Intestine and environment of the chicken as reservoirs for extraintestinal pathogenic Escherichia coli strains with zoonotic potential

Abstract

Although research has increasingly focused on the pathogenesis of avian pathogenic Escherichia coli (APEC) infections and the "APEC pathotype" itself, little is known about the reservoirs of these bacteria. We therefore compared outbreak strains isolated from diseased chickens (n = 121) with nonoutbreak strains, including fecal E. coli strains from clinically healthy chickens (n = 211) and strains from their environment (n = 35) by determining their virulence gene profiles, phylogenetic backgrounds, responses to chicken serum, and in vivo pathogenicities in a chicken infection model. In general, by examining 46 different virulence-associated genes we were able to distinguish the three groups of avian strains, but some specific fecal and environmental isolates had a virulence gene profile that was indistinguishable from that determined for outbreak strains. In addition, a substantial number of phylogenetic EcoR group B2 strains, which are known to include potent human and animal extraintestinal pathogenic E. coli (ExPEC) strains, were identified among the APEC strains (44.5%) as well as among the fecal E. coli strains from clinically healthy chickens (23.2%). Comparably high percentages (79.2 to 89.3%) of serum-resistant strains were identified for all three groups of strains tested, bringing into question the usefulness of this phenotype as a principal marker for extraintestinal virulence. Intratracheal infection of 5-week-old chickens corroborated the pathogenicity of a number of nonoutbreak strains. Multilocus sequence typing data revealed that most strains that were virulent in chicken infection experiments belonged to sequence types that are almost exclusively associated with extraintestinal diseases not only in birds but also in humans, like septicemia, urinary tract infection, and newborn meningitis, supporting the hypothesis that not the ecohabitat but the phylogeny of E. coli strains determines virulence. These data provide strong evidence for an avian intestinal reservoir hypothesis which could be used to develop intestinal intervention strategies. These strains pose a zoonotic risk because either they could be transferred directly from birds to humans or they could serve as a genetic pool for ExPEC strains.

FIG. 1.

Virulence gene patterns, sources of strains, phylogenetic groups, and results of serum tests for 347 E. coli strains. For VAGs the presence of a gene in a strain is indicated by a black bar. The sources of strains are indicated as follows: light gray, A_fecal strains; dark gray, A_environ strains; and light green, APEC strains. EcoR groups are indicated as follows: yellowish, group A; blue, group B1; red, group B2; and green, group D. The results of the serum test are indicated as follows: dark blue, resistant; light gray, intermediate resistance; cyan, sensitive. Genes not present in any of the strains (afa/dra, bfp, nfaE, stx₁, and stx₂) are not shown. epis., episomal; chr., chromosomal.

FIG. 2.

Organ lesion scores for groups of chickens 24 h after infection with APEC outbreak strain IMT5155 (gray bar), nonpathogenic control strain IMT11327 (open bar), A_fecal E. coli strains (dotted bars), and A_environ E. coli strains (striped bars). *, P ≤ 0.05; **, P ≤ 0.02; ***, P ≤ 0.001. IMT, Institut für Mikrobiologie und Tierseuchen (Institute of Microbiology and Epizootics).

FIG. 3.

Bacterial loads in (A) lungs, (B) spleens, and (C) kidneys of groups of chickens 24 h after intratracheal infection with APEC outbreak strain IMT5155 (gray bars), nonpathogenic control strain IMT11327 (open bars), A_fecal E. coli strains (dotted bars), and A_environ E. coli strains (striped bars). *, P ≤ 0.05; **, P ≤ 0.02; ***, P ≤ 0.001. IMT, Institut für Mikrobiologie und Tierseuchen (Institute of Microbiology and Epizootics).