拮抗细菌Enterobacter cloaeae B8的一株抗菌素抗性突变体(英文)

为了研究拮抗细菌Enterobacter cloacae B8在野外水稻叶面上的定殖情况及拮抗作用,由自然选择和Tn5诱变从B8产生了一株抗菌素抗性突变体BX8.BX8能在含利福平达400μg/ml或卡那霉素达300μg/ml的LB培养基中生长.实验表明该抗菌素抗性的产生没有降低BX8的拮抗活性.该抗性比较稳定,在无抗菌素培养基中培养.BX8至少分裂生长60代内抗性不变.     

有机磷农药降解菌—阴沟肠杆菌的生物学特性

【研究目的】对有机磷农药降解菌阴沟肠杆菌的生物学特性进行研究,为研究其降解特性机理及应用提供理论依据。【方法】测定阴沟肠杆菌生长曲线、生理生化特征,筛选出最适生长培养基,测定不同温度、pH值、摇床振数、装液量下的生长状况,确定最适生长条件,测定对乐果、甲胺磷、敌敌畏、敌百虫的耐受力和利用情况。【结果】阴沟肠杆菌培养3h后进入对数生长期,可以利用分解大部分含碳化合物,在营养贫瘠的条件上生长,生长温度范围为10~43℃,生长pH值范围为6~12,对乐果、甲胺磷、敌敌畏、敌百虫的降解利用必须建立在一定的营养物质的基础上。【结论】该菌的繁殖速度快,在营养条件较贫瘠的条件下就可以生存,是一株生存能力很强的菌株,适合应用于土壤、水体环境中的有机磷农药的降解。     

Phosphate-solubilizing microorganisms associated with the rhizosphere of mangroves in a semiarid coastal lagoon

 The phosphate-solubilizing potential of the rhizosphere microbial community in mangroves was demonstrated when culture media supplemented with insoluble, tribasic calcium phosphate, and incubated with roots of black (Avicennia germinans L.) and white [Laguncularia racemosa (L.) Gaertn.] mangrove became transparent after a few days of incubation. Thirteen phosphate-solubilizing bacterial strains were isolated from the rhizosphere of both species of mangroves: Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus atrophaeus, Paenibacillus macerans, Vibrio proteolyticus, Xanthobacter agilis, Enterobacter aerogenes, Enterobacter taylorae, Enterobacter asburiae, Kluyvera cryocrescens, Pseudomonas stutzeri, and Chryseomonas luteola. One bacterial isolate could not be identified. The rhizosphere of black mangroves also yielded the fungus Aspergillus niger. The phosphate-solubilizing activity of the isolates was first qualitatively evaluated by the formation of halos (clear zones) around the colonies growing on solid medium containing tribasic calcium phosphate as a sole phosphorus source. Spectrophotometric quantification of phosphate solubilization showed that all bacterial species and A. niger solubilized insoluble phosphate well in a liquid medium, and that V. proteolyticus was the most active solubilizing species among the bacteria. Gas chromatographic analyses of cell-free spent culture medium from the various bacteria demonstrated the presence of 11 identified, and several unidentified, volatile and nonvolatile organic acids. Those most commonly produced by different species were lactic, succinic, isovaleric, isobutyric, and acetic acids. Most of the bacterial species produced more than one organic acid whereas A. niger produced only succinic acid. We propose the production of organic acids by these mangrove rhizosphere microorganisms as a possible mechanism involved in the solubilization of insoluble calcium phosphate. Received: 21 April 1999     

百菌清降解菌BJQ2的分离、鉴定及影响因素研究

采用未有百菌清用药史的土壤定向培养49 d,分离出50株百菌清耐受菌株,经初筛、复筛后得到1株降解能力最强的细菌BJQ2,经形态学、生理生化试验、16S rDNA序列同源性比对及系统发育树的分析,初步确定为阴沟肠杆菌(Enterobacter cloacae B9),BJQ2可以以百菌清为唯一碳源于基础培养基中生长.BJQ2降解百菌清的最适宜条件为:培养温度30～35℃,初始pH值为7.0～8.0,百菌清的初始浓度为20 mg· L-1,选用菌体母液(OD600=0.35),以10％的接种量接种,6d后提取百菌清,利用气相色谱进行检测分析,计算后发现百菌清的降解率可以达到79.2％.     

