with high morbidity and mortality rates. This study analyzed carbapenem-resistant Pseudomonas aeruginosa (CRPA) at a tertiary hospital in Shandong, China, using whole-genome sequencing (WGS). The objective was to explore the mechanisms and molecular characteristics of carbapenem resistance. A retrospective analysis of 91 isolates from January 2022 to March 2023 was performed, which included strain identification and antimicrobial susceptibility testing. WGS was utilized to determine the genome sequences of these CRPA strains, and the species were precisely identified using average nucleotide identification (ANI), with further analysis on multilocus sequence typing and strain relatedness. Some strains were found to carry the ampD and oprD genes, while only a few harbored carbapenemase genes or related genes. Notably, all strains possessed the mexA, mexE, and mexX genes. The major lineage identified was ST244, followed by ST235. The study revealed a diverse array of carbapenem resistance mechanisms among hospital isolates, differing from previous studies in mainland China. It highlighted that carbapenem resistance is not due to a single mechanism but rather a combination of enzyme-mediated resistance, AmpC overexpression, OprD dysfunction, and efflux pump overexpression. This research provides valuable insights into the evolutionary mechanisms and molecular features of CRPA resistance in this region, aiding in the national prevention and control of CRPA, and offering references for targeting and developing new drugs.
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  • 文章类型: Journal Article
    变形链球菌是革兰氏阳性,兼性厌氧细菌,在牙齿表面形成生物膜后,会导致龋齿,同时产生有机酸,使牙釉质和牙本质脱矿。我们观察到多不饱和花生四烯酸(AA)(ω-6;20:4)具有抗变形链球菌的抗菌活性,这促使我们研究它的作用机制。在存在5%CO2的情况下,AA对变形链球菌的最低抑制浓度(MIC)为25μg/ml,而在不添加CO2的情况下,其降低至6.25-12.5μg/ml。抗菌作用是由于杀菌和抑菌作用的组合。最小生物膜抑制浓度(MBIC)与MIC相同,这表明抗生物膜作用的一部分是由于抗菌活性。基因表达研究显示生物膜相关基因的表达降低,表明AA还具有特定的抗生物膜作用。使用电位DiOC2(3)染料的流式细胞仪分析,荧光外排泵基板,和活/死SYTO9/碘化丙啶染色显示AA导致立即膜超极化,改变膜运输和外排泵活动,并随着随后的膜穿孔而增加膜的渗透性。高分辨率扫描电子显微镜(HR-SEM)显示爆发细菌的残留物。此外,使用氧化还原探针2的流式细胞仪分析,7'-二氯荧光素二乙酸酯(DCFHDA)表明AA以剂量依赖性方式充当抗氧化剂。α-生育酚,一种终止自由基链的抗氧化剂,抵消了AA的抗菌活性,表明细菌中AA的氧化会导致细胞毒性自由基的产生,从而导致细菌生长停滞和死亡。重要的是,即使在100μg/ml时,AA对正常Vero上皮细胞也没有毒性,它没有引起红细胞溶血。总之,我们的研究表明,AA是一种潜在安全的药物,可用于减少致龋变形链球菌的细菌负担。
    Streptococcus mutans is a Gram-positive, facultative anaerobic bacterium, which causes dental caries after forming biofilms on the tooth surface while producing organic acids that demineralize enamel and dentin. We observed that the polyunsaturated arachidonic acid (AA) (ω-6; 20:4) had an anti-bacterial activity against S. mutans, which prompted us to investigate its mechanism of action. The minimum inhibitory concentration (MIC) of AA on S. mutans was 25 μg/ml in the presence of 5% CO2, while it was reduced to 6.25-12.5 μg/ml in the absence of CO2 supplementation. The anti-bacterial action was due to a combination of bactericidal and bacteriostatic effects. The minimum biofilm inhibitory concentration (MBIC) was the same as the MIC, suggesting that part of the anti-biofilm effect was due to the anti-bacterial activity. Gene expression studies showed decreased expression of biofilm-related genes, suggesting that AA also has a specific anti-biofilm effect. Flow cytometric analyses using potentiometric DiOC2(3) dye, fluorescent efflux pump substrates, and live/dead SYTO 9/propidium iodide staining showed that AA leads to immediate membrane hyperpolarization, altered membrane transport and efflux pump activities, and increased membrane permeability with subsequent membrane perforation. High-resolution scanning electron microscopy (HR-SEM) showed remnants of burst bacteria. Furthermore, flow cytometric analysis using the redox probe 2\',7\'-dichlorofluorescein diacetate (DCFHDA) showed that AA acts as an antioxidant in a dose-dependent manner. α-Tocopherol, an antioxidant that terminates the radical chain, counteracted the anti-bacterial activity of AA, suggesting that oxidation of AA in bacteria leads to the production of cytotoxic radicals that contribute to bacterial growth arrest and death. Importantly, AA was not toxic to normal Vero epithelial cells even at 100 μg/ml, and it did not cause hemolysis of erythrocytes. In conclusion, our study shows that AA is a potentially safe drug that can be used to reduce the bacterial burden of cariogenic S. mutans.
