梭菌属的一个新种——产气梭菌

从豆腐废水为原料的厌氧降解反应器中,采用亨盖特氏厌氧技术,分离到两株厌氧生芽孢的杆菌,革兰氏染色通常为阴性。直杆或稍弯杆形,以周生鞭毛运动。芽孢椭圆或卵圆形,不膨胀细胞,通常次端生,最适生长温度为30—37℃,在45℃不生长。在pH 4．5或9．0也不生长。6．5％NaCl抑制其生长。能发酵多种碳水化合物。在PYG培养液中从葡萄糖产生大量的H2和CO2中量的乳酸和少量的乙酸、丁酸、乙醇和丁酵。不利用柠橡酸盐和丙酮酸盐。DNA中G+C含量为41.2mol％。经鉴定为棱菌属(又名校状芽孢杆菌属)的一个新种,命名为产气梭菌(Clostridium aerogenes sp.nov.)。     

粪便标本艰难梭菌三种培养方法的比较

目的 比较粪便标本中艰难梭菌三种培养方法的差异性,寻找适合临床实验室使用的艰难梭菌培养方法。方法 将健康儿童的300份粪便标本分别用快速芽孢孵育法、乙醇直接处理法、富集芽孢培养法进行培养,并对三种培养方法的差异性进行统计学比较。结果 300份粪便标本快速芽孢孵育法培养出艰难梭菌51例,检出率为17.0%;乙醇直接处理法培养出48例,检出率为16.0%;富集芽孢培养法培养出44例,检出率为14.7%。三种方法的检出率差异无统计学意义（P=0.7352,P>0.05）。结论 乙醇直接处理法、快速芽孢孵育法简单快速,适合临床实验室进行艰难梭菌的快速培养;富集芽孢培养法杂菌少,适合大量艰难梭菌培养便于结果的观察及进一步的处理。     

地衣芽孢杆菌原生质体电转化方法的研究

为了解决地衣芽孢杆菌(Bacillus licheniformis)工业菌株难以转化的问题,将原生质体制备、电穿孔和原生质体再生技术相结合,建立了一种地衣芽孢杆菌原生质体电击转化方法。在对菌体生长状态、溶菌酶作用时间、电转电压、渗透压保护剂等条件进行优化后,试验了将不同类型的表达载体,即游离型质粒p GJ103(3.3kb)和整合型质粒p AX01(9.3kb),分别转入两株地衣芽孢杆菌工业生产菌株B.licheniformis CICC 10181和B.licheniformis CICC 20204中。实验结果显示,对数生长期后期的菌体酶解40min后制备的地衣芽孢杆菌原生质体得率为96%,再生率达25%以上。原生质体与质粒DNA在最适电压0.6k V/mm下电击转化,并以0.5mol/L山梨醇或甘露醇作为渗透压保护剂进行再生培养后,最终游离型质粒的转化率可达0.88×102~1.1×102CFU/μg p GJ103,整合型质粒的转化率达到0.45×102~0.52×102CFU/μg p AX01。该方法为地衣芽孢杆菌野生工业菌株的遗传改造提供了一种新的、高效的转化手段。     

解纤维梭菌培养条件的优化

解纤维梭菌Clostridium cellulolyticum是产纤维小体的专性厌氧菌,由于其培养困难,目前仍难以实现高效培养.文中采用响应面法对产纤维小体的解纤维梭菌C.cellulolyticum高细胞密度培养的条件进行了优化.首先用Plackett-Burman实验设计对影响因素效应进行评价,筛选出的显著影响因素分别为:酵母提取物浓度、纤维二糖浓度及培养温度.之后用最陡爬坡实验设计逼近菌体最佳生长条件的区域范围.最后通过中心组合实验设计和响应面分析方法确定显著影响因素的水平和C.cellulolyticum的最优培养条件.优化后的显著影响因素酵母提取物浓度、纤维二糖浓度和培养温度分别为3 g/L、7 g/L和34℃.在最优条件下,摇瓶培养的菌体浓度OD600值由0.303提高到了0.586,增加了93.4％.在发酵罐批次培养条件下,菌体OD600值达到了3.432,比文献报道值高出了2.8倍.研究结果为C.cellulolyticum培养及应用研究提供了基础.     

