肠道病原菌的质粒研究

本文报告肠道病原菌质粒研究结果，对ＥＴＥＣ肠毒素质粒、ＥＩＥＣ侵袭性质粒，Ｃｏｌ质粒和Ｒ质粒进行检测和研究。Ｒ质粒、肠毒素质粒和Ｃｏｌ质粒通过接合传递，能将有关质粒传递给受体菌；ＥＴＥＣ肠毒素质粒不仅有试管内接合传递，并能在乳鼠、兔肠段、小白鼠体内接合传递；作者用植物消质灵消除细菌质粒，结果毒素质粒消除率为４６．０％，四环纱质粒为６８．０％，氯霉素为９９．０％。     

产毒大肠杆菌肠毒素质粒(Entg::Tn5)的转导实验研究

我们利用1株带有Km抗性基因的产毒大肠杆菌E·colikl2W1485(Ent::Tn5)作为P1增殖菌制备P1(LT)裂解液,11株分属E、coli、E1EC、S.dysentery、S、sonnei、S.boydii、Proteus菌株作为受体菌,进行试管内转导试验。结果表明11株受体菌中有9株发生质粒完全和部分转导,分别呈表型K_m~+LT~+和K_m~+LT~-。选c600/P1(LT)转导子进行质粒电泳检测,发现K_m~+LT~+转导子出现同P1(LT)增殖菌E、colikl2相同的质粒带,而K_m~+LT~-未出现质粒带。我们再从11株受体菌中选用7株进行兔肠内转导实验,结果发现有3株受体菌所在肠段明显肿胀;而另4株虽不能引起肠段肿胀,但也可分离出转导子。同时,选用10株受体菌进行小白鼠灌胃质粒转导实验,其中4组可分离出转导子。 试管内转导子保留半年后,除1株K_m~+LT~+转导子失去质粒外,其它均保留其原表型。说明转导子稳定性良好。据上述结果提示Ent质粒和R质粒可通过转导在自然界传播。     

棒状杆菌滤膜接合法质粒接合转移实验方法的研究

目的 建立棒状杆菌的滤膜接合法质粒接合转移实验方法 .方法 采用滤膜接合法行质粒接合转移实验.第一次异种属之间的质粒接合转移实验,使用琼脂稀释法筛选出3株高耐红霉素的粪肠球菌临床分离株,红霉素最低抑菌浓度(MIC)>256 mg/L,左氧氟沙星MIC≤8 mg/L,作为供体菌;3株棒状杆菌临床分离株的红霉素MIC≤32 mg/L,左氧氟沙星MIC为128 mg/L,作为受体菌.第二次同属不同种细菌之间的质粒接合转移实验.用第一次转移成功的3株接合子干燥棒状杆菌(红霍素MIC>256 mg/L,头孢他啶MIC为16 mg/L)作为供体,分别向6株不同种的棒状杆菌(红霉索MIC≤32 mg/L,头孢他啶MIC≥128 mg/L)进行质粒接合转移.结果异种属之间的质粒接合转移实验中,3株粪肠球菌作为供体分别向3株棒状杆菌进行9次质粒接合转移,有4株接合子成功转移了耐药表型和基因型,转移频率为44%.同属不同种细菌之间的质粒接合转移实验中,3株转移成功的干燥棒状杆菌作为供体分别向6株不同种的棒状杆菌进行18次质粒接合转移,有7株接合子成功转移了耐药表型,转移频率为39%.接合子不同程度地获得了对红霉素的耐药性,棒状杆菌获得了粪肠球菌的耐药性,并凡将耐药性再传递给了同属不同种的其他棒状杆菌.结论 滤膜接合法质粒接合转移实验可以用于研究棒状杆菌的耐药传递机制.     

种鸭大肠杆菌性生殖器官病的研究 3.大肠杆菌耐药性及其R质粒接合传递的研究

从患生殖器官病的病鸭生殖道和肠道中分离大肠杆菌530株,在用TC、SD、CM、KM、GM、SM、AP、Fu、Rif等9种药物进行细菌药敏试验的基础上,进行了R质粒接合传递试验和质粒DNA电泳分析。试验结果表明:大肠杆菌耐药率为59.4％,其中多重耐药菌株占56.5％,细菌对TC抗性最高(54.0％);耐药菌的R质粒接合传递率为42.6％,对AP抗性的菌株,其R质粒传递率达75.0％。本文还对抗菌药物在该菌R质粒形成与发展中起的作用、鸭生殖道致病性大肠杆菌的可能起源、K_(99)抗原基因与R质粒的连锁,以及细菌抗TC、AP、SD、CM、SM等几种药物基因的连锁遗传等问题作了初步探讨。     

