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。     

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。     

24株产不同抗生素的放线菌质粒的分离和鉴定

分离鉴定了中国微生物菌种保藏管理委员会保藏的24株产不同抗生素的放线菌的质粒。按改进的Kado和Liu快速法提取质粒DNA,经电泳、电镜的检测,在24株检测菌中的7株菌里分离出10个质粒,携带质粒的菌株约占检测菌株的29％。质粒DNA的分子量约为1.8—61.6 kb。通过消除质粒试验和致死接合(Ltz)反应,研究了质粒和抗生素产生的关系。     

肺炎杆菌质粒图谱的研究与分析

本文对临床分离的８９株肺炎杆菌进行了７种抗生素敏感性检测以及质粒图谱分析。结果,９１．２％的菌株均对２种或２种以上抗生素耐药,并呈９个型别的耐药谱。全部被检菌株共分１６种质粒图谱,其中主要为ＰＰⅠ、ＰＰⅡ、ＰＰⅢ、ＰＰⅣ及ＰＰⅤ。这５种主要质粒图谱型别所包含的５６株菌株中,９４．６％的菌株具有共同稳定相似的耐药谱,即耐羧苄青霉素、氨苄青霉素及灭滴灵。另外７６株检出质粒的菌株中,９１．７％的菌株含有分子量１２６×１０￣６的大质粒。     

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     

江苏部分地区食源性和人源沙门氏菌的多重耐药性研究

从江苏省部分地区收集了117个沙门氏菌分离株,其中食物源和人源菌株分别有81株和36株。16种抗生素敏感性试验表明,有111个分离株对2种或2种以上的抗生素有耐药性,人源沙门氏菌分离株的抗生素耐药率比食物源的高,单一抗生素以链霉素耐药率(92.3%,108/117)最高。对5种或5种以上抗生素耐药的分离株有59株(50.4%),其中对特定六种抗生素:氨苄青霉素、氯霉素、链霉素、磺胺、四环素和卡那霉素耐药(ACSSuTK,R型)的菌株有12株。设计18对耐药基因和I类整合子保守区的引物,对36株有不同来源和耐药特征的多重耐药菌株进行耐药基因和I类整合子的检测,PCR扩增结果与抗生素敏感性表型一致。有30株细菌携带有I类整合子,大小为0.3、0.6、1.0、1.2和1.6kb,其中1.6kb(aadA5-dfr17)大小的整合子在25株细菌中分布(24/36)。接合试验表明,氨苄青霉素、氯霉素、链霉素、甲氧苄氨嘧啶和四环素的耐药特性是由接合性质粒携带。结果显示,耐药基因多数由I类整合子和质粒携带,可以通过接合试验发生转移,可移动的DNA成分可能在耐药特性的转移和分布中起到重要作用。     

B-animalisV9质粒检测及抗生素敏感性试验

本实验对动物双歧杆菌Vg（B．animalisvg）菌株进行质粒检测及抗生素敏感性测试。结果表明,Baninalis V9菌株无质粒检出。该菌株对针对G＋菌的抗生素,如青霉素类（青霉素G、氨苄青霉素）和大环内酯类（氯霉素、红霉素）表现出高度敏感;对抑制细菌蛋白质合成的广谱（G＋、G-）抗生素（四环素、利福平、痢特灵）表现出中度敏感;对G-菌的抗生素,如氨基糖苷类（庆大霉素、链霉素）、喹诺酮类（诺氟沙星）及内酰胺类（卡那霉素、丁胺卡那霉素）表现出耐药性;而对广谱抗生素磺胺甲基异嗯唑也表现为耐药。     

鸡大肠杆菌的质粒及染色体DNA分析

对分离于诊断为大肠杆菌病鸡的心脏、肝脏及健康鸡粪便的56个菌株进行了生化试验、药物敏感试验、质粒图谱分析、质粒酶切图谱分析及染色体DNA酶切图谱分析。生化试验鉴定了分离的菌株为大肠杆菌。对其中32株大肠杆菌做了12种抗生素的药敏试验,结果显示大部分菌株对四环素、红霉素、氨苄青霉素、链霉素有耐药性,耐药比例分别为96.87％、81.25％、75.00％、84.38％。所分析的56株大肠杆菌均含有质粒,适于质粒分析。比较菌株的     

