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.     

Intermittent Escherichia coli O157:H7 colonisation at the terminal rectum mucosa of conventionally-reared lambs

In cattle, the lymphoid rich regions of the rectal-anal mucosa at the terminal rectum are the preferred site for Escherichia coli O157:H7 colonisation. All cattle infected by rectal swab administration demonstrate long-term E. coli O157:H7 colonisation, whereas orally challenged cattle do not demonstrate long-term E. coli O157:H7 colonisation in all animals. Oral, but not rectal challenge of sheep with E. coli O157:H7 has been reported, but an exact site for colonisation in sheep is unknown. To determine if E. coli O157:H7 can effectively colonise the ovine terminal rectum, in vitro organ culture (IVOC) was initiated. Albeit sparsely, large, densely packed E. coli O157:H7 micro-colonies were observed on the mucosa of ovine and control bovine terminal rectum explants. After necropsy of orally inoculated lambs, bacterial enumeration of the proximal and distal gastrointestinal tract did suggest a preference for E. coli O157:H7 colonisation at the ovine terminal rectum, albeit for both lymphoid rich and non-lymphoid sites. As reported for cattle, rectal inoculation studies were then conducted to determine if all lambs would demonstrate persistent colonisation at the terminal rectum. After necropsy of E. coli O157:H7 rectally inoculated lambs, most animals were not colonised at gastrointestinal sites proximal to the rectum, however, large densely packed micro-colonies of E. coli O157:H7 were observed on the ovine terminal rectum mucosa. Nevertheless, at the end point of the study (day 14), only one lamb had E. coli O157:H7 micro-colonies associated with the terminal rectum mucosa. A comparison of E. coli O157:H7 shedding yielded a similar pattern of persistence between rectally and orally inoculated lambs. The inability of E. coli O157:H7 to effectively colonise the terminal rectum mucosa of all rectally inoculated sheep in the long term, suggests that E. coli O157:H7 may colonise this site, but less effectively than reported previously for cattle.     

Detection of Escherichia coli O157 and Escherichia coli O157:H7 by the immunomagnetic separation technique and stx1 and stx2 genes by multiplex PCR in slaughtered cattle in Samsun Province, Turkey

This study was conducted to investigate the presence of Escherichia (E.) coli O157 and E. coli O157:H7 and stx1 and stx2 genes on cattle carcasses and in rectal samples collected from Samsun Province of Turkey. A total of 200 samples collected from cattle carcasses and the rectal contents of 100 slaughtered cattle from two commercial abattoirs were tested using the immunomagnetic separation technique and multiplex PCR methods. E. coli O157 and E. coli O157:H7 were detected in 52 of the 200 samples (26%) tested. Of the positive samples, 49 were E. coli O157 and three were E. coli O157:H7. The E. coli O157 strain was isolated from 24 carcasses and 25 rectal samples, while E. coli O157:H7 was isolated from two carcasses and one rectal sample. Of the 49 samples positive for E. coli O157, 32 were from the rectal and carcass samples of the same animal, while two E. coli O157:H7 isolates were obtained from rectal swabs and carcasses of the same animal. The stx1 and stx2 genes were both detected in 35 E. coli O157 isolates and one E. coli O157:H7 isolate, but the stx2 gene was only detected alone in two E. coli O157 isolates. Overall, 16 carcasses tested positive for E. coli O157 and one carcass tested positive for E. coli O157:H7 based on both carcass and rectal samples. Overall, the results of this study indicate that cattle carcasses pose a potential risk to human health due to contamination by E. coli O157 and E. coli O157:H7 in the feces.     

Occurrence of non-sorbitol fermenting, verocytotoxin-lacking Escherichia coli O157 on cattle farms

Escherichia coli O157 is often associated with hemorrhagic colitis and the hemolytic uremic syndrome (HUS). The verocytotoxins are considered to be the major virulence determinants. However, vt-negative E. coli O157 were recently isolated from patients with HUS. Several transmission routes to humans are described, but cattle feces are the primary source from which both the food supply and the environment become contaminated with E. coli O157.In a prevalence study performed on dairy, beef, mixed dairy/beef and veal farms in the summer of 2007, vt-negative isolates were detected on 11.8% (8/68) of the positive farms. From these eight farms, a total of 43 sorbitol-negative E. coli O157:H7 were collected. On five farms, only strains negative for the vt genes were present whereas both vt-negative and vt-positive strains could be detected on three other farms. Further characterization revealed that all isolates carried the eaeA and hlyA genes. Pulsed-field gel electrophoresis (PFGE) of all isolates resulted in nine different PFGE types and within the vt-negative strains, four different genotypes were identified, indicating that certain genetic clones are widespread over the cattle population.     