三带喙库蚊胸腔接种乙型脑炎减毒活疫苗病毒的感染动态及致病力观察

目的通过蚊虫胸腔接种乙脑病毒减毒活疫苗SA14-14-2株,了解该疫苗病毒在蚊虫体内的繁殖情况及其毒力稳定性,进一步评价该疫苗的安全性。方法建立三带喙库蚊的实验室种群,用SA14-14-2、SA14和中山株胸腔接种三带喙库蚊,感染后不同时间取一定数量的蚊虫,研磨制成悬液,用空斑试验检测蚊虫体内的病毒滴度。用感染蚊悬液接种乳鼠和感染蚊虫直接叮咬乳鼠的方法,观察对乳鼠的致病性。结果SA14-14-2、SA14和中山株病毒感染蚊虫后,第2~20d蚊虫体内均能检测到病毒,其中SA14-14-2株的滴度为2~3.72 logPFU/ml,SA14株为3~4.85 logPFU/ml,中山株为3~5.40 log-PFU/ml。中山株的感染滴度最高,其次是SA14株,SA14-14-2株的感染滴度最低,表明蚊虫对野毒株(中山株和SA14株)更为敏感。感染SA14-14-2病毒的三带喙库蚊悬液接种乳鼠,未能引起乳鼠发病或死亡。感染SA14-14-2病毒的三带喙库蚊叮咬乳鼠,未见乳鼠发病或死亡,也未从小白鼠血清中检测到乙脑病毒抗体。结论经胸腔接种,SA14-14-2病毒能在三带喙库蚊体内稳定繁殖。动物接种和蚊虫叮咬试验表明,经蚊体内繁殖的SA14-14-2病毒毒力仍保持原有的弱毒特性,表明该减毒活疫苗通过蚊虫体内繁殖后不会造成传播。     

肺炎克雷伯菌产CTX-M-14型超广谱β-内酰胺酶的基因环境研究

目的 为研究肺炎克雷伯菌产CTX-M-14型ESBL基因的基因环境及其传播机制。方法 本试验对产CTX-M-14型ESBL基因进行质粒接合传递试验;对阳性接合子进行质粒抽提并酶切,PBR322载体连接并转化,将转化子进行测序,进行Blast比对研究基因环境。结果 质粒接合试验表明,产CTX-M-14型ESBL基因的阳性菌株能通过质粒接合试验传递耐药性,CTX-M-14基因可经质粒传递,接合子的耐药表型与供体菌基本一致,具有与供体菌相似的耐药表型,仅个别药物的MIC值较供体菌有所降低。在含头孢噻肟平板上筛选的含CTX-M-14基因的阳性转化子对头孢噻肟耐药,而对其它大多数药物表现敏感。产CTX-M-14型ESBL基因的基因环境表明,阳性转化子含有2 637 bp的目的基因序列,经Blast比对,该转化子序列的基因环境结构元件包括上游插入序列ISEcp1、-35区和-10区、重复序列IRR、876 bp的CTX-M-14基因开放阅读框及下游插入序列IS903和IRL,上传NCBI获基因序列号为HQ650134。结论 产CTX-M-14型ESBL菌株的耐药情况较严重,临床菌可通过接合方式编码ESBLs的传播,临床应加强细菌耐药性监测,进行深入研究。     

我国鼠疫耶尔森菌毒力特征研究

目的 研究我国各疫源地分离的鼠疫耶尔森菌(以下简称鼠疫菌)毒力特征.方法 以1943-2012年我国11块鼠疫自然疫源地不同时间、地区、宿主、媒介体内分离的894株鼠疫菌作为研究对象.将每株菌的37℃24h培养物用灭菌生理盐水制成菌悬液,比浊后稀释为2×101、2×102、2×103、2× 104、2×l05、2× 106、2×107、2×108、2×109个/ml,分别取0.5 ml皮下注射于小白鼠鼠鼷部,每组5只动物,分笼饲养观察14 d,动物死亡后取淋巴、肝、脾、肺、心组织进行细菌培养,以分离出鼠疫菌为特异性死亡,并计算半数致死量(LD50).结果 894株代表性菌株中,87.36％(781/894)属于强毒菌,4.36％(39/894)为中等毒力株,8.28％(74/894)为弱毒菌;对不同生态型鼠疫菌株进行毒力比较,青藏高原喜马拉雅旱獭鼠疫自然疫源地的青藏高原型、祁连山型鼠疫菌绝大多数属于鼠疫强毒株,分别占94.42％(457/484)、93.10％(27/29).结论 我国各鼠疫自然疫源地鼠疫菌的毒力特征以鼠疫强毒株为主.     

