Contribution of AIDA-I to the pathogenicity of a porcine diarrheagenic Escherichia coli and to intestinal colonization through biofilm formation in pigs

In order to evaluate the role of the AIDA-I of porcine diarrheagenic Escherichia coli strain PD20 serogroup O143 (AIDA-I⁺, STb⁺), a mutant strain PD20M (AIDA-I⁻, STb⁺) was generated from strain PD20 by an allelic exchange procedure. In addition, the full-length aidA gene was reintroduced into strain PD20M to generate the complemented strain PD20C (pTaidA, AIDA-I⁺, STb⁺). A non-pathogenic E. coli strain PD71 was used as negative control. Each strain was inoculated to newborn pigs via stomach tube. Severity of diarrhea was evaluated clinically and intestinal colonization was assessed by histology, immunohistochemistry (IHC), and transmission electron microscopy (TEM) including immunogold electron microscopy (IGEM). The adhesion pattern to HeLa cells, bacterial auto-aggregation and biofilm formation were evaluated in vitro. Pigs infected with strains PD20 or PD20C developed diarrhea 16 and 28 h after inoculation, respectively, in contrast to pigs infected with strains PD20M or PD71. Histology, IHC, TEM and IGEM examinations showed heavy bacterial colonization with biofilm formation in the large intestine, and marked in vivo expression of AIDA-I protein in pigs infected with strains PD20 or PD20C in contrast to pigs infected with strains PD20M or PD71. The in vitro assays showed marked diffuse adherence to HeLa cells, enhanced bacterial auto-aggregation and significant biofilm formation (p < 0.05) by the AIDA-I⁺ strains, when compared to AIDA-I⁻ strains. These results demonstrate that expression of AIDA-I is essential for intestinal colonization and in vitro bacterial autoaggregation and biofilm formation. Thus, AIDA-I may be considered a significant virulence determinant in development of diarrhea caused by porcine diarrheagenic AIDA-I⁺ E. coli PD20 in piglets.     

猪大肠杆菌和葡萄球菌的分离鉴定及耐药性分析

对湘西北地区某猪场患病的仔猪的粪便及病变的脾脏、肝脏、淋巴结、肺脏、肾水肿液进行病原菌分离鉴定,并用Kirby-Bauer法测定分离菌株对15种抗菌药的敏感性。结果表明,患病仔猪分离到大肠杆菌和猪葡萄球菌两种细菌。其中大肠杆菌对庆大霉素、头孢呋肟、丁胺卡那霉素、四环素、先锋必素敏感,对氯霉素、氧哌嗪青霉素、先锋五号、头孢曲松钠、新霉素中介,对羧苄青霉素、强力霉素、青霉素、氨苄青霉素、红霉素有较高的耐药性。葡萄球菌对先锋霉素Ⅴ、丁胺卡那霉素敏感,对头孢曲松钠中介,对其他抗生素耐药。其中大肠杆菌对药物敏感率、中介率、耐药率均为33.33%;葡萄球菌的敏感率为13.33%,中介率为6.67%,耐药率为80.00%。     

石河子地区隐性乳房炎奶牛源大肠杆菌的分离鉴定及耐药性分析

为了解引起石河子地区规模化奶牛场隐性乳房炎奶牛源大肠杆菌的流行情况及其耐药性,本试验采用兰州乳房炎检测板(Lanzhou mastitis test,LMT)对石河子地区11个规模化奶牛场进行了检测,同时对无菌采集的检测为阳性的乳样进行了大肠杆菌分离鉴定及耐药性分析.结果显示,石河子地区11个规模化奶牛场的隐性乳房炎感染阳性率为44.56%(313/1950);对313份无菌采集的乳样进行细菌分离鉴定,结果共分离出病原菌108株,其中大肠杆分离率为14.81%(16/108);药敏试验结果显示16株大肠杆菌对氧氟沙星、头孢唑啉、克林霉素高度敏感,对庆大霉素、氨苄西林、链霉素中度敏感,对阿莫西林、四环素有耐药性.本试验为该地区大肠杆菌源性隐性乳房炎的防制提供科学依据.     

