Purpose: The objective of this study was to phenotypically and genomically characterize three novel, extrachromosomal, temperate phage-like plasmids designated AnCo1, AnCo2, and AnCo3.
Methods: Whole genome sequencing was performed on two antimicrobial resistant Escherichia coli isolated from bovine feedlot associated wildlife and a clinical isolate of Salmonella Derby. Bioinformatic analysis was performed using RAST, PHASTER, ResFinder, and MEGA7 for annotation, identification of prophage regions, identification of antibiotic resistance genes, and construction of a phylogenetic tree, respectively. The phage-like plasmids were induced by mitomycin-C and confirmed by PCR, targeting the integrase gene. Electron microscopy was conducted for morphological characterization.
Results: AnCo1 and AnCo2 from wildlife E. coli isolates were determined to be 112,201 bp and 109,071 bp, respectively, and carried the extended-spectrum β-Lactamase CTX-M-15 gene, which was flanked by the mobile genetic element, ISEcp1. AnCo3 from the Salmonella Derby isolate was 105,994 bp with homology to phage-like plasmid pSTM_phi, and did not encode any antibiotic resistance genes. Plasmid extraction, PCR and genomic analysis suggested that the chromosomes of AnCo1 and AnCo2 were circular and extrachromosomal and had homology to E. coli phage-like plasmid pECOH89. AnCo1 and AnCo2 were inducible with mitomycin-C and electron microscopy demonstrated that AnCo1 had a large head (110 nm in diameter) and a long noncontractile tail characteristic of the Siphoviridae family. A maximum-likelihood tree constructed using the integrase gene as a marker for temperate phage diversity revealed that AnCo1, AnCo2, AnCo3, and seven published phage-like plasmids were located within their own branch. This suggested that they represent a novel family of phages.
Significance: AnCo1, AnCo2, and AnCo3 are novel phage-like plasmids that could potentially spread antibiotic resistance determinants among foodborne bacteria along the farm-to-fork continuum.