All blood, No stool: enterohemorrhagic Escherichia coli O157:H7 infection

Enterohemorrhagic Escherichia coli serotype O157:H7 is a pathotype of diarrheagenic E. coli that produces one or more Shiga toxins, forms a characteristic histopathology described as attaching and effacing lesions, and possesses the large virulence plasmid pO157. The bacterium is recognized worldwide, especially in developed countries, as an emerging food-borne bacterial pathogen, which causes disease in humans and in some animals. Healthy cattle are the principal and natural reservoir of E. coli O157:H7, and most disease outbreaks are, therefore, due to consumption of fecally contaminated bovine foods or dairy products. In this review, we provide a general overview of E. coli O157:H7 infection, especially focusing on the bacterial characteristics rather than on the host responses during infection.     

Shiga toxin-producing Escherichia coli O157:H7 from extensive cattle of the fighting bulls breed

Shiga toxin-producing Escherichia coli (STEC) O157:H7 represents a major public health concern worldwide, with cattle recognized as their main natural reservoir. The aim of this work was to determine the prevalence and the pheno-genotypic characteristics of STEC O157:H7 in a herd with 268 cattle of the fighting bulls breed (De Lidia breed) managed under extensive conditions in the South-West of Spain. Rectal-anal swabs of all animals were collected and examined for STEC O157:H7 by performing an immunomagnetic concentration and separation procedure combined with PCR, and the resulting isolates were characterized by both phenotypic and genotypic methods. Overall, STEC O157:H7 was isolated from seven animals (2.6%) in the herd. The PCR procedure indicated that all seven isolates displayed stx₂, eae-γ1, ehxA, O157 rfbE, and fliCh7 genes. They belonged to phage types 4 (one isolate) and 42 (two isolates), and four isolates reacted with typing phages but did not conform to a recognized pattern. Among the seven isolates there were five indistinguishable PFGE patterns and other two which differed only in ?2 restriction fragments, supporting the existence of horizontal transmission among animals in the herd. The present study demonstrates that cattle managed under extensive conditions in Spain can excrete STEC O157:H7 with their faeces. To our knowledge this is the first isolation of this pathogen from De Lidia cattle.     

动物源E.coli O157∶H7 stx基因的检测与系统进化分析

为了解2009－2010年间在河南、甘肃地区分离鉴定的5株大肠埃希菌O157(E.coli O157)携带stx的情况及不同分离株间stx分子进化与变迁的情况,本研究利用PCR方法对分离株进行了stx基因检测,并完成了序列测定与系统演化分析。结果表明,5株不同动物源的分离株均含有stx1及stx2基因。序列分析结果显示5株分离株间stx1、stx2的核苷酸及氨基酸同源性均较高;stx1基因均与参考株中的山羊源和食品源E.coli O157菌株的同源性较高,进化树中遗传距离最近;分离株的stx2基因与多株牛源及少数人源参考株也具有较高的同源性,进化树中虽然5株分离菌均在一个大主干分支中,但分离株27与其他各分离株及参照株遗传距离最远,独自处于一次级分支中;分离株L37与W、12与50分别分布于牛源、人源E.coli O157小次级分支中;由此可推测,分离株所携带的stx1很有可能是经食品源或羊源E.coli O157传递而来;分离株L37与W、分离株12与50的stx2可能是由牛源、人源E .coli O157菌株传递而来,分离株27的stx2来源不清楚。研究结果表明,5株E.coli O157分离株均含有stx1、stx2基因,但两个基因的起源存在差异。     

Avian colibacillosis caused by an intestinal pathogenic Escherichia coli isolate from calf diarrhea

An intestinal pathogenic Escherichia coli isolate from calf diarrhea, containing the iutA, f17A, afa-8D, and cnf2 genes, was able to cause avian colibacillosis after experimental infection in chickens. Intra-tracheal inoculation and spray of this strain caused 10% of mortality and gross lesions, including airsacculitis, pericarditis, and perihepatitis. These results suggest that some bovine pathogenic E. coli can cause extra-intestinal infections in other animal species.     

