山东地区鸡源性大肠杆菌的耐药性与质粒图谱的监测分析

对来源于山东各地自然发病鸡群的19株鸡源致病性大肠杆菌进行了耐药性和质粒图谱检测,并对菌株耐药性与质粒图谱间关系进行了分析研究.结果表明:19株鸡源致病性大肠杆菌均具有多重耐药性,耐药图谱复杂;除3株菌没有提取到质粒外,其余菌株均提取到质粒条带1～5条;对比分析发现,菌株耐药性与所携带质粒的数量和大小并无直接的相关性.     

鸡致病性大肠杆菌生物学特性的相关性研究

对来源于河南省新郑地区的14株鸡致病性大肠杆菌进行了致病性试验、血清型鉴定、耐药性试验和质粒图谱检测,并对其相互关系进行了分析研究,结果表明:菌株的血清型与其致病性、耐药性、质粒图谱均无一定的相关性;菌株的致病性与其耐药性、质粒图谱有一定的关系,耐药种类越多的菌株其致病性越强,质粒图谱中含有相对分子量较大的质粒条带的菌株其致病性较强;并且耐药图谱的复杂性与其质粒图谱中的条带数无一定的相关性.     

河南省鸡致病性大肠杆菌耐庆大霉素质粒的检测分析

通过对12株100%耐庆大霉素的多重耐药菌株的质粒提取,电泳检测和转化试验,成功获得了2株由质粒介导的耐庆大霉素的多重耐药菌株。结果表明:同一质粒可编码一个至数个耐药性基因,不同质粒可以携带相同的耐药性基因,并且这些质粒可以传递多种耐药性。证明了菌株对庆大霉素的耐药性是由耐药性质粒介导的,细菌的抗药性与耐药性质粒的转移有很大关系。     

大肠杆菌耐药性抑制剂作用机制的初步研究

20株人源和动物源性大肠杆菌在体外经黄连提取物(命名为耐药性抑制剂IT2)作用后,对氟喹诺酮类抗菌药、庆大霉素和多西环素的耐药性明显被抑制.选取经抑制剂IT2作用后耐药性变化显著的5对菌株进行质粒提取和电泳检测,发现J0、H004、J3、J2 等4株菌作用前均有质粒带,与其相对应的耐药性抑制剂作用后的菌株ITJ0、ITH004、ITJ3、ITJ2质粒带全部消失,只有菌株J1经抑制剂IT2作用前后质粒带无变化.提取外膜蛋白进行SDS-PAGE电泳分析,菌株J1、J3、J0缺少OmpF、OmpA, J2仅缺少OmpA,经耐药性抑制剂作用后,J1、J3、J0的OmpF均由缺失恢复为表达,OmpC的表达量呈显著增加,H004没有变化.结果表明耐药性抑制剂IT2对大肠杆菌的部分耐药性具有良好的抑制作用,其作用与阻碍耐药质粒拷贝、恢复外膜通道蛋白正常表达关系密切.     

一株鸭致病性大肠杆菌的分离鉴定及耐药分析

对江西省樟树市某鸭养殖场送检的病死鸭进行病理剖检,通过细菌分离、生化鉴定得到1株致病性大肠杆菌。药敏试验结果表明,该菌株对头孢拉定、头孢唑啉、头孢曲松和阿莫西林等β-内酰胺类抗生素产生了严重的耐药。通过质粒提取和接合转移试验对分离菌株进行耐药性分析;提取得到一10 kb左右的质粒;质粒接合转移试验表明,成功将该质粒转化入感受态E.coli JM109,并能使E.coli JM109获得对β-内酰胺类抗生素耐药且与分离菌株的耐药谱一致。推测该菌株产β-内酰胺酶的基因存在于质粒,并且该耐药基因能随质粒的转移转化入敏感菌E.coli JM109。     

鸡致病性大肠埃希氏菌耐氧氟沙星质粒的检测

对分离的２０株鸡致病性大肠杆菌进行了药敏实验,筛选出１３株耐氧量沙星（ＯＦＬ）的菌株。经质粒提取,电泳和转化实验,获得一株 粒倡导 的耐煌多重耐药菌株。该菌株对参试的１１种抗生素全部耐药菌株其中ＯＦＬＭＩＣＭ〉５０μｇ／ｍｌ。该菌Ｒ质粒电泳可见６条带,转化大肠杆菌ＤＨ５α,在２０μｇ／ｍｌＯＦＬ的ＬＢ学板筛选得到的转化子,该转化子含有原菌株中２．７ｋｂ的质粒,获得原菌株部分耐药性。连续转化５次,均     

