人肠道梭菌和甲烷马赛球菌协同代谢甜菜碱和胆碱产甲烷研究

【目的】根据人肠道富含胆碱和甜菜碱,同时肠道微生物组中具有裂解胆碱和还原甜菜碱产三甲胺的细菌,以及利用三甲胺产甲烷的古菌,本研究探讨肠道细菌与古菌协同代谢甜菜碱和胆碱产甲烷的可能性。【方法】调查不同年龄段人群粪便中的16S rRNA基因多样性,分析肠道中古菌的菌群组成;利用定量PCR(quantitative PCR,qPCR)定量甲烷马赛球菌(Methanomassiliicoccus)特异的甲醇甲基转移酶基因mtaB和甲烷八叠球菌(Methanosarcina)及细菌的16S rRNA基因拷贝数,分析肠道中甲基营养型产甲烷古菌及总细菌的含量;宏基因组组装基因组(metagenome-assembled genomes,MAGs)分析携带甜菜碱还原酶基因grdH和胆碱裂解酶基因cutC的细菌组成。从粪便中分离代谢甜菜碱及胆碱产生三甲胺的细菌,并与分离自人肠道的甲烷马赛球菌构建共培养物,测定其协同转化甜菜碱和胆碱产甲烷的能力。【结果】年轻人粪便中含有甲烷杆菌科(Methanobacteriaceae,82.16%)的甲烷短杆菌属(Methanobrevibacter,49.18%)和甲烷杆菌属(Methanobacterium,33.34%)、甲基营养型的甲烷八叠球菌科(Methanosarcinaceae,5.67%)的甲烷八叠球菌属(Methanosarcina,5.70%),以及甲烷马赛球菌科(Methanomassiliicoccaceae,3.13%)的甲烷马赛球菌属(Methanomassiliicoccus,3.14%)。而中老年人粪便中的甲烷古菌多样性较低,也未检测到甲烷马赛球菌。qPCR定量分析显示年轻人比中老年人肠道的总古菌含量高3.11倍,其中甲烷马赛球菌高6.53倍、甲烷八叠球菌高5.52倍,总细菌含量高2.90倍。宏基因组分析组装了229个细菌基因组,其中42个携带基因grdH和cutC,这些细菌属于毛螺菌科(Lachnospiraceae)、肠杆菌科(Enterobacteriaceae)和梭菌科(Clostridiaceae)等。从粪便中分离到恶名梭菌(Clostridium malenominatum)B8,菌株B8与卢米尼甲烷马赛球菌(Methanomassiliicoccus luminyensis)B10共培养物可降解47.03%的甜菜碱和25.83%胆碱,并产生甲烷,在培养液中检测到三甲胺先积累后被降解。【结论】人肠道细菌恶名梭菌B8和卢米尼甲烷马赛球菌B10可协同代谢甜菜碱和胆碱产甲烷,推测它们在人肠道中可将部分食物中的甜菜碱和胆碱代谢产生甲烷。     

甲烷八叠球菌和甲烷丝菌的能量代谢与胞外电子传递

乙酸营养型产甲烷古菌是自然界甲烷生成的主要驱动力,约贡献全球生物甲烷的三分之二。本文系统综述了两类乙酸营养型产甲烷古菌——甲烷八叠球菌（Methanosarcina）和甲烷丝菌（Methanothrix）的生理及形态特征、底物谱与代谢途径、电子传递链与能量耦合机制;综合比较了两类古菌在乙酸活化策略、辅酶再生通路和能量合成方式的异同,揭示其在不同环境条件下的代谢适应性与生态位分化;进一步总结了甲烷八叠球菌与甲烷丝菌胞外电子传递的研究进展,涵盖种间电子传递、微生物电腐蚀、胞外呼吸等微生物过程,阐明代谢通路和能量耦合机制。文章为全面理解乙酸营养型产甲烷古菌的代谢网络与电子流调控提供整合性视角,并为未来研究其环境适应策略与代谢调控机制奠定理论基础。     

