土壤宏基因组文库来源酯酶的鉴定与表征

【目的】利用宏基因组学技术挖掘土壤微生物来源的新型酯酶。【方法】构建土壤微生物宏基因组文库,利用三丁酸甘油酯平板法对所构建的文库进行筛选,并对阳性克隆中鉴定出的酯酶基因进行异源表达和生物化学特性分析。【结果】通过筛选文库中的12万个克隆,获得了一个阳性克隆,对克隆中的DNA片段进行序列分析,发现了一个可能的酯酶基因,通过研究其表达产物,确定其最适pH为9.0,最适反应温度为56°C,在90°C下仍可保持20%的酶活性;能专一性水解短链脂类,对长链脂类无水解作用;对一定浓度范围内的有机试剂如二甲基亚砜、甲醇、乙醇有较好的耐受性,尤其当二甲基亚砜含量为10%(体积比)时,相对酶活可提高44%。【结论】不依赖于微生物可培养性的宏基因组学技术可以发现新的活性酶,本研究获得的对高温、有机试剂有较好耐受性的酯酶ESTYN1具有在工业生产中应用的潜力。     

牙菌斑培养菌群宏基因组文库构建及抗生素耐药基因筛选

[目的]构建牙菌斑培养菌群宏基因组文库,筛选牙菌斑生物膜中细菌的抗生素耐药基因.[方法]采集20例无龋健康人的集合牙菌斑并进行厌氧培养.提取牙菌斑培养菌群宏基因组构建Fosmid文库.用卡那霉素、四环素及氨苄西林对文库进行筛选,并对筛选到的抗性Fosmid克隆进行末端测序、亚克隆构建、亚克隆测序和序列分析.[结果]构建了牙菌斑培养菌群宏基因组Fosmid文库,插入片段长度在36-48 kb间约有15 120个克隆,插入片段长度小于36 kb的约有3 360个克隆.筛选获得一个氨基糖苷类双功能修饰酶AacA-AphD基因、一个核糖体保护蛋白型四环素耐药基因tet (M)及一个C家族β-内酰胺酶基因.[结论]证实了可以通过构建宏基因组文库的方法来筛选牙菌斑培养菌群中的抗生素耐药基因.     

替加环素与米诺环素治疗布鲁菌病的进展

布鲁菌病是由布鲁菌引起的人畜共患传染性疾病，由带菌动物通过多种途径传染给易感人群，可侵犯人体各器官系统。目前推荐的抗布鲁菌治疗方案为多西环素、利福平、喹诺酮类、氨基糖类等抗菌药物组合的二联或者三联抗菌方案，但治疗无效率和疾病复发率仍较高。替加环素和米诺环素作为四环素类药物，经体外实验证明均可有效抑制布鲁菌，且50%最小抑菌浓度（50% minimal inhibitory concentration，MIC50） 和 90%最小抑菌浓度（90% minimal inhibitory concentration，MIC90）均较低。临床研究亦证实，包含替加环素和米诺环素的联合抗菌方案具有较低的治疗无效率和疾病复发率。因此，替加环素和米诺环素可作为治疗布鲁菌病的候选用药。本文就布鲁菌病传统治疗方案以及替加环素和米诺环素的抗菌效果进行综述。     

替加环素结合舒巴坦对多重耐药鲍曼不动杆菌病原微生物的治疗效果观察

目的探讨重症多重耐药鲍曼不动杆菌肺炎对替加环素结合舒巴坦的疗效反应。方法选取98例自2011年6月到2013年6月收治于沈阳军区总医院的严重鲍曼不动杆菌肺炎的患者,数字表法随机分为对照组与观察组,对照组在常规治疗基础上行替加环素治疗,观察组在对照组的基础上行舒巴坦治疗,观察两组治疗后的总有效率与不良反应情况。结果观察组患者的ET、AngⅡ和NO水平分别为(0.35±0.10)mg/L、(146.3±28.6)μg/L和(37.8±8.7)μmol/L,均明显低于对照组的(0.40±0.12)mg/L、(185.7±35.3)μg/L和(48.6±10.5)μmol/L,对比差异具有统计学意义(P0.05)。与对照组相比,观察组的总有效率明显提高(81.63%vs 40.82%,P0.05),而不良反应显著降低(P0.05)。结论替加环素结合舒巴坦治疗多重耐药鲍曼不动杆菌肺炎安全有效,值得临床广泛关注和应用。     

