Canine Providencia alcalifaciens: virulence factors and phylogenetic analysis of an emerging enteropathogen

In 2019, an outbreak of acute haemorrhagic diarrhoea (AHD) in dogs in Norway was linked to Providencia alcalifaciens. A marked increase in diarrhoeal cases in dogs during the autumn, along with the frequent identification of P. alcalifaciens in affected dogs, supports its role as a causative agent in seasonal diarrhoea in dogs in Norway. Previous phylogenetic studies have revealed two major genetic lineages of P. alcalifaciens, one of which is associated with diarrhoeal disease and carries a plasmid-borne type 3 transport system (T3SS_1a). The aim of this study was to place Norwegian P. alcalifaciens isolates in a global phylogenetic context and to characterize the distribution of putative virulence-associated genes (VAGs) in isolates from dogs with AHD. Whole-genome sequencing was performed on 273 isolates, and 48 publicly available genomes were included for comparative analyses. Bioinformatic processing and phylogenetic analyses were conducted on the complete dataset of 321 P. alcalifaciens genomes. VAGs were identified using ABRicate and two virulence gene databases. Phylogenetic analyses revealed two main clusters, A and B. Most isolates from sick dogs belonged to cluster A, particularly to subcluster A-1 - the largest of five subclusters - which primarily contained isolates from sick dogs. All isolates in subclusters A-1 and A-4 harboured genes encoding T3SS_1a, a type II secretion system and the effector genes stcE, yopJ and cdtA-C. blast searches using closed reference genomes showed high sequence identity (>90%) to plasmid elements in 196 of 197 isolates from subclusters A-1 and A-4, suggesting plasmid-mediated acquisition of virulence factors. These findings support that specific genetic variants of P. alcalifaciens, particularly those in subclusters A-1 and A-4, exhibit increased virulence potential and are associated with AHD in dogs. The high proportion of isolates from clinically affected dogs, including many from the 2019 outbreak, further underscores the link between subcluster A-1 and disease.