新疆维、哈两民族粪样中韦荣球菌属和梭菌属水平与2型糖尿病的相关性研究

目的探讨目标差异肠道菌群与新疆维吾尔族、哈萨克族2型糖尿病(T2DM)的相关性。方法采用16S r DNA实时荧光定量PCR对新疆维吾尔族、哈萨克族正常糖耐量及2型糖尿病患者粪样中韦荣球菌属和梭菌属水平进行检测。两组间均数比较采用t检验,运用Pearson分析方法对差异肠道菌群与新疆维、哈两民族正常糖耐量及2型糖尿病人群的体检及生化指标进行相关性分析。结果 (1)16S r DNA实时定量PCR结果显示,韦荣球菌属及梭菌属水平在哈萨克族2型糖尿病组粪样中显著高于该民族正常糖耐量组(P=0.035,P=0.028)。(2)新疆维吾尔族、哈萨克族正常糖耐量人群和2型糖尿病人群粪样中韦荣球菌属水平与体重及体重指数(BMI)显著正相关(r=0.469,P=0.009;r=0.623,P=0.002),与高密度脂蛋白胆固醇(HDL-C)水平显著负相关(r=-0.466,P=0.025);梭菌属水平与体重、空腹血糖(FBG)、甘油三酯(TG)水平显著正相关(r=0.375,P=0.049;r=0.398,P=0.033;r=0.375,P=0.041)。结论肠道中Veillonella spp和C.coccoides subgroup水平的变化可能与新疆哈萨克族、维吾尔族2型糖尿病的发病有关,需进一步深入探讨。     

酪酸梭菌活菌片联合多烯磷脂酰胆碱对酒精性肝病患者肠道菌群及炎症因子的影响

目的 探讨酪酸梭菌活菌片联合多烯磷脂酰胆碱对酒精性肝病（ALD）患者肠道菌群及炎症因子的影响。方法 选取2015年1月至2018年1月进行治疗的ALD患者80例,随机分为观察组和对照组。两组患者均予以严格戒酒、高蛋白高热量低脂饮食、维生素及门冬氨酸钾镁等常规治疗。观察组患者在此基础上加用酪酸梭菌活菌片（0.7 g/次,3次/d,口服）和多烯磷脂酰胆碱胶囊（456 mg/次,3次/d,口服）联合治疗8周。对照组患者在常规治疗基础上单纯加用多烯磷脂酰胆碱片治疗,其剂量、用法及疗程与观察组相同。观察并比较治疗前后患者肝功能指标、肠道菌群数量及炎症因子的变化。结果 治疗8周后,两组患者血清ALT、AST和TBiL水平均较治疗前显著下降（P<0.05）,且观察组下降幅度较对照组更大（P<0.05）。两组患者肠道双歧杆菌和乳杆菌数量较治疗前明显上升,大肠埃希菌数量较治疗前明显下降（P<0.05）,且观察组变化幅度较对照组更大（P<0.05）。两组患者血清IL-6和TNF-α水平较治疗前明显下降（P<0.05）,且观察组下降幅度较对照组更大（P<0.05）。结论 酪酸梭菌活菌片联合多烯磷脂酰胆碱能有效改善ALD患者肝功能指标,促进肝功能恢复,其作用机制可能与其能纠正肠道菌群失调,促进有益菌的繁殖,同时下调血清炎症因子水平,抑制肝内炎症反应密切相关。     