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  • 文章类型: Journal Article
    银良好的抗菌性能使其广泛应用于食品中,医学,和环境应用。然而,随着银基抗菌剂的广泛使用,银基抗菌剂在环境中的释放和积累越来越多,抗银细菌的患病率正在增加。为了防止超级细菌的出现,有必要对毒品使用进行合理和严格的控制。细菌对银的抗性机制尚未完全阐明,本文就细菌抗银机制的研究进展作一综述。结果表明,细菌对银的抗性可以通过诱导银颗粒聚集和Ag+还原而发生,抑制银接触和进入细胞,细胞中银颗粒和Ag+的流出,并激活损伤修复机制。我们建议抗银的细菌机制涉及相关系统的组合。最后,我们将讨论这些信息如何用于开发下一代银基抗菌药物和抗菌疗法。并提出了一些抗菌策略,如“特洛伊木马”-伪装,使用外排泵抑制剂来减少银外排,与“扫雷艇”一起工作,银颗粒的固定。
    The good antimicrobial properties of silver make it widely used in food, medicine, and environmental applications. However, the release and accumulation of silver-based antimicrobial agents in the environment is increasing with the extensive use of silver-based antimicrobials, and the prevalence of silver-resistant bacteria is increasing. To prevent the emergence of superbugs, it is necessary to exercise rational and strict control over drug use. The mechanism of bacterial resistance to silver has not been fully elucidated, and this article provides a review of the progress of research on the mechanism of bacterial resistance to silver. The results indicate that bacterial resistance to silver can occur through inducing silver particles aggregation and Ag+ reduction, inhibiting silver contact with and entry into cells, efflux of silver particles and Ag+ in cells, and activation of damage repair mechanisms. We propose that the bacterial mechanism of silver resistance involves a combination of interrelated systems. Finally, we discuss how this information can be used to develop the next generation of silver-based antimicrobials and antimicrobial therapies. And some antimicrobial strategies are proposed such as the \"Trojan Horse\" - camouflage, using efflux pump inhibitors to reduce silver efflux, working with \"minesweeper\", immobilization of silver particles.