替加环素对多重耐药鲍曼不动杆菌引起重症肺炎患者的临床疗效

目的:探讨替加环素治疗多重耐药鲍曼不动杆菌引起的重症肺炎的临床疗效。方法:回顾性研究我院多重耐药鲍曼不动杆菌引起的重症肺炎60例,随机分为对照组和实验组,每组30例。对照组患者给予头孢哌酮舒巴坦治疗,实验组患者应用替加环素治疗。观察并比较两组患者治疗前后血清降钙素原(PCT)、C反应蛋白(CRP)及白细胞计数(WBC)的变化情况、不良反应情况、细菌清除情况及临床疗效。结果:与治疗前相比,两组患者血清PCT、CRP及WBC水平均降低(P0.05);与对照组相比,实验组患者血清PCT、CRP及WBC水平较低(P0.05);实验组不良反应发生率及再感染率低于对照组(P0.05);实验组细菌清除率及临床总有效率高于对照组(P0.05)。结论:对于由多重耐药鲍曼不动杆菌引起的重症肺炎,替加环素具有很好的临床疗效,值得在临床上推广使用。     

替加环素与多粘菌素B对泛耐药鲍曼不动杆菌体外研究

目的为治愈耐舒普深的泛耐药鲍曼不动杆菌（PDR-Ab）提供新药。方法替加环素采用二倍琼脂稀释法,多粘菌素B用E-test条法测分离目标菌株100株的最低抑菌浓度（M IC）,并用WHONET 5.4软件分析数据。结果多粘菌素B对耐舒普深的泛耐药鲍曼不动杆菌株的M IC值分布情况为：1.5 mg/L为2株,1.0 mg/L为9株,0.75 mg/L为9株,0.5 mg/L为38株,0.38 mg/L为34株,0.25 mg/L为8株,均为敏感;替加环素对耐舒普深的泛耐药鲍曼不动杆菌株的M IC值分布情况为：≥32 mg/L为0株、16 mg/L为2株、8 mg/L为3株、4 mg/L为4株、2 mg/L为6株、1 mg/L为9株、0.5 mg/L为30株、0.25 mg/L为33株、0.125 mg/L为9株、0.06 mg/L为2株、0.03 mg/L为2株,敏感率为95.0%,中敏率为3.0%,耐药率为2.0%。结论替加环素或多粘菌素B是目前对耐舒普深的泛耐药鲍曼不动杆菌最有效药物之一。     

基于宏基因组技术解析南极不同纬度地区土壤抗生素抗性基因的分布与迁移

[目的] 南极洲不同地区环境极端多样,且受人类活动影响不一。本研究旨在探究南极不同纬度地区土壤抗生素抗性基因（ARGs）的分布特征与迁移机制。[方法] 下载南极不同纬度地区及加拿大阿尔伯特地区养殖场附近土壤宏基因组数据集,利用MetaWRAP进行组装,使用CARD、PlasFlow和ICEberg数据库对ARGs与可移动遗传元件（MGEs）进行注释。[结果] 在南极不同纬度地区土壤中,优势菌门为变形菌门、放线菌门、拟杆菌门和厚壁菌门。共注释出25类406种ARGs,以多重耐药类、四环素类及氨基糖苷类抗生素抗性基因为主。NMDS分析结果表明,南极不同纬度地区与养殖场附近土壤中ARGs的分布特征显著不同（ANISOM,P=0.001）。南极高纬度地区ARGs占总基因数的比例为0.28%,显著低于低纬度地区（1.93%,P<0.01）。不同抗生素类型的ARGs呈现不同的区域分布模式,其中硝基咪唑类、氨基糖苷类、糖肽类与大环内酯类ARGs主要分布在南极高纬度地区,四环素类与磺胺类ARGs主要分布在南极低纬度地区（P<0.05）。南极土壤中ARGs的迁移研究表明,质粒携带的ARGs占检测到的ARGs的17%。同时,共发现163个整合与接合元件（ICEs）可携带多抗耐药类、肽类和四环素类等14类ARGs。这些携带ARGs的ICEs主要分布于α-、β-与γ-变形菌纲中。[结论] 南极高纬度与南极低纬度地区土壤中ARGs的分布存在差异性,质粒与ICEs共同介导ARGs的迁移。本研究为进一步了解抗生素时代之前的原始抗性组提供数据基础。     

