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

ARTICLE IN PRESS Plasmid ■■ (2015) ■■–■■

Contents lists available at ScienceDirect

Plasmid j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y p l a s

Different IncI1 plasmids from Escherichia coli carry ISEcp1-blaCTX-M-15 associated with different Tn2-derived elements Zhiyong Zong a,b, Andrew N. Ginn a, Hana Dobiasova a,c,d,1, Jonathan R. Iredell a, Sally R. Partridge a,* a Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, The University of Sydney and Westmead Hospital, Westmead, NSW 2145, Australia b Department of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, China c Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic d CEITEC VFU, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic

A R T I C L E

I N F O

Article history: Available online Keywords: IncI1 blaCTX-M-15 ISEcp1 Tn2 IS26 Recombination

A B S T R A C T

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. © 2015 Elsevier Inc. All rights reserved.

Communicated by Julian Rood. * Corresponding author. Centre for Infectious Diseases and Microbiology, Level 4, Westmead Millennium Institute, 176 Hawkesbury Road, Westmead, NSW 2145, Australia. Fax: +61 2 8672 3099. E-mail address: [email protected] (S.R. Partridge). 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.

1. 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

http://dx.doi.org/10.1016/j.plasmid.2015.04.007 0147-619X/© 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: Zhiyong Zong, Andrew N. Ginn, Hana Dobiasova, Jonathan R. Iredell, Sally R. Partridge, Different IncI1 plasmids from Escherichia coli carry IS Ecp1-blaCTX-M-15 associated with different Tn2-derived elements, Plasmid (2015), doi: 10.1016/j.plasmid.2015.04.007

ARTICLE IN PRESS 2

Z. Zong et al./Plasmid ■■ (2015) ■■–■■

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, 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 and 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 multilocus sequence type (ST) 131, particularly when carried on IncF plasmids, and with clonal complex CC405 (D’Andrea et al., 2013; Naseer and 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 bla CTX-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 bla CTX-M-15 contexts with those on IncF plasmids and the plasmid backbones with other IncI1 plasmids.

Fig. 1. Contexts of ISEcp1-blaCTX-M-15 transposition units. Selected genes are indicated by labelled arrows. IS are represented by boxes labelled with their name or number, with the pointed end indicating IRR. The 38 bp IR of Tn2 and Tn3 are shown as tall black bars, with other parts of these transposons shaded and the res site shown as a small black box. Sequences of 5 bp DRs are given. (A) Part of the K. ascorbata chromosome with blaCTX-M-3 and orf477. (B) The ISEcp1-blaCTX-M-15-orf477Δ transposition unit inserted in Tn2 truncated by IS26 in IncF plasmids such as pC15-1a. (C) The ISEcp1-blaCTX-M-15-orf477Δ transposition unit in an interrupted but complete Tn2 in IncI1 ST31 plasmids. The vertical arrow shows the site of insertion of IS1294b in pEC_Bactec. (D) The ISEcp1-blaCTX-M-15-orf477Δ transposition unit in the Tn2/Tn3 hybrid transposon with an internal deletion in pJIE139. The 129 bp immediately upstream of ISEcp1 exactly match both Tn2 and Tn3. The switch between Tn2 and Tn3 sequences occurs between positions 802 and 1228 bp from the outer end of IRtnp. (E) The longer ISEcp1-blaCTX-M-15-orf477 transposition unit inserted in Tn2 in pECN580 and pKo6. Diagrams are drawn from the following GenBank accession nos.: K. ascorbata chromosome, AJ632119; pC15-1a, AY458016; ST31 plasmids, see Table 1; pJIE139, EU418926; pECN580, KF914891; pKo6, KC958437.