大肠埃希菌和肺炎克雷伯菌质粒中AmpC酶基因型的检测

目的探讨台州地区大肠埃希菌和肺炎克雷伯菌临床分离株质粒介导的AmpC酶基因型流行状况。方法应用VITEK-60型全自动细菌鉴定仪鉴定细菌,按NCCLS推荐的确证试验测定β-内酰胺酶(ESBLs);采用头孢西丁纸片扩散法筛选疑产AmpC酶阳性菌株,并通过酶粗提物头孢西丁三维试验、接合试验和PCR测序等实验分析该菌株的基因型及基因表型。结果299株大肠埃希菌和肺炎克雷伯菌ESBLs检出率为19.73%(59/299),AmpC酶纸片扩散法筛选阳性率为12.04%(36/299),且36株AmpC酶筛选阳性菌株均为ESBLs阳性株;该菌株中有15株经三维试验证实为AmpC酶阳性,阳性率5.02%(15/299);15株产AmpC菌经接合试验得到15株接合子,经PCR及测序证实与原菌株表型基本一致。质粒型AmpC基因中13株为CIT型,2株为DHA型。结论台州地区大肠埃希菌和肺炎克雷伯菌临床分离株中已出现质粒携带的AmpC基因,基因型以CIT型为主,其次是DHA型。AmpC基因可能与编码ESBLs的基因存在于同一质粒上,编码的AmpC酶的质粒可在细菌间传递。     

河南省人源产ESBLs鼠伤寒沙门菌单相变异株的分子特征研究

目的 分析河南省腹泻病例粪便标本中产超广谱β内酰胺酶(ESBLs) 的鼠伤寒沙门菌单相变异株耐药情况,并研究其分子学特征。方法 对河南省腹泻粪便中分离的124株鼠伤寒沙门菌单相变异株沙门菌,通过肉汤稀释法进行抗生素敏感性试验并筛选产ESBLs菌株;PCR方法检测β-内酰胺酶编码基因携带情况,采用质粒接合试验分析耐药基因的水平转移情况,应用脉冲场凝胶电泳(PFGE)进行亲缘关系分析。结果 124株鼠伤寒沙门菌单相变异株沙门菌耐药严重,其中有16株为产ESBLs菌株。16株产ESBLs菌株均携带CTX-M型耐药基因,并检测出OXA型和TEM型耐药基因;其中9株菌可将CTX-M基因通过质粒转移到大肠埃希菌J53,药敏分析发现其他抗生素的耐药性可以发生共转移。16株产ESBLs 鼠伤寒沙门菌单相变异株沙门菌经XbaⅠ酶切后共分为14种带型,无明显的优势带型。结论 检出产ESBLs 的鼠伤寒沙门菌单相变异株沙门菌基因型具有多样性,耐药基因可通过接合性质粒在不同菌属间播散。     

RNA干扰选择性下调心肌细胞血管紧张素Ⅱ受体AT1a

目的用RNA干扰技术选择性下调乳鼠心肌细胞上血管紧张素Ⅱ1a型受体(AT1aR)的表达。方法用携带U6启动子和AT1a特异性短发夹RNA(shRNA)编码序列的质粒载体pAT1a-shRNA1,pAT1a-shRNA2,及含非特异性shRNA编码序列的对照质粒pGenesil-Con(pCon),转染原代培养的乳鼠心肌细胞,通过半定量RT-PCR和Western-blot法检测AT1a,AT2受体的表达情况,以内参照β-actin进行标化。结果pAT1a-shRNA1使AT1a的mRNA和蛋白质表达分别降低70%和67%,pAT1a-shRNA2使之分别降低66%和52%,转染对照质粒组较空白对照组AT1a受体表达及各组AT2受体表达无显著差异。结论RNA干扰技术能选择性下调乳鼠心肌细胞AT1a受体的表达,为心血管疾病基因治疗的研究提供了新的思路。     

Resistance to antimicrobial agents, hemolytic activity and plasmids in Aeromonas species

