双歧杆菌质粒的检测及其对抗生素敏感性

本文应用ＬｅＢＬａｎｃ法提取６种２０株双歧杆菌菌株的质粒,发现共有４个种的６株菌株存在着１～３个质粒,多数质粒的分子量小于６．０ｋｂ。存在质粒的双歧杆菌包括分离自人的短双歧杆菌、青春双歧杆菌、两歧双岐杆菌和婴儿双歧杆菌。用固体培养基滤纸片抑菌圈法测定双歧杆菌对１５种常用抗生素的敏感性,结果表明,测试的２０株双歧杆菌菌株对所检测的１５种抗生素的敏感性无规律可循,而且质粒消除实验表明质粒的存在与所检测的抗生素抗性无相关性。     

双歧杆菌耐药性与质粒

随着微生态学理论与实践的不断发展,人们对维持正常菌群的生态平衡的重要性的认识越来越深刻［１］。根据微生态学的有关理论,多种活菌制剂已经被开发出来,在临床被用于恢复人体的正常菌群,用来治疗各种因素导致的肠道菌群失调［２］。现就与双歧杆菌活菌制剂研制和临...     

双歧杆菌DM9227和乳酸杆菌DM8121质粒的检测及药敏试验

应用分子克隆常规质粒提取方法（略作修改）,对一株Ｂｉｆ．ｉｎｆａｎｔｉｓＤＭ９２２７及另一株Ｌａｃｔｏ．ｃａｓｉｅ．ＤＭ８１２１进行质粒提取及分析,并用固体培养基药敏纸片法测定此两株菌对１７种常用抗生素的敏感性。结果发现,ＤＭ８１２１及ＤＭ９２２７中无质粒的存在。药敏结果显示,ＤＭ８１２１对强力霉素、链霉素、庆大霉素较敏感,而对头孢菌素、氨苄青霉素、青霉素耐药。ＤＭ９２２７对头孢菌素、先锋霉素、庆大霉素、卡那霉素较敏感,对氨苄青霉素、羧苄青霉素、青霉素、强力霉素等耐药。     

益生芽隐杆菌对抗生素的敏感性及其质粒的检测

采用滤纸片抑菌圈法测定３０株益生芽直菌对１５种抗生素的敏感性。结果９０％菌抗磺胺嘧啶,对其余抗生素大部分菌株敏感。用改良碱裂解法提取３０株菌质粒,只有６１２９菌株含有一个大约４．３ｋｂ的质粒,质粒消除南粒与磺胺嘧啶抗性无关。     

益生芽孢杆菌对抗生素的敏感性及其质粒的检测

采用滤纸片抑菌圈法测定３０株益生芽孢杆菌对１５种抗生素的敏感性。结果９０％菌株抗磺胺嘧啶,对其余抗生素大部分菌株敏感。用改良碱裂解法提取３０株菌质粒,只有６１２９菌株含有一个大约４３ｋｂ的质粒,质粒消除实验证明该质粒与磺胺嘧啶抗性无关。     

双歧杆菌的耐药性与质粒

目的：研究双歧杆菌的耐药性与质粒的关系。方法 对１７株５种来自微生态制剂的双歧杆菌进行抗生素药敏试验和质粒检测,利用溴化乙锭消除其质粒;比较质粒消除前后耐药性的改变。结果１７株双歧杆菌对氨基糖式类和多肽类抗生素呈强抗性;除１株短型双歧杆菌Ｂ１５７存有２．７Ｋｂ和５．６Ｋｂ两种质粒外,其余菌株均未质粒,消除后持粒的Ｂ１５７株菌对抗生素的敏感性并未改变。结论 此１７株双歧杆菌的耐药性与质粒无直接相关性     

一株双歧杆菌质粒聚合酶基因的PCR扩增和鉴定

目的PCR扩增人双歧杆菌天然质粒的聚合酶基因。方法用改良型MRS双歧杆菌选择培养基,从人新鲜粪便分离长双歧杆菌,PCR扩增长双歧杆菌质粒聚合酶(Bifidobacterium plasmid polymerase,BPP)基因,对BPP基因检测阳性的PCR产物通过序列分析,进行鉴定。结果人长双歧杆菌天然质粒的聚合酶基因PCR扩增后,经1.0%琼脂糖凝胶电泳,测得BPP基因的相对分子质量约为1.9 kb。通过BLAST序列比对分析与GenBank中相应基因同源性为96%。结论成功克隆了1株双歧杆菌天然质粒的聚合酶基因,为构建与双歧杆菌宿主质粒相适应的载体奠定了基础。     