ARTICLE IN PRESS Z. Zong et al./Plasmid ■■ (2015) ■■–■■

2. Materials and methods 2.1. 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) of isolates was carried out using the scheme formerly available at http://mlst.ucc.ie/mlst/ dbs/Ecoli, now at http://mlst.warwick.ac.uk/mlst/dbs/Ecoli (Wirth et al., 2006). For each of the four isolates, a single transconjugant in E. coli DH5αRf demonstrated to carry blaCTX-M-15 and an IncI1 replicon by PCR (Zong et al., 2008) was selected for further study. 2.2. Plasmid analysis S1 nuclease followed by PFGE (Barton et al., 1995; Partridge et al., 2011b) was used to estimate plasmid sizes. Plasmids were initially compared by digestion with PstI according to the manufacturer’s instructions (New England Biolabs, Ipswich, MA) and plasmid multi-locus sequence typing (pMLST) using published primers (Garcia-Fernandez et al., 2008), with alleles and sequence types designated by http://pubmlst.org/plasmid. 2.3. PCR amplification PCR primers designed as part of this study and selected published primers are given in Table S1. Crude lysates were prepared for use as templates as described previously (Partridge et al., 2011b). Long range PCR was carried out using the Expand Long Template PCR system (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. PCR amplicons were purified for sequencing using a PureLink PCR Purification Kit (Invitrogen, Carlsbad, CA, USA). 2.4. Initial mapping of blaCTX-M-15 contexts Initially, a combination of methods was used to determine the contexts of blaCTX-M-15, including PCR mapping (Fig. S1). Whole plasmid DNA from alkaline lysis preparations was sequenced directly following treatment with polyethylene glycol 8000, as described previously (Partridge et al., 2011b). Sanger sequencing was carried out on an ABI PRISM 3100 Genetic Analyzer at the Westmead DNA facility or by Macrogen (Seoul, Korea). Sequences obtained generally allowed identification of a matching region in GenBank by BLAST searching. Primers (Table S1) were then designed to confirm links between regions using conventional or long range PCR and selected regions sequenced. The blaCTX-M-15 region of pJIE242 was cloned by ligating XhoIdigested plasmid DNA to pBC SK + phagemid (Stratagene, La Jolla, CA, USA) digested with SalI and transforming into E. coli DH5α with selection on nutrient agar (Oxoid, Basingstoke, UK) plates containing chloramphenicol (Sigma,

3

St Louis, MO, USA; 20 μg/ml) and cefotaxime (Sigma; 8 μg/ml). Plasmid DNA from a transformant confirmed to carry blaCTX-M-15 by PCR was prepared for sequencing using a PureLink Quick Plasmid Miniprep kit (Invitrogen). 2.5. Illumina sequencing of plasmids Plasmid DNA was purified from E. coli DH5αRf transconjugants of clinical isolates using the HiSpeed Plasmid MIDI kit (Qiagen, Vic, Australia) following the manufacturer’s instructions. Libraries were prepared from 1 ng of DNA using a Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, USA). Raw reads from paired-end 250-bp MiSeq (Illumina) sequencing (Australian Genomics Research Facility) were pre-processed using FLASH (Magoc and Salzberg, 2011) and assembled into contigs using a combination of Velvet 1.2.09 (Zerbino and Birney, 2008), SPAdes 2.5.0 (Bankevich et al., 2012), CLC Genomics Workbench (CLC bio, Aarhus, Denmark) and/or Geneious R7 (Biomatters, New Zealand). Scaffolding was performed using SSPACE 2.0 (Boetzer et al., 2011). Contigs were assembled and analysed using Geneious R7, Lasergene (DNASTAR Inc., Madison, WI) and Gene Construction Kit (Textco BioSoftware Inc, West Lebanon, NH, USA). Sequences were annotated using RAST (Overbeek et al., 2014) and by comparison with well-characterised plasmids R64 (IncI1; GenBank accession no. AP005147; Sampei et al., 2010) and R621a (IncIγ; AP011954; Takahashi et al., 2011). Similarity searches were carried out using programs available through http://www.ncbi.nlm.nih.gov/. Single nucleotide differences between closely related plasmids were checked in raw reads using Geneious and in some cases the relevant regions were also amplified (Table S1) and sequenced. 2.6. Nucleotide sequence accession numbers The complete sequences of the plasmids analysed here have been submitted to GenBank under the following accession nos.: pJIE113, EU418923; pJIE139, EU418926; pJIE174, EU418931. The sequences submitted each represent only one possible arrangement of shufflon segments, as noted in the GenBank entries. 3. Results and discussion 3.1. 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, 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 infections in the USA (Manges et al., 2001) and since identified in several other countries, including Australia (Platell et al., 2010). blaCTX-M-15 may have been previously indentified



4

Table 1 Characteristics of isolates and related IncI1 plasmids carrying blaCTX-M-15. Isolatea