Acquired antimicrobial resistance in the intestinal microbiota of diverse cat populations

The aim of this study was to investigate the prevalence of acquired antimicrobial resistance in the resident intestinal microbiota of cats and to identify significant differences between various cat populations. Escherichia coli, Enterococcus faecalis, E. faecium and Streptococcus canis were isolated as faecal indicator bacteria from rectal swabs of 47 individually owned cats, 47 cattery cats and 18 hospitalised cats, and submitted through antimicrobial sensitivity tests. The results revealed that bacteria isolated from hospitalised and/or cattery cats were more frequently resistant than those from individually owned cats. E. coli isolates from hospitalised cats were particularly resistant to ampicillin, tetracycline and sulfonamide. Both enterococci and streptococci showed high resistance to tetracycline and in somewhat lesser extent to erythromycin and tylosin. Most E. faecium isolates were resistant to lincomycin and penicillin. One E. faecalis as well as one E. faecium isolate from hospitalised cats showed 'high-level resistance' (MIC > 500 microg/ml) against gentamicin, a commonly used antimicrobial agent in case of human enterococcal infections. The results of this research demonstrate that the extent of acquired antimicrobial resistance in the intestinal microbiota of cats depends on the social environment of the investigated population. It is obvious that the flora of healthy cats may act as a reservoir of resistance genes.     

Oral antimicrobials increase antimicrobial resistance in porcine E. coli – A systematic review

Administration of antimicrobials to livestock increases the risk of antimicrobial resistance (AMR) in commensal bacteria. Antimicrobials in pig production are usually administered per pen via feed which implies treatment of sick alongside with healthy animals. The objective of this systematic literature review was to investigate the effect of orally administered antimicrobials on AMR in Escherichia coli of swine.     

Gene markers of generic Escherichia coli associated with colonization and persistence of Escherichia coli O157 in cattle

Enterohemorrhagic Escherichia coli (EHEC) O157 are important foodborne pathogens whose major reservoir are asymptomatic cattle. There is evidence suggesting that nonpathogenic E. coli and bacteriophages in the gastro-intestinal tract can influence the pathogenicity of EHEC O157. The factors contributing to the onset and persistence of shedding EHEC O157 in cattle are not completely elucidated. This study used Bayesian network analysis to identify genetic markers of generic E. coli associated with shedding of EHEC O157 in cattle from data generated during an oral experimental challenge study in 4 groups of 6 steers inoculated with three different EHEC O157 strains. The quantification of these associations was accomplished using mixed effects logistic regression. The results showed that the concurrent presence of generic E. coli carrying the prophage marker R4-N and the virulence marker stx2 increased the odds of the onset of EHEC O157 shedding. The presence of prophage markers z2322 and X011C increased, while C1.N decreased the odds of shedding EHEC O157 two days later. A significant antagonist interaction effect between the presence of the virulence marker stx2 on the day of shedding EHEC O157 and two days before shedding was also found. In terms of the persistence of EHEC O157 shedding, the presence of prophage marker R4-N (OR = 16, and 95% confidence interval (CI): 1.1, 252) was found to increase the odds of stopping EHEC O157 shedding, whereas prophage marker C1.N (OR = 0.16, CI: 0.03, 0.7) and the enterohemolysin gene hly (OR = 0.03, CI: 0.001, 0.8) were found to significantly decrease the odds of stopping EHEC O157 shedding. In conclusion, the study found that the presence of certain genetic markers in the generic E. coli genome can influence the pathogenicity of EHEC O157.     

