Divergent Evolution of the repFII Replicon of IncF Plasmids Carrying Cytotoxic Necrotizing Factor cnf2 , Cytolethal Distending Toxin cdtIII , and f17Ae Fimbrial Variant Genes in Type 2 Necrotoxigenic Escherichia coli Isolates from Calves

Abstract

dAmong the pathovars of Escherichia coli in cattle, necrotoxigenic E. coli (NTEC) is defined by the production of cytotoxic necrotizing factors (CNFs). In particular, type 2 NTEC (NTEC2) strains are frequent in diarrheic and septicemic calves and usually coproduce CNF type 2 (CNF2), cytolethal distending toxin type III (CDTIII), and fimbrial adhesins of the F17 family, whose genetic determinants have frequently been reported on the same Vir-like plasmid. In this study, we investigated the genetic environment of the cnf2, f17Ae, and cdtIII genes in a collection of fecal E. coli isolates recovered from 484 French and 58 Iranian calves. In particular, we highlighted the spread of cnf2, f17Ae, and cdtIII on similar 150-kb IncF plasmids harboring the newly assigned repFII replicon allele F74 in NTEC2 isolates. Interestingly, this 150-kb IncF plasmid differed from the 140-kb IncF plasmid harboring the newly assigned repFII replicon allele F75 and carrying cnf2 alone. These results suggest two divergent lineages of cnf2-carrying IncF plasmids depending on the presence of the f17Ae and cdtIII genes. This partition was observed in E. coli strains of unrelated backgrounds, suggesting two different evolutionary paths of cnf2-carrying IncF plasmids rather than divergent evolutions of NTEC2 clones. The driving forces for such divergent evolutions are not known, and further studies are required to clarify the selection of plasmid subtypes spreading virulence determinants in E. coli, in particular, plasmids of the IncF family. Escherichia coli is considered a dominant member of the normal microflora of mammals but may also be responsible for intestinal and extraintestinal infections (1). Accordingly, several pathovars of E. coli have been reported over time both in humans and in animals (1, 2). In general, those infections result from a combination of host susceptibility and biological functions expressed by the pathogenic E. coli strain, such as an enhanced ability to colonize the host or the production of toxigenic factors.To adhere to host cells, pathogenic E. coli strains usually produce fimbrial or afimbrial adhesins. In cattle, E. coli isolates from diarrheic and septicemic calves often produce F17 fimbriae (3, 4), which bind N-acetyl-D-glucosamine (GlcNAc)-containing receptors present on host intestinal epithelial cells (4, 5). F17 fimbriae consist of fine filamentous heteropolymers composed of two main subunits: the pilin F17-A subunit, which forms the bulk of the fimbriae, and the adhesin F17-G subunit (4). Several variants have been reported: F17a-A, F17b-A, F17c-A, F17d-A, F17e-A, and F17f-A for the F17-A pilin (6-10) and F17-G1, F17-G2, and F17-G3 for the F17-G adhesin (10, 11). In particular, the recently reported F17e-A variant was abundantly found in E. coli isolates from diarrheic calves in Iran and was found to be associated with F17-G3 on a pathogenicity or a fitness island in Shiga toxin-producing E. coli (STEC) strain MHI813 (10).Among the virulence determinants reported in cattle-pathogenic E. coli strains...

F74 plasmids are major vectors of virulence genes in bovine NTEC2

“…This is true in France, where they have been circulating for more than 10 years and have already been described (Bihannic et al . 2016), but also in England and in the United States, as shown from sequences extracted from the public databases (Table 1). In France, divergent plasmid evolution was observed, with the presence of F74 plasmids in isolates carrying plasmid‐borne cdtIII and F17 genes, and the presence of F75 plasmids when the cdtIII gene was missing.…”

“…More recently, NTEC2 isolates co‐carrying the cnf2 , cdtIII and F17 genes were described in France, with cnf2 alternatively found on IncF belonging to the F74 allelic variant when cdtIII was carried on the plasmid, and to F75 when cdtIII was absent or located on the chromosome (Bihannic et al . 2016).…”

“…Sequence analysis revealed the presence the cnf2 and cdt-ABC genes, as well as a fimbrial F17 operon, the outer membrane protein ompP and a truncated haemolysin operon hly-AC flanked by inverted copies of ISEc8, clustered together on a pathogenic island (PAI). More recently, NTEC2 isolates co-carrying the cnf2, cdtIII and F17 genes were described in France, with cnf2 alternatively found on IncF belonging to the F74 allelic variant when cdtIII was carried on the plasmid, and to F75 when cdtIII was absent or located on the chromosome (Bihannic et al 2016).…”

Insight Into the Virulence Related Secretion Systems, Fimbriae, and Toxins in O2:K1 Escherichia coli Isolated From Bovine Mastitis

“…Although its upstream four genes showed a high sequence identity with the well-known hemolysin genes hlyCABD and really function for β-hemolysin, the downstream cnf2 gene has never been found in the human ExPEC. It should be noted that cnf2 gene was extensively encoded in the strains isolated from newborn calves with the significant extraintestinal infection ( 34 ) and significantly involved in the bacterial virulence, suggesting this plasmid encoding hemolysin- cnf2 operon may be closely related to the bovine infection, including cows and calves. In fact, similar plasmid harbored by E. coli isolates from the septicemic calves and lambs ( 34 , 70 ) was designated as Vir plasmid, which was reported to propagate the cnf2 and f17A genes in NTEC2 cattle isolates ( 71 ) and other toxic genes, such as cdtIII ( 72 ).…”

“…Numerous toxins, including α-hemolysin, cytotoxic necrotizing factor 1 (CNF1), secreted autotransporter toxin (Sat), and protease involved in colonization (Pic) (29)(30)(31)(32)(33), have been identified to play the key roles during ExPEC infection. It should be noted that many necrotoxigenic E. coli (NTEC) strains isolated from newborn calves carry CNF2 and cytolethal distending (Cdt III) toxins (34), which may be also prevalent in the bovine mastitis E. coli.…”