Pyrosequencing of the Vir plasmid of necrotoxigenic Escherichia coli

Necrotoxigenic Escherichia coli, or NTEC, are defined as E. coli producing the toxin known as cytotoxic necrotizing factor, or CNF. NTEC are responsible for various diseases of humans and animals, including urinary tract infection, septicemia, and diarrhea. A subgroup of NTEC known as NTEC-2 produce a variant of CNF (CNF-2) whose gene is located on a plasmid known as Vir. Because of its involvement in NTEC-2 pathogenesis and its broad distribution among production animals, a Vir plasmid from a bovine NTEC-2 strain was sequenced and analyzed. This plasmid was found to belong to the RepFIB and RepFIIA replicon types, and it totaled 138,682 base pairs in size. Within this plasmid was an approximately 60-kb pathogenicity island, defined by its possession of multiple virulence factors within distinct genetic regions of lower G+C content bounded by inverted repeats. Within this PAI were a variety of putative virulence factors, including F17b fimbrial genes, genes of a novel fimbrial operon, tibAC, hemolysins, and the cnf2 and cdt toxin-encoding genes. The occurrence of this plasmid's virulence- and replication-associated genes was sought among a collection of 96 CNF-2-positive isolates. The most prevalent genes among this collection included repA (RepFIB), cnf2, an ompP homolog, and the tib-. AC genes encoding for aggregation and biofilm formation. The Vir plasmid has evolved from an IncFIB ancestral backbone, with the RepFIB locus apparently driving the acquisition of its accessory virulence-associated elements via site-specific recombination. Overall, the completed sequence of a Vir plasmid increases our understanding of NTEC-2 pathogenomics and IncFIB plasmid evolution.