Escherichia coli O104 in Feedlot Cattle Feces: Prevalence, Isolation and Characterization

Citation: Shridhar, P. B., Noll, L. W., Shi, X. R., Cernicchiaro, N., Renter, D. G., Bai, J., & Nagaraja, T. G. (2016). Escherichia coli O104 in Feedlot Cattle Feces: Prevalence, Isolation and Characterization. Plos One, 11(3), 17. doi:10.1371/journal.pone.0152101Escherichia coli O104:H4, an hybrid pathotype of Shiga toxigenic and enteroaggregative E. coli, involved in a major foodborne outbreak in Germany in 2011, has not been detected in cattle feces. Serogroup O104 with H type other than H4 has been reported to cause human illnesses, but their prevalence and characteristics in cattle have not been reported. Our objectives were to determine the prevalence of E. coli O104 in feces of feedlot cattle, by culture and PCR detection methods, and characterize the isolated strains. Rectal fecal samples from a total of 757 cattle originating from 29 feedlots were collected at a Midwest commercial slaughter plant. Fecal samples, enriched in E. coli broth, were subjected to culture and PCR methods of detection. The culture method involved immunomagnetic separation with O104-specific beads and plating on a selective chromogenic medium, followed by serogroup confirmation of pooled colonies by PCR. If pooled colonies were positive for the wzx(O104) gene, then colonies were tested individually to identify wzx(O104)-positive serogroup and associated genes of the hybrid strains. Extracted DNA from feces were also tested by a multiplex PCR to detect wzx(O104)-positive serogroup and associated major genes of the O104 hybrid pathotype. Because wzx(O104) has been shown to be present in E. coli O8/O9/O9a, wzx(O104)-positive isolates and extracted DNA from fecal samples were also tested by a PCR targeting wbdD(O8/O9/O9a), a gene specific for E. coli O8/O9/O9a serogroups. Model-adjusted prevalence estimates of E. coli O104 (positive for wzx(O104) and negative for wbdD(O8/O9/O9a)) at the feedlot level were 5.7% and 21.2%, and at the sample level were 0.5% and 25.9% by culture and PCR, respectively. The McNemar's test indicated that there was a significant difference (P < 0.01) between the proportions of samples that tested positive for wzx(O104) and samples that were positive for wzx(O104), but negative for wbdD(O8/O9/O9a) by PCR and culture methods. A total of 143 isolates, positive for the wzx(O104), were obtained in pure culture from 146 positive fecal samples. Ninety-two of the 143 isolates (64.3%) also tested positive for the wbdD(O8/O9/O9a), indicating that only 51 (35.7%) isolates truly belonged to the O104 serogroup (positive for wzx(O104) and negative for wbdD(O8/O9/O9a)). All 51 isolates tested negative for eae, and 16 tested positive for stx1 gene of the subtype 1c. Thirteen of the 16 stx1-positive O104 isolates were from one feedlot. The predominant serotype was O104:H7. Pulsed-field gel electrophoresis analysis indicated that stx1-positive O104:H7 isolates had 62.4% homology to the German outbreak strain and 67.9% to 77.5% homology to human diarrheagenic O104:H7 strains. The 13 isolates obtained from the same feedlot were of the same PFGE subtype with 100% Dice similarity. Although cattle do not harbor the O104:H4 pathotype, they do harbor and shed Shiga toxigenic O104 in the feces and the predominant serotype was O104:H7

Analysis of virulence potential of Escherichia coli O145 isolated from cattle feces and hide samples based on whole genome sequencing.

Escherichia coli O145 serogroup is one of the big six non-O157 Shiga toxin producing E. coli (STEC) that causes foodborne illnesses in the United States and other countries. Cattle are a major reservoir of STEC, which harbor them in their hindgut and shed in the feces. Cattle feces is the main source of hide and subsequent carcass contaminations during harvest leading to foodborne illnesses in humans. The objective of our study was to determine the virulence potential of STEC O145 strains isolated from cattle feces and hide samples. A total of 71 STEC O145 strains isolated from cattle feces (n = 16), hide (n = 53), and human clinical samples (n = 2) were used in the study. The strains were subjected to whole genome sequencing using Illumina MiSeq platform. The average draft genome size of the fecal, hide, and human clinical strains were 5.41, 5.28, and 5.29 Mb, respectively. The average number of genes associated with mobile genetic elements was 260, 238, and 259, in cattle fecal, hide, and human clinical strains, respectively. All strains belonged to O145:H28 serotype and carried eae subtype γ. Shiga toxin 1a was the most common Shiga toxin gene subtype among the strains, followed by stx2a and stx2c. The strains also carried genes encoding type III secretory system proteins, nle, and plasmid-encoded virulence genes. Phylogenetic analysis revealed clustering of cattle fecal strains separately from hide strains, and the human clinical strains were more closely related to the hide strains. All the strains belonged to sequence type (ST)-32. The virulence gene profile of STEC O145 strains isolated from cattle sources was similar to that of human clinical strains and were phylogenetically closely related to human clinical strains. The genetic analysis suggests the potential of cattle STEC O145 strains to cause human illnesses. Inclusion of more strains from cattle and their environment in the analysis will help in further elucidation of the genetic diversity and virulence potential of cattle O145 strains

Genetic Analysis of Virulence Potential of Escherichia coli O104 Serotypes Isolated From Cattle Feces Using Whole Genome Sequencing

Escherichia coli O104:H4, a Shiga toxin-producing hybrid pathotype that was implicated in a major foodborne outbreak in Germany in 2011, has not been detected in cattle. However, serotypes of O104, other than O104:H4, have been isolated from cattle feces, with O104:H7 being the most predominant. In this study, we investigated, based on whole genome sequence analyses, the virulence potential of E. coli O104 strains isolated from cattle feces, since cattle are asymptomatic carriers of E. coli O104. The genomes of ten bovine E. coli O104 strains (six O104:H7, one O104:H8, one O104:H12, and two O104:H23) and five O104:H7 isolated from human clinical cases were sequenced. Of all the bovine O104 serotypes (H7, H8, H12, and H23) that were included in the study, only E. coli O104:H7 serotype possessed Shiga toxins. Four of the six bovine O104:H7 strains and one of the five human strains carried stx1c. Three human O104 strains carried stx2, two were of subtype 2a, and one was 2d. Genomes of stx carrying bovine O104:H7 strains were larger than the stx-negative strains of O104:H7 or other serotypes. The genome sizes were proportional to the number of genes carried on the mobile genetic elements (phages, prophages, transposable elements and plasmids). Both bovine and human strains were negative for intimin and other genes associated with the type III secretory system and non-LEE encoded effectors. Plasmid-encoded virulence genes (ehxA, epeA, espP, katP) were also present in bovine and human strains. All O104 strains were negative for antimicrobial resistance genes, except one human strain. Phylogenetic analysis indicated that bovine E. coli O104 strains carrying the same flagellar antigen clustered together and STEC strains clustered separately from non-STEC strains. One of the human O104:H7 strains was phylogenetically closely related to and belonged to the same sequence type (ST-1817) as the bovine O104:H7 STEC strains. This suggests that the bovine feces could be a source of human illness caused by E. coli O104:H7 serotype. Because bovine O104:H7 strains carried virulence genes similar to human clinical strains and one of the human clinical strains was phylogenetically related to bovine strains, the serotype has the potential to be a diarrheagenic pathogen in humans

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

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

<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