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  • 文章类型: Journal Article
    四环素(TCs)在农业和医药上的广泛利用,导致四环素耐药性在人类中无边界传播,动物,和环境,对生态系统和人类社会都构成了巨大的风险。官能团修饰的变化导致新一代TC具有更高的抑菌功效,但是它们对抗生素抗性基因(ARGs)的出现和进化的影响尚不清楚。为此,选择了三代的四个TC,比较了它们对影响土壤微生物群落中ARGs进化的结构效应。研究结果表明,低世代TC,如四环素和土霉素,与高代替加环素相比,显示出更大的刺激ARGs产生和增殖的倾向。分子对接分析表明,D环官能团的修饰决定了TC与转录调节因子和外排泵的底物结合袋的结合能力,主要涉及耐药性。这可以通过逆转录定量聚合酶链反应定量和细胞内抗生素积累评估进一步证明。本研究从化合物-蛋白质结合的角度揭示了抗生素诱导ARG产生的结构效应的机制。从而为控制抗生素耐药性的传播提供理论支持。
    The widespread utilization of tetracyclines (TCs) in agriculture and medicine has led to the borderless spread of tetracycline resistance in humans, animals, and the environment, posing huge risks to both the ecosystem and human society. Changes in the functional group modifications resulted in a higher bacteriostatic efficacy of the new generation of TCs, but their effect on the emergence and evolution of antibiotic resistance genes (ARGs) is not yet known. To this end, four TCs from three generations were chosen to compare their structural effects on influencing the evolution of ARGs in soil microbial communities. The findings revealed that low-generation TCs, such as tetracycline and oxytetracycline, exhibited a greater propensity to stimulate the production and proliferation of ARGs than did high-generation tigecycline. Molecular docking analysis demonstrated that modifications of the D-ring functional group determined the binding capacity of TCs to the substrate-binding pocket of transcriptional regulators and efflux pumps mainly involved in drug resistance. This can be further evidenced by reverse transcription-quantitative polymerase chain reaction quantification and intracellular antibiotic accumulation assessment. This study sheds light on the mechanism of the structural effect of antibiotic-induced ARG production from the perspective of compound-protein binding, therefore providing theoretical support for controlling the dissemination of antibiotic resistance.
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  • 文章类型: Journal Article
    金属纳米粒子(MNPs)由于其广谱的应用前景,近年来受到了广泛的关注,特别是在生物医学应用中。这里,我们发现,长期暴露于铂纳米颗粒(PtNPs)会增加铜绿假单胞菌PAO1对亚胺培南和环丙沙星的敏感性。我们将PAO1暴露于PtNP(一系列剂量,从0.125到35μg/mL)持续60天,并对进化菌株(ES)进行了表征,并与野生型(WT)进行了比较,以了解敏感性提高的机制。我们发现,oprD的过表达和mexEF-oprN的下调促进了抗生素的细胞内积累,从而增加敏感性。此外,在调节因子lasR和mexT中发现了功能丧失突变。将完整的lasR从野生型(WT)克隆到ES中略微改善亚胺培南抗性。引人注目的是,将mexT从WT克隆到ES中可以将亚胺培南和环丙沙星的抗性恢复到原始水平。简而言之,由mexT控制的膜通透性的增加使得PAO1对亚胺培南和环丙沙星非常敏感,lasR介导的群体感应降低使PAO1对亚胺培南略有敏感。总的来说,这些结果揭示了长期暴露于MNPs的抗生素敏感性机制,这提供了一种有希望的方法来防止抗生素耐药性。
    Metal nanoparticles (MNPs) have recently gained extensive attention due to their broad-spectrum prospect, particularly in biomedical application. Here, we reveal that long-term exposure to platinum nanoparticles (Pt NPs) increases the susceptibility of Pseudomonas aeruginosa PAO1 to imipenem and ciprofloxacin. We exposed PAO1 to Pt NPs (a series of doses, varying from 0.125 to 35 μg/mL) for 60 days and characterized the evolved strains (ES) and compared with wild type (WT) to understand the mechanism of heightened sensitivity. We found that overexpression of oprD and downregulation of mexEF-oprN facilitate the intracellular accumulation of antibiotic, thus increasing susceptibility. Furthermore, loss-of-function mutations were discovered in regulators lasR and mexT. Cloning intact lasR from wild-type (WT) into ES slightly improves imipenem resistance. Strikingly, cloning mexT from WT into ES reverts the imipenem and ciprofloxacin resistance to the original level. Briefly, the increase of membrane permeability controlled by mexT made PAO1 greatly susceptible to imipenem and ciprofloxacin, and the decrease of quorum sensing mediated by lasR made PAO1 slightly susceptible to imipenem. Overall, these results reveal an antibiotic susceptibility mechanism from prolonged exposure to MNPs, which provides a promising approach to prevent antibiotic resistance.
