Canine uropathogenic and avian pathogenic Escherichia coli harboring conjugative plasmids exhibit augmented growth and exopolysaccharide production in response to Enterococcus faecalis

by Grayson K. Walker, M. Mitsu Suyemoto, Megan E. Jacob, Siddhartha Thakur, Luke B. Borst

Uropathogenic Escherichia coli (UPEC) and avian pathogenic Escherichia coli (APEC) are extraintestinal pathogenic Escherichia coli (ExPEC) that infect dogs and poultry. These agents occur both as single-species infections and, commonly, in co-infection with Enterococcus faecalis (EF); however, it is unclear how EF co-infections modulate ExPEC virulence. Genetic drivers of interspecies interactions affecting virulence were identified using macrocolony co-culture, chicken embryo co-infection experiments, and whole-genome sequence analysis of ExPEC and EF clinical isolates. Ten of 11 UPEC strains originally co-isolated with EF exhibited a growth advantage when co-cultured with EF on iron-limited, semi-solid media in contrast to growing alone (P < 0.01). Phylogenetic analyses of these UPEC and 18 previously screened APEC indicated the growth-response phenotype was conserved in ExPEC despite strain diversity. When genomes of EF-responsive ExPEC were compared to non-responsive ExPEC genomes, EF-induced growth was associated with siderophore, exopolysaccharide (EPS), and plasmid conjugative transfer genes. Two matched pairs of EF-responsive and non-responsive ExPEC were selected for further characterization by macrocolony proximity and chicken embryo lethality assays. EF-responsive ExPEC produced 5 to 16 times more EPS in proximity to EF and were more lethal to embryos alone and during co-infection with EF compared to non-responsive ExPEC (P < 0.05). A responsive APEC strain cured of its conjugative plasmid lost the enhanced growth and EPS production response to EF. These data demonstrate that ExPEC growth augmentation by EF occurs in UPEC and APEC strains and is linked to conjugative virulence plasmids and EPS production, which are widely conserved ExPEC virulence determinants.