替加环素不敏感鲍曼不动杆菌的分子流行病学及耐药机制

为探讨替加环素不敏感鲍曼不动杆菌Acinetobacter baumannii的耐药机制,为院内感染控制及临床合理用药提供理论依据,采用琼脂稀释法和微量肉汤稀释法检测全国多中心12个城市20家医院临床分离的94株非重复的替加环素不敏感鲍曼不动杆菌的最低抑菌浓度(Minimum inhibitory concentration,MIC),应用多位点序列分型(Multilocus sequence typing,MLST)技术进行分子流行病学研究,应用eBURST软件对MLST结果进行分析;用PCR和测序技术分析常见耐药基因(bla_(OXA-40-like)、bla_(OXA-58-like)、bla_(OXA-23-like)、bla_(OXA-51-like)、bla_(NDM-1)),与替加环素耐药相关的外排泵调控基因adeR和adeS的突变位点、trm的突变位点。经检测94株鲍曼不动杆菌除对多粘菌素B 100%敏感、对米诺环素敏感率25.5%外,其他抗菌药物的敏感率均低于3.5%,亚胺培南和美罗培南敏感率均只有1.1%。MLST分型得到12种ST分型,以ST195(45株,47.9%)、ST208(19株,20.2%)和ST457(10株,10.6%)为主,eBURST分析发现其中8个ST型均属于克隆复合体92(Clonal Complex 92,CC92);99%菌株bla_(OXA-23-like)型碳青霉烯酶基因阳性;均未扩增出bla_(NDM-1)基因;外排泵调控基因adeR和adeS的检出率分别是73.4%和91.5%,Asp26Asn和Ala97Glu分别为adeR和adeS的高频突变位点;在12株鲍曼不动杆菌中检测到了adeS基因的ISAba1,以北部地区为主;trm基因均在第240位核苷酸发生缺失突变。综上所述,替加环素不敏感鲍曼不动杆菌对除多粘菌素B外的大多数抗菌药物具有很高的耐药性,AdeABC外排泵上游的双组分调控系统adeR和adeS的缺失和突变,trm缺失突变是导致鲍曼不动杆菌对替加环素敏感性降低的主要原因。     

利用土壤宏基因组技术定向筛选抗生素产生菌的方法学建立

为了快速从土壤中定向筛选抗生素产生菌, 本研究利用宏基因组技术进行了土壤中抗生素产生菌快速筛选方法的探索。对6 种不同土质的土壤利用液氮冷冻法提取土壤基因组DNA 并用PVPP柱层析法进行纯化, 每克湿土可得到32.25?61.25 μg DNA, 片段大小为23 kb左右, 说明宏基因组DNA样品完整, 16S rDNA的 PCR结果证明了该基因组DNA纯度可用于后期的分子操作。利用Herbimycin产生菌掺入法进行了抗生素产生菌筛选方法学的验证。结果证明, 每克湿土掺入103个Herbimycin产生菌时, 利用Herbimycin合成基因簇的特定引物即可从所提取的土壤基因组DNA中扩增出目标条带, 并且可以利用传统菌种分离方法从土壤中重新分离出来被掺入到土壤的Herbimycin产生菌。这些结果证明本研究建立的定向抗生素产生菌筛选方法, 具有快捷、灵敏的特点, 大大减少传统筛选方法的工作强度和时间, 为微生物新药的研发建立一种全新的研究方法。     

替加环素与5种抗生素对多重耐药鲍曼不动杆菌体外联合药敏实验的研究

分析亚胺培南、头孢哌酮-舒巴坦、头孢曲松、左氧氟沙星、庆大霉素5种临床常用鲍曼不动杆菌治疗的抗生素单用和分别与替加环素联用的体外敏感性实验的研究,以期发现较好的联合用药方案,为临床合理使用抗生素提供用药参考。采用微量肉汤稀释法测定5种抗生素对鲍曼不动杆菌的MIC值,再采用棋盘法测定5种抗生素分别与替加环素联用MIC值,并计算FIC指数。结果显示,替加环素与亚胺培南、头孢哌酮舒巴坦、左氧氟沙星、头孢曲松具有协同和相加作用,替加环素与庆大霉素具有拮抗作用。临床在选择抗生素治疗鲍曼不动杆菌所引起的重症感染时,可根据该药敏实验结果与亚胺培南或头孢哌酮舒巴坦或左氧氟沙星或头孢曲松与替加环素联合使用,但应避免与庆大霉素联合使用。     