Molecular characterization of Escherichia coli O157:H7 strains isolated from different sources and geographic regions

Escherichia (E.) coli serotype O157:H7 is a globally distributed human enteropathogen and is comprised of microorganisms with closely related genotypes. The main reservoir for this group is bovine bowels, and infection mainly occurs after ingestion of contaminated water and food. Virulence genetic markers of 28 O157:H7 strains were investigated and multilocus enzyme electrophoresis (MLEE) was used to evaluate the clonal structure. O157:H7 strains from several countries were isolated from food, human and bovine feces. According to MLEE, O157:H7 strains clustered into two main clonal groups designated A and B. Subcluster A1 included 82% of the O157:H7 strains exhibiting identical MLEE pattern. Most enterohemorrhagic E. coli (EHEC) O157:H7 strains from Brazil and Argentina were in the same MLEE subgroup. Bovine and food strains carried virulence genes associated with EHEC pathogenicity in humans.     

0157:H7型大肠杆菌感染与乳业生产的关系及调控措施

随着乳业生产规模的扩大和乳品消费人群的增加．由乳业生产和乳品消费而引发的人兽共患病引起了人们的高度重视。其中,因摄入被O157：H7型大肠杆菌污染的乳制品而引起的人消化道疾病是较常见且较严重的一种。奶牛是O157：H7型大肠杆菌的主要宿主之一,可在无症状的情况下向环境中排出致病菌,因此,国外乳业发达国家的牛场都特别重视O157：H7型大肠杆菌在牛群中的感染状况。而国内目前在此方面的研究还相对较薄弱,有待进一步深入研究。本文对O157：H7型大肠杆菌的传播特点、调控措施等作了简要综述,为我国开展此方面研究提供参考。     

牛源大肠杆菌O157:H7的分离鉴定

为调查新疆部分地区E.coli O157：H7的感染情况和菌株致病性,从新疆阿克苏、伊犁、塔城3个地区的牛场采集新鲜粪样564份,对E.coli O157：H7进行分离与鉴定。利用E.coli营养肉汤（EC肉汤）对样品进行增菌后,用山梨醇麦康凯培养基（SMAC）平板选择性培养,再经过4-甲基伞形酮-β-D葡萄糖醛酸苷培养基（MUG）的筛选,对疑似菌株进行生化和PCR鉴定,并将分离鉴定到的菌株进行小鼠攻毒试验。结果显示,从伊犁地区采集的样品中共分离出2株E.coli O157：H7（Y166和Y226）,其检出率为0.88%;小鼠攻毒试验中,Y166和Y226试验组小鼠在48 h内全部死亡,具有一定致病性;从阿克苏、塔城所采样品中未分离到E.coli O157：H7。     

Interaction between attaching and effacing Escherichia coli serotypes O157:H7 and O26:K60 in cell culture

Ruminants harbour both O157:H7 and non-O157 Attaching Effacing Escherichia coli (AEEC) strains but to date only non-O157 AEEC have been shown to induce attaching effacing lesions in naturally infected animals. However, O157 may induce lesions in deliberate oral inoculation studies and persistence is considered dependent upon the bacterially encoded locus for enterocyte effacement. In concurrent infections in ruminants it is unclear whether non-O157 AEEC contribute either positively or negatively to the persistence of E. coli O157:H7. To investigate this, and prior to animal studies, E. coli O157:H7 NCTC 12900, a non-toxigenic strain that persists in conventionally reared sheep, and non-toxigenic AEEC O26:K60 isolates of sheep origin were tested for adherence to HEp-2 tissue culture alone and in competition one with another. Applied together, both strains adhered in similar numbers but lower than when either was applied separately. Pre-incubation of tissue culture with either one strain reduced significantly (P < 0.05) the extent of adherence of the strain that was applied second. It was particularly noticeable that AEEC O26 when applied first reduced adherence and inhibited microcolony formation, as demonstrated by confocal microscopy, of E. coli O157:H7. The possibility that prior colonisation of a ruminant by non-O157 AEEC such as O26 may antagonise O157 colonisation and persistence in ruminants is discussed.     