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  • 文章类型: Journal Article
    重金属抗性细菌分泌细胞外蛋白(e-PNs)。然而,e-PN在重金属抗性中的作用仍然难以捉摸。这里傅里叶变换红外光谱暗示N-H,C=O和NH2-R在模型生物Cuprividuspauculus1490中Ni2的吸附和抗性中起着至关重要的作用(C。pauculus)。蛋白酶K处理降低了C.pauculus的Ni2抗性,强调了e-PN的重要作用。进一步的三维激发-发射基质荧光光谱分析表明,随着Ni2处理,作为e-PN一部分的色氨酸蛋白显着增加。蛋白质组学和定量实时聚合酶链反应数据表明,响应Ni2在C的代谢中引起了主要变化。在这些脂多糖生物合成中,一般分泌途径,Ni2+相关转运蛋白和多药外排在Ni2+耐药中起重要作用。总之,结果提供了一个概念模型,用于理解e-PN如何促进细菌耐药性和Ni2吸附。
    Heavy metal-resistant bacteria secrete extracellular proteins (e-PNs). However, the role of e-PNs in heavy metal resistance remains elusive. Here Fourier Transform Infrared Spectroscopy implied that N-H, C = O and NH2-R played a crucial role in the adsorption and resistance of Ni2+ in the model organism Cuprividus pauculus 1490 (C. pauculus). Proteinase K treatment reduced Ni2+ resistance of C. pauculus underlining the essential role of e-PNs. Further three-dimension excitation-emission matrix fluorescence spectroscopy analysis demonstrated that tryptophan proteins as part of the e-PNs increased significantly with Ni2+ treatment. Proteomic and quantitative real-time polymerase chain reaction data indicated that major changes were induced in the metabolism of C. pauculus in response to Ni2+. Among those lipopolysaccharide biosynthesis, general secretion pathways, Ni2+-affiliated transporters and multidrug efflux play an essential role in Ni2+ resistance. Altogether the results provide a conceptual model for comprehending how e-PNs contribute to bacterial resistance and adsorption of Ni2+.
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  • 文章类型: Journal Article
    替加环素耐药肠杆菌已成为公众关注的焦点,和移动tet(X)变体和tmexCD-toprJ外排泵主要负责替加环素耐药性的传播。医院污水被认为是抗生素耐药性的重要蓄水池,而在这一生态位中对替加环素的耐药性研究不足。
    在这项研究中,从一组替加环素耐药肠杆菌中筛选出5株大肠埃希菌和6株肺炎克雷伯菌,通过药敏试验进行进一步调查,共轭,全基因组测序,和生物信息学分析。
    所有五种大肠杆菌菌株都带有tet(X4),它位于不同的质粒上,包括新型的IncC/IncFIA(HI1)/IncHI1A/IncHI1B(R27)杂化构造。此外,携带tet(X4)的质粒能够通过缀合转移并在不存在抗生素的情况下在受体中稳定。tmexCD1-toprJ1在两种肺炎克雷伯菌(LZSFT39和LZSRT3)中被鉴定,它由一种新型的多药耐药转座子携带,在新的IncR/IncU杂交质粒上命名为Tn7368。此外,我们发现两种肺炎克雷伯菌(LZSFZT3和LZSRT3)表现出过表达外排基因acrB和oqxB,分别,这很可能是由ramR和oqxR的突变引起的。
    总而言之,这项研究的发现扩大了我们对携带替加环素抗性基因的遗传元件的认识,建立了研究人类结构多样性和进化轨迹的基线,动物,和环境替加环素抗性体。
    UNASSIGNED: The tigecycline-resistant Enterobacterales have emerged as a great public concern, and the mobile tet(X) variants and tmexCD-toprJ efflux pump are mainly responsible for the spread of tigecycline resistance. Hospital sewage is considered as an important reservoir of antimicrobial resistance, while tigecycline resistance in this niche is under-researched.