四川规模化鸡场鸡致病性E.coli血清型、耐药性和质粒图谱的研究

从四川10个规模化鸡场分离的经生化鉴定、动物回归试验获得46株鸡致病性E.coli。对46株鸡致病性E.coli进行了耐药性、血清型鉴定和质粒DNA分析的研究。试验结果表明：鸡致病性大杆菌易产生耐药性，且多为多重耐药；鉴定出36株大肠杆菌的O血清型共16种，占鉴定菌株的78.3%；流行的主要血清为O89、O141、O119、O127和O131，共22株，占定型菌株的61.6%，鸡致病性大肠杆菌血清型多，各地血清型各类差异大；质粒得率为100％，来源相同的菌株有相同或相似的质粒图谱和酶切图谱，来源不同的菌株质粒图谱一般不同，同一血清型可以有不同的质粒图谱。     

云南多重耐药猪源大肠杆菌的分离鉴定及其耐药机制初步研究

从云南省3个规模化猪场仔猪黄白痢病猪病例分离到7株革兰氏阴性小杆菌,用生理生化鉴定、药敏试验和致病性试验对分离菌株进行鉴定,并提取细菌的质粒,分析质粒与细菌耐药性之间的相关性。结果表明:7株分离株经生理生化试验鉴定为仔猪黄白痢大肠杆菌,对小白鼠有强致病性,分离株对15种抗生素表现为多重耐药,其中14耐1株、13耐2株、11耐2株、8和8耐以下2株,头孢类抗生素是治疗仔猪黄白痢的首选药物,表明云南猪大肠杆菌已经呈现出十分严重的耐药性。质粒图谱分析表明,4株分离株均携带分子量约为5000bp的一个质粒,而另外3株不携带任何质粒。质粒转化实验表明,分离株所携带的质粒为非耐药性质粒,与细菌耐药性无直接相关性。     

鹌鹑肠道微生物总DNA的最佳提取方法

为了获取高质量、较完整的肠道菌群基因组DNA,试验采用常规的饱和苯酚/氯仿法(方法一)、牛瘤胃DNA的提取法(方法二)和对上述两种方法进行优化后得到的肠道微生物DNA的提取法(方法三)对鹌鹑肠道微生物总DNA进行了提取,并对结果进行了比较。结果表明:应用方法一和方法二提取的DNA浓度比较低,电泳显示样品DNA条带没有方法三明亮;以方法三提取的肠道总DNA为模板,采用细菌通用引物,对其16S rDNA V3可变区进行PCR扩增,得到较清晰的图谱,条带整齐。说明采用方法三提取鹌鹑肠道微生物DNA较完整,可用于后续的分子生物学试验研究。     

鸡源大肠杆菌耐药性分析及中药对大肠杆菌耐药性消除作用的研究

研究鸡源大肠杆菌耐药性及中药对大肠杆菌耐药性消除作用。选择22种抗生素采用Kirby-Bauer法对山西省部分地区分离的20株致病性鸡源大肠杆菌进行耐药性检测,对分离菌株进行质粒分析和耐药质粒转化试验,并分析多种中药对大肠杆菌耐药性的消除作用。结果显示,20株试验菌对测试抗生素均存在不同程度的耐药性,其中5耐及5耐以上的菌株占分离菌株的90%,试验菌对青霉素耐药率最高(90%),其次为恩诺沙星(85%),而对黏杆菌素和呋喃妥因的耐药性最低;分离出12种质粒谱型,同一地区、同一鸡场菌株的质粒图谱相同或相似,不同地区菌株的质粒图谱不同,仅有部分相同或相近的流行质粒共存;对其中5株菌株进行耐药质粒的转化试验,发现同一质粒可编码1个至数个耐药性基因,不同质粒可以携带相同的耐药性基因;中药单剂或合剂对大肠杆菌的耐药性有一定的消除作用,其中黄芩的消除效果最好,中药处理后部分菌株质粒图谱无变化,只有黄连和双黄连造成2条质粒带丢失;对中药提取物作用后的消除子进行药敏试验,结果发现大肠杆菌对环丙沙星等多种抗生素恢复了敏感性。研究结果提示联合使用中药治疗鸡源大肠杆菌病有显著效果。     

鸡源大肠杆菌的耐药性监测及生物学特性研究

从山东省诸城地区6个集约化养鸡场采集50只病死鸡病料,从中分离鉴定得到32株大肠杆菌,对其进行了12种常规药敏试验,发现分离的菌株有不同程度的耐药性。对14株优势血清型菌株进行了质粒DNA提取分析,然后用Hind Ⅲ限制性内切酶对质粒进行了酶切。结果表明,质粒的得率为100%,来源相同的菌株具有相同或相似的质粒图谱,也就是说它们有共同的起源;来源不同的菌株的质粒图谱一般不同,即有不同的起源。将血清型和质粒图谱比较,结果发现,同一血清型可以有不同的质粒图谱,不同血清型可以有相似的质粒图谱,血清型与质粒图谱没有直接的联系。     