产气荚膜梭菌双圈溶血现象之培养及其毒素分析

通过实验介绍了产气荚膜梭菌在血琼脂平板上经数次传代培养后恢复毒力产生双圈溶血现象的培养方法,并阐明了导致该现象产生的两种毒素(θ毒素和α毒素)的作用机理。     

蜜蜂肠道菌群的培养方法及特性研究进展

肠道菌群在蜜蜂的消化、营养和抗病性等方面发挥了很多潜在的益生作用。为了加深对以蜜蜂为主的传粉昆虫肠道菌群的了解,本文综述了蜜蜂肠道菌群的人工厌氧培养方法及特性,重点综述了Snodgrassella属、Gilliamella属、Frischella属、Lactobacillus属、Bifidobacterium属和Alpha-1,Alpha-2厌氧细菌类群。旨在通过特定的培养方法获取纯培养的细菌,以供进一步研究特定肠道菌群与特定功能的直接联系。希望这些培养技术能帮助大家提升对蜜蜂肠道共生菌在蜜蜂营养和健康方面所起的作用有所了解。     

复合碳源培养对酒糟厌氧消化液中丙酸互营氧化菌及群落结构的影响

丙酸累积是影响厌氧消化系统稳定性的主要因素,为了考察酒糟厌氧消化过程中间代谢产物的累积情况,以总固体含量(TS)（质量分数） 5%和7%的白酒糟为发酵原料进行了批次试验。结果表明,乙酸(最高浓度33~129 mmol/L)、丙酸(39~61 mmol/L)、丁酸(5~44 mmol/L)和15种氨基酸(0.01~0.3 mmol/L)为主要中间代谢产物。为了探究其中关键的丙酸降解菌群,以酒糟原始沼液JO为植种源,10 mmol/L丙酸和0.1 mmol/L混合氨基酸为复合碳源进行富集培养,获得中温厌氧丙酸-氨基酸培养系JO-AP。高通量测序分析表明,互营丙酸降解菌与厚壁菌门（Firmicutes）的丙酸厌氧降解菌（Pelotomaculum schinkii）近缘,16S rRNA基因相似性100%,占细菌总丰度的16.7%。对比酒糟原始沼液JO、丙酸培养系JO-P及丙酸-氨基酸培养系JO-AP中的主要功能菌群,发现采用单一碳源和复合碳源获得的优势互营丙酸降解菌不同;传统培养与分子生物学技术相结合可以更全面地掌握系统中的微生物群落组成。     

艰难梭菌实验室快速鉴定方法检验效能的比较

目的评价3种艰难梭菌实验室鉴定方法的快速性及准确性。方法 2013年6月至2013年11月,收集136名ICU患者共248份标本进行24 h、48 h厌氧培养,并进行鉴定,同时对大便标本提取DNA进行tcd B毒素基因检测。以48 h培养结果为参考标准,评价24小时培养法和PCR扩增tcd B基因法鉴定艰难梭菌的灵敏度、特异度、阳性预测值和阴性预测值。结果 136名ICU发生腹泻的患者中,11名患者共有12份标本48 h培养为艰难梭菌阳性,艰难梭菌感染率为8.09%(11/136),标本阳性率为4.84%(12/248)。24小时培养法及PCR扩增tcd B基因法的灵敏度、特异度、阳性预测值、阴性预测值分别为75.00%、100%、100%、98.74%和83.33%、99.15%、83.33%、99.15%。结论提取大便DNA tcd B毒素基因是最快速、灵敏度和特异度均较高的方法;48小时培养法时间相对较长,但稳定可靠,并可以保留菌株进行后续试验;24小时培养法,时间短、简便但会漏诊某些阳性病例。     

玉米黄浆水在丙酮-丁醇厌氧梭菌发酵中的应用

为降低丙酮-丁醇厌氧梭菌发酵生产丁醇的成本,研究了不同添加量玉米黄浆水对发酵的影响。与葡萄糖培养基相比,在发酵培养基中添加少量玉米黄浆水对发酵产量无显著影响。当添加体积分数为25％的玉米黄浆水时,丙酮、丁醇和乙醇的最终质量浓度分别是0．31、2．70和8．00g/L,总溶剂量为11．01g／L。通过成本核算,每生产1kg溶剂,添加体积分数25％的玉米黄浆水可比葡萄糖培养基节约成本2．11元。     

