Attaching And Effacing Escherichia Coli Strains Research Articles

Effect of dietary laminarin and fucoidan on selected microbiota, intestinal morphology and immune status of the newly weaned pig.

A 2 × 2 factorial experiment was conducted to investigate the interactions between laminarin (LAM; 0 and 300 parts per million (ppm)) and fucoidan (FUC; 0 and 240 ppm) levels on intestinal morphology, selected microbiota and inflammatory cytokine gene expression in the weaned pig. There was an interaction between LAM and FUC supplementation on the Enterobacteriaceae population (P< 0·05) and the abundance of attaching and effacing Escherichia coli (AEEC) strains (P< 0·05) in the colon. Pigs offered the FUC diet had a reduced Enterobacteriaceae population compared with pigs offered the basal diet. However, the effect of FUC on the Enterobacteriaceae population was not observed when combined with LAM. Pigs offered the LAM diet had reduced abundance of AEEC strains compared with pigs offered the basal diet. However, there was no effect of LAM on the abundance of AEEC strains when combined with FUC. There was an interaction between LAM and FUC supplementation on villous height (P< 0·01) and the villous height:crypt depth ratio (P< 0·01) in the duodenum. Pigs offered the LAM or FUC diet had an increased villous height and villous height:crypt depth ratio compared with pigs offered the basal diet. However, there was no effect of the LAM and FUC combination diet on intestinal morphology. Pigs offered the LAM-supplemented diets had a lower IL-6 (P< 0·05), IL-17A (P< 0·01) and IL-1β (P< 0·01) mRNA expression in the colon compared with pigs offered the diets without LAM. In conclusion, supplementation with either LAM or FUC alone modified intestinal morphology and selected intestinal microbiota, but these effects were lost when offered in combination.

Attaching and effacing Escherichia coli and Shiga toxin-producing E. coli in children with acute diarrhoea and controls in Teresina/PI, Brazil

This 3.5-year prospective study was conducted to ascertain the level of attaching and effacing Escherichia coli (AEEC) associated diarrhoea in children from Teresina, a northeastern state of Brazil. Passed faecal specimens from 400 patients (250 with and 150 without diarrhoea) up to 60 months of age attending from 2004 to 2007 at two public hospitals were investigated. Conventional microbiology methods and PCR were employed. Escherichia coli was isolated from 390 children, 240 of them with diarrhoea. A total of 117 AEEC strains were cultivated from specimens from 63 children, 37 with and 26 without diarrhoea. No association between AEEC and diarrhoea was observed. Atypical enteropathogenic E. coli (a-EPEC) (79.4%) was more commonly found than typical EPEC (t-EPEC). Association between EPEC and EPEC subtypes and diarrhoea was not detected. Mixed infection by t-EPEC and a-EPEC and infection by Shiga toxin-producing E. coli (STEC) were rare. Enteropathogenic E. coli was more common in males and in children aged less than 12 months. Correlation between serotyping and PCR results was 0.19. High resistance rates of AEEC to ampicillin, cephalotin, and trimethoprim-sulfamethoxazole were found. In conclusion, EPEC is very common in children with diarrhoea and controls in the population we studied, with a-EPEC predominating. This diarrhoeagenic E. coli (DEC) pathotype is more common in infant males and is resistant to drugs frequently used in clinical practice.

Comparison of ruminant and human attaching and effacing Escherichia coli (AEEC) strains

The presence of 12 genes associated with virulence in human attaching and effacing Escherichia coli (AEEC) was studied within a collection of 20 enterohemorrhagic E. coli (EHEC) and 206 atypical enteropathogenic E. coli (EPEC) isolated from ruminants. In addition, virulence genes and the clonal relationship of 49 atypical EPEC O26 strains isolated from humans and ruminants were compared to clarify whether ruminants serve as a reservoir of atypical EPEC for humans. A great diversity in the content of virulence gene was found. Thus, the espH, espG and map genes were detected in more than 85% of ruminant AEEC strains; the tccP2, espI, efa1/lifA, ehxA and paa genes were present in 50–70% of strains; and other genes such as tccP, espP, katP and toxB were detected in <25% of strains. EHEC strains contained more virulence genes than atypical EPEC strains. Our results suggest for the first time that the efa1/lifA gene is associated with diarrhea in newborn ruminants and that the AEEC strains with the H11 flagellar antigen are potentially more virulent than the non-H11 AEEC strains. Importantly, we identified a new intimin variant gene, eaeρ, in three ruminant atypical EPEC strains. The comparison of ruminant and human EPEC O26 strains showed that some ruminant strains possess virulence gene profiles and pulse-field gel electrophoresis pulsotypes similar to those of human strains. In conclusion, our data suggest that atypical EPEC is a heterogeneous group with different pathogenic potential and that ruminants could serve as a reservoir of atypical EPEC for humans.

