Project description:Cattle are the asymptomatic reservoirs of Escherichia coli O157:H7 (O157) that preferentially colonizes the bovine recto-anal junction (RAJ). Understanding the influence of O157 on the diversity of the RAJ microbiota could give insights into its persistence at the RAJ in cattle. Hence, we compared changes in bovine RAJ and fecal microbiota following O157 challenge under experimental conditions. Cattle were either orally challenged (n = 4) with1010 CFU of a streptomycin-resistant O157 strain 86-24, or mock-challenged (n = 4) with phosphate buffered saline. Rectoanal mucosal swab (RAMS) and fecal samples were collected at different time points for analysis. Alpha diversity measures (Chao1 species richness and Shannon diversity index) were found to be significantly different between RAMS and fecal samples but not influenced by O157 challenge. The Firmicutes to Bacteroidetes (F: B) ratio was higher in RAMS samples from O157 colonized animals and this may have influenced the consistent yet decreased O157 colonization at the RAJ. Specific bacterial genera that were present in relative low abundance in fecal and RAMS microbiota did not affect overall microbial diversity but were associated with O157 colonization. Differential abundance analysis (DAA) of genera in samples from O157 shedding cattle indicated significantly higher relative abundance of Paenibacillus and Fusobacterium in RAMS, and Tyzzerella in fecal samples. Mock-challenged cattle showed higher relative abundance of Intestinimonas and Citrobacter in RAMS samples, and Succinivibrio, and Prevotella 1 in fecal samples. These results suggest that O157 challenge exerts transient influence on the intestinal microbial community which in turn might promote O157 colonization in a site-specific manner.
Project description:Enterohaemorrhagic Escherichia coli (EHEC), like E. coli O157:H7 are frequently detected in bovine faecal samples at slaughter. Cattle do not show clinical symptoms upon infection, but for humans the consequences after consuming contaminated beef can be severe. The immune response against EHEC in cattle cannot always clear the infection as persistent colonization and shedding in infected animals over a period of months often occurs. In previous infection trials, we observed a primary immune response after infection which was unable to protect cattle from re-infection. These results may reflect a suppression of certain immune pathways, making cattle more prone to persistent colonization after re-infection. To test this, RNA-Seq was used for transcriptome analysis of recto-anal junction tissue and ileal Peyer's patches in nine Holstein-Friesian calves in response to a primary and secondary Escherichia coli O157:H7 infection with the Shiga toxin (Stx) negative NCTC12900 strain. Non-infected calves served as controls.In tissue of the recto-anal junction, only 15 genes were found to be significantly affected by a first infection compared to 1159 genes in the ileal Peyer's patches. Whereas, re-infection significantly changed the expression of 10 and 17 genes in the recto-anal junction tissue and the Peyer's patches, respectively. A significant downregulation of 69 immunostimulatory genes and a significant upregulation of seven immune suppressing genes was observed.Although the recto-anal junction is a major site of colonization, this area does not seem to be modulated upon infection to the same extent as ileal Peyer's patches as the changes in gene expression were remarkably higher in the ileal Peyer's patches than in the recto-anal junction during a primary but not a secondary infection. We can conclude that the main effect on the transcriptome was immunosuppression by E. coli O157:H7 (Stx-) due to an upregulation of immune suppressive effects (7/12 genes) or a downregulation of immunostimulatory effects (69/94 genes) in the ileal Peyer's patches. These data might indicate that a primary infection promotes a re-infection with EHEC by suppressing the immune function.