艰难梭菌相关性腹泻的治疗进展及对我国的启示

艰难梭菌相关性腹泻（Clostridium difficile associated diarrhea,CDAD）是艰难梭菌感染（Clostridium difficile Infection,CDI）引起的一种机体严重疾病。随着艰难梭菌感染率的增加及高毒力株027／NAP1／BI的出现,导致该病在全球特别是北美、及欧洲等地区爆发性流行,对于该病的控制引起全球研究者的高度重视。本文就其治疗进展进行综述,并结合我国实际分析该病防治中存在的问题并提出建议。     

Electrochemical analysis of Clostridium propionicum and its acrylic acid production in microbial fuel cells

Currently, acrylic acid is produced at a low yield by the resting cells of Clostridium propionicum with the supplement of extra electron acceptors. As an alternative way, acrylic acid production coupled with electricity generation was achieved by C. propionicum‐based microbial fuel cells (MFCs). Electricity was generated in the salt‐bridge MFCs with cysteine and resazurin in the anode chamber as mediators, and K₃Fe(CN)₆ as the cathode electron acceptor. Power generation was 21.78 mW/m² with an internal resistance of 9809 Ω. Cyclic voltammograms indicated the main mechanism of power production was the electron transfer facilitated by mediators in the system. In the salt‐bridge MFC system, 0.694 mM acrylic acid was produced together with electricity generation.     

Toxic effects of acrylic acid on Clostridium propionicum and isolation of acrylic acid‐tolerant mutants for production of acrylic acid

This work presents a systematic investigation of the toxic effects of acrylic acid on the growth of Clostridium propionicum and the isolation of acrylic acid‐tolerant mutants. The results suggest that addition of acrylic acid prolonged the lag phase of the fermentation and reduced the initial‐specific growth rate, as well as the final cell concentration. Moreover, the toxic effect of acrylic acid was sensitive to the pH value. The minimal inhibition concentration of acrylic acid increased from 1.11 to 31.25 mM when the pH value rose from 5.8 to 7.4. In addition, the molar concentration ratio of products (acetic acid:propionic acid) was enhanced with the supplementation of acrylic acid. The highest ratio was 0.7:1 when acrylic acid was 20.83 mM at pH 7.4. Two acrylic acid‐tolerant mutants were isolated, which could still grow at a high concentration (43.06 mM) of acrylic acid. These strains could be instrumental for improved bioproduction of acrylic acid.     

A simple method for differentiation of Propionibacterium acnes and Propionibacterium propionicum

Abstract TLC glycolipid profiles of several culture collection and clinical strains of Propionibacterium acnes and Propionibacterium propionicum were examined. The former were characterized by weak orcinol-positive minor glycolipids of type g, while the others had mainly strong orcinol-positive major glycolipids of type G. The simple and rapid small scale procedure seemed to be useful for differentiation of these phenotypically similar and genotypically closely related species irrespective of their serotypes.     

Plasmid transformation of Clostridium perfringens by electroporation methods

Two electroporation methods were compared and modified to improve the frequencies of transfer of plasmid DNA into Clostridium perfringens. A plasmid shuttle vector, pSB92A2, containing chloramphenicol and ampicillin resistance genes and a clostridial origin of replication isolated from a cryptic C. perfringens plasmid, was constructed and successfully introduced into C. perfringens by both electrotransformation methods. Modifications which improved frequencies by 15-28 fold are described and may improve frequencies sufficiently for some vector/host combinations to consider the future use of more direct cloning strategies for the clostridia.     

Isolation of Clostridium propionicum strain 19acry3 and further characteristics of the species

Two mixed cultures able to ferment acrylate to equimolar acetate and propionate were enriched from anaerobic sediments. From one of these mixed cultures a pure culture of a Gram-positive, obligately anaerobic bacterium was isolated. This strain, designated 19acry3 (= DSM 6251) was identified as belonging to the species Clostridium propionicum. Only a narrow range of organic compounds supported growth, including acrylate and lactate. Acrylate and lactate were fermented to acetate and propionate in a 1:2 molar ratio. When co-cultured with the non-acrylate-fermenting Campylobacter sp. strain 19gly1 (DSM 6222), the fermentation balance shifted to almost equimolar acetate and propionate. Strain 19acry3 was compared with Clostridium propionicum type strain X2 (DSM 1682). The two strains displayed similar phenotypic properties. The mol% G+C of DNA isolated from both strains was 36–37 (by thermal denaturation). Both strains displayed a characteristic fluorescence when observed by fluorescence microscopy. Cell-free extracts of both strains were examined by spectrophotofluorimetry. In both strains, two excitation peaks were observed at 378 and 470 nm. Excitation at either of these wavelengths resulted in an emission maximum at 511 nm.     