Isolation date

Phylo group

JIE113 Bactec 2011C-3493 HUSEC2011 JIE139 JIE242 JIE174 ESBL-12

22/07/06

B1

448

D D D

678 678 69 69 2495

05/2011 2011 05/06/06 11/01/07 05/10/06

STb

Plasmida

Size (kb)

pST

GenBank

pJIE113 pEC_Bactec pESBL-EA11 pHUSEC2011-1 pJIE139 pJIE242 pJIE174 pESBL-12

91.205 92.970 88.544 88.546 87.925 ~92 96.452 96.463

31 31 31 31 88 37 37 37

EU418923 GU371927 CP003290 HE610900 EU418926 EU418931 CP008735

a

Names of isolates and plasmids characterised here are in bold typeface. 2011C-3493 and HUSEC2011 were identified as ST678 from the sequences available in GenBank accession nos. CP003289 and HF572917, respectively. ST448 shares four of seven alleles with 678, but has 2–12 differences in the remaining three. The ST of the isolates carrying pEC_Bactec and pESBL-12 were not reported. b

in ST69 (Brolund et al., 2014) but does not appear to have been seen in ST448. S1 nuclease/PFGE (Fig. S2A) of one transconjugant carrying blaCTX-M-15 from each of the four isolates suggested that a single plasmid (designated pJIE followed by the original isolate number) was present in each case. pJIE174 and pJIE242 gave similar PstI restriction patterns, but pJIE113 and pJIE139 gave patterns that were distinct from these and from each other (Fig. S2B). pJIE113 was identified as IncI1 pMLST ST31. The 21 other IncI1 ST31 plasmids listed at http://pubmlst.org/plasmid (April 2015) all carry blaCTX-M-15 and all but one (Shigella sonnei from the USA) are from E. coli isolates from various western European countries. IncI1 pMLST types ST8 and ST68 each have only one difference from ST31 in a single allele (ardA and rep, respectively) and are considered part of clonal complex (CC) 31. The three listed ST8 plasmids, from Salmonella from the UK, and two listed ST68 plasmids, from E. coli in France, also all carry blaCTX-M-15, leading to the suggestion that this gene has been acquired by the CC31 lineage (Johnson et al., 2011). pJIE174 and pJIE242 are both ST37 and the three other ST37 plasmids listed at http://pubmlst.org/plasmid, from E. coli from the Netherlands or UK, also all carry blaCTX-M-15. pJIE139, with three novel alleles, was designated ST88 (Table 1). pJIE113, pJIE139 and pJIE174 were sequenced, assembled, annotated and analysed. 3.2. pJIE113, pJIE139 and pJIE174 have typical IncI1 plasmid organisation Raw reads for each of the three plasmids were assembled into ~6–9 contigs (>1000 fold coverage), but different assembly methods often gave different contig boundaries. The combined contigs for each plasmid were assembled using information from mapping of the resistance regions to close gaps. Four shufflon segments (A, B, C, D), corresponding to those in R64, were identified among contigs for each plasmid, but these contigs could not be definitively assembled into any particular order (Brouwer et al., 2015). PCR (Table S1) across the shufflon region amplified several bands, including one of the expected size (2.1 kb) for a complete shufflon. Sequencing of this amplicon gave mixed bases beyond the first shufflon repeat in all cases, suggesting that multiple arrangements are present in the plasmid populations of these

isolates. Shufflon segments were assembled in the same order as in R64 to close the plasmid sequences. All three plasmids have the same overall backbone organisation as other IncI1 plasmids, corresponding to replication, leading and transfer regions, but with different insertions and/or deletions and sequence differences. These backbones were compared with those of the archetypal IncI1 plasmid R64 (Sampei et al., 2010) and the IncIγ plasmid R621a (Takahashi et al., 2011), the most extensively analysed of the available sequences of related plasmids. Some regions of the plasmids sequenced here are more closely related to R64, other regions to R621a and some regions differ from both of these plasmids (Fig. 2), suggesting that these IncI1 backbones are mosaics, presumably generated by recombination between related plasmids. Most hypothetical genes annotated in R64 and/or R621a could be identified in the three pJIE plasmids, with some exceptions due to frameshifts in genes overlapping repeat regions. The replication regions of the three pJIE plasmids each include an inc gene (encoding antisense RNA required for regulation of plasmid copy number and the basis of replicon incompatibility) identical to R64 and different from R621a, which is known to be compatible with R64 (Takahashi et al., 2011). repY (required for activation of repZ translation) of all three plasmids is identical to both R621a and R64. repZ (encoding the replication initiation protein) of pJIE139 and pJIE174 is almost identical (3 differences) to that of R64, while repZ of pJIE113 is closer (14 differences) to that of R621a. A recent transposon mutagenesis study proposed that the hmoA gene, present in pJIE113 and pJIE174 but not R64, R621a or pJIE139 (Fig. 2), may have a role in plasmid replication or segregation (Yamaichi et al., 2015). The leading region in all three plasmids includes the impABC genes, thought to be involved in induced mutagenesis of host cells, the psiAB (plasmid SOS inhibition) genes and the ardA gene (antirestriction). None of the three pJIE plasmids have the parAB genes found in R64, and a different parAB gene pair found inserted in the R621a leading region is present in pJIE174 only. These pairs of genes are listed as encoding a Type I partitioning system in Table 1 of Sampei et al. (2010) and Table 1 of Takahashi et al. (2011), respectively, but both actually appear to encode a Type II system (Gerdes et al., 2000). Like R621a, all three pJIE plasmids encode a proposed partitioning system consisting of