Phenotypic and Genotypic Characterization of Antimicrobial Resistance in Enterohemorrhagic Escherichia Coli and Atypical Enteropathogenic E. Coli Strains from Ruminants

Two hundred and twenty-six attaching and effacing Escherichia coli (AEEC) strains (20 enterohemorrhagic E. coli and 206 atypical enteropathogenic E. coli) isolated from calves, lambs, and goat kids with diarrhea and from healthy cattle, sheep, and goats were tested for their resistance to 10 antimicrobial agents by the disc diffusion method. Resistant and intermediate strains were analyzed by polymerase chain reaction for the presence of the major resistance genes. The overall percentage of resistant strains to tetracycline, streptomycin, erythromycin, and sulfamethoxazole was very high (>65%). Moreover, a high level of resistance (approximately 30%) to ampicillin, chloramphenicol, trimethoprim, and trimethoprim-sulfamethoxazole was also detected. The AEEC strains were very susceptible (>90%) to gentamicin and colistin. Because AEEC from ruminants can cause diseases in human beings, the high frequency of antimicrobial resistance detected in the current study is a source of concern. For each antimicrobial agent, the predominant resistance genes in the resistant strains were ampicillin, bla(TEM) (97.1%); tetracycline, tetA (76.7%); gentamicin, aac(3)II (80%); streptomycin, strA/strB (76.7%) and aadA (71.7%); chloramphenicol, catI (85.1%); trimethoprim, dhfrI (76.3%); and sulfamethoxazole, sul1 (60%) and sul2 (63.3%). In the majority of cases, resistance to a given antimicrobial, except for streptomycin, was caused by a single gene. A negative association between tetA and tetB, between aac(3)II and aac(3)IV, and between dhfrI and dhfrV was observed. The present study gives baseline data on frequency and molecular basis of antimicrobial resistance in AEEC strains from ruminants.

Characterization of attaching and effacing Escherichia coli (AEEC) isolated from pigs and sheep

BackgroundAttaching and effacing Escherichia coli (AEEC) are characterized by their ability to cause attaching-and-effacing (A/E) lesions in the gut mucosa of human and animal hosts leading to diarrhoea. The genetic determinants for the production of A/E lesions are located on the locus of enterocyte effacement (LEE), a pathogenicity island that also contains the genes encoding intimin (eae). This study reports data on the occurrence of eae positive E. coli carried by healthy pigs and sheep at the point of slaughter, and on serotypes, intimin variants, and further virulence factors of isolated AEEC strains.ResultsFaecal samples from 198 finished pigs and 279 sheep were examined at slaughter. The proportion of eae positive samples was 89% for pigs and 55% for sheep. By colony dot-blot hybridization, AEEC were isolated from 50 and 53 randomly selected porcine and ovine samples and further characterized. Strains of the serotypes O2:H40, O3:H8 and O26:H11 were found in both pigs and sheep. In pigs O2:H40, O2:H49, O108:H9, O145:H28 and in sheep O2:H40, O26:H11, O70:H40, O146:H21 were the most prevalent serotypes among typable strains. Eleven different intimin types were detected, whereas γ2/θ was the most frequent, followed by β1, ε and γ1. All but two ovine strains tested negative for the genes encoding Shiga toxins. All strains tested negative for the bfpA gene and the EAF plasmid. EAST1 (astA) was present in 18 of the isolated strains.ConclusionOur data show that pigs and sheep are a source of serologically and genetically diverse intimin-harbouring E. coli strains. Most of the strains show characteristics of atypical enteropathogenic E. coli. Nevertheless, there are stx-negative AEEC strains belonging to serotypes and intimin types that are associated with classical enterohaemorrhagic E. coli strains (O26:H11, β1; O145:H28, γ1).

