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. 2006 Aug;74(8):4778-92.
doi: 10.1128/IAI.00067-06.

Adherent and invasive Escherichia coli is associated with granulomatous colitis in boxer dogs

Kenneth W Simpson  1 , Belgin Dogan, Mark Rishniw, Richard E Goldstein, Suzanne Klaessig, Patrick L McDonough, Alex J German, Robin M Yates, David G Russell, Susan E Johnson, Douglas E Berg, Josee Harel, Guillaume Bruant, Sean P McDonough, Ynte H Schukken
Affiliations

Adherent and invasive Escherichia coli is associated with granulomatous colitis in boxer dogs

Kenneth W Simpson et al. Infect Immun. 2006 Aug.

Abstract

The mucosa-associated microflora is increasingly considered to play a pivotal role in the pathogenesis of inflammatory bowel disease. This study explored the possibility that an abnormal mucosal flora is involved in the etiopathogenesis of granulomatous colitis of Boxer dogs (GCB). Colonic biopsy samples from affected dogs (n = 13) and controls (n = 38) were examined by fluorescent in situ hybridization (FISH) with a eubacterial 16S rRNA probe. Culture, 16S ribosomal DNA sequencing, and histochemistry were used to guide subsequent FISH. GCB-associated Escherichia coli isolates were evaluated for their ability to invade and persist in cultured epithelial cells and macrophages as well as for serotype, phylogenetic group, genome size, overall genotype, and presence of virulence genes. Intramucosal gram-negative coccobacilli were present in 100% of GCB samples but not controls. Invasive bacteria hybridized with FISH probes to E. coli. Three of four GCB-associated E. coli isolates adhered to, invaded, and replicated within cultured epithelial cells. Invasion triggered a "splash"-type response, was decreased by cytochalasin D, genistein, colchicine, and wortmannin, and paralleled the behavior of the Crohn's disease-associated strain E. coli LF 82. GCB E. coli and LF 82 were diverse in serotype and overall genotype but similar in phylogeny (B2 and D), in virulence gene profiles (fyuA, irp1, irp2, chuA, fepC, ibeA, kpsMII, iss), in having a larger genome size than commensal E. coli, and in the presence of novel multilocus sequence types. We conclude that GCB is associated with selective intramucosal colonization by E. coli. E. coli strains associated with GCB and Crohn's disease have an adherent and invasive phenotype and novel multilocus sequence types and resemble E. coli associated with extraintestinal disease in phylogeny and virulence gene profile.