引起院内感染的肠球菌和肠道中的肠球菌耐药性差异的研究

目的　研究临床分离的院内感染的肠球菌对常用抗生素的耐药性及与健康人肠道中的肠球菌的耐药性比较。方法　应用琼脂稀释法检测从临床分离的５２ 株肠球菌和健康人肠道中分离的肠球菌的最低抑菌浓度（ＭＩＣ） 。结果　肠球菌对临床常用的１１ 种抗生素的耐药性以万古霉素最低,ＭＩＣ５０ 为２ ,ＭＩＣ９０ 为４ 。屎肠球菌和粪肠球菌的耐药性对万古霉素有明显差异。院内感染分离的肠球菌和健康人肠道中的肠球菌的耐药性亦有显著的差异。结论　肠球菌对抗生素的敏感性以万古霉素最敏感,肠球菌应鉴定到种的水平以便更好地监控抗生素的耐药性。     

粪便pH和涂片检查肠道菌群失调的研究及意义

目的探讨粪便pH和涂片检查细菌球/杆在菌群失调诊断中的价值。方法对腹泻3 d以上或疾病中并发腹泻的194例患者为研究对象。采集新鲜粪便(10 min内),观察其性状、颜色、测pH、粪便常规,涂片革兰染色查细菌总数、形态特点、革兰球菌/杆菌及细菌培养。另选同期住院的非腹泻患者、职工家属和到保健科定期体检的小儿80例作为正常对照。结果经培养确诊为菌群失调的有118例,其中男76例,女42例,年龄最小45天,最大5岁。粪便pH失调组分布在6~8,≥7者占78.8%,对照组分布在5~6.5,多集中在≤5.5,占85%,相比差异有显著性(u=12.02,P<0.01)。涂片查球菌/杆菌失调组为3/7~8/2,球菌≥40%者106例(89.8%),对照组为3/7~1/9,球菌≥40%者无1例,两者比较差异有显著性(u=11.19,P<0.01)。两种快速诊断法的灵敏度分别为94.9%和100%,特异度均为95%,诊断界值pH为6.5,球/杆为3/7。结论粪便pH和涂片检查肠道菌群失调方法简便、快捷、经济实用,能在15 min内作出诊断,尤对急重症患者可赢得治疗时机,值得推广。     

一株人消化道中韦荣氏球菌的分离、鉴定及其代谢特性研究

【目的】分离与鉴定人肠道乳酸利用菌,研究其乳酸利用的代谢特性。【方法】利用Hungate滚管技术从人粪便中分离厌氧细菌,通过形态、生化和16S r RNA基因鉴定;通过体外发酵技术研究乳酸代谢,并且与乳酸产生菌共培养,研究二者之间交互饲喂。【结果】验证了乳酸可作为代谢中间产物被人肠道混合菌群利用;分离到一株乳酸利用菌,在24 h内消耗乳酸超过50 mmol/L,经鉴定为韦荣氏球菌,并将其形成乙酸和丙酸。当和肠球菌共培养时,可以有效地减缓乳酸的积累。【结论】本株乳酸利用菌可以作为潜在的益生菌,和乳酸菌一起调节人肠道的乳酸动态平衡。     

实时荧光定量PCR法研究结直肠癌患者肠道拟杆菌属、梭杆菌属和梭菌属量的变化

目的通过研究结、直肠癌患者肠道拟杆菌属、梭杆菌属和梭菌属量的变化,揭示肠道相关菌群改变在大肠癌发病中的作用及意义。方法收集术前结、直肠癌患者粪便标本40例及正常对照标本40例,根据细菌的靶基因序列设计特异性引物。提取待测粪便标本细菌DNA,应用SYBR Green I实时荧光定量PCR测定不同细菌的数量。结果正常对照组与实验组粪便中细菌数量分别为拟杆菌属(8.76±0.77;9.85±0.88)、梭杆菌属(7.94±1.25;10.0±1.65)、梭菌属(3.54±0.70;6.56±0.68),拟杆菌属中的脆弱拟杆菌为(2.12±0.48;4.07±1.77)、梭杆菌属中的坏死梭杆菌为(2.31±0.26;7.62±2.68)及梭菌属中的肉毒梭菌为(2.76±1.16;5.43±1.21),实验组数量均明显增多(P0.05)。结论结、直肠癌患者粪便中拟杆菌属、梭杆菌属和梭菌属的数量较正常对照明显增多,提示结、直肠癌的发生发展与肠道菌群有明显关系。     