Pathotypic Characterization of Enterocyte Effacement-related LEE Genes in EHEC and EPEC Isolated from Diarrheal Patients

Attaching and effacing Escherichia coli (AEEC) cause enteric infections in humans and animals. Attaching indicates the intimate attachment of bacteria to the enterocyte, and effacing relates to the localized effacement of brush border microvilli. Enteropathogenic (EPEC) and enterohemorrhagic Escherichia coli (EHEC) infections are characterized by the formation of attaching and effacing (AE) lesion on the intestinal epithelial cells. Therefore, they are often grouped together as AEEC. Development of multiplex PCR allowed us to type five of the most important genes implicated in the formation of the AE lesion. A total of 60 AEEC strains isolated from diarrheal patients were investigated by multiplex PCR for the presence of the insertion site of locus of enterocyte effacement (LEE) and LEE-related (eae, tir, espA, espB, and espD) genes. Associating the results of LEE genes typing in the AEEC strains, three different pathotypes are determined: eae γ -tir γ -espA γ -espB γ -espD γ (O157:H7), eae β -tir β -espA β -espB β -espD β (O26:H11), and eae α -tir α -espA α -espB α -espD α (O55:H6). These results indicate that AEEC are a heterogenous groups of organisms.

Use of virulence factor-specific egg yolk-derived immunoglobulins as a promising alternative to antibiotics for prevention of attaching and effacingEscherichia coliinfections

Using a porcine ileal in vitro organ culture model, we have demonstrated that egg yolk-derived antibodies specific for the attaching and effacing Escherichia coli (AEEC) virulence factors intimin and translocated intimin receptor (Tir), but not those specific for the AEEC-secreted proteins EspA, EspB and EspD, significantly reduced the bacterial adherence of the porcine enteropathogenic E. coli strain ECL1001, formerly 86-1390. Moreover, antibodies specific for intimin and Tir also significantly reduced bacterial adherence of heterologous AEEC strains, including human, bovine and canine enteropathogenic E. coli strains, as well as of O157:H7 Shiga toxin-producing E. coli strains in this model. In addition, we demonstrated that the oral administration of these anti-intimin antibodies significantly reduced the extent of attaching and effacing lesions found in the small intestine of weaned pigs challenged with the porcine enteropathogenic E. coli strain ECL1001. Overall, our results underline the potential use of specific egg yolk-derived antibodies as a novel approach for the prevention of AEEC infections.

Investigation of domestic animals and pets as a reservoir for intimin- ( eae) gene positive Escherichia coli types

Domestic animals belonging to seven different species (cattle, sheep, dogs, cats, pigs, chicken and goats) were investigated as natural reservoirs for attaching and effacing Escherichia coli (AEEC). For this, 2165 E. coli strains from faeces of 803 animals were examined for the presence of the intimin -( eae) gene as a characteristic of AEEC strains. Ten percent of the animals were found to excrete AEEC, most frequently found in sheep (19.2%) and pigs (17.6), followed by cattle (10.4%), dogs (7.2%), cats (6.5%) and poultry (2.3%). The 97 AEEC strains from animals were grouped into 44 serotypes. Only four E. coli serotypes (O2:H8, O26:[H11], O109:[H25] and O145:[H28] were found in more than one animal host species. AEEC O26:[H11] strains were most frequently isolated (13.4%) being present in cattle, poultry, pigs and sheep. A search for virulence markers associated with enterohemorrhagic E. coli (EHEC) revealed Shiga-toxin genes in three (3.1%) AEEC strains from sheep. Bundle forming pili genes as a trait of typical enteropathogenic E. coli (EPEC) were detected in four (4.1%) strains from dogs and cats. The remaining 90 AEEC strains were classified as atypical EPEC. Typing of intimin genes revealed intimin β being present in 51.5% of the strains, followed by intimins θ (23.7%), ɛ (6.2%), κ (5.2%), ζ (5.2%), α, η and ι (each 1.0%). Our data indicate that domestic animals and pets constitute an important natural reservoir of AEEC strains, and some of these (O26:[H11], O103:H2, O128:H2, O145:[H28] and O177:[H11]) are known to occur as pathogens in humans.