Project description:Escherichia coli O157:H7 is a foodborne pathogen that colonizes ruminants. Cattle are considered the primary reservoir of E. coli O157:H7 with super-shedders, defined as individuals excreting > 104 E. coli O157:H7 CFU g-1 feces. The mechanisms leading to the super-shedding condition are largely unknown. Here, we used 16S rRNA gene pyrosequencing to examine the composition of the fecal bacterial community in order to investigate changes in the bacterial microbiota at several locations along the digestive tract (from the duodenum to the rectal-anal junction) in 5 steers previously identified as super-shedders and 5 non-shedders. The overall bacterial community structure did not differ by E. coli O157:H7 shedding status; but several differences in the relative abundance of taxa and OTUs were noted between the two groups. The genus Prevotella was most enriched in the non-shedders while the genus Ruminococcus and the Bacteroidetes phylum were notably enriched in the super-shedders. There was greater bacterial diversity and richness in samples collected from the lower- as compared to the upper gastrointestinal tract (GI). The spiral colon was the only GI location that differed in terms of bacterial diversity between super-shedders and non-shedders. These findings reinforced linkages between E. coli O157:H7 colonization in cattle and the nature of the microbial community inhabiting the digestive tract of super-shedders.
Project description:The increased summertime prevalence of cattle carriage of enterohemorrhagic Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) is associated with the increased summertime incidence of human infection. The mechanism driving the seasonality of STEC O157 carriage among cattle is unknown. We conducted experimental challenge trials to distinguish whether factors extrinsic or intrinsic to cattle underlie the seasonality of STEC O157 colonization. Holstein steers (n = 20) exposed to ambient environmental conditions were challenged with a standardized pool of STEC O157 strains four times at 6-month intervals. The densities and durations of rectoanal junction mucosa (RAJ) colonization with STEC O157 were compared by season (winter versus summer), dose (10(9) CFU versus 10(7) CFU), and route of challenge (oral versus rectal). Following summer challenges, the RAJ STEC O157 colonization density was significantly lower (P = 0.016) and the duration was shorter (P = 0.052) than for winter challenges, a seasonal pattern opposite to that observed naturally. Colonization was unaffected by the challenge route, indicating that passage through the gastrointestinal microbiome did not significantly affect the infectious dose to the RAJ. A 2-log reduction of the challenge doses in the second-year trials was accompanied by similarly reduced RAJ colonization in both seasons (P < 0.001). These results refute the hypothesis that cattle are predisposed to STEC O157 colonization during the summer months, either due to intrinsic factors or indirectly due to gastrointestinal tract microbiome effects. Instead, the data support the hypothesis that the increased summertime STEC O157 colonization results from increased seasonal oral exposure to this pathogen.
Project description:Escherichia coli O157:H7 causes hemorrhagic colitis and the life-threatening hemolytic-uremic syndrome in humans and transiently colonizes healthy cattle at the terminal rectal mucosa. To investigate the role of the O antigen in persistence and colonization in the animal host, we generated an E. coli O157:H7 mutant defective in the synthesis of the lipopolysaccharide side chain (O antigen) by deletion of a putative perosamine synthetase gene (per) in the rfb cluster. The lack of O antigen was confirmed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and anti-O157 antibody. The growth rate and cell membrane permeability of the Deltaper mutant were similar to the growth rate and cell membrane permeability of the wild type. Changes in membrane and secreted proteins were observed, but the expression of intimin, EspA, and EspB, implicated in bacterial intestinal colonization, was not altered, as determined by immunoblotting and reverse transcription-PCR. Similar to other O-antigen deletion mutants, the Deltaper mutant was pleiotropic for autoaggregation and motility (it was FliC negative as determined by immunoblotting and flagellum negative as determined by electron microscopy). The abilities of the mutant and the wild type to persist in the murine intestine and to colonize the bovine terminal rectal mucosa were compared. Mice fed the Deltaper mutant shed lower numbers of bacteria (P < 0.05) over a shorter time than mice fed the wild-type or complemented strain. After rectal application in steers, lower numbers of the Deltaper mutant than of the wild type colonized the rectoanal junction mucosa, and the duration of the colonization was shorter (P < 0.05). Our previous work showed that flagella do not influence E. coli O157:H7 colonization at the bovine terminal rectal mucosa, so the current findings suggest that the O antigen contributes to efficient bovine colonization.