Bacteria inhabiting artificially inoculated xylem of Picea abies

Bacteria inhabiting the xylem of Norway spruce (Picea abies (L.) H. Karsten) were investigated. The trees had been wounded and artificially inoculated with fungi and bacteria obtained from wounds of naturally infected spruce. One and five growing seasons after inoculation the Gram‐negative bacterial population present in the stem of inoculated trees were analysed.

The Gram‐negative bacteria isolated from the trees were identified on the basis of morphological, biochemical and physiological tests and whole‐cell fatty acid composition. The predominant strains were Enterobacteriaceae fermenter strains (E. agglomerans or E. sakazakii), fluorescent and yellow pigmented Pseudomonas, Acinetobacter and Moraxella spp. All Gram‐positive bacteria were Bacillus species.

The Gram‐negative bacteria of Norway spruce differed from the Gram‐positive species in possessing stronger lipolytic activity and in their ability to utilize pine resins for growth. Gram‐positive bacteria were generally able to utilise cellulose and hemicellulose, whereas among the Gram‐negative bacteria only one xylanolytic (yellow Pseudomonas) strain was found.     

鸡病原肠杆菌的鉴定

为明确两起鸡细菌性感染症的病原种类,本研究对11株分离菌进行了形态特征、理化特性等主要表观性状的鉴定,测定了代表菌的16S rRNA基因序列,构建了系统发育树;以代表菌株对健康鸡做人工感染试验,测定其致病作用;对分离菌以琼脂扩散(K-B)法进行对抗菌类药物的敏感性测定.结果表明11株菌7株为阴沟肠杆菌(Enterobacter cloacae),4株为产气肠杆菌(Enterobacter aerogenes).其中所测序的代表菌分离株HQ040619-1的16S rRNA基因序列长度1421 bp(EU073021)、HC050612-1为1449 bp(EU047701)及HC050612-4为1451 bp (EU047702);人工感染试验表明这些分离株均具有较强的致病作用;药物敏感性试验结果显示,在不同分离菌株间的敏感与耐药等差异不明显.     

阴沟肠杆菌的Hy3AL和hycA部分基因克隆及序列分析

为了克隆氢化酶3大亚基（HyA3L）和甲酸氢酶抑制因子（hycA）,研究其在阴沟肠杆菌产氢代谢中的作用和机制,笔者利用CODEHOP设计阴沟肠杆菌NRRL B-414 HyA3L和hycA简并引物,选用引物HyA3J和HycAJ扩增基因组DNA,分别得到约1500、150 bp的PCR产物,该产物连接pMD18-T载体,并克隆转化至E.coli DH5α感受态细胞中,经筛选后测序。推导的HyA3J扩增产物氨基酸序列与Enterobacter sp. 638的HyA3L蛋白一致性最高达到99%,仅有3个氨基酸的差异,表明其为阴沟肠杆菌的HyA3L;推导的HycAJ扩增产物氨基酸序列与Enterobacter cloacae subsp. cloacae ATCC 13047的HycA蛋白一致性最高达到92%,仅有4个氨基酸的差异,表明其为阴沟肠杆菌的hycA。通过以上分析可得出,采用CODEHOP程序设计的简并引物可信性强,阳性率高。阴沟肠杆菌NRRL B-414的氢化酶3大亚基和甲酸氢酶抑制因子部分基因的成功克隆,为通过代谢工程手段敲除氢化酶3大亚基和甲酸氢酶抑制因子的基因全长克隆奠定基础,不但增加了这2个基因的资源,也可为其基因敲除等代谢工程研究提供科学依据和工作基础。     