Typing of the eae and espB genes of attaching and effacing Escherichia coli isolates from ruminants

The types of the eae and espB genes of 178 attaching and effacing Escherichia coli (AEEC) strains isolated from diarrhoeic and healthy ruminants were investigated by PCR. Six types of the eae gene: β (beta), γ1 (gamma-1), γ2 (gamma-2), ε (epsilon), ζ (zeta) and ι (iota), and three types of the espB gene: α, β and γ were identified in the strains studied. Moreover, three strains were negative to all the types of the eae gene tested. The types β and γ2 in healthy cattle, β, γ2 and ε in healthy sheep and goats, and β in diarrhoeic calves, lambs and goat kids were the most frequent types of the eae gene among the strains studied. Although the eaeβ gene was the most prevalent among AEEC from healthy and diarrhoeic ruminants, the percentages of AEEC strains with this type found in this study in diarrhoeic animals (66.7–100%) were higher than those found in healthy animals (33.3–40.6%). Thus, these data suggest that AEEC strains with the eaeβ gene are associated with neonatal diarrhoea in ruminants. The eaeγ1, eaeζ and eaeι genes were found in low percentages in the strains studied (4.5, 2.8 and 7.3%, respectively). All the types of the eae gene, except the type ι, showed a close correlation with the types of the espB gene: the eaeβ and eaeε genes with the espBβ gene, the eaeγ2 and eaeζ genes with the espBα gene and the eaeγ1 gene with the espBγ gene.

HEp-2 cell adherence, actin aggregation, and intimin types of attaching and effacing Escherichia coli strains isolated from healthy infants in Germany and Australia.

Fecal samples from healthy children under 2 years of age living in Berlin, Germany (205 infants), and Melbourne, Australia (184 infants), were investigated for the presence of attaching and effacing (AE) Escherichia coli (AEEC) strains by screening for eae (intimin) genes. Twenty-seven AEEC strains were isolated from 14 children (7.6%) from Melbourne and from 12 children (5.9%) from Berlin. The 27 AEEC strains were classified as enterohemorrhagic E. coli (one strain, producing Shiga toxin 1), typical enteropathogenic E. coli (EPEC) (one strain carrying an EPEC adherence factor [EAF] plasmid), and atypical EPEC (25 strains negative for Shiga toxins and EAF plasmids). The AEEC were divided into 18 different serotypes, O-nontypeable and O-rough strains. Typing of their intimin genes revealed the presence of intimin alpha in 6 strains, intimin beta in 11 strains, intimin gamma in 7 strains, intimin zeta in 2 strains, and intimin eta in one strain. Analysis of HEp-2 cell adherence showed diffuse adherence or localized adherence-like patterns in 26 AEEC strains; local adherence was found only with the EAF-positive strain. Ten AEEC strains showed an AE property with the fluorescent actin staining (FAS) test. The introduction of an EAF plasmid (pMAR7) converted 11 FAS-negative AEEC strains to FAS positive and increased the FAS reaction in six FAS-positive AEEC strains, indicating that the genes needed for the AE phenotype were functional in these strains. Our finding indicates that atypical EPEC strains could play a double role as strains that naturally immunize against intimin in humans and as reservoirs for new emerging human pathogenic EPEC strains.

Association between intimin (eae) and EspB gene subtypes in attaching and effacing Escherichia coli strains isolated from diarrhoeic lambs and goat kids.

