Sequences of Two Related Multiple Antibiotic Resistance Virulence Plasmids Sharing a Unique IS26-Related Molecular Signature Isolated from Different Escherichia coli Pathotypes from Different Hosts

Abstract

Enterohemorrhagic Escherichia coli (EHEC) and atypical enteropathogenic E. coli (aEPEC) are important zoonotic pathogens that increasingly are becoming resistant to multiple antibiotics. Here we describe two plasmids, pO26-CRL125 (125 kb) from a human O26:H- EHEC, and pO111-CRL115 (115kb) from a bovine O111 aEPEC, that impart resistance to ampicillin, kanamycin, neomycin, streptomycin, sulfathiazole, trimethoprim and tetracycline and both contain atypical class 1 integrons with an identical IS26-mediated deletion in their 3´-conserved segment. Complete sequence analysis showed that pO26-CRL125 and pO111-CRL115 are essentially identical except for a 9.7 kb fragment, present in the backbone of pO26-CRL125 but absent in pO111-CRL115, and several indels. The 9.7 kb fragment encodes IncI-associated genes involved in plasmid stability during conjugation, a putative transposase gene and three imperfect repeats. Contiguous sequence identical to regions within these pO26-CRL125 imperfect repeats was identified in pO111-CRL115 precisely where the 9.7 kb fragment is missing, suggesting it may be mobile. Sequences shared between the plasmids include a complete IncZ replicon, a unique toxin/antitoxin system, IncI stability and maintenance genes, a novel putative serine protease autotransporter, and an IncI1 transfer system including a unique shufflon. Both plasmids carry a derivate Tn21 transposon with an atypical class 1 integron comprising a dfrA5 gene cassette encoding resistance to trimethoprim, and 24 bp of the 3´-conserved segment followed by Tn6026, which encodes resistance to ampicillin, kanymycin, neomycin, streptomycin and sulfathiazole. The Tn21-derivative transposon is linked to a truncated Tn1721, encoding resistance to tetracycline, via a region containing the IncP-1α oriV. Absence of the 5 bp direct repeats flanking Tn3-family transposons, indicates that homologous recombination events played a key role in the formation of this complex antibiotic resistance gene locus. Comparative sequence analysis of these closely related plasmids reveals aspects of plasmid evolution in pathogenic E. coli from different hosts.

Elucidation of quantitative structural diversity of remarkable rearrangement regions, shufflons, in IncI2 plasmids

“…Shufflons have also been identified in IncI1γ 3 , IncI2 4, 5 , IncK 6, 7 and IncZ plasmids 8, 9 . Shufflons generate variants of the PilV protein, a minor component of the thin pilus.…”

IncI shufflons: Assembly issues in the next-generation sequencing era

“…Shufflons have now also been identified in more distantly related IncK (Cottell et al, 2014) and IncZ (Venturini et al, 2013) plasmids. The shufflon is a site-specific recombination system that generates variants of the PilV protein, the minor component of the thin pilus.…”

Porcine commensalEscherichia coli: A reservoir for class 1 integrons associated with IS26

“…The deletion event in the 3`-CS of the integron depicted in 4E (dfrA5-IS26) may serve as another genetic signature for tracking resistance genes, and bacteria that carry them, through different hosts and environments , Dawes et al, 2010, Levings et al, 2005. Previously we observed the 4E integron structure on plasmids carrying virulence genes in atypical EPEC strains isolated from cattle with gastrointestinal disease and E. coli strains linked to EHEC O26:H-isolated from humans with haemorrhagic colitis (Venturini et al, 2010, Venturini et al, 2013. In each of these earlier cases, the IS26 that interrupted the 3`-CS of the integron formed part of the left boundary of Tn6026, an IS26-flanked, globally disseminated transposon that harbours multiple ARGs (Cain et al, 2010, Venturini et al, 2010, Venturini et al, 2013.…”

“…Horizontally acquired DNA, mediated by mobile genetic elements, plays an important role in the evolution of the species. Commensal or pathogenic bacteria may, in a single horizontal gene transfer event, acquire a mobile genetic element carrying multiple ARGs, virulence genes, and other genetic cargo that encode traits that offer a niche advantage (Kingsley et al, 2009, Levings et al, 2008, Levings et al, 2005, Levings et al, 2007, Liu et al, 2015, Moran et al, 2016, Venturini et al, 2010, Venturini et al, 2013. Furthermore, indirect selection pressure can, in the absence of antibiotic use, lead to the persistence of transferred genes.…”

“…IS26 is prominent in this regard because it has no target site specificity and can mobilise an increasing variety of ARGs (Canton and Coque, 2006, Dionisi et al, 2009, Dropa et al, 2016, Fournier et al, 2006, Harmer et al, 2014, Liu et al, 2017, Toleman and Walsh, 2011. Furthermore, IS26 is recognised to play a key role in: i) the evolution of plasmids and genomic islands that carry combinations of virulence and antimicrobial resistance genes (Doublet et al, 2009, Siebor and Neuwirth, 2013, Venturini et al, 2010, Venturini et al, 2013; ii) driving the formation of cointegrate plasmids encoding virulence and antibiotic resistance genes (Mangat et al, 2017) and;iii) initiating deletions in large multidrug resistance plasmids that enhance plasmid stability and expand host range (Porse et al, 2016).…”