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.     

Staphylococcus aureus and Escherichia coli elicit different innate immune responses from bovine mammary epithelial cells

Escherichia coli and Staphylococcus aureus are the most important pathogenic bacteria causing bovine clinical mastitis and subclinical mastitis, respectively. However, little is known about the molecular mechanisms underlying the different host response patterns caused by these bacteria. The aim of this study was to characterize the different innate immune responses of bovine mammary epithelium cells (MECs) to heat-inactivated E. coli and S. aureus. Gene expression of Toll-like receptor 2 (TLR2) and TLR4 was compared. The activation of nuclear factor kappa B (NF-κB) and the kinetics and levels of cytokine production were analyzed. The results show that the mRNA for TLR2 and TLR4 was up-regulated when the bovine MECs were stimulated with heat-inactivated E. coli, while only TLR2 mRNA was up-regulated when the bovine MECs were stimulated with heat-inactivated S. aureus. The expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and IL-8 increased more rapidly and higher when the bovine MECs were stimulated with heat-inactivated E. coli than when they were stimulated with heat-inactivated S. aureus. E. coli strongly activated NF-κB in the bovine MECs, while S. aureus failed to activate NF-κB. Heat-inactivated S. aureus could induce NF-κB activation when bovine MECs cultured in medium without fetal calf serum. These results were confirmed using TLR2- and TLR4/MD2-transfected HEK293 cells and suggested that differential TLR recognition and the lack of NF-κB activation account for the impaired immune response elicited by heat-inactivated S. aureus.     

Phylogenetic distribution of virulence genes in Escherichia coli isolated from bovine mastitis in Iran

One hundred and twenty seven Escherichia coli isolates from bovine mastitis were examined to detect the phylogenetic group/subgroups and a selection of virulence associated genes. Forty nine (38.58%) isolates belonged to group B1 the remaining isolates fell into four phylogenetic subgroups: A₀ (18.11%), A₁ (26.77%), D₁ (6.29%) and D₂ (10.23%). None of the isolates belonged to B2 group. Forty seven (37.00%) isolates were positive for at least one virulence gene, among them f17A was the most common gene, found in 20.47% of the isolates. Among the E. coli isolates, 11.81% had iucD, 9.44% f17c-A, 9.44% cnf2, 7.87% f17b-A, 6.29% afaD-8 and afaE-8, 3.14% f17d-A, 0.78% cnf1 and 0.78% clpG genes. All of the detected virulence genes were present alone or in combination with each other except clpG and f17d-A genes that were only found alone. None of the isolates contained the genes for F17a-A, intimin, P or S fimbriae.     

Effect of susceptibility to enterotoxigenic Escherichia coli F4 and of dietary tryptophan on gut microbiota diversity observed in healthy young pigs

Healthy weaned pigs susceptible to enterotoxigenic Escherichia coli F4 (ETEC) require more tryptophan (Trp) to maximize their performance. This may be related to an effect on intestinal microbiota. We studied the intestinal bacterial diversity of healthy pigs with different susceptibility to ETEC and fed different Trp levels. Thirty-six littermate weaned pigs were selected to obtain a set potentially formed of 50% ETEC-susceptible and 50% non-susceptible pigs, based on a Mucin 4 gene polymorphism. Pigs were fed a diet with 0.17 (TrpL) or 0.22 (TrpH) standardized ileal digestible Trp:Lys ratio for 21 days. Slaughtered pigs were classified into non-susceptible, mildly susceptible, and susceptible, by testing ETEC adhesion to intestinal villi. Bacterial diversity in jejunum content was assessed by the 16S rRNA gene-targeted denaturing gradient gel electrophoresis (DGGE) fingerprinting analysis and expressed by the Shannon index. Susceptible pigs had a reduced bacterial diversity, particularly with TrpL diet (p = 0.003). The ETEC adhesion class affected the quantification of enterobacteria DNA (p = 0.027). One DGGE band, which referred to Clostridium bartlettii, was not evidenced in all the susceptible pigs; less DNA from this microbe was quantified by RT-PCR in the jejunum from TrpH susceptible pigs (p = 0.025) compared to TrpL. The gene expression for β-galactoside α-2,3-sialyltransferase 1 was higher in jejunal tissue of ETEC-susceptible pigs (p = 0.019). In studies on pig gut microbiota, the presence of intestinal receptors for ETEC should be considered because of their contribution to a reduced bacterial diversity. This effect could be partially reversed by dietary Trp addition.     

