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. 2000 Dec;38(12):4343-50.
doi: 10.1128/JCM.38.12.4343-4350.2000.

Citrobacter rodentium, the causative agent of transmissible murine colonic hyperplasia, exhibits clonality: synonymy of C. rodentium and mouse-pathogenic Escherichia coli

S A Luperchio  1 , J V Newman, C A Dangler, M D Schrenzel, D J Brenner, A G Steigerwalt, D B Schauer
Affiliations

Citrobacter rodentium, the causative agent of transmissible murine colonic hyperplasia, exhibits clonality: synonymy of C. rodentium and mouse-pathogenic Escherichia coli

S A Luperchio et al. J Clin Microbiol. 2000 Dec.

Abstract

Citrobacter rodentium (formerly Citrobacter freundii biotype 4280 and Citrobacter genomospecies 9) was described on the basis of biochemical characterization and DNA-DNA hybridization data and is the only Citrobacter species known to possess virulence factors homologous to those of the human pathogens enteropathogenic Escherichia coli and enterohemorrhagic E. coli. These virulence factors are encoded on the locus of enterocyte effacement (LEE), a pathogenicity island required for the characteristic attaching and effacing (AE) pathology seen in infection with these three pathogens. C. rodentium, which apparently infects only mice, provides a useful animal model for studying the molecular basis of AE pathology. No work has been done to assess differences in pathogenicity between C. rodentium isolates from diverse sources. Here, we report the examination of 15 C. rodentium isolates using a battery of genetic and biochemical approaches. No differences were observed between the isolates by repetitive-element sequence-based PCR analysis, biochemical analysis, and possession of LEE-specific virulence factors. These data suggest that members of the species are clonal. We further characterized an atypical E. coli strain from Japan called mouse-pathogenic E. coli (MPEC) that, in our hands, caused the same disease as C. rodentium. Applying the same battery of tests, we found that MPEC possesses LEE-encoded virulence factors and is indistinguishable from the previously characterized C. rodentium isolate DBS100. These results demonstrate that MPEC is a misclassified C. rodentium isolate and that members of this species are clonal and represent the only known attaching and effacing bacterial pathogen of mice.

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Figures

FIG. 1
FIG. 1
Clustal alignment of full-length Tir proteins, showing that C. rodentium Tir is most similar to that of the human EPEC strain E2348/69. When carboxyl-terminal intimin peptides are similarly analyzed, C. rodentium clusters with the rabbit EPEC strain RDEC-1, demonstrating the difficulty in identifying a precursor organism for the C. rodentium LEE. The tree was arbitrarily anchored with the C. rodentium EspB protein, as Tir does not show appreciable homology to any protein previously reported. GenBank accession numbers are AF125993 (EHEC EDL933), AF022236 (EPEC E2348/69), AF045568 (RDEC-1), AJ223063 (EHEC 413/89-1), and AF177537 (C. rodentium EspB).
FIG. 2
FIG. 2
Transmission electron micrographs showing AE pedestal induction on mouse colonic epithelial cells by (A) CDC1843-73T (original magnification, ×12,000; inset, ×20,000) and (B) MPEC (original magnification, ×6,000; inset, ×25,000). Bars, 1 μm.
FIG. 3
FIG. 3
Rep-PCR analysis of whole-genome DNA from Citrobacter species type strains (A) and C. rodentium isolates (B) using the REP primer pair. All C. rodentium isolates produced an identical banding pattern (B), which is in contrast to the Citrobacter species type strains, for which no two species exhibit an identical profile (A). Lane M, 1-kb ladder. (A) Lane numbers represent the Citrobacter species numbers given in Table 1. (B) Lane numbers represent the 16 C. rodentium isolates in the order listed in Table 1.
FIG. 4
FIG. 4
Graphic representation of histologic scores for hyperplasia (A) and necrosis (B) of H&E-stained colonic tissue, showing that all three strains were able to induce similar levels of hyperplasia while only DBS100 (labeled 100) induced necrosis. Each dot represents one animal. The y axis represents an increasing scale of severity from 0 to 4, as described in Materials and Methods. The Mann-Whitney U test was used to calculate P values. The dagger indicates a statistically significant difference (P < 0.05) between CDC1843-73T (CDC) and MPEC. All groups in both panels are statistically different from the applicable LB controls (P < 0.05).
FIG. 5
FIG. 5
H&E-stained colon sections. (A) Section from a mock-infected (sterile broth) mouse showing normal colonic crypt architecture. (B) Section from an MPEC-infected mouse showing crypt hyperplasia, goblet cell depletion, and mild inflammation. (C) DBS100-induced necrosis showing epithelial damage, severe mucosal inflammation with an accompanying inflammatory exudate, and submucosal edema. All panels are at an identical level of magnification.
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