2011年黄三角地区鸭源大肠杆菌的分离鉴定

本试验从黄河三角洲区域2011年规模化肉鸭商品鸭场送检的病料中,通过细菌分离、培养特性观察、生化试验和致病性试验,分离到54株鸭源高致病性大肠杆菌。通过药敏试验,发现分离菌对多种抗生素存在多重耐药,其中对链霉素、氨苄西林和克林霉素3种药物的耐药性达到90%以上。敏感性较高的药物有阿米卡星、头孢噻肟、头孢曲松等药物。对分离菌进行了O抗原血清型分型,初步鉴定有12种血清型存在,其中O78、O35、O2、O18分别有12株、8株、6株和5株,为该区域优势血清型。本试验为了解和掌握该区域鸭源大肠杆菌病的流行情况,进一步研究和防控该病提供了参考依据。     

曲靖地区仔猪腹泻细菌性病原的分离鉴定与耐药性分析

为了解曲靖地区仔猪出现腹泻的细菌性病因,对5个养殖场148份出现仔猪腹泻症状的样品进行细菌分离培养及耐药性分析,结果显示,检出致病性大肠杆菌(E.coli)114份,阳性率为77.03%;沙门氏菌(Salmonella)26份,阳性率为17.57%,混合感染率为17.57%.致病性大肠杆菌和沙门氏菌对多种抗生素产生了耐药性,其中114株致病性大肠杆菌中97株呈多重耐药性,占总菌数的85.09%,26株沙门氏菌中20株呈多重耐药性,占总菌数的76.92%.结果表明,致病性大肠杆菌是导致当地哺乳仔猪腹泻的主要细菌性病原,部分仔猪继发感染沙门氏菌,两种细菌对多种抗生素产生了耐药性,多重耐药现象较严重.     

肠出血性大肠杆菌Ⅲ型分泌系统调控因素的研究进展

Ⅲ型分泌系统(Typr Ⅲ secretion system,T3SS)是一种将细菌蛋白通过膜屏障注入宿主细胞的装置,Ⅲ型分泌系统(T3SS)是肠出血性大肠杆菌定植反刍动物储存宿主所必需的,也是引起人致病的重要原因之一。E. coli 注射蛋白质进入上皮细胞使得细菌可以黏附并且促使其长时间定植在动物体内。在此,对关于EHEC Ⅲ型分泌系统调控的研究近况、效应蛋白表达的共调控进行了综述。     

牦牛产志贺毒素大肠杆菌主要黏附因子相关基因的分子流行病学调查

为了解中国牦牛产志贺毒素的大肠杆菌(Shiga toxin-producing Escherichia coli, STEC)中主要黏附因子的流行情况,采用PCR方法对来自四川甘孜阿坝等地区健康牦牛的70株STEC的eae、saa、iha 3种与黏附相关的毒力基因进行检测,并对部分含有相关黏附因子的阳性分离株的毒力基因进行了克隆及序列分析。结果显示,牦牛STEC中saa、iha的阳性率分别为71.42%(50/70)和78.57%(55/70),无eae基因序列(0/70),saa、iha的测序结果与GenBank上序列的同源性分别为100%和93%~99%。健康牦牛分离的STEC无LEE毒力岛编码eae,其他的一些与黏附相关的主要毒力基因saa、iha的携带率较高。     

Genomic Description of Antibiotic Resistance in Escherichia coli and Enterococci Isolates from Healthy Lusitano Horses

Antibiotic resistance is a global problem, and it is known that commensal bacteria can act as reservoir of antibiotic resistance genes of clinical importance. The aim of the present study was to determine the antibiotic resistance phenotype and mechanisms implicated in resistance of Escherichia coli and Enterococcus spp. isolates collected from fecal samples of 90 Lusitano horses from Portugal. Sixteen of the 71 E. coli isolates (22.5%) recovered showed resistance to at least one of the antibiotics tested. The number of E. coli isolates resistant to streptomycin, tetracycline, chloramphenicol, ampicillin, trimethoprim-sulfamethoxazole, and gentamicin was 9, 7, 6, 3, 2, and 1, respectively. The bla_TEM-1 and bla_OXA-1 genes were detected in ampicillin-resistant isolates and the sul2 and dfrA1 genes in trimethoprim-sulfamethoxazole-resistant, while the aac(3)-I, floR and tet(A) were found in the gentamicin, chloramphenicol and tetracycline-resistant isolates, respectively. Twenty-two of the 71 (31%) recovered enterococci showed antibiotic resistance for at least one of the tested antibiotics, and resistant isolates were identified as Enterococcus faecium (n = 14), E. faecalis (n = 3), E. hirae (n = 2), and Enterococcus spp. (n = 3). The erm(B) and erm(C) genes were identified in erythromycin-resistant enterococci and the tet(M) and/or tet(L) genes in tetracycline-resistant isolates. The slight prevalence of antibiotic resistance among commensal bacteria of healthy Lusitano horses can improve the treatment of upcoming infections in these horses because these microorganisms can be considered as antimicrobial indicator bacteria.     