Method for electroporation for the early chick embryo

In vitro whole-embryo culture of chick embryos, originally invented by New, has been widely used for studies of early embryogenesis. Here, a method for electroporation using the New culture and its derivatives is described, to achieve misexpression of exogenous gene in a temporally and spatially controlled manner in gastrulating chick embryos. Detailed information for the devices and procedures, and some experimental examples are presented.     

Introduction of plasmids into whole cells of Clostridium acetobutylicum by electroporation

Abstract A method is presented for the introduction of plasmids into Clostridium acetobutylicum ATCC 8052 by electroporation. A plasmid shuttle vector, pMTL500E, which contains the erythromycin resistance gene and replication machinery of plasmid pAMβ1, was constructed and introduced into C. acetobutylicum by electroporation. The vector was then used to introduce a 2.2 kb Cla I/ Sph I chromosomal fragment from C. pasteurianum into a leucine requiring mutant of C. acetobutylicum , SBA9, where complementation of auxotrophy was observed. Plasmid DNA indistinguishable from that introduced, on the basis of agarose gel electrophoresis, was observed in transformants containing either plasmid.     

Transferring genes into cultured mammalian embryos by electroporation

Mammalian whole embryo culture (WEC) was developed long before transgenic and gene targeted animals are widely used. Electroporation (EP) into cultured rodent embryos has expanded the potential to analyze gene functions in mammalian embryos by transferring exogenous plasmid vectors or small nucleotides in region- and stage-specific ways. This method is quite simple, and therefore enables us to analyze gene functions more quickly than genetic manipulation. In this review, we introduce combinatorial methods of WEC and EP, and summarize various applications in developmental neurobiology.     

A型产气荚膜梭菌经小鼠传代的毒力变化

目的:探讨A型产气荚膜梭菌经小鼠腹腔传代后的毒力变化。方法:选择10只小鼠,腹腔注射浓度为5.0×108 cfu/mL的A型产气荚膜梭菌菌液1 mL,记录第一代小鼠存活时间;收集第一代死亡最早的小鼠腹腔内的细菌,经血平板厌氧培养24 h后配制5.0×108 cfu/mL浓度的菌液,并腹腔注射10只小鼠,每只1 mL,记录第二代小鼠存活时间;重复实验并记录第三代、第四代小鼠的存活时间;死亡小鼠腹腔收集的细菌以革兰染色涂片镜检,血平板培养,用API 20A试剂条进行生化鉴定;用SPSS 13.0软件分析每代小鼠的存活时间组间差异。结果:小鼠腹腔收集的标本经革兰染色后均可见两端钝圆棒状的革兰阳性杆菌,血平板厌氧培养均为边缘呈锯齿状有双溶血环的较大菌落,API 20A的结果为产气荚膜梭菌;小鼠存活时间随小鼠腹腔传代数不断延长,第一代与第二代、第三代、第四代之间的存活时间差异有统计学意义,第二代、第三代与第四代两两之间的存活时间差异无统计学差异。结论:A型产气荚膜梭菌经小鼠腹腔第一次传代后毒力显著减弱,随后传代中毒力基本不变,与传统的细菌经动物腹腔培养后毒力增强的观点不一致。     

Clostridium paraputrificum M-21发酵制氢培养条件研究

利用类腐败梭状芽孢杆菌M-21(Clostridium paraputn M-21),在37℃、150r／min条件下发酵制氢,以葡萄糖为碳源,蛋白胨为氮源,lmol的葡萄糖可以产生1．05mol的氢气,最终所产气体中有70％H2和30％c02(体积百分数)。最优初始pH范围为7．0—7．5,少量乙酸的存在对氢气的生成有促进作用,若大量存在,会严重抑制茵的生长。在C／N质量比为1．0时,所产氢气体积最多,1g葡萄糖可产生75mL Hz(常温常压)。在高温83℃下对种茵预处理30s促进孢子的萌发,会缩短发酵产氢的时间。以淀粉为碳源,所产氢气体积略微高于葡萄糖,1g淀粉可产生78mL H2(常温常压);以蔗糖为碳源,所产氢气体积略微低于葡萄糖,1g蔗糖可产生72mL H2(常温常压)。该茵不能降解利用羧甲基纤维素,木质素磺酸钠,及纸浆等。