    UNASSIGNED: In this study, five Escherichia coli and six Klebsiella pneumoniae strains were selected from a collection of tigecycline-resistant Enterobacterales for further investigation by antimicrobial susceptibility testing, conjugation, whole-genome sequencing, and bioinformatics analysis.
    UNASSIGNED: All five E. coli strains harbored tet(X4), which was located on different plasmids, including a novel IncC/IncFIA(HI1)/IncHI1A/IncHI1B(R27) hybrid structure. In addition, tet(X4)-bearing plasmids were able to transfer by conjugation and be stabilized in the recipient in the absence of antibiotics. tmexCD1-toprJ1 was identified in two K. pneumoniae (LZSFT39 and LZSRT3) and it was carried by a novel multidrug-resistance transposon, designated Tn7368, on a novel IncR/IncU hybrid plasmid. In addition, we found that two K. pneumoniae (LZSFZT3 and LZSRT3) showed overexpression of efflux genes acrB and oqxB, respectively, which was most likely to be caused by mutations in ramR and oqxR.
    UNASSIGNED: In conclusion, the findings in this study expand our knowledge of the genetic elements that carry tigecycline resistance genes, which establishes a baseline for investigating the structure diversity and evolutionary trajectories of human, animal, and environmental tigecycline resistomes.
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  • 文章类型: Journal Article
    目的:耳念珠菌是最近出现的一种致病性真菌,因其对常规抗真菌药物的耐药性而受到全球关注。本研究采用全基因组方法来探索C.auris如何克服常见抗真菌药物氟康唑的生长抑制作用。我们关注的是一种名为转座子的“跳跃遗传元件”引起的基因破坏,导致氟康唑耐药。我们发现了两个基因的突变,每个编码Ubr2/Mub1泛素连接酶复合物的成分,这标志着转录调节因子Rpn4的降解。当任何一种蛋白质都不存在时,稳定的Rpn4在细胞中积累。我们发现Rpn4通过与启动子中的PACE元件结合来激活其自身以及主要药物外排泵基因CDR1的表达。此外,我们在许多耐药临床分离株中发现了Ubr2的氨基酸变化,有助于Rpn4稳定和增加氟康唑耐药性。
    OBJECTIVE: Candida auris is a recently emerged pathogenic fungus of grave concern globally due to its resistance to conventional antifungals. This study takes a whole-genome approach to explore how C. auris overcomes growth inhibition imposed by the common antifungal drug fluconazole. We focused on gene disruptions caused by a \"jumping genetic element\" called transposon, leading to fluconazole resistance. We identified mutations in two genes, each encoding a component of the Ubr2/Mub1 ubiquitin-ligase complex, which marks the transcription regulator Rpn4 for degradation. When either protein is absent, stable Rpn4 accumulates in the cell. We found that Rpn4 activates the expression of itself as well as the main drug efflux pump gene CDR1 by binding to a PACE element in the promoter. Furthermore, we identified an amino acid change in Ubr2 in many resistant clinical isolates, contributing to Rpn4 stabilization and increased fluconazole resistance.