5

Fig. 2. Maps representing pJIE IncI1 plasmid backbones in comparison with R621a and R64. The shufflon has been omitted, but its position is indicated. Horizontal arrows show the extent and direction of selected genes. Arrow colours are designed to illustrate the mosaic nature of the backbones: black, identical or a few random differences across multiple sequences; purple, more closely related to R621a; red, more closely related to R64; cyan or gold, significantly different from R621a and R64 and from each other (from a MAUVE alignment in Geneious). Blue vertical lines indicate repeats identified by Sampei et al. (2010) and/or Takahashi et al. (2011), some of which are numbered. The positions of various insertions are indicated above each diagram, with * indicating the position of the different Tn2/ISEcp1-blaCTX-M-15 insertions (see Figs 1 and 3). This figure is based on Fig. 2 in (Takahashi et al., 2011) and diagrams are approximately to scale.

proteins named Soj and YfhA (a Spo0J homolog). This is listed as Type II system in Table 1 of Takahashi et al. (2011), but the Soj protein actually appears to have Walker-type ATPase motifs characteristic of Type I systems (Gerdes et al., 2000; Koonin, 1993). Repeat regions flanking the soj gene in R621a (Takahashi et al., 2011) are actually made up of tandemly repeated 8-bp sequences (usually AAGATAAC) present in both orientations. Different numbers of these repeats are present in IncI1 plasmids that carry soj (Fig. S3), but whether these repeats are involved in plasmid partitioning and/or whether the different numbers have any effect remains to be determined. The transfer region in all three pJIE plasmids included the expected IncI1 tra/trb conjugation genes. As previously noted for another ST31 plasmid pEC_Bactec (Takahashi et al., 2011), the excA and traY genes of pJIE113 are more similar to R621a than R64, as are those of pJIE174. In contrast, excA and traY of pJIE139 are more closely related to R64. The products of these genes are involved in entry exclusion (Sakuma et al., 2013) and may affect plasmid compatibility (Takahashi et al., 2011). A pil operon, for thin pilus formation, is present in all three pJIE plasmids, but pJIE113 and pJIE139 appear to have a 295 bp insertion between pilL and pilK compared with R64, R621a and pJIE174 and to lack pilJ. All three pJIE plasmids include the traABC genes that may be involved in expression of transfer genes but, like R621a, pJIE113 and pJIE139 lack both traD found in R64 and trcD found in ColIbP9 (Takahashi et al., 2011), while pJIE174 has trcD. The functions of traD and trcD have not yet been determined, so any biological relevance of these differences is presently unclear. An oriT site with 17 bp (repeat 1) and 8 bp (repeat 2) inverted repeats identical to R64 and R621a (pJIE174) or with complementary single nucleotide differences in the

two copies of repeat 1 (pJIE113, pJIE139) is present upstream of nikA. All three pJIE plasmids carry a group II intron inserted at the same position, 39 bp after the start of the ssb gene encoding a single-stranded DNA binding protein (Golub and Low, 1985). The intron in pJIE139 has 18 nucleotide differences from the identical introns in pJIE113 and pJIE174. The same intron (1–3 nucleotide differences from pJIE113/174) is found in the same location in IncI1 plasmids pESBL-12 (blaCTX-M-15; ST37; CP008735), pESBL-283 (blaCTX-M-1; ST7; CP008736) and pH2291-112 (blaCTX-M-1; ST3; KJ484629). Insertional inactivation of ssb of the IncI1 plasmid ColIb-P9 suggested that this gene is not required for conjugation or plasmid stability, but effects on the downstream psiAB were seen (Howland et al., 1989). A recent transposon mutagenesis study also suggested that the ssb gene in pESBL-EA11 is not required for maintenance or transfer (Yamaichi et al., 2015). The same intron is also found in the ssb gene (~88% identical to the IncI1 ssb) in the IncF plasmids pEK499 (blaCTX-M-15; EU935739), pEC958 (blaCTX-M-15; HG941719) and pH2291-144 (KJ484628).