60株大肠杆菌的分离与致病性鉴定

大肠杆菌(Escherichia coli,E.coli)是鸡肠道常在菌,部分菌株具有致病性。鸡大肠杆菌病就是由大肠杆菌中的某些致病性菌株引起的鸡感染性疾病。致病性大肠杆菌均携带毒力岛基因ChuA。为鉴定分离自临床病例的鸡大肠杆菌的致病性,本试验采用PCR法检测ChuA基因进行分子生物学鉴定,60株大肠杆菌中有31株属于致病性大肠杆菌,总阳性率为51.67%。     

The food contaminant fumonisin B1 reduces the maturation of porcine CD11R1+ intestinal antigen presenting cells and antigen-specific immune responses, leading to a prolonged intestinal ETEC infection

Consumption of food or feed contaminated with fumonisin B₁ (FB₁), a mycotoxin produced by Fusarium verticillioides, can lead to disease in humans and animals. The present study was conducted to examine the effect of FB₁ intake on the intestinal immune system. Piglets were used as a target and as a model species for humans since their gastro-intestinal tract is very similar. The animals were orally exposed to a low dose of FB₁ (1 mg/kg body weight FB₁) for 10 days which did not result in clinical signs. However, when compared to non-exposed animals, FB₁-exposed animals showed a longer shedding of F4⁺ enterotoxigenic Escherichia coli (ETEC) following infection and a lower induction of the antigen-specific immune response following oral immunization. Further analyses to elucidate the mechanisms behind these observations revealed a reduced intestinal expression of IL-12p40, an impaired function of intestinal antigen presenting cells (APC), with decreased upregulation of Major Histocompatibility Complex Class II molecule (MHC-II) and reduced T cell stimulatory capacity upon stimulation. Taken together, these results indicate an FB₁-mediated reduction of in vivo APC maturation.     

Low numbers of intestinal Shiga toxin-producing E. coli correlate with a poor prognosis in sheep infected with bovine leukemia virus

Healthy ruminants carry intestinal Shiga toxin (Stx)-producing Escherichia coli (STEC). Stx has antiviral activities in vitro and STEC numbers correlate with reduced early viremia in sheep experimentally infected with bovine leukemia virus (BLV). This study assessed the impact of intestinal STEC on BLV-induced disease for one year post-BLV-challenge. High STEC scores (CFU/g feces × frequency of STEC-positive samples) correlated with good health, whereas poor weight gain, distress, and tumor development occurred only among animals with low STEC scores. STEC carriage was associated with increased percentages of B cells in peripheral blood.     

Isolation and characterization of antimicrobial-resistant Escherichia coli from national horse racetracks and private horse-riding courses in Korea

Limited information is available regarding horse-associated antimicrobial resistant (AR) Escherichia (E.) coli. This study was designed to evaluate the frequency and characterize the pattern of AR E. coli from healthy horse-associated samples. A total of 143 E. coli (4.6%) were isolated from 3,078 samples collected from three national racetracks and 14 private horse-riding courses in Korea. Thirty of the E. coli isolates (21%) showed antimicrobial resistance to at least one antimicrobial agent, and four of the AR E. coli (13.3%) were defined as multi-drug resistance. Most of the AR E. coli harbored AR genes corresponding to their antimicrobial resistance phenotypes. Four of the AR E. coli carried class 1 integrase gene (intI1), a gene associated with multi-drug resistance. Pulsed-field gel electrophoretic analysis showed no genetic relatedness among AR E. coli isolated from different facilities; however, cross-transmissions between horses or horses and environments were detected in two facilities. Although cross-transmission of AR E. coli in horses and their environments was generally low, our study suggests a risk of transmission of AR bacteria between horses and humans. Further studies are needed to evaluate the risk of possible transmission of horse-associated AR bacteria to human communities through horse riders and horse-care workers.     