Using cellular fitness to map the structure and function of a major facilitator superfamily effluxer

The major facilitator superfamily (MFS) effluxers are prominent mediators of antimicrobial resistance. The biochemical characterization of MFS proteins is hindered by their complex membrane environment that makes in vitro biochemical analysis challenging. Since the physicochemical properties of proteins drive the fitness of an organism, we posed the question of whether we could reverse that relationship and derive meaningful biochemical parameters for a single protein simply from fitness changes it confers under varying strengths of selection. Here, we present a physiological model that uses cellular fitness as a proxy to predict the biochemical properties of the MFS tetracycline efflux pump, TetB, and a family of single amino acid variants. We determined two lumped biochemical parameters roughly describing K_m and V_max for TetB and variants. Including in vivo protein levels into our model allowed for more specified prediction of pump parameters relating to substrate binding affinity and pumping efficiency for TetB and variants. We further demonstrated the general utility of our model by solely using fitness to assay a library of tet(B) variants and estimate their biochemical properties.     

Cloning,sequencing and analysis of the pucC genes from Rubrivivax gelatinosus strain 151 and Rhodopseudomonas acidophila strain 10050

The pucC genes of Rubrivivax gelatinosus strain 151 and Rhodopseudomonas acidophila strain 10050 have been identified, cloned and sequenced. In Rubrivivax gelatinosus the arrangement of the pucC gene with regard to the pucBA genes was shown to differ from that found in other species of photosynthetic bacteria. The Rhodopseudomonas acidophila pucC was found downstream of four new pucBA gene pairs, bringing the sequenced pucBA pairs to a total of eight in this strain. The predicted PucC protein sequences were compared to those of PucC from other species and showed high similarity. Similarity was also seen to more distantly related proteins LhaA and orf428 of Rhodobacter capsulatus, orf G115 of Rhodospirillum rubrum and `orf428' from Synechocystis sp. PCC6803. An analysis of the predicted secondary structure of these proteins is given, and their structural similarity to proteins in the Major Facilitator Superfamily is discussed with regard to their possible function. This revised version was published online in June 2006 with corrections to the Cover Date.     

油藏样本潜在核心功能微生物群的宏基因组挖掘

[背景]识别出具有驱油功能的内源性微生物群是当前设计高效微生物采油技术的迫切需求.[目的]通过宏基因组分析找出原位油藏微生物中具有产酸/产气功能的潜在核心微生物群.[方法]利用室内模拟体系,对原位油藏微生物进行有机营养(从有氧到无氧)的激活,运用生物信息、多元统计分析以及网络分析等方法,寻找潜在内源性核心功能微生物群....     

Insertional inactivation of a Tet(K)/Tet(L) like transporter does not eliminate tetracycline resistance in Bacillus cereus

Bacillus cereus ATCC 10987 and ATCC 14579 can be induced to high levels of resistance to tetracycline. The chromosomal B. cereus gene bctl encodes a transmembrane protein with homology to Gram-positive tetracycline efflux proteins and relation to other members of the major facilitator superfamily of transport proteins. A mutant strain containing an insertionally inactivated bctl gene did not show impaired tetracycline resistance. No additional altered phenotype was observed in the mutant. Accumulation studies suggested that the resistance mechanism involves a reduced sensitivity to intracellular tetracycline.     

北京东灵山树线处土壤细菌的PICRUSt基因预测分析

树线变动会对生物多样性分布以及生态系统功能的维持造成深远影响,研究树线处土壤微生物群落及其功能,对预测高海拔生态系统响应气候变化具有重要价值。采用Mi Seq高通量测序技术及PICRUSt基因预测分析方法,对北京东灵山辽东栎林及树线之上亚高山草甸的土壤细菌群落及功能进行对比研究,结果表明:土壤细菌物种多样性在树线处没有发生显著的变化,沿海拔也没有呈现出明显趋势,但细菌群落结构以及预测功能基因均发生了变化。在39个二级预测功能分类中有10个子功能的相对多度在森林和草甸中具有明显差异。其中,其他次生产物代谢的生物合成、转录、多糖生物合成和代谢、酶家族、信号分子及交互作用、环境适应、细胞生长和死亡等的功能基因在森林中明显高于草甸中;而维他命及辅因子代谢、膜运输、内分泌系统等的功能基因在草甸中偏高。     