人鼻病毒1B感染人扁桃体上皮细胞和人肺支气管上皮细胞的比较代谢组学研究

人鼻病毒(Human rhinovirus,HRV)是呼吸道感染的主要病原体之一,明确HRV的致病机制能为有效防控该病毒感染提供科学依据.为确定1B型HRV(human rhinovirus type 1B,HRV1B)感染致宿主细胞的代谢组改变及差异性,本文采用非靶向代谢组学技术研究HRV1B感染人扁桃体上皮细胞UT-SCC-60B和人肺支气管上皮细胞BEAS-2B后代谢组的改变情况.HRV1B感染UT-SCC-60B细胞6h和12h分别有21个差异显著代谢产物(differentially significant metabolites,DSMs)(上调13个、下调8个)和51个DSMs(上调42个、下调9个),HRV1B感染UT-SCC-60B和BEAS-2B细胞6h和12h后,比较分析发现分别有303个DSMs(上调69个,下调234个)和324个DSMs(上调88个,下调236个),未知DSMs占据比例较大.脂肪酸、脂质、氨基酸、核苷酸和糖类的比例随着感染时间的延长而增加,7-酮基脱氧胆酸、溶血磷脂酰胆碱、垂盆草甙、组氨酸-甘氨酸、腺苷酸等涉及到胆汁酸代谢、脂肪酸和脂质代谢、糖代谢、氨基酸代谢和核苷酸代谢.因此,细胞水平表明HRV1B感染改变了人上皮细胞的脂肪酸、脂质、氨基酸、核苷酸和糖类的代谢水平.     

正常和异常的人精浆中甘油-3-磷酸胆碱、总唾液酸及化学元素含量的比较研究

用酶法和过碘酸 -硫代巴比妥酸法对正常男性和异常男性患者精浆甘油 3 磷酸胆碱(GPC)、总唾液酸 (TSA)量进行了测定 .通过电感耦合等离子直读光谱仪 (ICP)测定精浆中多种化学元素的含量 .结果表明 :正常男性与异常男性患者精浆中GPC的含量有显著性差异 (P <0 0 5) ;正常男性与异常男性患者精浆中TSA的含量无显著性差异 ;正常男性与异常男性患者精浆中钙、镁、磷、硒和锌元素的含量具有显著差异 ,而正常男性与异常男性患者精浆中铝、钡、铁和铅元素的含量无显著差异 .这些结果提示精浆中GPC、钙、镁、磷、硒和锌元素的含量可能与男性不育有关 ,而精浆中TSA、铝、钡、铁和铅元素与男性不育关系还需进一步探讨 .     

Purification of bacterial L-methionine gamma-lyase

A chromatographic procedure using sequential ion-exchange columns is described for separating choline, trimethylamine, trimethylamine oxide, and betaine extracted from marine fish tissues; added exogenous carnitine can also be separated by the system. Choline with its positive charge binds to the AG 50W-X8 (Na+, pH 9) column. The column is first eluted with 0.1 N NaOH to collect trimethylamine, trimethylamine oxide, and betaine; choline is then eluted with 0.5 N NaOH. The amines collected with 0.1 N NaOH are subsequently separated using an AG 50W-X8 (H+, pH 4) column eluted with a linear 0-1 M NaC1 gradient.     

