Serum resistance and aerobactin iron uptake in avian Escherichia coli mediated by conjugative 100-megadalton plasmid.

A total of 115 strains of Escherichia coli isolated from chickens with colisepticemia in Japan were examined for chicken lethality and virulence factors. It was found that serum resistance and aerobactin-mediated iron uptake are the most prevalent characteristics in these strains. Among them, S-20, a representative virulent strain of serotype O2, was further studied. S-20 harbored a conjugative 100-megadalton (Mdal) plasmid, designated pKI100. Curing and reintroduction experiments showed that pKI100 encodes both serum resistance and aerobactin-mediated iron uptake, and the diminished virulence of the pKI100-cured strain was fully restored by the reintroduction of the plasmid. These results demonstrated that pKI100 is the virulence plasmid of the S-20 strain, and that serum resistance and aerobactin-mediated iron uptake are the virulence factors in E. coli strains which cause avian colibacillosis.     

大肠埃希氏菌多重耐药性与整合子的研究

整合子目前被认为是耐药基因在水平传播的重要因子,由Hall等命名,由两部分组成,5'与3'端保守区域(conserved segments简称cs)以及中间的基因盒,具有特异重组位点,能够将携带有耐药基因的基因盒选择性地整合到整合子上,获得耐药性[1].     

Vacuolating cytotoxin produced by avian pathogenic Escherichia coli

The purpose of this study was to determine whether avian pathogenic Escherichia coli produced cytotoxic activity. Culture supernatants of 20 E. coli strains isolated from cellulitis lesions in chickens, five E. coli strains from avian septicemia, five from swollen head syndrome, and five from the feces of healthy chickens were incubated with primary chicken embryo fibroblast (CEF) cells, primary chicken kidney (PCK) cells, a quail fibroblast cell line (QT-35), and four mammalian cell lines (human epithelioid cervical carcinoma, African green monkey kidney, Chinese hamster ovary, and human larynx epidermoid carcinoma). Cytotoxicity was observed with supernatants from the 30 avian pathogenic strains on the two primary chicken cells (CEF and PCK). The highest dilution of culture supenatant that induced cytotoxic changes in 50% of the cells was 1/64. Supernatants from the five strains from normal feces were noncytotoxic, and none of the supernatants was cytotoxic for the QT-35 or the four mammalian cell lines. The cytotoxic effect, which was observed as early as 2 hr after exposure of the cells, was maximal at 6 hr and was evident as vacuolation, morphologically indistinguishable from that previously reported for culture supernatants of Helicobacter pylori. Like the activity in H. pylori, the cytotoxicity of the avian pathogenic strains was destroyed by heating at 70 C for 30 min and by exposure to proteolytic enzymes and was retained by filtration with a 100,000 molecular weight cut-off ultrafilter. Supernatants of two vacuolating cytotoxin-positive cultures of H. pylori failed to induce vacuolation of the CEF and PCK cells but caused the characteristic vacuolation in HeLa and Vero cells. The observations suggest that avian pathogenic E. coli produce a cytotoxin that is similar to the cytotoxin of H. pylori but may be specific for avian cells.     

Adhesive properties and iron uptake ability in Escherichia coli lethal and nonlethal for chicks

Escherichia coli that are virulent for poultry usually result in a respiratory disease, which is frequently followed by a general infection. Adhesiveness of E. coli to epithelial cells and iron-uptake ability of E. coli could be involved in different steps of the disease. These properties were studied in 59 E. coli strains originating from poultry, with reference to lethality for day-old chicks. Adhesive properties were found in 64% of the lethal strains and in only 23% of the nonlethal strains. The ability to grow in limited iron conditions was strongly correlated with lethality. Fifty-two percent of the lethal E. coli strains, but none of the nonlethal strains, possessed both adhesive and iron-uptake abilities. It is suggested that these two properties play a role in the virulence of E. coli for poultry.     

