Variability in the Occupancy of Escherichia coli O157 Integration Sites by Shiga Toxin-Encoding Prophages.

Escherichia coli O157:H7 strains often produce Shiga toxins encoded by genes on lambdoid bacteriophages that insert into multiple loci as prophages. O157 strains were classified into distinct clades that vary in virulence. Herein, we used PCR assays to examine Shiga toxin (Stx) prophage occupancy in yehV, argW, wrbA, and sbcB among 346 O157 strains representing nine clades. Overall, yehV was occupied in most strains (n = 334, 96.5%), followed by wrbA (n = 213, 61.6%), argW (n = 103, 29.8%), and sbcB (n = 93, 26.9%). Twelve occupancy profiles were identified that varied in frequency and differed across clades. Strains belonging to clade 8 were more likely to have occupied sbcB and argW sites compared to other clades (p < 0.0001), while clade 2 strains were more likely to have occupied wrbA sites (p < 0.0001). Clade 8 strains also had more than the expected number of occupied sites based on the presence of stx variants (p < 0.0001). Deletion of a 20 kb non-Stx prophage occupying yehV in a clade 8 strain resulted in an ~18-fold decrease in stx2 expression. These data highlight the complexity of Stx prophage integration and demonstrate that clade 8 strains, which were previously linked to hemolytic uremic syndrome, have unique Stx prophage occupancy profiles that can impact stx2 expression.

Differences in adherence and virulence gene expression between two outbreak strains of enterohaemorrhagic Escherichia coli O157 : H7.

The Escherichia coli O157 : H7 TW14359 strain was implicated in a multi-state outbreak in North America in 2006, which resulted in high rates of severe disease. Similarly, the O157 : H7 RIMD0509952 (Sakai) strain caused the largest O157 : H7 outbreak to date. Both strains were shown to represent divergent phylogenetic lineages. Here we compared global gene expression patterns before and after epithelial cell exposure, as well as the ability to adhere to and invade epithelial cells, between the two outbreak strains. Epithelial cell assays demonstrated a 2.5-fold greater adherence of the TW14359 strain relative to Sakai, while whole-genome microarrays detected significant differential expression of 914 genes, 206 of which had a fold change >/=1.5. Interestingly, most locus of enterocyte effacement (LEE) genes were upregulated in TW14359, whereas flagellar and chemotaxis genes were primarily upregulated in Sakai, suggesting discordant expression of these genes between the two strains. The Shiga toxin 2 genes were also upregulated in the TW14359 strain, as were several pO157-encoded genes that promote adherence, including type II secretion genes and their effectors stcE and adfO. Quantitative RT-PCR confirmed the expression differences detected in the microarray analysis, and expression levels were lower for a subset of LEE genes before versus after exposure to epithelial cells. In all, this study demonstrated the upregulation of major and ancillary virulence genes in TW14359 and of flagellar and chemotaxis genes in Sakai, under conditions that precede intimate bacterial attachment to epithelial cells. Differences in the level of adherence to epithelial cells were also observed, implying that these two phylogenetically divergent O157 : H7 outbreak strains vary in their ability to colonize, or initiate the disease process.

Effect of feeding a direct-fed microbial on total and antimicrobial-resistant fecal coliform counts in preweaned dairy calves.

OBJECTIVE: To determine whether feeding a direct-fed microbial (DFM) to dairy calves would reduce total and antimicrobial-resistant coliform counts in feces and affect average daily gain (ADG). ANIMALS: 21 preweaned Holstein heifer calves. PROCEDURES: The study had a randomized complete block design. Within each block, 3 consecutively born calves were randomly assigned to 1 of 3 treatment groups within 24 hours after birth (day 0). Calves were fed the DFM at 1.0 g (DFM1; n = 7) or 0.5 g (DFM2; 7) twice daily or no DFM (control; 7) from days 0 through 29. A fecal sample was collected from each calf daily on days 0 through 3 and then every other day through day 29. Fecal samples were cultured, and mean numbers of total coliforms and coliforms resistant to ampicillin, ceftiofur, and tetracycline were compared among the 3 treatment groups. Calves were weighed on days 0 and 29 to calculate ADG. RESULTS: Mean total fecal coliform counts did not differ significantly among the 3 treatment groups. Mean ceftiofur-resistant and tetracycline-resistant coliform counts for the control group were significantly lower, compared with those for the DFM1 and DFM2 groups. Mean ADG did not differ significantly between the DFM1 and DFM2 groups; however, the mean ADG for all calves fed the DFM was 0.15 kg less than that for control calves. CONCLUSION AND CLINICAL RELEVANCE: Results suggested that the DFM fed to the preweaned calves of this study did not reduce total or antimicrobial-resistant coliform counts in feces.

Increased adherence and expression of virulence genes in a lineage of Escherichia coli O157:H7 commonly associated with human infections.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) O157:H7, a food and waterborne pathogen, can be classified into nine phylogenetically distinct lineages, as determined by single nucleotide polymorphism genotyping. One lineage (clade 8) was found to be associated with hemolytic uremic syndrome (HUS), which can lead to kidney failure and death in some cases, particularly young children. Another lineage (clade 2) differs considerably in gene content and is phylogenetically distinct from clade 8, but caused significantly fewer cases of HUS in a prior study. Little is known, however, about how these two lineages vary with regard to phenotypic traits important for disease pathogenesis and in the expression of shared virulence genes. METHODOLOGY/PRINCIPAL FINDINGS: Here, we quantified the level of adherence to and invasion of MAC-T bovine epithelial cells, and examined the transcriptomes of 24 EHEC O157:H7 strains with varying Shiga toxin profiles from two common lineages. Adherence to epithelial cells was >2-fold higher for EHEC O157:H7 strains belonging to clade 8 versus clade 2, while no difference in invasiveness was observed between the two lineages. Whole-genome 70-mer oligo microarrays, which probe for 6088 genes from O157:H7 Sakai, O157:H7 EDL 933, pO157, and K12 MG1655, detected significant differential expression between clades in 604 genes following co-incubation with epithelial cells for 30 min; 186 of the 604 genes had a >1.5 fold change difference. Relative to clade 2, clade 8 strains showed upregulation of major virulence genes, including 29 of the 41 locus of enterocyte effacement (LEE) pathogenicity island genes, which are critical for adherence, as well as Shiga toxin genes and pO157 plasmid-encoded virulence genes. Differences in expression of 16 genes that encode colonization factors, toxins, and regulators were confirmed by qRT-PCR, which revealed a greater magnitude of change than microarrays. CONCLUSIONS/SIGNIFICANCE: These findings demonstrate that the EHEC O157:H7 lineage associated with HUS expresses higher levels of virulence genes and has an enhanced ability to attach to epithelial cells relative to another common lineage.