Number of fecal samples from feedlot cattle positive for <i>E</i>. <i>coli</i> O104 and O8/O9/O9a based on PCR assays of <i>wzx</i>_O104 and <i>wbdD</i>_O8/O9/O9a.

<p>Number of fecal samples from feedlot cattle positive for <i>E</i>. <i>coli</i> O104 and O8/O9/O9a based on PCR assays of <i>wzx</i>_O104 and <i>wbdD</i>_O8/O9/O9a.</p

Virulence gene profiles of strains of <i>Escherichia coli</i> O104 isolated from feedlot cattle feces.

<p>Virulence gene profiles of strains of <i>Escherichia coli</i> O104 isolated from feedlot cattle feces.</p

Model-adjusted prevalence estimates of fecal samples from feedlot cattle positive for <i>wzx</i>_O104/<i>wbdD</i>_O8/O9/O9a and <i>wzx</i>_O104 at the feedlot-, lot- and sample- levels.

<p>Model-adjusted prevalence estimates of fecal samples from feedlot cattle positive for <i>wzx</i>_O104/<i>wbdD</i>_O8/O9/O9a and <i>wzx</i>_O104 at the feedlot-, lot- and sample- levels.</p

<i>In silico</i> restriction fragment length polymorphism (RFLP) subtyping of Shiga toxin genes of O104 isolates.

<p>(A) RFLP pattern of Shiga toxin of an O104 isolate; (B) RFLP pattern of <i>stx</i>1c of a reference sequence (Accession no. DQ449666.1); (C) RFLP pattern of <i>stx</i>1a of a reference sequence (Accession no. M16625.1); (D) RFLP pattern of <i>stx</i>1d of a reference sequence (Accession no. AY170851.1)</p

Pulsed-field gel electrophoresis-based clustering of <i>Escherichia coli</i> O104 strains from cattle feces and human clinical strains (O104:H4; O104:H21; and O104:H7).

<p>Pulsed-field gel electrophoresis-based clustering of <i>Escherichia coli</i> O104 strains from cattle feces and human clinical strains (O104:H4; O104:H21; and O104:H7).</p

Number of fecal samples from feedlot cattle positive for <i>wzx</i>_O104 possessing <i>E</i>. <i>coli</i> (serogroups O104 and/or O8/O9/O9a) based on the culture method and <i>wzx</i>_O104-positive isolates that tested positive for <i>wbdD</i>_O8/O9/O9a (serogroups O8/O9/O9a) or negative for <i>wbdD</i>_O8/O9/O9a (serogroup O104).

<p>Number of fecal samples from feedlot cattle positive for <i>wzx</i>_O104 possessing <i>E</i>. <i>coli</i> (serogroups O104 and/or O8/O9/O9a) based on the culture method and <i>wzx</i>_O104-positive isolates that tested positive for <i>wbdD</i>_O8/O9/O9a (serogroups O8/O9/O9a) or negative for <i>wbdD</i>_O8/O9/O9a (serogroup O104).</p

Target genes, primer sequences used and size of amplicons in PCR assays.

<p>Target genes, primer sequences used and size of amplicons in PCR assays.</p

Detection of the six major non-O157 serogroups of <i>Escherichia coli</i>, based on a culture- or multiplex PCR-based method, in fecal samples of feedlot cattle (n = 576).

<p>*Denotes significant difference in proportions within serogroups (<i>P</i> < 0.05).</p

DNA microarray-based assessment of virulence potential of Shiga toxin gene-carrying <i>Escherichia coli</i> O104:H7 isolated from feedlot cattle feces

<div><p><i>Escherichia coli</i> O104:H4, a hybrid pathotype reported in a large 2011 foodborne outbreak in Germany, has not been detected in cattle feces. However, cattle harbor and shed in the feces other O104 serotypes, particularly O104:H7, which has been associated with sporadic cases of diarrhea in humans. The objective of our study was to assess the virulence potential of Shiga toxin-producing <i>E</i>. <i>coli</i> (STEC) O104:H7 isolated from feces of feedlot cattle using DNA microarray. Six strains of STEC O104:H7 isolated from cattle feces were analyzed using FDA-<i>E</i>. <i>coli</i> Identification (ECID) DNA microarray to determine their virulence profiles and compare them to the human strains (clinical) of O104:H7, STEC O104:H4 (German outbreak strain), and O104:H21 (milk-associated Montana outbreak strain). Scatter plots were generated from the array data to visualize the gene-level differences between bovine and human O104 strains, and Pearson correlation coefficients (r) were determined. Splits tree was generated to analyze relatedness between the strains. All O104:H7 strains, both bovine and human, similar to O104:H4 and O104:H21 outbreak strains were negative for intimin (<i>eae</i>). The bovine strains were positive for Shiga toxin 1 subtype c (<i>stx</i>1c), enterohemolysin (<i>ehxA</i>), tellurite resistance gene (<i>terD</i>), IrgA homolog protein (<i>iha</i>), type 1 fimbriae (<i>fimH</i>), and negative for genes that code for effector proteins of type III secretory system. The six cattle O104 strains were closely related (r = 0.86–0.98) to each other, except for a few differences in phage related and non-annotated genes. One of the human clinical O104:H7 strains (2011C-3665) was more closely related to the bovine O104:H7 strains (r = 0.81–0.85) than the other four human clinical O104:H7 strains (r = 0.75–0.79). Montana outbreak strain (O104:H21) was more closely related to four of the human clinical O104:H7 strains than the bovine O104:H7 strains. None of the bovine <i>E</i>. <i>coli</i> O104 strains carried genes characteristic of <i>E</i>. <i>coli</i> O104:H4 German outbreak strain and unlike other human strains were also negative for Shiga toxin 2. Because cattle <i>E</i>. <i>coli</i> O104:H7 strains possess <i>stx</i>1c and genes that code for enterohemolysin and a variety of adhesins, the serotype has the potential to be a diarrheagenic foodborne pathogen in humans.</p></div

Genotype analysis of the O104 strains from bovine and clinical origins.

<p>(A) Hierarchical cluster dendrogram generated by array probeset differences for 241 strains including the diarrheagenic <i>Escherichia coli</i> collection representing the broad diversity across the <i>E</i>. <i>coli</i> species. Phylogroups are highlighted with color shading. The strains reported in this study from human clinical and bovine origin are highlighted in red and green boxes, respectively. Additionally, O104:H4 strains from the German and Georgian outbreaks [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196490#pone.0196490.ref015" target="_blank">15</a>] (indicated with asterisk) are used as reference. The scale bar represents the number of probeset differences. (B) Enlargement of the cluster region containing bovine and human <i>E</i>. <i>coli</i> O104:H7 strains from our study.</p