Project description:In 2011, a severe outbreak of hemolytic-uremic syndrome was caused by an unusual, highly virulent enterohemorrhagic E. coli (EHEC) O104:H4 strain, which possessed EHEC virulence traits in the genetic background of human-adapted enteroaggregative E. coli. To determine magnitude of fecal shedding and site of colonization of EHEC O104:H4 in a livestock host, 30 (ten/strain) weaned calves were inoculated with 10(10) CFU of EHEC O104:H4, EHEC O157:H7 (positive control) or E. coli strain 123 (negative control) and necropsied (4 or 28 d.p.i.). E. coli O157:H7 was recovered until 28 d.p.i. and O104:H4 until 24 d.p.i. At 4 d.p.i., EHEC O104:H4 was isolated from intestinal content and detected associated with the intestinal mucosa. These results are the first evidence that cattle, the most important EHEC reservoir, can also carry unusual EHEC strains at least transiently, questioning our current understanding of the molecular basis of host adaptation of this important E. coli pathovar.
Project description:Cattle are the primary carrier of Escherichia coli O157:H7, a foodborne human pathogen, and those shedding >104 CFU/gram of feces of E. coli O157:H7 are defined as supershedders (SS). This study investigated the rectoanal junction (RAJ) mucosa-associated microbiota and its relationship with host gene expression in SS and in cattle from which E. coli O157:H7 was not detected (nonshedders [NS]), aiming to elucidate the mechanisms involved in supershedding. In total, 14 phyla, 66 families, and 101 genera of RAJ mucosa-associated bacteria were identified and Firmicutes (61.5 ± 7.5%), Bacteroidetes (27.9 ± 6.4%), and Proteobacteria (5.5 ± 2.1%) were the predominant phyla. Differential abundance analysis of operational taxonomic units (OTUs) identified 2 OTUs unique to SS which were members of Bacteroides and Clostridium and 7 OTUs unique to NS which were members of Coprococcus, Prevotella, Clostridium, and Paludibacter Differential abundance analysis of predicted microbial functions (using PICRUSt [phylogenetic investigation of communities by reconstruction of unobserved states]) revealed that 3 pathways had higher abundance (log2 fold change, 0.10 to 0.23) whereas 12 pathways had lower abundance (log2 fold change, -0.36 to -0.20) in SS. In addition, we identified significant correlations between expression of 19 differentially expressed genes and the relative abundance of predicted microbial functions, including nucleic acid polymerization and carbohydrate and amino acid metabolism. Our findings suggest that differences in RAJ microbiota at both the compositional and functional levels may be associated with E. coli O157:H7 supershedding and that certain microbial groups and microbial functions may influence RAJ physiology of SS by affecting host gene expression.IMPORTANCE Cattle with fecal E. coli O157:H7 at >104 CFU per gram of feces have been defined as the supershedders, and they are responsible for the most of the E. coli O157:H7 spread into farm environment. Currently, no method is available for beef producers to eliminate shedding of E. coli O157:H7 in cattle, and the lack of information about the mechanisms of supershedding greatly impedes the development of effective methods. This study investigated the role of the rectoanal junction (RAJ) mucosa-associated microbiome in E. coli O157:H7 shedding, and our results indicated that the compositions and functions of RAJ microbiota differed between supershedders and nonshedders. The identified relationship between the differentially abundant microbes and 19 previously identified differentially expressed genes suggests the role of host-microbial interactions involved in E. coli O157:H7 supershedding. Our findings provide a fundamental understanding of the supershedding phenomenon which is essential for the development of strategies, such as the use of directly fed microbials, to reduce E. coli O157:H7 shedding in cattle.
