Horsing around: Escherichia coli ST1250 of equine origin harbouring epidemic IncHI1/ST9 plasmid with bla CTX-M-1 and an operon for short-chain fructooligosaccharides metabolism

Abstract

The relatedness of the equine-associated Escherichia coli ST1250 and its single- and double-locus variants (ST1250-SLV/DLV), obtained from horses in Europe, was studied by comparative genome analysis. A total of 54 isolates of E. coli ST1250 and ST1250-SLV/DLV from healthy and hospitalized horses across Europe [Czech Republic (n=23), the Netherlands (n=18), Germany (n=9), Denmark (n=3) and France (n=1)] from 2008-2017 were subjected to whole-genome sequencing. An additional 25 draft genome assemblies of E. coli ST1250 and ST1250-SLV/DLV were obtained from the public databases. The isolates were compared for genomic features, virulence genes, clade structure and plasmid content. The complete nucleotide sequences of eight IncHI1/ST9 and one IncHI1/ST2 plasmids were obtained using long-read sequencing by PacBio or MinION. In the collection of 79 isolates, only 10 were phylogenetically close (<8 SNP). The majority of isolates belonged to phylogroup B1 (73/79, 92.4%) and carried bla _CTX-M-1 (58/79, 73.4%). The plasmid content of the isolates was dominated by IncHI1 of ST9 (56/62, 90.3%) and ST2 (6/62, 9.7%), while 84.5% (49/58) bla _CTX-M-1 genes were associated with presence of IncHI1 replicon of ST9 and 6.9% (4/58) with IncHI1 replicon of ST2 within the corresponding isolates. The operon for the utilization of short chain fructooligosaccharides (fos operon) was present in 55 (55/79, 69.6%) isolates, and all of these carried IncHI1/ST9 plasmids. The eight complete IncHI1/ST9 plasmid sequences showed the presence of bla _CTX-M-1 and the fos operon within the same molecule. Sequences of IncHI1/ST9 plasmids were highly conserved (>98% similarity) regardless of country of origin and varied only in the structure and integration site of MDR region. E. coli ST1250 and ST1250-SLV/DLV are phylogenetically-diverse strains associated with horses. A strong linkage of E. coli ST1250 with epidemic multi-drug resistance plasmid lineage IncHI1/ST9 carrying bla _CTX-M-1 and the fos operon was identified.

FIG 1

Phylogenetic tree depicting the diversity of E. coli ST1250 and ST1250-SLV/DLV isolates with highlighted clusters A, BI, BII, and C. Isolates marked *M or *PB were sequenced on MinION or PacBio, respectively. Metadata for country of origin, year of isolation, source, ST, phylogroup, and serotype are included. Colored boxes indicate the presence of the IncHI1 plasmid and its ST, the fos operon, and ESBL genes. The designations of physically obtained isolates are set in boldface, and data sets from EnteroBase start with “ESC.” N/A, not available; E, equine; Env, equine clinic environment; H, human; NT, not typeable; NL, The Netherlands; DK, Denmark; CZ, Czech Republic; DE, Germany; CH, Switzerland; US, United States; GP, Guadeloupe; FR, France; CA, Canada.

FIG 2

BRIG comparison of IncHI1/ST9 plasmids including draft genome assemblies with pEQ1 as a reference. The fos operon and the bla_CTX-M-1 gene are annotated in red. The origins of the samples are color-coded as follows: light blue, Czech Republic; yellow, Denmark; light green and dark green, The Netherlands (different studies); dark blue, France; purple; Germany; turquoise, Switzerland.

FIG 3

Comparison of two variants of the MDR regions occurring in the HI1/ST9 plasmids that were studied. Diagrams of two representative plasmids are shown: p15S04714-1 (top) and p99063 (bottom). Plasmid backbone gene products are shown in blue, antimicrobial resistance gene products in red, transposases in orange, and hypothetical proteins (HP) in gray, unless they are part of the backbone (blue). An asterisk at an insertion sequence (IS) indicates <100% nucleotide identity with the reference IS.