Elsevier

Plasmid

Volume 80, July 2015, Pages 118-126
Plasmid

Different IncI1 plasmids from Escherichia coli carry ISEcp1-blaCTX-M-15 associated with different Tn2-derived elements

https://doi.org/10.1016/j.plasmid.2015.04.007Get rights and content

Highlights

  • We sequenced 3 different IncI1 plasmids encoding the important CTX-M-15 β-lactamase.

  • All three plasmids have a typical IncI1 organisation, but one is a quite novel type.

  • Detailed comparative analysis reveals more subtle differences in IncI1 backbones.

  • blaCTX-M-15 is inserted in different Tn2 derivatives in the same backbone region.

  • Tn2- and IS26-mediated events may contribute to the spread of blaCTX-M-15.

Abstract

The blaCTX-M-15 gene, encoding the globally dominant CTX-M-15 extended-spectrum β-lactamase, has generally been found in a 2.971-kb ISEcp1-blaCTX-M-15-orf477Δ transposition unit, with ISEcp1 providing a promoter. In available IncF plasmid sequences from Escherichia coli, this transposition unit interrupts a truncated copy of transposon Tn2 that lies within larger multiresistance regions. In E. coli, blaCTX-M-15 is also commonly associated with IncI1 plasmids and here three such plasmids from E. coli clinical isolates from western Sydney 2006–2007 have been sequenced. The plasmid backbones are organised similarly to those of other IncI1 plasmids, but have insertions and/or deletions and sequence differences. Each plasmid also has a different insertion carrying blaCTX-M-15. pJIE113 (IncI1 sequence type ST31) is almost identical to plasmids isolated from the 2011 E. coli O104:H4 outbreak in Europe, where the typical blaCTX-M-15 transposition unit interrupts a complete Tn2 inserted directly in the plasmid backbone. In the novel plasmid pJIE139 (ST88), ISEcp1-blaCTX-M-15-orf477Δ lies within a Tn2/3 hybrid transposon. Homologous recombination could explain movement of ISEcp1-blaCTX-M-15-orf477Δ between copies of Tn2 on IncF and IncI1 plasmids and generation of the Tn2/3 hybrid. pJIE174 (ST37) is almost identical to pESBL-12 from the Netherlands and in these plasmids blaCTX-M-15 is flanked by two copies of IS26 that truncate the transposition unit within a larger region bounded by the ends of Tn2. blaCTX-M-15 and the associated ISEcp1-derived promoter may be able to move from this structure by the actions of IS26, independently of both ISEcp1 and Tn2.

Introduction

The CTX-M extended spectrum β-lactamases (ESBL), particularly CTX-M-15, have become dominant worldwide in recent years (D'Andrea et al., 2013). blaCTX-M-3, the apparent progenitor of blaCTX-M-15, appears to have been captured from the Kluyvera ascorbata chromosome by the insertion sequence ISEcp1 (Rodríguez et al., 2004). ISEcp1 is bounded by 14-bp inverted repeats (IRL and IRR) but uses IRL in conjunction with alternative sequences resembling these IR to mobilise adjacent regions, creating 5-bp direct repeats (DR) of flanking sequence on transposition and ISEcp1 also provides a promoter for expression of adjacent genes (Poirel et al, 2003, Poirel et al, 2005).

blaCTX-M-15 has generally been found 48 bp beyond the IRR end of ISEcp1 in a 2.971-kb transposition unit containing a 1.315-kb fragment of the K. ascorbata chromosome that also includes a partial open reading frame, designated orf477Δ (Fig. 1A). In IncF plasmids, which commonly carry blaCTX-M-15 in Escherichia coli, this transposition unit is inserted in the transposon Tn2, which carries blaTEM-1b (Bailey et al, 2011, Partridge, Hall, 2005). Tn2 also includes tnpA (transposase) and tnpR (resolvase) genes and a resolution (res) site and is flanked by 38-bp inverted repeats, designated IRtnp and IRTEM here. In these IncF plasmids, the IRtnp end of Tn2 is truncated by IS26 (Fig. 1B) and this structure has been found as part of large multiresistance regions (MRR) that also include different combinations of the blaOXA-30, aac(3)-IIe (Partridge, 2011), aac(6′)-Ib-cr and tetA(A) genes and sometimes a class 1 integron and other resistance genes (Boyd et al, 2004, Partridge et al, 2011b, Woodford et al, 2009). blaCTX-M-15 is often associated with E. coli multi-locus sequence type (ST) 131, particularly when carried on IncF plasmids, and with clonal complex CC405 (D'Andrea et al, 2013, Naseer, Sundsfjord, 2011).

blaCTX-M-15 has also been found on IncI1 plasmids, but generally with fewer additional resistance genes than in IncF plasmids (e.g. Hopkins et al., 2006) and a few IncI1 plasmids carrying blaCTX-M-15 have now been completely sequenced. A survey demonstrating the dominance of blaCTX-M genes in clinical isolates from western Sydney, Australia in 2005–2007 revealed that five E. coli with four different pulsed-field gel electrophoresis (PFGE) profiles carried blaCTX-M-15 on an IncI1 plasmid (Zong et al., 2008). Here, we have completely sequenced three of these plasmids to compare the blaCTX-M-15 contexts with those on IncF plasmids and the plasmid backbones with other IncI1 plasmids.

Section snippets

Bacterial strains

Five E. coli (JIE113, JIE139, JIE174, JIE236 and JIE242) carrying blaCTX-M-15 and an IncI1 replicon isolated in June 2006–January 2007 from urine samples from different patients were from a larger collection of Enterobacteriaceae clinical isolates referred for microbiological testing at Westmead Hospital, Sydney, Australia (Zong et al., 2008). As the PFGE fingerprints of JIE236 and JIE242 were identical (Zong et al., 2008), only JIE242 was studied further. Multi-locus sequencing typing (MLST)

blaCTX-M-15 is carried by different strains and different IncI1 plasmids

By MLST, JIE113 is ST448, both JIE139 and JIE242 are ST69, and JIE174 is the novel ST2495. This contrasts with the isolates from the same collection with blaCTX-M-15 on IncF or IncX4 plasmids, which were either ST131 (n = 8) or ST405 (n = 4) (Partridge et al, 2011a, Partridge et al, 2011b), although ST2495 differs from ST405 by only 3 nucleotide changes in a single allele, gyrB. ST69 is part of E. coli “clonal group A” (CgA), originally identified in a community outbreak of urinary tract

Conclusions

Comparison of the plasmids analysed here with the well-characterised R64 and R621a builds on the conclusions of Takahashi et al. (2011) about a conserved overall organisation of IncI1 plasmids with various insertions, deletions and substitutions that may influence compatibility and other functions. The ST31 and ST37 sequences also suggest that, unlike the case of ST2 plasmids carrying blaCMY-2 (Tagg et al., 2014), plasmids assigned to at least some other pMLST groups include backbones that are

Acknowledgments

Z.Z. was supported by an Endeavour International postgraduate Research Scholarship from the Australian Government Department of Education, Science and Training. Plasmid sequencing was funded by a Research Grant from the Australian Society for Antimicrobials. This work was supported by grants G1001021, G1002076 and G1046886 from the Australian National Health and Medical Research Council. We thank Carola Venturini for preparing plasmid DNA, Joey Lai for library preparation and Michael Roper and

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    Communicated by Julian Rood.

    1

    Permanent address: Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic; CEITEC VFU, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic.

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