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  • 文章类型: Journal Article
    目的:吲哚金杆菌是一种临床相关的微生物,一直在上升,报道了多药耐药(MDR)菌株。已证明携带tet(X2)的吲哚基因对抗生素替加环素具有抗性,然而,对四环素家族的所有其他成员敏感。抗性的这种不一致性促使人们对tet(X2)对吲哚梭菌中替加环素抗性的贡献进行研究。材料和方法:在这项研究中,我们报告了对替加环素耐药但对四环素敏感的MDRC.indologies菌株(CI3125)中替加环素耐药的基因组机制的综合分析,多西环素,还有米诺环素.我们用全基因组测序,定量逆转录PCR,蛋白质印迹,抗生素降解试验,和外排泵抑制试验,以揭示吲哚梭菌对替加环素的耐药机制,并阐明四环素家族抗生素耐药机制的不一致。结果:我们的研究结果表明,CI3125携带60个抗生素抗性基因,分布在6个不同的遗传岛(GI)上,具有水平转移的潜力。值得注意的是,tet(X2)基因位于CI3125的GI06上。tet(X2)的遗传环境分析表明,黄杆菌属和拟杆菌属中的所有tet(X2)基因在上游共享一个保守且功能性的核糖体结合位点。与预期相反,我们的RT-qPCR显示,在CI3125中,tet(X2)未被转录,而Western印迹表明,在CI3125中,tet(X2)蛋白不存在。相反,我们证明,在存在五种不同的外排泵抑制剂[1-(1-萘基-甲基)-哌嗪,苯基-精氨酸-β-萘胺,维拉帕米,利血平,和羰基氰化物3-氯苯腙]。这一发现为外排泵参与替加环素耐药性提供了证据,这可能是C.indologenes之间的普遍机制。我们的研究表明,CI3125中对四环素家族的耐药性不一致可能归因于tet(X2)的沉默和替加环素外排泵的功能。结论:总体而言,我们的研究结果强调了基因组方法在理解临床相关微生物中抗生素耐药的潜在机制方面的重要性.当CI3125中的tet(X2)保持沉默时,我们的研究结果表明,它可能通过GI水平传播。因此,我们的研究结果对于在临床环境中处理吲哚梭菌感染具有重要意义.
    Purpose: Chryseobacterium indologenes is a clinically relevant microorganism that has been on the rise, with multidrug-resistant (MDR) strains being reported. C. indologenes carrying tet(X2) has been demonstrated to be resistant to the antibiotic tigecycline, yet, sensitive to all other members of the tetracycline family. This inconsistency in resistance prompts an inquiry into the contribution of tet(X2) to tigecycline resistance in C. indologenes. Materials and Methods: In this study, we report on a comprehensive analysis of the genomic mechanisms underlying tigecycline resistance in a MDR C. indologenes strain (CI3125) that was resistant to tigecycline but sensitive to tetracycline, doxycycline, and minocycline. We used whole-genome sequencing, quantitative reverse transcription PCR, Western blot, antibiotic-degrading tests, and efflux pump inhibiting tests to reveal the mechanism of tigecycline resistance in C. indologenes and elucidate the inconsistency in the antibiotic resistance mechanism for the tetracycline family. Results: Our findings demonstrate that CI3125 carries 60 antibiotic resistance genes distributed on 6 different genetic islands (GIs), with the potential for horizontal transfer. Notably, the tet(X2) gene is located on GI06 of CI3125. Genetic environment analysis of tet(X2) showed that all tet(X2) genes in Flavobacterium and Bacteroides share a conservative and functional ribosome-binding site upstream. Contrary to expectation, our RT-qPCR showed that tet(X2) was not transcribed in CI3125, and Western blot suggested the absence of tet(X2) protein in CI3125. Rather, we demonstrate that minimum inhibitory concentration values for tigecycline decreased two- to eight-folds in the presence of five different efflux pump inhibitors [1-(1-naphthyl- methyl)-piperazine, phenyl-arginine-β-naphthylamide, verapamil, reserpine, and carbonyl cyanide 3-chlorophenylhydrazone]. This finding provides evidence for the involvement of efflux pumps in tigecycline resistance, which is likely to be a universal mechanism among C. indologenes. Our study proposes that the inconsistency in resistance to the tetracycline family in CI3125 may be ascribed to the silence of tet(X2) and the functions of efflux pumps for tigecycline. Conclusions: Overall, our results highlight the importance of genomic approaches in understanding the underlying mechanisms of antibiotic resistance in clinically relevant microorganisms. While tet(X2) in CI3125 is silent, our findings suggest that it may be horizontally spread through GIs. Hence, our findings have significant implications for the management of C. indologenes infections in clinical settings.