海水养殖生境中硫酸盐还原菌活性的抑制机制

【目的】海水养殖生境中的硫化物(H₂S)严重损害养殖生物健康,控制该条件下硫酸盐还原菌(sulfate-reducing bacteria,SRB)的代谢活性是有效抑制H₂S产生的重要途径。【方法】本研究利用稀释涂布-叠皿夹法对海水养殖生境底泥中SRB进行富集筛选,获得SRB菌株,通过投加硝酸盐对菌株产H₂S的活性进行抑制。【结果】获得的2株SRB Desulfovibrio sp.NY-1和Clostridium sp.NH-1,能够在35℃、pH为7.0及盐度为20–30 mg/L条件下,分别积累高达435和150 mg/L H₂S。硝酸盐不能有效抑制NY-1产H₂S的活性,基因调控作用以及缺乏将硝酸盐作为电子受体的酶体系是其不能被抑制的主要原因。硝酸盐对NH-1 H₂S产生活性有可逆性抑制,其具有硝酸盐异化还原成铵(dissimilatory nitrate reduction to ammonium,DNRA)的能力,优先利用硝酸盐作为电子受体。DNRA作用下的中间代谢产物亚硝酸盐是有效抑制菌株NH-1产H₂S活性的主要原因,其抑制机理主要为抑制菌株的生长繁殖。【结论】硝酸盐对不同SRB菌株具有不同的抑制机制和效果,在进行硫化物污染控制前需要对产生硫化物的SRB菌群进行分析判别。     

Heterologous expression of the<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> alcohol acetyltransferase genes in<Emphasis Type="Italic"> Clostridium acetobutylicum</Emphasis> and<Emphasis Type="Italic"> Escherichia coli</Emphasis> for the production of isoamyl acetate

Esters are formed by the condensation of acids with alcohols. The esters isoamyl acetate and butyl butyrate are used for food and beverage flavorings. Alcohol acetyltransferase is one enzyme responsible for the production of esters from acetyl-CoA and different alcohol substrates. The genes ATF1 and ATF2, encoding alcohol acetyltransferases from the yeast Saccharomyces cerevisiae have been sequenced and characterized. The production of acids and alcohols in mass quantities by the industrially important Clostridium acetobutylicum makes it a potential organism for exploitation of alcohol acetyltransferase activity. This report focuses on the heterologous expression of the alcohol acetyltransferases in Escherichia coli and C. acetobutylicum. ATF1 and ATF2 were cloned and expressed in E. coli and ATF2 was expressed in C. acetobutylicum. Isoamyl acetate production from the substrate isoamyl alcohol in E. coli and C. acetobutylicum cultures was determined by head-space gas analysis. Alcohol acetyltransferase I produced more than twice as much isoamyl acetate as alcohol acetyltransferase II when expressed from a high-copy expression vector. The effect of substrate levels on ester production was explored in the two bacterial hosts to demonstrate the efficacy of utilizing ATF1and ATF2 in bacteria for ester production.     

Removal of wastewater pathogen indicators in a constructed wetland in Leon, Spain

This paper studies the seasonal presence and removal of the pathogenous micro-organisms Escherichia coli, total coliforms (TC), Clostridium perfringens (Cp), faecal streptococci (FS), Giardia cysts, Cryptosporidium oocysts and helminth eggs, in a constructed wetland treatment system. The removal efficiency of this system with respect to the indicator micro-organisms achieved maximum values in spring and autumn at 99.9% for E. coli and TC, respectively, in winter at 97.0% for FS, in summer at 100% for Clostridium and throughout the year, also at 100%, in the case of Giardia cysts, Cryptosporidium oocysts and helminth eggs. In general, very low protozoan and helminth egg counts were found, and the system demonstrated efficient reduction of the wastewater indicator pathogens.     

Studies on the characterization of rat prostate androgen receptors

Summary Choline used as the sole carbon or carbon and nitrogen source induces in Pseudomonas aeruginosa an active transport system. The induction of the choline uptake is repressed by succinate independently of the presence of ammonium ion in the culture medium. The repression mediated by succinate was insensitive to cyclic AMP. Substitution for dibutyryl-cyclic AMP was without effect. Choline metabolites that also support the growth of Pseudomonas aeruginosa were poor inducer agents of the choline transport. Kinetic evidence and the employment of choline metabolites as effectors indicated that the choline uptake system of this bacterium is formed by at least two components: one of high affinity (Km=3 µM) and another of low affinity (Km=400 µM). Contrary to what occurs in the synaptosome system, the high affinity form for the choline uptake was not dependent on Na⁺ ions and is not inhibited by hemicholinium-3. Since Pseudomonas aeruginosa can utilize choline as the sole carbon and nitrogen source, the induction of the choline transport with two components in this bacterium may be related to its own strategy to survive and grow in an adverse environment.     