A total of 174 Aeromonas isolates consisting of 100 strains from patients with diarrhea being mainly overseas travellers nd healthy subjects, and 74 strains from environmental sources including foods, fish, fresh water, sea water and river soil collected in the area of Tokyo Metropolis and Kanagawa Prefecture was examined for the antimicrobial resistance, presence of plasmids and hemolytic activity. Almost all the isolates (99.4%) were resistant to aminobenzyl penicillin. The isolation frequency of chloramphenicol- or tetracycline-resistant strain was low. Most environmental isolates of A. hydrophila were resistant to multiple antimicrobial agents. Thirty-seven percent of environmental isolates and 39% of human fecal ones carried plasmids. In environmental isolates, seven A. hydrophila and three A. sobria strains carried 63- to 150-kilobase pair (kb) conjugative R plasmids. Two A. hydrophila strains from both the healthy subject and domestic case with diarrhea carried 58- to 90-kb conjugative R plasmids, respectively. None of the isolates from the feces of overseas traveller's diarrhea carried the plasmid. Irrespective of the sources. A. hydrophila showed the highest hemolytic activity among three Aeromonas species. Eighty percent or more of A. hydrophila isolates were of hemolysin positive. The hemolytic titer of A. hydrophila strains from human feces was higher than that of the strains from environmental sources.     

Induced cell aggregation and mating in Streptococcus faecalis: evidence for a bacterial sex pheromone.

Recipient strains of Streptococcus faecalis produce a trypsin sensitive, heat resistant, nuclease resistant factor, designated clumping-inducing agent (CIA) which causes strains carrying certain conjugative plasmids to aggregate. RNA and protein synthesis but not DNA synthesis are required for aggregation to occur. Recipient filtrates that contain CIA activity also induce donors to mate at high frequencies. Introduction of a transferable plasmid into strains producing CIA dramatically reduces the amount of CIA activity produced by the strain but allows the strain to respond to exogenously added CIA. Our data suggest that CIA represents a bacterial sex hormone (pheromone).     

Disrupting antibiotic resistance propagation by inhibiting the conjugative DNA relaxase

Conjugative transfer of plasmid DNA via close cell-cell junctions is the main route by which antibiotic resistance genes spread between bacterial strains. Relaxases are essential for conjugative transfer and act by cleaving DNA strands and forming covalent phosphotyrosine linkages. Based on data indicating that multityrosine relaxase enzymes can accommodate two phosphotyrosine intermediates within their divalent metal-containing active sites, we hypothesized that bisphosphonates would inhibit relaxase activity and conjugative DNA transfer. We identified bisphosphonates that are nanomolar inhibitors of the F plasmid conjugative relaxase in vitro. Furthermore, we used cell-based assays to demonstrate that these compounds are highly effective at preventing DNA transfer and at selectively killing cells harboring conjugative plasmids. Two potent inhibitors, clodronate and etidronate, are already clinically approved to treat bone loss. Thus, the inhibition of conjugative relaxases is a potentially novel antimicrobial approach, one that selectively targets bacteria capable of transferring antibiotic resistance and generating multidrug resistant strains.     

断乳前后兔肠道正常菌群的生态变化及菌群调整疗法

先后采集22只断乳前(1～2月龄)健康乳兔粪便,进行了四类菌群的分类计数,每克粪便平均含菌数(对数值)分别为:分叉杆菌9.72±0.93,乳酸杆菌8.58±0.56,肠球菌5.69±1.06,大肠杆菌6.36±1.57。与此同时,采集18只断乳后5～8天仔兔粪便,进行三类菌的分离计数,每克粪便平均含菌数(对数值)分别为:乳酸杆菌7.33±0.87,肠球菌4.56±0.41,大肠杆菌7.73±0.49。 为了调整兔肠道的菌群失调,我们用从健康乳兔分离的K_4等乳酸杆菌菌株制成的菌剂,先后对39只腹泻病兔进行治疗,治愈35只,治愈率89.74％,对照组12只(未作治疗),死亡9只。初步观察,该菌剂的疗效极为显著(P<0.01)。     

Antimicrobial resistance and conjugative R plasmids in Escherichia coli strains isolated from animals in Peninsular Malaysia

Fifteen independent E. coli strains of avian, bovine and porcine origin in Peninsular Malaysia were tested for antibiotic resistance and conjugative R plasmids. Eight (53%) isolates were found to be antibiotic resistant. Among them, 37.5% were mono-resistant and 62.5% were resistant to three or more antibiotics, i.e., multi-resistant. All of them were resistant to Tc and sensitive to Gm and Nx. Three of the eight antibiotic resistant strains were able to transfer all or part of their resistance to an E. coli K12 recipient by conjugation. The transfer frequencies of Km, Sm and Tc resistance of the three donors varied between 4.5 X 10(-8) to 6.8 X 10(-7). Analysis of the plasmid profiles of all the three donors and their respective transconjugants after agarose gel electrophoresis provided conclusive evidence that the transferable resistance traits were plasmid-mediated.     