奶粉中阪崎肠杆菌检测方法的比较研究

奶粉中的阪崎肠杆菌对人类的危害性很大,采用荧光PCR检测法、传统检验法和FDA法3种不同的检测方法对贝因美A婴儿奶粉进行了检测,将检测结果进行比较分析得出,荧光PCR检测法所用时间最短,灵敏度较高。     

阪崎肠杆菌单克隆抗体制备及ELISA检测方法的建立

试验旨在制备抗阪崎肠杆菌的单克隆抗体,初步建立其ELISA检测方法。以灭活的阪崎肠杆菌全菌体为抗原免疫BALB/c小鼠,筛选血清效价高的小鼠脾细胞与SP2/0骨髓瘤细胞进行细胞融合,制备杂交瘤细胞,并用间接ELISA法选取阳性杂交瘤细胞,扩大培养后测定单克隆抗体的效价,进行特异性及抗体间的配对,使用mAb亚类检测试剂盒鉴定单克隆抗体的亚型,并利用得到的抗体建立双抗体夹心ELISA检测方法。本试验得到3株具有良好特异性能稳定分泌单克隆抗体的阳性细胞株5C10、2B6和Ab02,经两两配对,据阳性D_{450 nm}值及P/N值选择1：20000稀释的5C10作为包被抗体,1：40000稀释的Ab02作为酶标二抗,建立ELISA检测法,应用建立的方法与荧光定量PCR方法检测动物实验室保存的20份进出口送检奶粉样品,结果显示试验结果一致。本试验成功制备阪崎肠杆菌的单克隆抗体并建立其ELISA检测法,为大批量快速检测阪崎杆菌奠定了基础。     

猪阴沟肠杆菌16SrRNA甲基化酶耐药基因分析

 【目的】分析猪阴沟肠杆菌GZL41B中4种质粒介导的16SrRNA甲基化酶耐药基因及其水平传播方式;【方法】采用PCR及序列分析方法检测鉴定猪阴沟肠杆菌中16SrRNA甲基化酶耐药基因。以大肠杆菌E.coli Rif+488(耐利福平)为受体菌进行接合试验研究16SrRNA甲基化酶耐药基因的水平传播方式。用微量稀释法测定原菌株及其接合子对18种抗菌药物的敏感性。用PCR及序列测定法分析比较来自同一猪腹泻直肠拭子阴沟肠杆菌GZL41B和大肠杆菌GZL41H的侧翼序列并对172株动物源大肠杆菌中16SrRNA甲基化酶耐药基因流行情况进行调查。【结果】从阴沟杆菌中成功扩增出目的片段,该片段经限制性内切酶HindⅢ酶切后,出现与预期的531 bp和194 bp相符的两段片段,序列测定结果证实该基因为rmtB,GenBank登录号为EF017943。以E.coli Rif+488(耐利福平)为受体菌,成功地获得了接合子,该接合子经鉴定为大肠杆菌。原菌株和接合子对卡那霉素、阿米卡星、妥布霉素、西梭米星、萘替米星、庆大霉素等4,6-二脱氧链霉胺类高度耐药,其MIC均≥256 μg?mL-1。大肠杆菌GZL41H中,rmtB的上游为Tn3转座子的部分序列,下游为肠炎沙门氏菌基因岛SGI1上融合酶编码基因groEL/intI1的部分序列orf1,而来源相同的阴沟肠杆菌GZL41B未扩增到与大肠杆菌GZL41H相同的侧翼序列。172株动物源大肠杆菌中,25株菌(15%)检出rmtB,1株菌(0.6%)检出armA;【结论】16SrRNA甲基化酶耐药基因rmtB首次出现在猪阴沟肠杆菌中,该基因介导猪阴沟肠杆菌对4,6-二脱氧链霉胺类氨基糖苷抗生素的高度耐药,并能通过接合方式在不同种属细菌间进行传播。rmtB在阴沟肠杆菌和大肠杆菌中传播的遗传背景存在差异。动物源大肠杆菌中rmtB广泛流行。