Occurrence of weak mutators among avian pathogenic Escherichia coli (APEC) isolates causing salpingitis and peritonitis in broiler breeders

A collection of 46 avian pathogenic Escherichia coli (APEC) isolates was examined for the presence of mutators by determining the rate of mutation to rifampicin resistance. The collection included 34 E. coli isolates obtained in pure culture from chronic lesions of salpingitis and peritonitis in 34 broiler breeders, of which 12 were associated with the development of secondary septicemia. Twelve additional isolates were obtained from a clonal outbreak (ST95) of E. coli peritonitis syndrome (EPS), the lesions of which changed gradually over time into a subacute/chronic form. The hypothesis of the present study was that mutation rates would be higher for chronic infection isolates than for isolates from acute infections/exacerbations. The distribution of mutation rates followed a pattern similar to that found for other clinical isolates of E. coli, with a modal/median value of 1.47 × 10⁻⁸. Of the 46 isolates, 24% (n = 11) were weakly hypermutable (2.00 × 10⁻⁸ ≤ μ < 2.00 × 10⁻⁷), however, no strong mutators were detected (μ ≥ 2.00 × 10⁻⁷). Chronic salpingitis isolates had the highest proportion (45%, P = 0.001) of weak mutators and also, significantly higher mutation rates (P = 0.003) compared to isolates that caused septicemia (4%). In addition, mutation rates were significantly lower among ST95 isolates (P < 0.0005), and among isolates from the same clonal group as ST95 (P = 0.027), when compared to isolates from other groups. Although a clear association with the time phase of infection (as lesions of EPS became more chronic) could not be observed (ρ = 0.523, P = 0.081), a higher frequency of weak mutators among chronic infection isolates suggests that increased mutation rates play a role in adaptation of APEC to long-term persistence in an infected host environment.     

Detection and characterisation of Shiga toxin-producing Escherichia coli other than Escherichia coli O157:H7 in wild ruminants

Shiga toxin-producing Escherichia coli (STEC) are an important group of emerging pathogens, with ruminants recognised as their main natural reservoir. The aim of this work was to establish the prevalence of non-O157 STEC in free-ranging wild ruminants in the Extremadura region of Spain and to characterise them phenogenotypically. Faecal samples were collected from 243 wild ruminants, including Cervus elaphus, Capreolus capreolus, Dama dama and Ovis musimon and were examined for STEC using both phenotypic (Vero cells) and genotypic (PCR and PFGE) methods.Shiga toxin-producing Escherichia coli were isolated from 58 (23.9%) of the samples and a total of 65 isolates were characterised. A PCR method indicated that 11 (16.9%) strains carried the stx₁ gene, 44 (67.7%) carried the stx₂ gene and 10 (15.4%) carried both these genes. The ehxA gene was detected in 37 (57%) of the isolates but none contained either the eae or saa genes. The isolates were from a total of 12 ‘O’ serogroups, although 80% were restricted to the O2, O8, O128, O146, O166 and O174 serogroups. The most commonly isolated STEC bacteria, which were from the O146 serogroup, exhibited a high degree of polymorphism as indicated by PFGE. Shiga toxin-producing Escherichia coli isolates of serogroups O20, O25, O166, O171, O174 and O176 had not previously been found in wild ruminants. This is the first study to confirm that wild ruminants in Spain are a reservoir of STEC and are thus a potential source of human infection.     

大肠杆菌O157:H7 rfbE基因和fliC基因的克隆与序列分析

根据已发表的E．coli O157：H7 EDL933株的rfbE基因和fliC基因，设计了两对引物，分别对两个O157：H7，菌株的rfbE基因和一个菌株的fliC基因进行PCR，并将PCR产物克隆于pMD18-T栽体质粒。测序结果表明，获得到了与理论相符的582bp rfbE基因片段和802bp fliC基因片段。一株菌的rfbE基因存在无意义突变（两个）。而另一株菌fliC基因有一个有意义突变（aac→gac）