多重耐药伤寒沙门菌耐药基因的研究

目的 检测多重耐药伤寒沙门菌对抗菌药物的敏感性及其耐药基因定位。方法 随机选取实验室保存的5株耐药菌和1株敏感菌,用纸片扩散法检测对12种抗菌药物的敏感性。用利舍平抑制试验检测菌株是否存在药物外排系统。PCR法检测TEM型β-内酰胺酶基因、aac(6′)-Ⅰb和aac3-Ⅱ型氨基糖苷类修饰酶基因、qacEΔ1-sul1耐消毒剂和磺胺基因、catA和catB氯霉素乙酰基转移酶基因以及cmlA氯霉素外排泵蛋白基因等7种耐药基因。结果 5株多重耐药菌对氨苄西林、哌拉西林、头孢呋辛、头孢西丁、卡那霉素、庆大霉素、复方磺胺甲噁唑及氯霉素等8种抗菌药物全部耐药,对美罗培南、头孢哌酮、头孢吡肟全部敏感;对头孢噻吩中度敏感。1株无质粒pRST98的菌株对上述药物全部敏感。利舍平抑制试验均为阴性。4株耐药菌TEM型β-内酰胺酶基因检测为阳性。全部耐药菌株aac3-Ⅱ、qacEΔ1-sul1、catA基因均为阳性,而aac(6′)-Ⅰb、catB和cmlA基因均为阴性。 结论 多重耐药伤寒沙门菌质粒pRST98上同时存在多种耐药基因, 是导致菌株同时对多种结构各异的抗生素耐药的原因。     

不同地区宋内志贺菌耐药性及脉冲场凝胶电泳分型分析

摘要:【目的】通过对不同地区的宋内志贺菌株进行药物敏感性检测、耐药基因的扩增以及基因分型,了解不同地区宋内志贺菌的耐药情况与流行趋势。【方法】使用微量肉汤稀释法测定了54 株宋内志贺菌对21种药物的敏感性,用PCR方法扩增相关耐药基因,利用脉冲场凝胶电泳(Pulsed Field Gel Electrophoresis,PFGE)技术进行宋内志贺菌的基因分型,最后采用BioNumerics分析软件对所有菌株进行聚类,分析其相似度。【结果】实验菌株对于甲氧苄氨嘧啶/磺胺甲噁唑、四环素、替卡西林/棒酸、氨苄西林、庆大霉素5种抗生素普遍耐药,对亚胺培南、头孢吡肟、左氟沙星、诺氟沙星、阿米卡星5种抗生素全部表现为敏感。共检测出包括blaTEM,blaCTX以及整合子在内的7 种不同的耐药基因。全部实验菌株可分为26个不同的PFGE带型,分型后表现出较高的基因同源性。宋内志贺菌的耐药性、携带基因与带型具有一定地域相关性。【结 论】目前各地区流行的宋内志贺菌株对甲氧苄氨嘧啶/磺胺甲噁唑与四环素已经普遍耐药,不同地区出现相同聚类分型的菌株表明存在跨区域流行的宋内志贺菌群。因此,加强对不同地区宋内志贺菌的监控对减少多重耐药菌株的产生具有重要意义。     

Antibiotic formation in plasmid and plasmid-free strains]

Antibiotic activity of three plasmid and three plasmid-free strains of Bacillus pumilus was studied. The antibiotic activity was found in one plasmid and two plasmid-free strains. It was supposed that both the chromosomic and extrachromosomic genes could participate in regulation of the antibiotic production in Bac. pumilus. It was shown that the antibiotics produced by Bac. pumilus had a very narrow spectrum and inhibited multiplication only of several grampositive bacteria.     

Plasmid transformation of Bacillus cereus protoplasts

The process of polyethyleneglycol-induced plasmid transformation of Bacillus cereus protoplasts was studied. Plasmid transfer into Bacillus cereus strains was demonstrated with the frequencies 1.3.10(1)-1.6.10(2) transformants per 1 mkg of plasmid DNA. The plasmids transferred are stably inherited by Bacillus cereus cells causing tetracycline resistance (pBC16) or kanamycin resistance (pUB110 and pBD64). The proposed method can be used for construction of Bacillus cereus strains having the plasmid determined characteristics.     