papA gene of avian pathogenic Escherichia coli

P fimbrial adhesins may be associated with the virulence of avian pathogenic Escherichia coli (APEC). However, most APECs are unable to express P fimbriae even when they are grown under conditions that favor P fimbrial expression. This failure can be explained by the complete absence of the pap operon or the presence of an incomplete pap operon in Pap-negative APEC strains. In the present study, we analyzed the pap operon, specifically the papA gene that encodes the major fimbrial shaft, to better understand the pap gene cluster at the genetic level. First, by PCR, we examined a collection of 500 APEC strains for the presence of 11 genes comprising the pap operon. Except for papA, all the other genes of the operon were present in 38% to 41.2% of APEC, whereas the papA was present only in 10.4% of the APEC tested. Using multiplex PCR to probe for allelic variants of papA, we sought to determine if the low prevalence of papA among APEC was related to genetic heterogeneity of the gene itself. It was determined that the papA of APEC always belongs to the F11 allelic variant. Finally, we sequenced the 'papA region' from two papA-negative strains, both of which contain all the other genes of the pap operon. Interestingly, both strains had an 11,104-bp contig interruptingpapA at the 281-bp position. This contig harbored a streptomycin resistance gene and a classic Tn10 transposon containing the genes that confer tetracycline resistance. However, we noted that the papA gene of every papA-negative APEC strain was not interrupted by an 11,104-bp contig. It is likely that transposons bearing antibiotic resistance genes have inserted within pap gene cluster of some APEC strains, and such genetic events may have been selected for by antibiotic use.     

禽致病性大肠杆菌生物被膜的形成及其影响因素

为确定影响细菌生物被膜(BF)的形成条件,本研究采用BF体外定性观察和定量粘附性检测两种方法对1株野生禽致病性大肠杆菌(E.coli)在不同环境(培养时间、培养基类型、引导载体类型、营养条件)下产生BF的差异进行了研究。结果显示野生禽致病性E.coli其宿主体外BF最适形成条件是培养时间为48h,培养基为5%TSB、引导载体为平底96孔聚苯乙烯微孔板(美国Corning Costar)。其生长周期为8h时开始起始粘附、24h形成若干微菌落、36h微菌落粘连、48h形成完整的BF、72h细菌脱落开始下一轮的BF生长。糖分和适量的无机盐都可以在一定程度上促进BF的形成和被膜内细菌的粘附性提高。该研究表明禽致病性E.coliBF的形成周期,并为抑制其形成提供了实验依据。     

鸭致病性大肠杆菌的分离与鉴定

对成都华阳地区两个种鸭场的鸭大肠杆菌病流行病学进行调查,并分析其发病特点。从这两个种鸭场的粪便和其中一个鸭场死亡鸭胚中共分离出19株大肠杆菌,我们对其培养特性、形态特征、生化特性、致病性及药物敏感性进行了研究,同时对分离的19株大肠杆菌的0群抗原进行了鉴定,其结果为O141 4株、O138 4株、O101 2株、O154 1株和O78 4株,还有4株未定型,有待于进一步的血清型鉴定。动物实验表明,已鉴定出血清型的15株大肠杆菌均有不同程度的致病性,药物敏感性实验表明,不同菌株对药物敏感性差异较大,但对多数抗菌药还没有产生抗药性。最后针对这两个种鸭场对该病的防治提出了对策。     

噬菌体控制致病性大肠杆菌的研究进展

大肠杆菌是引起细菌类食物中毒的主要原因之一,近年来,由大肠杆菌引起的食品安全事件频发,严重危害人类健康。各国科学家相继从肉类、蔬菜水果和牛奶等食品,或养殖场中分离得到了一些血清型的致病性大肠杆菌,其中最常见、流行最广泛的有O157:H7,K88,K99,O149等。然而,由抗生素滥用导致的耐药菌株出现,以及在动物饲养方面,抗生素种类和用量上的限制,使得人们对致病性大肠杆菌的控制更加困难。噬菌体是细菌的天敌,具有感染并裂解细菌的功能,与抗生素相比,噬菌体制剂具有特异性强、自我增殖快、研发时间短等优点,因此近年来噬菌体作为控制致病性大肠杆菌的研究受到广泛关注。本文就噬菌体在致病性大肠杆菌的生物防控方面的应用研究进展作一综述,以期为噬菌体及其制剂在保障动物性食品安全及动物疾病治疗领域的深入研究提供一定的参考。     