Project description:Escherichia coli O157:H7, a major Shiga toxin-producing pathogen, has a low infectious dose and causes serious illness in humans. The gastrointestinal tract of cattle is the primary reservoir of E. coli O157:H7, and thus, it is critical to eliminate or reduce E. coli O157:H7 gut colonization. Given that E. coli O157:H7 produces effectors that attenuate inflammatory signaling, we hypothesized that the host inflammatory response acts to perturb E. coli O157:H7 intestinal colonization. Tumor necrosis factor alpha (TNF-?) treatment of HT-29 cells resulted in increased expression of inflammatory cytokine interleukin 1? (IL-1?), IL-8, and TNF-? genes and increased IL-8 protein and resulted in decreased adhesion of E. coli O157:H7. Similarly, E. coli O157:H7 adhesion to cattle colonic explants was reduced by TNF-? treatment. Irrespective of the presence of E. coli O157:H7, TNF-? enhanced activation of p65, the key mediator of NF-?B inflammatory signaling, whereas E. coli O157:H7 infection suppressed this pathway by inhibiting p65 activation in HT-29 cells. To further explore the mechanisms linking the inflammatory response to attenuated E. coli O157:H7 adhesion, mucin 2 (MUC2) expression was analyzed, considering that the intestinal mucus layer is the first defense against enteric pathogens and MUC2 is the major secretory mucin in the intestine. MUC2 expression in HT-29 cells was increased by TNF-? treatment and by E. coli O157:H7 infection. However, reducing mucin expression by blocking mitogen-activated protein kinase (MAPK) extracellular signal-regulated protein kinases 1/2 (ERK1/2) and/or phosphatidylinositol 3-kinase (PI3K)/Akt signaling increased E. coli O157:H7 adherence to HT-29 cells. These data suggest that the inflammatory cytokine response acts to protect host epithelial cells against E. coli O157:H7 colonization, at least in part, by promoting mucin production.
Project description:Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a foodborne pathogen which causes illness in humans. Ruminants are the main reservoirs and EHEC predominantly colonizes the epithelium of the recto-anal junction of cattle. Immunosuppression by EHEC promotes re-infection of cattle. However, bovine lactoferrin (bLF) apparently can overrule the immunosuppression by inducing EHEC-specific IgA responses at the mucosal site. The IgA responses are significantly correlated with reduced EHEC shedding and the absence of colonization at the rectal mucosa following re-infection. Therefore, to examine the interaction between bLF and bovine rectal epithelial cells, we first developed a method to establish a primary cell culture of epithelial cells of the rectum of cattle. Furthermore, we used LC-MS/MS to demonstrate the presence of secreted lactoferrin in bovine milk and the absence of a "delta" isoform which is known to translocate to the nucleus of cells. Nevertheless, lactoferrin derived from bovine milk was internalized by rectal epithelial cells and translocated to the nuclei. Moreover, nuclear translocation of bLF was significantly enhanced when the epithelial cells were inoculated with EHEC, as demonstrated by confocal fluorescence microscopy and confirmed by Raman microscopy and 3D imaging.
Project description:Escherichia coli O157:H7 is a major foodborne human pathogen causing disease worldwide. Cattle are a major reservoir for this pathogen and those that shed E. coli O157:H7 at >104 CFU/g feces have been termed "super-shedders". A rich microbial community inhabits the mammalian intestinal tract, but it is not known if the structure of this community differs between super-shedder cattle and their non-shedding pen mates. We hypothesized that the super-shedder state is a result of an intestinal dysbiosis of the microbial community and that a "normal" microbiota prevents E. coli O157:H7 from reaching super-shedding levels. To address this question, we applied 454 pyrosequencing of bacterial 16S rRNA genes to characterize fecal bacterial communities from 11 super-shedders and 11 contemporary pen mates negative for E. coli O157:H7. The dataset was analyzed by using five independent clustering methods to minimize potential biases and to increase confidence in the results. Our analyses collectively indicated significant variations in microbiome composition between super-shedding and non-shedding cattle. Super-shedders exhibited higher bacterial richness and diversity than non-shedders. Furthermore, seventy-two operational taxonomic units, mostly belonging to Firmicutes and Bacteroidetes phyla, were identified showing differential abundance between these two groups of cattle. The operational taxonomic unit affiliation provides new insight into bacterial populations that are present in feces arising from super-shedders of E. coli O157:H7.