一株携带质粒的人两歧双歧杆菌的分离与鉴定

目的:分离携带天然质粒的人双歧杆菌.方法:用自制的改良型Blb双歧杆菌选择培养基,从人新鲜粪便分离双歧杆菌,对初步质粒检测阳性的单菌落通过糖发酵试验、(G C)mol%测定和16S rDNA序列分析,进行菌株鉴定.结果:筛选到一株携带天然质粒的人双歧杆菌,编号B200304,在1.0%琼脂糖凝胶上,测得质粒的相对分子质量约为22 kb.通过对该菌株的形态学观察和糖发酵试验等生理生化特征研究,证明该菌株为两歧双歧杆菌(Bifidobacterium bifidum);HPLC法测得其(G C)mol%为55.6,16SrDNA序列分析进一步证实该菌株为两歧双歧杆菌.结论:分离得到一株携带天然质粒的人两歧双歧杆菌新菌株.     

产亚硝酸优杆菌的抗生素敏感性试验

从临床34例胃癌患者的胃粘膜组织分离出产亚硝酸化杆菌19株,检出率55.88％,采用纸片琼脂扩散法,参照美国临床实验室标准委员会规定的标准判定药敏结果,选用10种抗生素纸片,对产亚硝酸优杆菌进行了体外药敏测定,其结果氯霉素、红霉素和庆大霉素对产亚硝酸优杆菌呈敏感性,卡那霉素、链霉素和四环素呈中度敏感,而青霉素类全部耐药。     

双歧杆菌质粒聚合酶基因对质粒载体稳定性的影响

目的探讨双歧杆菌天然质粒的聚合酶基因（Bifidobacterium plasmid polymerase,BPP）对人工构建的大肠埃希菌～双歧杆菌穿梭质粒载体（shuttle vector）在长双歧杆菌中稳定性的影响。方法首先电转化质粒pBADs—A和pBADs-BPP至长双歧杆菌,培养鉴定后,接种含质粒pBADs—A和pBADs-BPP的双歧杆菌于AMP^-和AMP^＋的MRS培养液中。厌氧培养后,将样品涂布于AMP^＋的MRS固体培养板上计数菌落数。结果相同条件下质粒pBADs·BPP组菌落数高于质粒pBADs—A组（P〈0．05）。结论BPP可以增加质粒载体的稳定性。     

益生菌B. animalis V9冻干粉安全性毒理学研究

目的对益生菌B.animalis V9冻干菌粉进行安全性毒理学评价,确保该菌株在发酵乳制品及保健类食品中的应用安全。方法连续14d对小鼠灌胃不同剂量的(0.5、5和15g/kg体重)B.animalis V9菌粉,然后执行以下指标的测量,包括每天测体重,观察不同时间点(3、7和14d)小鼠生长状况,血液细胞成分、肝脏功能、脏器指数变化及脂类代谢情况等。在显微镜下进行肝、肾形态学观察。结果 B.animalis V9对小鼠的半数致死量(LD50)为15g/kg体重,灌胃14d小鼠未出现中毒症状,更无死亡现象,且对小鼠的生长发育无不良影响。小鼠体重变化与对照组比较差异无统计学意义(P0.05);血常规,肝功能、脏器指数、血清脂类等各项指标测定结果与对照组比较差异无统计学意义(P0.05);通过对小鼠肝脏、肾脏的染色镜检可知,肝脏、肾脏均无异常改变。结论急性毒性试验及相关指标检测结果显示B.animalis V9菌粉无毒副作用。     