A family of lysozyme-like virulence factors in bacterial pathogens of plants and animals.

We describe a conserved family of bacterial gene products that includes the VirB1 virulence factor encoded by tumor-inducing plasmids of Agrobacterium spp., proteins involved in conjugative DNA transfer of broad-host-range bacterial plasmids, and gene products that may be involved in invasion by Shigella spp. and Salmonella enterica. Sequence analysis and structural modeling show that the proteins in this group are related to chicken egg white lysozyme and are likely to adopt a lysozyme-like structural fold. Based on their similarity to lysozyme, we predict that these proteins have glycosidase activity. Iterative data base searches with three conserved sequence motifs from this protein family detect a more distant relationship to bacterial and bacteriophage lytic transglycosylases, and goose egg white lysozyme. Two acidic residues in the VirB1 protein of Agrobacterium tumefaciens form a putative catalytic dyad, Each of these residues was changed into the corresponding amide by site-directed mutagenesis. Strains of A. tumefaciens that express mutated VirB1 proteins have a significantly reduced virulence. We hypothesize that many bacterial proteins involved in export of macromolecules belong to a widespread class of hydrolases and cleave beta-1,4-glycosidic bonds as part of their function.     

氟哌酸和黄连素对志贺氏菌耐药性质粒消除研究

用氟哌酸和黄连素对２０株携有接合传递耐药性质粒（Ｒ质粒）及１９株携有非接合传递耐药性质粒（ｒ质粒）志贺氏菌进行体外消除研究。结果显示，可被消除的耐药性质粒大多数在２４小时内被消除。氟哌酸对Ｒ质粒及ｒ质粒消除率分别为５％和５２．６％，黄连素对Ｒ质粒及ｒ质粒消除率分别为１０％和５７．９％，两种药物对ｒ质粒消除率明显优于对Ｒ质粒消除率，其原因可能与ｒ质粒分子量小，非传递性及在药物作用下易于丢失有关。氟哌酸和黄连素均为广谱抗菌药物，因此在临床上治疗感染的同时又能消除耐药性质粒，阻止耐性扩散。     

Food poisoning caused by enteroinvasive Escherichia coli (O164:H-)--a case in which the causative agent was identified]

Food poisoning due to "Godofu (Sasayuki tofu)" as a main causative foodstuff which broke out on July 14, 1988. There were 670 out of 918 persons who ingested this food who became ill (incidence 73.0%). The main symptoms were diarrhea (93.4%), fever (77.5%), abdominal pain (64.5%), and vomiting (19.9%). A high degree of fever and watery diarrhea were characteristic of this poisoning. The average latent period was 35 hours with a range of one to 156. The O164:H- strains of enteroinvasive Escherichia coli (EIEC) were detected from 22 of the 32 fecal samples collected from the patients, five of ten samples collected from workers engaged in tofu making, and one sample of left-over Godofu. The virulence of EIEC strains isolated from the patients, workers, and left-over food was confirmed by invasion into HeLa and HEp-2 cells, Sereny test, and ELISA test to detect invasive plasmid-derived protein of the organism (conducted at Tokyo Metropolitan Research Laboratory of Public Health). These EIEC strains were sensitive (less than or equal to 0.19 to 6.25 micrograms/ml) to GM, ABPC, CBPC, CER, CET, NA, PB, MINO, TC and CP as well as KM and OFLX which were used for treatment. However, their susceptibility to FOM varied to some extent (6.25 to 25.0 micrograms/ml) and one strain isolated from a tofu worker was resistant to MINO, TC, FOM and CP (25 to greater than or equal to 100 micrograms/ml). Since investigation revealed that Godofu was left at room temperature about 29 degrees C until ingested, we did a experiment to check the bacterial growth in Godofu under similar conditions at the time of outbreak.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of Streptococcus faecalis sex pheromones after acquisition of plasmid DNA.

Recipient strains of Streptococcus faecalis excrete multiple, peptide sex pheromones that induce mating responses in donors harboring certain conjugative plasmids. Acquisition of plasmid DNA leads to a "shutting off" of pheromone excretion, and such cells become responsive to exogenous pheromone. Data are presented showing that donors excrete low levels of a modified, inactive form of the pheromone. This substance, when mixed in excess with active pheromone, inhibits pheromone activity (probably by competition for a receptor site on the donor). Modified forms of both cPD1 and cAD1 were revealed, and each appeared to have a mass about 350-400 daltons larger than the active pheromone. In both cases, pheromone activity could be regenerated by treatment with phosphodiesterase II.