Virulence factors of avian Escherichia coli

A total of 45 strains of Escherichia coli isolates from chickens with colisepticemia were examined for virulence factors commonly found in pathogenic groups of E. coli. These strains were studied for the following: pathogenicity in 1-day-old chicks; toxin, hemolysin, and colicin production; cell invasiveness and adherence; hemagglutination for fimbriae detection; serum resistance; aerobactin production in iron-limited conditions; and plasmid content. The characteristics exhibited by virulent strains were invasion for HeLa and chicken fibroblast cells, serum resistance, colicin V, and aerobactin production. None of the isolates were toxigenic or positive in hemagglutination tests. The molecular genetic studies of the virulence factors by agarose electrophoresis showed that the plasmids of these strains are of high molecular weight.     

Effect of EDTA-tris on an Escherichia coli isolate containing R plasmids

Solutions of ethylenediaminetetraacetate (EDTA)-tris combined with antibiotics have been shown to be effective in treating selected cases of persistent bacterial infections. Basic techniques in microbial genetics, including mating frequencies, chemical elimination of R plasmids, isolation of plasmid DNA and agarose gel electrophoresis, were used to determine if EDTA-tris has a curing effect on an R plasmid as part of its clinical action. Results of this study indicated that EDTA-tris by itself eliminated an antibiotic resistance marker from a clinical isolate of Escherichia coli and when combined with another chemical curing agent altered the isolate's mating frequency.     

Preliminary characterization of Escherichia coli isolated from pigs with diarrhoea in Venezuela

The aetiology of neonatal porcine diarrhoea was studied in 15 different herds located in the north-western region of Venezuela. Of 56 strains of Escherichia coli analyzed, 16 (28.6%) were shown to produce heat-stable (STa) enterotoxin, as detected by infant mouse assay. Only four of these STa+ isolates also possessed the K88 pilus antigen, two were 987P+ and none possessed the K99 antigen, leaving 10 STa+ samples in which no pilus antigen was identified. Among the 40 STa negative samples were six K88+ specimens, one K99+, four 987P+, one which reacted as K88+ + K99+ and one K88+ + 987P+. Considering as pathogenic any strain showing at least one of the characters studied, pathogenic E. coli were detected with an overall frequency of 42.9%, being more prevalent during the second week of life. An electrophoretic analysis of the plasmid content of the field isolates of E. coli, revealed the presence of numerous species of extrachromosomal DNA, although no direction association could be made between a particular plasmid and any of the pathogenic characteristics identified. Results of Southern blot analysis indicate that the STa enterotoxin was preferentially encoded within an endemic plasmid of 4.9 Md. Other plasmids present in the E. coli isolates could be related to antibiotic resistance. With the exception of one strain, all E. coli isolates were resistant to more than one of the nine drugs tested; multiresistant E. coli were frequently isolated, including four strains which were resistant to seven antibiotics.     

Similar cefoxitin-resistance plasmids circulating in Escherichia coli from human and animal sources

The aim of this study was to determine the molecular epidemiology of cefoxitin-resistance Escherichia coli identified in cattle entering feedlots and determine if there were any similarities to E. coli causing human infections in Canadian hospitals. A total of 51 E. coli were isolated from a total of 2483 cattle entering four feedlots in southern Alberta, Canada. DNA fingerprinting using pulsed-field gel electrophoresis revealed thirty-two unique patterns with two major clusters observed comprised of Cluster A (11 strains) and Cluster B (7 strains). PCR and sequence analysis revealed 38 isolates (74.5%) harboured bla(CMY-2), whereas the remainder were found to contain mutations in the promoter region of the chromosomal ampC gene, which has been previously associated with cefoxitin resistance. No resistance to nalidixic acid, ciprofloxacin, or amikacin was observed in the clinical isolates. bla(CMY-2) harbouring plasmids were transferred to E. coli DH10B. All of the plasmids carrying bla(CMY-2) contained the A/C replicon and also harboured other resistance genes. Plasmid fingerprinting using BglII revealed 17 unique patterns with all but one clustering within 70% similarity. Comparison of the plasmid fingerprints to those isolated from human clinically significant E. coli in Canada during a similar time period [Mulvey, M.R., Bryce, E., Boyd, D.A., Ofner-Agostini, M., Land, A.M., Simor, A.E, Paton, S., 2005. The Canadian Hospital Epidemiology Committee, and The Canadian Nosocomial Infection Surveillance Program, Health Canada. Molecular characterization of cefoxitin resistant Escherichia coli from Canadian hospitals. Antimicrob. Agents Chemother. 49, 358-365] revealed four strains that harboured bla(CMY-2) A/C replicon type plasmid with fingerprint similarities of greater than 90% to the ones identified in E. coli from the cattle in this study. These findings highlight the potential linkage of multidrug resistant organisms in food producing animals and human infections in Canadian hospitals. The plasmids conferred resistance to multiple antibiotics which could limit options for the treatment of infections caused by these strains.     