B．animalis V9对腹泻动物的保护性作用及其机制研究

目的研究益生菌B.animalis V9对腹泻动物的保护作用及其对腹泻致病菌的拮抗效果。方法采用自制的腹泻SPF鼠,喂服B．animalis V9,观察并检测腹泻致病菌,并在体外试验益生菌B．animalisV9对腹泻致病菌的拮抗作用。结果SPF鼠致泻动物模型喂食B．animalisV9,剂量为1．0×10^8CFU/（只·d）,3d后腹泻治愈率为90％,死亡率为9％,对照组自然恢复率为16％,死亡率为41％,其余仍有不同程度的腹泻现象。B．animalis V9在体外对志贺痢疾杆菌、沙门菌、铜绿假单胞菌和大肠埃希菌有不同程度的拮抗作用,共同培养60h后均未检测到上述4种致病菌。结论B．animalisV9可以有效的对腹泻小鼠进行治疗,其机制在于B．animalisV9对致泻菌株具有一定程度的拮抗作用。     

蒙古族儿童源益生特性双歧杆菌的筛选及鉴定

【目的】双歧杆菌在人和动物胃肠道中发挥着重要的生理作用,然而双歧杆菌能否耐受胃酸、肠液及胆汁酸是影响活菌制剂益生效果的关键因素,本研究旨在从蒙古族儿童粪便中分离筛选出具有良好益生特性的双岐杆菌。【方法】本文采用双岐杆菌选择性培养基对样品进行分离纯化,并对菌株进行生理生化鉴定,以耐受人工胃肠液、耐受牛胆盐为手段对各菌株的益生特性进行评价,并且对B. animalisV9进行了16S rDNA分子生物学鉴定。【结果】本文从12位健康蒙古族儿童粪便中分离得到11株双歧杆菌,经传统生理生化试验鉴定为5株B. adolescentis：A1、H3、G4、A8、V10;3株B. longum：C6、C7、D11;1株B. pseudocatenlatum：B2;1株B. bifidum：G5;1株B. animalis：V9。B. animalis V9具有较强的耐酸性,在pH2.0的人工胃液中厌氧培养3h后存活率为92.4%,而其它10株双歧杆菌在此条件下的存活率均小于31.25%;B. animalis V9在pH2.0的人工胃液中厌氧培养3h后接入pH8.0的人工肠液中消化8h,存活率为99.7%,并且可以耐受0.3%的牛胆盐。进一步对V9菌株进行16S rDNA分子生物学鉴定,发现与Bifidobacterium animalis subsp. lactic BB12的同源性为99%。【结论】本研究结果显示B. animalis V9来源安全,并且具有良好耐酸、耐胆盐益生特性,有望在乳制品及保健类产品中得到广泛的应用。     

利用胞内代谢物变化预测大肠埃希菌耐药性

目的利用胞内代谢物量的差异达到预测大肠埃希氏菌的药物敏感性结果。方法收集临床分离大肠埃希菌120株,在头孢他啶存在条件下培养4 h。收集菌体,测定细胞内化合物然后进行多变量分析。结果 120株大肠埃希菌的聚类分析结果很好地体现了药物敏感性特征,57株耐药菌中,有5株被误判,准确率为91%;47株中敏菌株中,有8株被误判,准确率为83%;16株敏感菌中有2株被误判;准确率为88%。结论该方法能够有效预测菌株的药敏结果,而且快速可靠。     

Plasmid profiles and antibiotic susceptibility of Haemophilus pleuropneumoniae serotypes 1, 3, 5, and 7

Abstract This paper describes the plasmid profiles obtained for 73 of 96 field isolates of Haemophilus pleuropneumoniae serotypes 1, 3, 5, and 7. We also characterized the antibiotic susceptibilities of these 96 isolates. Because of the high proportion of isolates resistant to some of the antibiotics, no conclusions can be drawn as to the role of plasmids in antibiotic resistance.     

62株阴沟肠杆菌的生化特性和药敏结果

目的探讨临床分离阴沟肠杆菌的实用鉴定方法.方法对VITEK-32微生物分析仪鉴定所得的62株阴沟肠杆菌的生化反应和药敏结果作回顾性分析.结果 62株阴沟肠杆菌用鉴定卡鉴定的可信度在94%～99%,其中51株为99%、占82.3%,4株为95%、占6.5%,其他7株、占11.2%.阴沟肠杆菌对除外亚胺培南的20种抗生素均有不同程度的耐药.结论脲酶、侧金盏花醇、精氨酸水解和赖氨酸脱羧等试验对肠杆菌属中5个常见菌种(阴沟、坂崎、产气、聚团和格高菲肠杆菌)的鉴别有重要意义.     