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Figures

FIG. 1.
FIG. 1.
Clinical and pathological features of granulomatous colitis of Boxer dogs. A. Colonoscopy shows a diffusely thickened and ulcerated mucosa (arrow). B. Histologically, there is severe loss of glandular structure and cellular infiltration (H&E; magnification, ×200). C. Mucosal infiltration with PAS-staining foamy macrophages (inset) is a dominant feature (magnification, ×200).
FIG. 2.
FIG. 2.
Mucosa-associated flora in Boxer dogs with granulomatous colitis. A. Fluorescence in situ hybridization with probe Cy3-EUB-338 (red) shows that the bacterial flora (arrows) in the normal colon is confined to superficial mucus and glands. DAPI-stained nuclei are blue (magnification, ×400). B. In granulomatous colitis of Boxer dogs, clusters of bacteria that hybridize with Cy3-EUB-338 (red) are located within the mucosa (magnification, ×400). C. Intracellular bacteria visualized with Cy3-EUB-338 (red) and FITC-antivimentin (green). DAPI stained DNA (blue). n, nucleus (magnification, ×600). D. Gram staining reveals that invasive bacteria are gram-negative coccobacilli within macrophages (arrows) (magnification, ×1,000).
FIG. 3.
FIG. 3.
16S rDNA libraries constructed from colonic biopsy samples of a Boxer dog with granulomatous colitis before (A) and after (B) clinical remission induced by enrofloxacin. The sequences of bacteria cultured from the biopsy sample are indicated by the suffix KD or GCB. Database sequences with the highest nucleotide homology are indicated by accession numbers.
FIG. 4.
FIG. 4.
Fluorescence in situ hybridization of GCB mucosa with probes against Enterobacteriaceae and E. coli/Shigella. A. Multifocal clusters of bacteria (long arrow) that hybridize with probes E. coli 1531 (red; Enterobacteriaceae) and EUB-338 (green) are evident within the mucosa. A mixed population of Enterobacteriaceae (orange) and other bacteria (green) is visible on the luminal surface (small arrow). DAPI stained DNA (blue) (magnification, ×400). B. Selective mucosal invasion by clusters of bacteria (long arrow) that hybridize with a probe against E. coli/Shigella (red) but not probe non-EUB-338 (green). DAPI stained DNA (blue) (magnification, ×400). C. The mucosally invasive flora is composed of coccobacilli 1 to 3 μm long that hybridize with a Cy3-E. coli/Shigella-specific probe but not non-EUB-338 (green). DAPI stained DNA (blue) (magnification, ×600).
FIG. 5.
FIG. 5.
Adhesion and invasion of cultured epithelial cells by E. coli associated with GCB (KD1 to -4) and Crohn's disease (LF 82), commensal E. coli (DH5α), and Salmonella enterica serovar Typhimurium. The results of statistical analysis are indicated by the letters A through D. Strains with different letters are significantly different from each other (P < 0.05). A. Adhesion to MAC-T epithelial cells. B. Invasion of MAC-T epithelial cells. C. Invasion of Caco-2 epithelial cells.
FIG. 6.
FIG. 6.
Microscopic examination of cultured epithelial cells infected with canine GCB-associated E. coli strain KD-2. A. Invasion of E. coli was confirmed by differential staining of intracellular and extracellular bacteria with differently labeled secondary antibodies before (FITC, green) and after (TRITC, red) permeabilization. Extracellular bacteria are yellow (red plus green); intracellular bacteria are red. DAPI stained DNA (blue). B. Transmission electron microscopy of cultured epithelial cells infected with GCB-associated strain KD-2 reveals bacteria adhering to the cell surface. Note the filopodia formed close to the bacterium (arrows). C. Filopodia (arrows) persist during entry into the cell. D. An oblique section shows a dense patch, possibly polymerized actin, subtending the bacterium during entry (arrow). E. The membrane “ruffle” (arrow) persists even after the bacterium has entered the epithelial cell. These findings are consistent with a trigger type of endocytosis.
FIG. 7.
FIG. 7.
Effects of inhibitors of microfilaments (cytochalasin D), microtubules (colchicine), tyrosine kinase (genistein), and PI 3-kinase (wortmannin) on invasion by IBD-associated E. coli and Salmonella enterica serovar Typhimurium. Invasion (percent of invasion in the absence of inhibitors) by GCB-associated E. coli (KD1 and KD2) was reduced by all inhibitors (P < 0.05). The results for LF 82 and the selective inhibition of Salmonella invasion by cytochalasin D and genistein but not colchicine or wortmannin are consistent with the literature. Results are expressed as means plus standard deviations.
FIG. 8.
FIG. 8.
Survival of GCB-associated (KD1 to -4) and Crohn's disease-associated (LF 82) E. coli in cultured epithelial cells and primary bone marrow-derived macrophages. A. Survival of E. coli over a 48-h period in cultured MAC-T epithelial cells, expressed as a percentage of the CFU recovered at 2 h. Strains KD1 to -3 and LF 82 replicate (**, P < 0.05), while DH5α and KD-4 decline. B. Survival of E. coli over a 72-h period in cultured primary bone marrow-derived macrophages. GCB- and CD-associated and commensal (DH5α) E. coli strains are not significantly different from each other and survive less well than Salmonella (P < 0.05).
FIG. 9.
FIG. 9.
Genetic diversity of GCB- and CD-associated E. coli strains. RAPD-PCR of genomic DNA extracted from E. coli strains associated with Crohn's disease (LF 82, lanes 1), and GCB (KD1 to -4, lanes 2 to 5) with RAPD primers 1254, 1281, and 1283.
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