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  • 文章类型: Journal Article
    替加环素是临床治疗多药耐药菌感染的重要抗菌药物,和替加环素耐药的金黄色葡萄球菌(TRSA)近年来的报道越来越多。值得注意的是,只有rpsJ和mepA与金黄色葡萄球菌的替加环素耐药性相关。mepA基因编码MepA外排泵,mepA的过度表达已被证实与替加环素耐药性直接相关。尽管MepA的突变广泛发生,TRSA与MepA突变之间的关联尚不清楚.在这项研究中,我们从各种来源探索了mepA基因的突变。然后,替加环素耐药相关突变T29I,E287G,并鉴定了MepA中的T29I+E287G,并通过突变体缺失和互补来评估它们的效果,替加环素积累试验,和分子对接实验。结果表明,替加环素的MIC,庆大霉素,和阿米卡星在特殊的互补转化体中增加,并在添加外排泵抑制剂羰基氰化物3-氯苯腙(CCCP)后恢复。mepA缺失突变株及其互补转化体的替加环素积累试验表明,T29I,E287G,T29I+E287G突变促进替加环素外排,分子对接显示突变T29I,E287G,和T29I+E287G降低了结合能并有助于配体结合。此外,我们推断了替加环素在体外选择压力下金黄色葡萄球菌的进化轨迹。总的来说,我们的研究表明,MepA突变在金黄色葡萄球菌的替加环素耐药中起重要作用.重要性先前的分析表明,除rpsJ突变外,MepA的过表达是涉及替加环素抗性的确切机制,通常取决于mepR突变体。然而,尚无研究评估MepA中TRSA中发现的多种突变的影响.这项研究表明,MepA中的突变赋予了对替加环素的抗性而没有过表达,并为鉴定TRSA提供了基因型参考。尽管在本研究中鉴定的MepA中的替加环素耐药相关突变在临床分离株中尚未观察到,鉴于金黄色葡萄球菌菌株在环境中普遍存在,因此应该探索其机制。在MepA中与替加环素抗性相关的突变流行之前,应在时间窗口内采取措施以包含TRSA。
    Tigecycline is an important antibacterial drug for treating infection by clinical multidrug-resistant bacteria, and tigecycline-resistant Staphylococcus aureus (TRSA) has been increasingly reported in recent years. Notably, only rpsJ and mepA are associated with the tigecycline resistance of S. aureus. The mepA gene encodes MepA efflux pumps, and the overexpression of mepA has been confirmed to be directly related to tigecycline resistance. Although the mutations of MepA widely occur, the associations between TRSA and mutations of MepA are still unclear. In this study, we explored mutations in the mepA genes from various sources. Then, tigecycline resistance-associated mutations T29I, E287G, and T29I+E287G in MepA were identified, and their effects were evaluated through mutant deletion and complementation, tigecycline accumulation assay, and molecular docking experiments. Results showed that the MICs of tigecycline, gentamicin, and amikacin increased in special complementary transformants and recovered after the addition of the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The tigecycline accumulation assay of the mepA-deleted mutant strain and its complementary transformants showed that T29I, E287G, and T29I+E287G mutations promoted tigecycline efflux, and molecular docking showed that mutations T29I, E287G, and T29I+E287G decreased the binding energy and contributed to ligand binding. Moreover, we inferred the evolutionary trajectory of S. aureus under the selective pressure of tigecycline in vitro. Overall, our study indicated that mutations in MepA play important roles in tigecycline resistance in S. aureus. IMPORTANCE Previous analysis has shown that overexpression of MepA is an exact mechanism involved in tigecycline resistance apart from the rpsJ mutation and is usually dependent on the mutant mepR. However, no research has evaluated the effects of diverse mutations discovered in TRSA in MepA. This study demonstrates that the mutations in MepA confer resistance to tigecycline without overexpression and provides genotypic references for identifying TRSA. Although tigecycline resistance-associated mutations in MepA identified in this study have not been observed in clinical isolates, the mechanism should be explored given that S. aureus strains are prevalent in the environment. Measures should be implemented to contain TRSA within the time window before tigecycline resistance-associated mutations in MepA are prevalent.
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