Homologous and heterologous transcription of the Cry+-plasmid in Bacillus thuringiensis

The possibility of homologous and heterologous transception of Cry+ plasmids in Bacillus thuringiensis is demonstrated. Cry+ plasmids from crystal bearing strain of Bacillus thuringiensis were transferred into acrystalline strain belonging to H5 serotype by mutual incubation. The donor strain was previously marked by the transmissive plasmid pAM beta 1 coding for erythromycin and lincomycin resistance. The transcipients having acquired the ability to synthesize delta-endotoxin were referred to H5 serotype due to their phenotype. By analogous method Cry+ plasmid was transferred from Bacillus thuringiensis to Bacillus cereus. Bacillus cereus strain GP7 was used as a recipient strain resistant to tetracycline. The presence of delta-endotoxin in transcipients was confirmed by bioprobes and immunoenzyme assay. To prove the transfer of Cry+ plasmid the plasmid profiles of the parent strains and transcipients have been analyzed. The formation of cellular contacts during mutual incubation of Bacillus thuringiensis and Bacillus cereus strains was demonstrated by electron microscopic study of ultrafine cuts.     

球形芽孢杆菌的抗药标记及外源DNA的转化与表达

用亚硝基胍（NTG）对球形芽孢杆菌（Bacillussphaericus）进行化学诱变,筛选到利福平（Rif）和链霉素（Sm）二个标记菌株。抗药浓度均达100u／ml培养基。其抗药性状能够获得较好地遗传。用含溶葡球菌酶基因的质粒DNA对RifR菌株进行原生质体转化,酶基因在该抗药菌株中获得了高效表达。经摇瓶发酵试验,溶葡球菌酶的活性约为122u／ml培养液。     

沈阳地区铜绿假单胞菌药物敏感性测定及质粒图谱分析

目的 测定铜绿假单胞菌对22种药物的敏感性,帮助临床选择用药。并对分离株进行质粒图谱分析以了解耐药菌株的流行情况。方法 药物敏感性实验采用纸片琼脂扩散法,质粒指纹图谱分析采用碱变性法提取质粒DNA,限制性内切酶切割后进行凝胶电泳分析。结果 铜绿假单胞菌对头胞哌酮、氧派酸、丁胺卡那霉素、壮观霉素、多粘菌毒和头孢三嗪的敏感率在84％－100％之间。所有菌株对其他16种抗性素均有不同程度的耐药。质粒DNA图谱分析显示,12株被检测菌株中有11株含有质粒DNA,其中8株含有23kb质粒DNA。结论 铜绿假单胞菌对头胞哌铜、氟派酸、丁胺卡那霉素、壮观霉素、多粘菌素敏感;多数耐药菌株含23kb质粒DNA。     

Transfer of kanamycin resistance among familial strains of Enterobacteriaceae isolated from a chronic bacterial carrier of Salmonella schottmuelleri

Escherichia coli, Enterobacter aerogenes and S. schottmuelleri were isolated from the large intestine of a bacteriocarrier. E. coli and E. aerogenes strains proved to be resistant to a number of antibiotics. Plasmids were detected in DNA preparations obtained from E. coli strains. After the hybridization of these E. coli strains with E. coli C600 5K and S. schottmuelleri at 28 degrees C the transfer of resistance to kanamycin was found to occur. From some of the transconjugates thus obtained resistance to kanamycin was transferred to E. aerogenes. This resistance was found to be controlled by the plasmid with a molecular weight exceeding 2 Md. The fact that S. schottmuelleri in the carrier's body retained their sensitivity to antibiotics can be explained by the absence of the transfer of plasmid Kmr at a temperature exceeding 28 degrees C and by the existence of the infective agent in an ecological niche other than that of E. coli.     

大熊猫肠道芽孢杆菌的分离鉴定及部分生物学特性

【目的】分析大熊猫肠道中芽孢杆菌的种类、纤维素分解能力、抗微生物作用和常用抗生素药物敏感性。【方法】利用芽孢耐高温特性分离菌株,基于16S r RNA基因序列构建系统发育树,通过测量芽孢杆菌在刚果红纤维素培养基上的分解圈分析其纤维素分解能力,采用牛津杯法测定芽孢杆菌的抑菌能力,结合软件分析抑菌能力和进化树之间的关系,通过PCR调查芽孢杆菌的抗菌肽分布规律,最后通过药敏试验检测芽孢杆菌是否对常用抗生素敏感。【结果】共分离得到21株芽孢杆菌;进化树显示,这些芽孢杆菌分为6个类别(Category);羧甲基纤维素钠水解结果显示,所有菌株均能分解纤维素;大部分芽孢杆菌菌株对3种肠道病原菌有较强的抑制能力,聚类分析表明,菌株的抗菌能力与基于16S r RNA基因的分类有一定的关联性;66.67%(14/21)的菌株中可以检测到2个或3个抗菌肽基因;药敏试验结果显示,菌株整体药物耐受率低,仅为7.54%(19/264),但仍有少数菌株对抗生素耐受。【结论】分离菌株种类丰富,分布平均,且均具有纤维素分解能力。21株菌株都含有抗菌肽基因,代谢产物对3种肠道病原菌具有明显抑制作用。常用抗生素耐受性低,对规范临床用药具有指导性。     