A novel antibiotic free plasmid selection system: advances in safe and efficient DNA therapy

The presence of antibiotic resistance genes in the delivered plasmids is one of the drawbacks of modern gene therapy and DNA vaccine applications. Here, we describe a strategy that allows for plasmid selection in bacterial hosts, without the requirement of any selection marker. Several bacterial strains were modified, so that the plasmid's replicational inhibitor RNA I could suppress the translation of a growth essential gene by RNA-RNA antisense reaction. An essential gene (murA) was modified such that a repressor protein (tetR) would hamper its expression. Only in the presence of plasmid and, hence, RNA I, was tetR turned down and murA expressed. Different commercially available plasmids could be selected by various modified Escherichia coli strains. We further designed a minimalistic plasmid devoid of any selection marker. All of the clones (n=6) examined, when the modified strain JM109-murselect was used for selection, contained plasmids. Thus, we have designed bacterial host strains that for the first time serve to select and maintain plasmids without the use of any selection marker or other additional sequence on the plasmid. Consequently, such plasmids may not only be safer, but due to their decreased size, advantages for the manufacturer and higher transfection efficiencies are anticipated.     

临床分离病原菌构成及对抗生素敏感性分析

目的 回顾性分析汕头大学医学院第一附属医院2004年（1月～10月）临床标本病原菌构成及对抗生素的敏感性,为治疗感染性疾病提供参考依据。方法 细菌鉴定及药敏试验采用VITEK-60全自动微生物鉴定仪。结果 共检出细菌1136株,革兰阴性杆菌占61．3％,革兰阳性球菌占38．7％,常见病原菌为金黄色葡萄球菌（224株）、铜绿假单胞菌（162株）、大肠埃希菌（152株）、肺炎克雷伯菌（128株）、不动杆菌属（76株）、溶血葡萄球菌（57株）、嗜麦芽窄食单胞菌（44株）、表皮葡萄球菌（37株）和阴沟肠杆菌（30株）。大肠埃希菌、肺炎克雷伯菌和阴沟肠杆菌产ESBL分别是60％、44％和46％。细菌对抗生素均呈不同程度耐药,敏感株所占比例极少,亚胺培南对肠杆菌科细菌的抗菌活性强于非发酵菌,未检出耐万古霉索葡萄球菌和肠球菌。结论 细菌构成与国内其他地区相近,但产酶现象比其他地区较为严重,常见细菌多呈多重耐药性,建议临床根据药敏结果选用抗生素,以期减少耐药菌株产生。     

Conjugal transfer of antibiotic resistances and plasmids from Campylobacter jejuni clinical isolates

Six Campylobacter jejuni clinical isolates were examined for the occurrence of plasmids in association with antibiotic resistances as well as conjugal transfer. All the isolates were found to carry three similar plasmids of 78 kb, 12.6 kb and 3.3 kb in size. Multiple resistance to at least three of the antibiotics tested was observed with resistance to tetracycline most common. En bloc transfer of donor resistances at frequencies ranging from 10(-8) to 10(-4) were seen in all but one of the isolates during conjugation. The conjugal transfer of erythromycin, neomycin and streptomycin were observed to occur at frequencies similar to that of chloramphenicol, kanamycin and tetracycline. In isolate ABA94, three different antibiotic resistance phenotypes of the transconjugants were seen. In addition to en bloc transfer of the donor resistances, in approximately 10% of the transconjugants the streptomycin resistance was lost although these transconjugants carried the donor complement of three plasmids. In a further 1% of the transconjugants, resistance to kanamycin only was detected and these transconjugants did not carry any plasmids.     

An inexpensive infrared growth sensor array for detection of bacterial antibiotic susceptibility

Abstract An inexpensive infrared sensor was constructed and used for the rapid testing of bacterial antibiotic susceptibility by detection of changes in absorbance at 950 nm. By comparing cultures of clinical isolates together with control strains ( Escherichia coli NCTC 10418, Staphylococcus aureus NCTC 6571 or Pseudomonas aeruginosa NCTC 10662) after addition of an antibiotic, results on susceptibility were obtained within 3–5 h from the original plate culture. Representative strains of E. coli, P. aeruginosa , and S. aureus were tested successfully against ampicillin, penicillin, gentamicin or ciprofloxacin.