Sequence analysis of bands associated with AE-positive and AE-negative ligated ileal loops of pigs inoculated with <i>E</i>. <i>coli</i> O157:H7.

<p>Sequence analysis of bands associated with AE-positive and AE-negative ligated ileal loops of pigs inoculated with <i>E</i>. <i>coli</i> O157:H7.</p

Alteration of the Microbiota and Virulence Gene Expression in <i>E</i>. <i>coli</i> O157:H7 in Pig Ligated Intestine with and without AE Lesions

<div><p>Background</p><p>Previously we found that <i>E</i>. <i>coli</i> O157:H7 inoculated into ligated pig intestine formed attaching and effacing (AE) lesions in some pigs but not in others. The present study evaluated changes in the microbial community and in virulence gene expression in <i>E</i>. <i>coli</i> O157:H7 in ligated pig intestine in which the bacteria formed AE lesions or failed to form AE lesions.</p><p>Methodology/Principal Findings</p><p>The intestinal microbiota was assessed by RNA-based denaturing gradient gel electrophoresis (DGGE) analysis. The DGGE banding patterns showed distinct differences involving two bands which had increased intensity specifically in AE-negative pigs (AE- bands) and several bands which were more abundant in AE-positive pigs. Sequence analysis revealed that the two AE- bands belonged to <i>Veillonella caviae</i>, a species with probiotic properties, and <i>Bacteroides</i> sp. Concurrent with the differences in microbiota, gene expression analysis by quantitative PCR showed that, compared with AE negative pigs, <i>E</i>. <i>coli </i>O157:H7 in AE positive pigs had upregulated genes for putative adhesins, non-LEE encoded <i>nleA</i> and quorum sensing <i>qseF</i>, acid resistance gene <i>ureD</i>, and genes from the locus of enterocyte effacement (LEE).</p><p>Conclusions/Significance</p><p>The present study demonstrated that AE-positive pigs had reduced activities or populations of <i>Veillonella caviae</i> and <i>Bacterioides</i> sp. compared with AE-negative pigs. Further studies are required to understand how the microbiota was changed and the role of these organisms in the control of <i>E</i>. <i>coli</i> O157:H7.</p></div

DGGE analysis of the bacterial community in the pig ileal loops with or without attaching and effacing (AE) lesion after inoculation of <i>E</i>. <i>coli</i> O157:H7 strain 86–24.

<p>The bacterial community profiles were generated by targeting 16S rRNA gene from reverse transcribed total intestinal bacterial RNA of the ileal contents. Figures A and B are overlapped top and lower portions of the same DGGE profiles but from different DGGE pictures with different resolutions. The profiles included control ileal loops inoculated with EMEM (control-loops), ileal loops without AE lesion and with AE lesion after <i>E</i>. <i>coli</i> O157:H7 inoculation (ileum-loops AE^- and ileum-loops AE⁺ respectively), and <i>E</i>. <i>coli</i> O157:H7 strain 86–24 grown <i>in vitro</i> before inoculation (In vitro).</p