Attaching and effacing Escherichia coli (AEEC) strains isolated from diarrhoeic lambs and goat kids were characterized for intimin (eae) and EspB (espB) gene subtypes by PCR and sequencing, and for genetic relatedness by PFGE. Fifty (23 ovine and 27 caprine) AEEC strains of 398 (246 ovine and 152 caprine) analysed were detected by colony blot hybridization. These strains were epidemiologically unrelated since they were isolated from different outbreaks of neonatal diarrhoea over a long period. Ovine AEEC strains belonged to serogroups O2, O4, O26, O80, O91 or were untypable, and caprine strains belonged to serogroups O3, O153 and O163. Two intimin subtypes were detected among the ovine and caprine strains studied. Most of the strains (43/50) had the beta type intimin gene, but seven ovine strains possessed a variant gamma type intimin gene (gamma(V)). Analysis of deduced amino acid sequences of the eae gene revealed that the sequences of beta intimin of ovine and caprine strains were virtually identical to those of beta intimin of rabbit EPEC, human EPEC clone 2 and swine AEEC, whereas the gamma(V) intimin present in seven ovine strains had 75-76% identity with gamma intimin of human EHEC clone 1 strains, and 96% of identity with intimin of the human EHEC strain 95NR1 of serotype O111:H-. A PCR test was developed to identify the three different espB gene subtypes, espB of human EPEC clone 1 (espBalpha), espB of human EHEC clone 1 (espBgamma) and espB of rabbit EPEC and human EPEC clone 2 (espBbeta). There was close correlation between the intimin beta type and the espBbeta gene subtype in the ovine and caprine AEEC strains. The seven ovine strains possessing the gamma(V) intimin gene possessed the espBalpha gene subtype. None of the strains studied possessed the espBgamma gene found in human O157:H7 EHEC strains. PFGE analysis of genomic DNA of selected strains showed a great diversity among strains. Cluster analysis of PFGE patterns showed greater divergence between strains with the gamma(V) intimin gene than between strains with the beta intimin gene. This study showed that most of the AEEC strains isolated from diarrhoeic lambs and goat kids possessed beta intimin and espB genes identical to those of rabbit EPEC, and they may be associated with enteric disease in small ruminants.

Presence of the LEE (locus of enterocyte effacement) in pig attaching and effacing Escherichia coli and characterization of eae, espA, espB and espD genes of PEPEC (pig EPEC) strain 1390

In the present study, attaching and effacing Escherichia coli (AEEC) O45 isolates from post-weaning pigs with diarrhoea were examined for the presence of the LEE (locus of enterocyte effacement) using various DNA probes derived from the LEE of human enteropathogenic E. coli (EPEC) strain E2348/69. The LEE fragment was conserved among the eae -positive pig isolates. The attaching and effacing activity of PEPEC (pig EPEC) O45 isolates is highly correlated with the presence of the LEE. Nevertheless, for some PEPEC isolates, the insertion site of the LEE is different or has diverged during evolution. The presence of the LEE fragment in PEPEC isolates provides further evidence that the LEE region is conserved among AEEC of different animal origins. In addition, the nucleotide sequence of the region containing the eae gene and esp genes of a pig AEEC isolate, strain 1390, was determined. Among examined Eae proteins, Eae of strain 1390 showed the highest similarity with Eae belonging to the β intimin group such as the Eae of rabbit AEEC. Moreover, all pig strains that produced attaching and effacing lesions in piglets and pig ileal explants belonged to the β intimin group. The deduced amino acid sequences of the EspA, EspB and EspD proteins of strain 1390 showed particularly strong homology to those of AEEC strains presenting a β intimin allele. Thus, pig AEEC possess the LEE sequences, and for the strain 1390, sequences of theeae and esp regions are related to those of other AEEC, in particular, strains presenting a β intimin allele, such as the rabbit AEEC.

Comparison of eae, tir, espA and espB genes of bovine and human attaching and effacing Escherichia coli by multiplex polymerase chain reaction.

Attaching and effacing Escherichia coli (AEEC) virulence genes include the eae, the tir, the espA and the espB genes. These genes have been sequenced from several AEEC strains. The sequences alignments revealed the presence of constant and variable regions. Multiplex polymerase chain reactions were developed, in order to determine the subtype of each gene present in a particular isolate. AEEC strains isolated from calves dead of diarrhea, from healthy calves and from infected humans were compared. The same pathotypes were found in sick and healthy calves but in inverted proportion. These pathotypes were also found in human AEEC. Although, the human EHEC strains from serotype O157 possessed their own pathotype.

Comparison of eae, tir, espA and espB genes of bovine and human attaching and effacing Escherichia coli by multiplex polymerase chain reaction

Attaching and effacing Escherichia coli (AEEC) virulence genes include the eae, the tir, the espA and the espB genes. These genes have been sequenced from several AEEC strains. The sequences alignments revealed the presence of constant and variable regions. Multiplex polymerase chain reactions were developed, in order to determine the subtype of each gene present in a particular isolate. AEEC strains isolated from calves dead of diarrhea, from healthy calves and from infected humans were compared. The same pathotypes were found in sick and healthy calves but in inverted proportion. These pathotypes were also found in human AEEC. Although, the human EHEC strains from serotype O157 possessed their own pathotype.