鸡致病性大肠杆菌O_1、O_2和O_(78)Ⅰ型菌毛结构基因的克隆、序列测定及同源性分析

提取鸡致病性大肠杆菌分离株O1、O2和O78的基因组DNA作模板,用TD-PCR技术从其中分别扩增出0.55 KB的I型菌毛结构基因(piliA).将扩增得到的piliA基因片段,用TA克隆的方法分别克隆进pGEM-T栽体中,转化至受体菌JM109中,用Amp/IPTG/X-gal琼脂平板蓝白菌落筛选法,得到舍阳性重组子的菌株,提取质粒用PstI单酶切及NcoI和PstI双酶切鉴定,结果证实,所构建的克隆质粒中均含有相应piliA基因.经DNA序列分析,其结构基因阅读框架大小为549 bp,但其中O1菌株Ⅰ型菌毛基因在第72位发生突变,有6个碱基插入.经DNAStar核酸分析软件分析,3个基因同源性为89.9%～92.0%.     

不同地区101个禽源性大肠杆菌分离株的致病性试验

从国内１８个省、市、自治区大肠杆菌病疑似病禽中分离并鉴定出大肠杆菌５９５株,鉴定出其中４４０个分离株的血清型。采用Ｒｏｓｅｎｂｅｒｇ氏报道的方法,测定了来自不同地区的６０个优势血清型分离株对ＳＰＦ鸡、３０个其他常见血清型及１１个国内少见血清型分离株对普通易感鸡的致病性。结果表明：所测定的１０１个分离株均为致病株,且９０％以上属高、中度致病株。     

Probable transmission between animals of a plasmid encoding aminoglycoside 3-N-acetyltransferase IV and dihydrofolate reductase I

Ten aminoglycoside-resistant Escherichia coli isolated from the faeces of healthy or diarrhoeic animals reared in the same herd were studied. These strains were resistant to high levels of apramycin and low levels of gentamicin. They were also resistant to streptomycin, tetracycline, trimethoprim and some to ampicillin, chloramphenicol, kanamycin or nalidixic acid. Two strains, isolated from a calf and a lamb, respectively, belonged to the same biotype. All the transconjugants resistant to gentamicin-apramycin were also resistant to streptomycin, tetracycline and trimethoprim. In all cases, these resistances were encoded by plasmids of 100 kb. Analysis of these plasmids by agarose gel electrophoresis after digestion by EcoRI or BamHI revealed their similarity. Hybridization with a 500-bp HpaI insert of plasmid pFE872 was observed with DNA from field strains and their transconjugants, demonstrating the presence on the 100-kb plasmids of the gene coding for a dihydrofolate reductase I. A single plasmid, designated pIP1831, could be observed in identical or different strains isolated from calves or lambs, suggesting the transmission of strains and plasmids between animals of different species.     

规模化鸡场大肠杆菌的分离鉴定及耐药性监测

为了对鸡致病性大肠杆菌的流行情况及耐药性调查分析,从山东省部分地区的60个鸡场采集了116份鸡内脏病料,结合培养特性对其进行了细菌的分离和生化鉴定,结果分离到大肠杆菌95株,其中致病性大肠杆菌76株。对这76株致病性大肠杆菌进行了12种抗生素敏感性试验,发现分离的菌株对12种药物均有不同程度的耐药性,青霉素对其完全没有抑制作用;新研制的利福平复合新药抑制作用最强,64.5%为高度敏感(49/76),34.2%中度敏感(26/76),表明该药对鸡大肠杆菌具有显著的效果;其中,丁胺卡那霉素(63.2%)、氟哌酸(55.3%)也有良好的抑菌作用。     

鸡致病性大肠杆菌肠毒素(LT)的鉴定及其基因的克隆

鸡致病性大肠杆菌分离株O78经家兔肠袢结扎试验(RILT)证实,该菌株产生热敏性肠毒素(LT)。用PCR技术从该菌株中扩增出1.2kb的LT基因,然后将纯化的PCR产物克隆到pGEM-T载体中,转化至受体菌JM109中。用Amp/IPTG/X-gal琼脂平板蓝白菌落筛选得到阳性重组菌株,提取质粒用SphI和SalI双酶切鉴定,结果证实,构建的克隆质粒pXCLT1含有LT基因。