产肠毒素大肠杆菌感染的分子致病机制

产肠毒素大肠杆菌（enterotoxigenic Escherichia coli,ETEC）是发展中国家人群腹泻的主要原因,一直是西方国家旅行者腹泻的最常见病因,也是引起动物（尤其是幼龄动物）腹泻的主要病原菌。根据ETEC产生热敏和（或）热稳定肠毒素特性可以将其分类成不同致病型。针对这类重要病原体的疫苗研发目前仍然面临着艰难的挑战。本文综述了病原体-宿主相互作用的分子机制,从基因组学和蛋白质组学两方面介绍致病菌的多种毒力因子以及作为靶标研发疫苗的潜力,分析了病原与不同宿主易感性的差异,为针对ETEC新型疫苗的研发和有效预防ETEC感染提供理论依据。     

微生态制剂对鸡体内大肠埃希氏菌的拮抗试验

试验比较了微生态制剂和喹乙醇对鸡体内大肠埃希氏菌的拮抗效果。结果表明，微生态制剂用于预防鸡大肠杆菌病的效果理想，但发病时的疗效却不及药物治疗效果。     

Carriage of potentially pathogenic Escherichia coli in chickens

DNA-DNA hybridization, cultured cell lines, and transmission electron microscopy were used to study pathogenicity traits of 64 Escherichia coli isolated from apparently healthy chickens from 18 small-scale farms in Thika District, Kenya. A total of 39 (60.9%) isolates hybridized with the eae gene probe for enteropathogenic E. coli (EPEC) whereas another 16 (25%) hybridized with the lt and st gene probes and were categorized as enterotoxigenic E. coli. Electron microscopic examination of the eae probe-positive E. coli cultures with the HT-2919A cell line confirmed that they were able to attach intimately and produced effacement typical of EPEC. In addition, negative stain electron microscopy showed that the EPEC strains produced pili that have previously been associated with increased virulence of E. coli infections in chickens. This study has also demonstrated that apparently healthy chickens may carry enteropathogenic E. coli strains.     

Differentiation of pathogenic and nonpathogenic Escherichia coli isolated from poultry

Twenty-one isolates of Escherichia coli recovered from chickens and turkeys were evaluated for pathogenicity in 1-week-old chicks. Fifteen produced coli-septicemia (pathogenic) and six were innocuous (nonpathogenic). Both pathogenic and nonpathogenic E. coli were tested for their ability to selectively absorb Congo red (CR) dye incorporated into agar medium. Eight of 15 pathogenic E. coli (somatic antigen types O1, O78, O11, O88, and OX9) absorbed the dye and produced red colonies (CR+) between 48 to 72 hours of incubation. All serotypes of E. coli with homologous somatic antigen O78 were CR+, while those of O2 antigen were CR- (white colonies). Five of six nonpathogenic E. coli also were CR+. In contrast to pathogenic E. coli, however, nonpathogenic isolates absorbed CR early, between 18 to 24 hours of incubation. Although CR dye binding did not correlate well with pathogenicity, it may be an identifiable property of some serotypes of E. coli.     

江西地区猪源致病性大肠杆菌的耐药性试验

对采自江苏周边地区规模化猪场的137株大肠杆菌耐药情况进行调查研究。通过分离纯化、生化鉴定以及致病性试验,得到67株致病性大肠杆菌。采用微量稀释法测定了8种药物对67株致病性大肠杆菌的最低抑菌浓度。结果显示,细菌对8种临床常用药物表现出较高的耐药性,多数细菌对5种药物耐药,甚至有些细菌对其中7种药物产生耐药性,其中细菌对头孢曲松、头孢曲松-舒巴坦和庆大霉素的耐药率最高,分别为100%、100%和97.01%。说明该地区采集的养殖场细菌耐药现象较为严重。     

Gastrointestinal colonization by salmonellae and pathogenic Escherichia coli in monoxenic and holoxenic chicks and poults

Chicks monocolonized by either salmonellae or pathogenic strains of Escherichia coli had persistent and undiminished colonization of all levels of the gastrointestinal tract and frequently had bacteremia during test periods ranging to 35 days. Poults monocolonized by salmonellae or Arizona hinshawii 7:"1,7,8 developed a similar pattern of colonization. Conventionally reared chicks and poults had rather variable colonization by these pathogens, and it was most persistent in the ceca. Groups treated with a native protective microflora were infrequently colonized. Differences in colonization are explainable by lack of competing bacteria in the monocolonized group and by various degrees of protection provided by microflora colonizing the other groups.     