乳酸菌和大肠杆菌对固定化犬肠道黏蛋白模型的黏附性

采用固定黏蛋白模型,结合细菌同位素γ-32P-ATP标记法,探讨犬源肠球菌E01和E16、犬源大肠杆菌DE、鸡源德氏乳酸杆菌德氏亚种N11及猪源约氏乳杆菌JJB3和致病性大肠杆菌ATCC25922和CVCC2060对本地犬各肠段黏蛋白的黏附能力;肠球菌E16和大肠杆菌DE各自的黏附机制;2株犬源肠球菌以3种作用方式（排斥、竞争、取代）对2株致病性大肠杆菌的黏附抑制作用。结果表明,各测试菌株在犬各肠段黏蛋白模型上的黏附值均不同;2株犬源肠球菌和1株猪源乳酸菌的黏附性能显著优于犬源大肠杆菌DE和2株致病性大肠杆菌;乳酸肠球菌E16表面的碳水化合物结构和糖蛋白参与了黏附过程,大肠杆菌菌体蛋白在黏附过程中发挥一定作用;乳酸肠球菌的黏附受体对高碘酸钠和胰蛋白酶处理不敏感,大肠杆菌DE的黏附受体具有碳水化合物结构,可能为糖蛋白类物质;在肠球菌与病源菌的3种作用方式中,取代和排斥方式对病原菌黏附抑制效果较好。这表明2株犬源肠球菌具开发为益生菌的潜力。     

Attenuation of virulence of Lawsonia intracellularis after in vitro passages and its effects on the experimental reproduction of porcine proliferative enteropathy

Non-pathogenic Lawsonia intracellularis variants have been obtained through multiple passages in cell culture but there is no information regarding the number of passages necessary to attenuate a pathogenic isolate. The present study evaluated the susceptibility of pigs to L. intracellularis after 10, 20 and 40 passages in vitro. Three groups (six animals/group) were inoculated with pure culture of L. intracellularis on passage 10, 20 or 40 and one group with placebo. The animals were monitored for clinical signs, fecal shedding and serological IgG response during 28 days post-inoculation. Gross and histologic lesions and the level of infection based on the amount of L. intracellularis-specific antigen in the intestinal mucosa identified by immunohistochemistry were evaluated in two animals from each group on days 14, 21 and 28. Animals inoculated with passages 10 and 20 demonstrated proliferative lesions typical of porcine proliferative enteropathy associated with the presence of Lawsonia-specific antigen in the intestinal mucosa. Passage 40-inoculated pigs did not show proliferative lesions or presence of Lawsonia antigen at any time point throughout the study. Similar patterns of the fecal shedding were observed in passage 10 and 20-infected pigs but those infected with passage 40 shed for a short period. Serological IgG responses in passage 10 and 20-inoculated pigs were detected from day 14 post-infection but not at all in passage 40-inoculated animals. These results demonstrate attenuation of the virulence properties of L. intracellularis between 20 and 40 cell passages in vitro. This information will be valuable for design of future experimental models and for studying the mechanisms involved in the attenuation of L. intracellularis virulence.     

Detection of genes encoding for virulence and adherence factors in Escherichia coli isolated in slaughtered Sarda breed sheep

In order to investigate the pathogenic profile of Escherichia coli hosted in “Sarda” sheep, autochthonous race present in Sardinia, thirty-seven E. coli strains collected from different sources (fleeces, carcass swabs and gut mucosa) of pre-chill slaughtered sheep (ewes and lambs) were serotyped using pheno- and genotypic methods. Furthermore, the presence of genes encoding for virulence factors and mediating for localized mucosal adherence factors was investigated, and pulsed-field gel electrophoresis (PFGE) characterization was performed.