CgMFS1, a Major Facilitator Superfamily Transporter,Is Required for Sugar Transport,Oxidative Stress Resistance,and Pathogenicity of Colletotrichum gloeosporioides from Hevea brasiliensis

Colletotrichum gloeosporioides is the main causal agent of anthracnose in various plant species. Determining the molecular mechanisms underlying the pathogenicity and fungicide resistance of C. gloeosporioides could help build new strategies for disease control. The major facilitator superfamily (MFS) has multiple roles in the transport of a diverse range of substrates. In the present study, an MFS protein CgMFS1 was characterized in C. gloeosporioides. This protein contains seven transmembrane domains, and its predicted 3D structure is highly similar to the reported hexose transporters. To investigate the biological functions of CgMFS1, the gene knock-out mutant ΔCgMFS1 was constructed. A colony growth assay showed that the mutant was remarkably decreased in vegetative growth in minimal medium supplemented with monosaccharides and oligosaccharides as the sole carbon sources, whereas it showed a similar growth rate and colony morphology as wild types when using soluble starch as the carbon source. A stress assay revealed that CgMFS1 is involved in oxidative stress but not in the fungicide resistance of C. gloeosporioides. Furthermore, its pathogenicity was significantly impaired in the mutant, although its appressorium formation was not affected. Our results demonstrate that CgMFS1 is required for sugar transport, resistance to oxidative stress, and the pathogenicity of Colletotrichum gloeosporioides from Hevea brasiliensis.     

云南腾冲热泉土壤微生物基因组文库的构建与分析

采用冻融、蛋白酶K、SDS-高盐-加热处理法联合的方法,直接从云南腾冲地区的一个弱碱性高温热泉沉积样品中提取和分离环境混合基因组DNA,产量为每克样品1~2μg DNA,用Promega试剂盒纯化后进行PstⅠ部分酶切处理,电泳回收3~8kb的片段后,构建了pSK( )为载体的基因组文库,共获得25000个阳性克隆,平均插入片段长度为4.6kb。通过随机DNA序列测定和基因注释,发现外源插入片段含有未见报道的序列。     

Identification of genes conferring arsenic resistance to Escherichia coli from an effluent treatment plant sludge metagenomic library

The majority of bacteria elude culture in the laboratory. A metagenomic approach provides culture-independent access to the gene pool of the whole bacterial community. A metagenomic library was constructed from an industrial effluent treatment plant sludge containing about 1.25 Gb of microbial community DNA. Two arsenic-resistant clones were selected from the metagenomic library. Clones MT3 and MT6 had eight- and 18-fold higher resistance to sodium arsenate in comparison with the parent strain, respectively. The clones also showed increased resistance to arsenite but not to antimony. Sequence analysis of the clones revealed genes encoding for putative arsenate reductases and arsenite efflux pumps. A novel arsenate resistance gene ( arsN ) encoding a protein with similarity to acetyltransferases was identified from clone MT6. ArsN homologues were found to be closely associated with arsenic resistance genes in many bacterial genomes. ArsN homologues were found fused to putative arsenate reductases in Methylibium petroleiphilum PM1 and Anaeromyxobacter dehalogenans 2CP-C and with a putative arsenite chaperone in Burkholderia vietnamiensis G4. ArsN alone resulted in an approximately sixfold higher resistance to sodium arsenate in wild-type Escherichia coli W3110.     

Identification and functional characterization of Penicillium marneffei major facilitator superfamily (MFS) transporters

ABSTRACT

Penicillium marneffei is a thermally dimorphic fungus that causes penicilliosis, and become the third-most-common opportunistic fungal infection in immunocompromised patients in Southeast Asia. Azoles and amphotericin B have been introduced for the treatment, however, it is important to investigate possible mechanisms of azole resistance for future treatment failure. We identified 177 putative MFS transporters and classified into 17 subfamilies. Among those, members of the Drug:H⁺ antiporter 1 subfamily are known to confer resistance to antifungals. Out of 39 paralogs, three (encoded by PmMDR1, PmMDR2, and PmMDR3) were heterologously overexpressed in S. cerevisiae AD? conferred resistance to various drugs and compounds including azoles, albeit to different degrees. PmMDR1-expressing strain showed resistance to the broadest range of drugs, followed by the PmMDR3, and PmMDR2 conferred weak resistance to a limited range of drugs. We conclude that PmMDR1 and PmMDR3, may be able to serve as multidrug efflux pumps.