Propanol as an end product of threonine fermentation

Clostridium sp. strain 17cr1 was able to ferment l-threonine to propionate and propanol. Electrons arising in the oxidation of 2-oxobutyrate to propionyl-CoA were apparently used in reductive pathway leading to propanol formation. Part of the propionyl-CoA was used to form propionate in an ATP-forming pathway via a propionate kinase, so that the final ATP yield was 0.5 mol per mol of l-threonine metabolised. Other growth substrates were fermented mainly to acetate and butyrate, and the reductive formation of butyrate, from 2 mol of acetyl-CoA or from crotonate or 3-hydroxybutyrate, was the main route for recycling reduced electron carriers arising during oxidative pathways for most substrates.     

Influence of Culture Parameters on Biological Hydrogen Production by Clostridium saccharoperbutylacetonicum ATCC 27021

Summary Various medium components (carbon and nitrogen sources, iron, inoculum size) and environmental factors (initial pH and the agitation speed) were evaluated for their effects on the rate and the yield of hydrogen production by Clostridium saccharoperbutylacetonicum. Among the carbon sources assessed, cells grown on disaccharides (lactose, sucrose and maltose) produced on the average more than twice (2.81 mol-H2/mol sugar) as much hydrogen as monosaccharides (1.29 mol-H2/mol sugar), but there was no correlation between the carbon source and the production rate. The highest yield (2.83 mol/mol) was obtained in lactose and sucrose but the highest production rate (1.75 mmol/h) in sucrose. Using glucose as carbon source, yeast extract was the best nitrogen source. A parallel increase between the production rate and the yield was obtained by increasing glucose concentration up to 40 g/l (1.76 mol-H2/mol, 3.39 mmol/h), total nitrogen as yeast extract up to 0.1% (1.41 mol/mol, 1.91 mmol/h) and agitation up to 100 rev/min (1.66 mol-H2/mol, 1.86 mmol/h). On the other hand, higher production rates were favoured in preference to the yield at a neutral initial pH 7 (2.27 mmol/h), 1000 mg iron/l or more (1.99 mmol/h), and a larger inoculum size, 10%, (2.36 mmol/h) whereas an initial alkaline pH of 8.5 (1.72 mol/mol), a lower iron concentration of 25 mg/l (1.74 mol/mol) and smaller inoculum size, 1%, (1.85 mol/mol) promoted higher yield over production rate.     

泸型酒窖泥中梭菌的分离及代谢产物分析

[目的] 分离窖泥中的梭菌微生物并对其代谢产物进行评估。[方法] 对窖泥中梭菌群落的16S rRNA基因进行高通量测序;利用高丰度的梭菌OTU序列在KOMODO数据库进行培养基的预测,定向分离窖泥中梭菌菌株;采用顶空固相微萃取结合气相色谱质谱联用仪对窖泥和代表性梭菌菌株的挥发性代谢产物进行检测。[结果] 利用KOMODO数据库预测的梭菌培养基共计筛选到31株梭菌微生物,分属于梭菌属的14个种;根据风味代谢特性,这些菌株主要分为两大类,一是C.carboxidivorans、C.sporogenes和C.tyrobutyricum等产酸为主的梭菌,二是C.beijerinckii、C.butyricum和C.sphenoides等产醇为主的梭菌。[结论] 利用测序序列预测培养基有助于从窖泥中分离获得丰富的梭菌菌株,其物种和代谢能力的多样性对解析白酒复杂风味形成机理奠定了一定的基础。