Antimicrobial susceptibility and molecular characterization of avian pathogenic Escherichia coli isolates

Ninety-five avian pathogenic Escherichia coli (APEC) isolates recovered from diagnosed cases of avian colibacillosis from North Georgia between 1996 and 2000 were serotyped and examined for typical virulence-factors, susceptibility to antimicrobials of human and veterinary significance, and genetic relatedness. Twenty different serotypes were identified, with O78 being the most common (12%). The majority of the avian E. coli isolates (60%), however, were non-typeable with standard O antisera. Eighty-four percent of isolates were PCR positive for the temperature-sensitive hemagglutinin (tsh) gene and 86% positive for the increased serum survival (iss) gene. Multiple antimicrobial-resistant phenotypes (> or =3 antimicrobials) were observed in 92% of E. coli isolates, with the majority of isolates displaying resistance to sulfamethoxazole (93%), tetracycline (87%), streptomycin (86%), gentamicin (69%), and nalidixic acid (59%). Fifty-six E. coli isolates displaying resistance to nalidixic acid were co-resistant to difloxacin (57%), enrofloxacin (16%), gatifloxacin (2%), and levofloxacin (2%). DNA sequencing revealed point mutations in gyrA (Ser83-Leu, Asp87-Tyr, Asp87-Gly, Asp87-Ala), gyrB (Glu466-Asp, Asp426-Thr), and parC (Ser80-Ile, Ser80-Arg). No mutations were observed in parE. Twelve of the quinolone-resistant E. coli isolates were tolerant to cyclohexane, a marker for upregulation of the acrAB multi-drug resistance efflux pump. Quinolone-resistant isolates were further genetically characterized via ribotyping. Twenty-two distinct ribogroups were identified, with 61% of isolates clustering into four major ribogroups, indicating that quinolone resistance has emerged among multiple avian pathogenic E. coli serogroups and chromosomal backgrounds.     

禽病原性大肠杆菌温度敏感性血凝素(Tsh)突变株的构建

运用基因重组方法将庆大霉素抗性基因(GM)连接到PCR扩增的tsh两端区域产生的2个目的基因片段之间,并共同插入到pUC18载体的多克隆位点中,构建出带GM标志的载体pUC18-tshFRGM,从中切下目的片段,再将之克隆到pMEG-375自杀性载体中,构建出自杀性载体pMEG375-tshFRGM,将突变载体转化到含tsh基因的受体APECE037株中,根据同源重组原理,筛选出tsh基因缺失的E037突变株。E037和E037(Δtsh)株的LD50分别为105.6CFU和109.0CFU,动物感染性试验表明,E037(Δtsh)株在内脏器官和血液中的感染能力和大肠杆菌病变程度均有了明显降低。     

Prevalence of pathogenic Escherichia coli in the broiler house environment

Matched sampling of Escherichia coli from broiler house litter and bird lesions of either cellulitis or colibacillosis was conducted to investigate the relationship of pathogenic E. coli to those found in the environment. Isolates were collected from six broiler flocks representing six geographically disparate ranches. Isolates were compared by flock for similarity in serotype and genotyped by pulsed-field gel electrophoresis. Serotyping revealed a considerable dissociation between the two groups of isolates. The prevalence of pathogenic E. coli that matched the environmental isolates from the same house was 0 to 3%. Statistical analysis of the serotype data showed a strong dependence of serotype on isolate source, indicating a high probability that a particular serotype would be found among lesions or litter but not in both groups. Genotyping of isolates on two farms supported the results of serotyping and provided differentiation of isolates that could not by typed by serology. These results suggested that the prevalence of pathogenic E. coli in the broiler house was independent of the prevalence of other commensal or environmental E. coli. Understanding the composition of E. coli populations in commercial poultry production may have bearing on the epidemiology and control of E. coli related diseases.