Plasmid transduction by Bacillus anthracis bacteriophage CP54

Possibility of plasmid transduction in Bacillus anthracis vaccine strains Sterne and STI-1 by bacteriophage CP54ant having an increased ability of adsorbtion and a shortened period of latent development in Bacillus anthracis cells has been isolated. The main parameters of plasmid transduction by the bacteriophage have been established for the plasmid pTG141 (TcR). They include the effect of multiplicity of infection, the level of UV-inactivation of bacteriophage, the presence of antiphage serum in the incubation medium. Plasmid transduction by the mutant phage CP54ant was found to be more efficient as compared with the one by the parent phage. The isolated transductants served as donors of the transduced plasmid for Bacillus anthracis and Bacillus thuringiensis strains.     

绿色荧光蛋白基因原核表达载体的构建及其在昆虫肠道短短芽孢杆菌和苏云金芽孢杆菌中的表达

【目的】构建带有苏云金芽孢杆菌cry3a基因非芽孢依赖启动子和绿色荧光蛋白基因gfp(Green Fluorescent Protein)的原核表达载体,并转化从桑粒肩天牛幼虫肠道分离的两株常驻细菌短短芽孢杆菌CQUBb和苏云金芽孢杆菌CQUBt,以检测cry3a启动子在昆虫肠道常驻菌中的启动子活性,获得GFP标记菌株,为常驻菌在昆虫幼虫肠道中的定殖情况和杀虫工程菌的构建奠定基础。【方法】采用重叠延伸PCR将cry3a基因启动子和gfp基因进行融合,并与pHT304载体连接构建重组质粒pHT3AG,获得的重组质粒以电脉冲转化肠道常驻菌短短芽孢杆菌CQUBb和苏云金芽孢杆菌CQUBt,于可见光和荧光显微镜下观察荧光并通过SDS-PAGE分析重组菌株的蛋白表达情况,然后对重组菌株进行生长动力学分析和稳定性测试。【结果】重组菌在营养期大量组成型表达GFP,经电泳分离在凝胶上出现约29kDa的特异蛋白条带;重组菌生长曲线与出发菌没有显著差异,说明外源质粒未对宿主菌的生长带来明显不利影响;抗性条件下传代30次后两菌株外源质粒稳定性仍可达95%、67%;两个菌株比较,CQUBb比CQUBt质粒转化率高、重组菌GFP表达时间长、表达量大,并且重组菌株稳定性好。【结论】成功地将cry3a基因核心启动子和gfp基因转入桑粒肩天牛幼虫肠道常驻菌,实现了该启动子在Bt之外的菌株中发挥作用,构建了两个GFP标记菌株;重组基因工程菌株表达量大,稳定性好,可以用作昆虫肠道内微生态研究和芽孢杆菌表达系统以及杀虫菌株的构建。     

Occurrence of antibiotic and metal resistance and plasmids in Bacillus strains isolated from marine sediment

Eleven hundred Bacillus strains isolated from marine sediment from the Minas Basin, Nova Scotia, Canada, were purified on LB agar supplemented with ampicillin, chloramphenicol, erythromycin, streptomycin, tetracycline, or mercuric chloride. Seventy-seven isolates were examined for plasmid DNA, and for resistance to 11 antibiotics, HgCl2, and phenylmercuric acetate. Minimum inhibitory concentrations of Ag, Cd, Co, Cu, and Zn were also determined. Forty-three percent of antibiotic- and mercury-resistant strains contained one or more plasmids ranging from 1.9 to 210 MDa. Fifty-four percent carried plasmids greater than 20 MDa, and 97% were resistant to two or more metals. There was no correlation between plasmid content and resistance either to antibiotics or to mercurial compounds in these strains. Mercury-resistant isolates were unable to transform Hg2+ to volatile Hg0 by virtue of a mercuric reductase enzyme system (mer). Strains resistant to Hg2+ were investigated for their ability to produce H2S and intracellular acid-labile sulfide when grown in the absence and presence of HgCl2. Lower levels of H2S and intracellular sulfide were detected only in metal-resistant strains grown in the presence of HgCl2, suggesting that cellular sulfides complexed with Hg2+ in these strains.