3.3. pJIE113 and related ST31 plasmids carry ISEcp1-blaCTX-M-15 within a complete Tn2 pJIE113 (91.205 kb) is almost identical to ST31 plasmids including pEC_Bactec, isolated from a horse in Belgium (Smet et al., 2010), and those from the 2011 O104:H4 outbreak in Europe, such as pESBL-EA11 (Ahmed et al., 2012) and pHUSEC2011-1 (GenBank accession no. HE610900) and others that have not been fully assembled and/or are not available in GenBank (e.g. Mellmann et al., 2011; Rohde et al.,


ARTICLE IN PRESS 6

Z. Zong et al./Plasmid ■■ (2015) ■■–■■

2011; Tietze et al., 2015). The sequences of the four plasmids listed above differ at a few positions, some of which could be sequencing errors in homopolymeric T regions (Table S2). pEC_Bactec alone carries an IS1294b element (1.713 kb) and an ISCro1-like element, here designated ISCro1b (2.699 kb), while only pJIE113 has the intron (section 3.1), explaining the differences in plasmid sizes (Table 1). In all four ST31 plasmids, a complete Tn2 carrying the 2.971-kb ISEcp1-blaCTX-M-15-orf477Δ transposition unit is inserted in the soj repeat region, flanked by DR (TTATC; Fig. 1C). The insertion of ISEcp1-blaCTX-M-15-orf477Δ in the tnpA transposition gene of Tn2 might be expected to prevent transposition. However, as both 38-bp IR and the res site are intact, it is possible that TnpA provided by another Tn3like transposon could mediate movement of this interrupted Tn2. If so, this configuration could be the predecessor of the structure found in the MRR of IncF plasmids, where the IRtnp end of Tn2 is truncated by IS26 (Fig. 1A). Alternatively, doublecrossover between an intact copy of Tn2 and the Tn2 segments flanking ISEcp1-blaCTX-M-15-orf477Δ could be responsible for moving a region that includes this transposition unit between different copies of Tn2. In this case exchange of ISEcp1-blaCTX-M-15-orf477Δ, either from IncI1 to IncF plasmids or from IncF to IncI1 plasmids, would be possible. 3.4. pJIE139 is a novel plasmid carrying ISEcp1-blaCTX-M-15 in a hybrid Tn2/Tn3 transposon The backbone of pJIE139 (87.925 kb) is ≤99% identical to the most closely related IncI1 plasmids, with insertions of the group II intron (section 3.1) and a Tn2-derived transposon inserted in the yagA hypothetical gene found adjacent to soj, flanked by DR (TAAGA). The typical 2.971-kb ISEcp1-blaCTX-M-15-orf477Δ transposition unit is found within this transposon, with the same orf477Δ/Tn2 boundary downstream of blaCTX-M-15 as in pJIE113 and MRR on IncF plasmids. However, only a 129-bp fragment of the IRTEM end of Tn2, which does not include blaTEM-1b, is present immediately upstream of ISEcp1 and part of the IRtnp end of the transposon matches Tn3 rather than Tn2 (Fig. 1D) (Bailey et al., 2011; Partridge and Hall, 2005). Searches with the boundary between Tn2 and IRL of ISEcp1 revealed a match to several plasmids, including the closely related IncN plasmids pECN580 (Chen et al., 2014) and pKo6 (KC958437). The ISEcp1 transposition unit in these plasmids carries a 1.761kb fragment of the K. ascorbata chromosome, with a complete orf477 and a 127 bp ‘spacer’ between IRR of ISEcp1 and the start codon of blaCTX-M-3, inserted in a different place in Tn2 (Fig. 1E). Recombination in ISEcp1 could explain linkage of the 2.971-kb ISEcp1-blaCTX-M-15-orf477Δ transposition unit to the 129-bp transposon region found in pECN580/pKo6 and the creation of the Tn2/Tn3 hybrid structure. The sequences of Tn2 and Tn3 are identical over the 129 bp fragment at the IRTEM end present in pJIE139, so it is not possible to determine the origin of this part of the transposon. The hybrid transposon in pJIE139 is missing the res site in addition to part of tnpA which would prevent res-mediated resolution even if it could be transposed by TnpA supplied by another transposon. However, it is flanked by 5-bp DRs and it is possible that an intact Tn2/Tn3 hybrid structure was created

elsewhere and inserted in this location by transposition and then modified.

3.5. pJIE174 carries blaCTX-M-15 flanked by directly oriented copies of IS26 pJIE174 (96.452 kb) is almost identical to the recently reported IncI1 ST37 plasmid pESBL-12 (96.463 kb) from E. coli from human urine from The Netherlands (Brouwer et al., 2014). There is only one nucleotide difference between their backbones: a G in pJIE174 at position 19305 (confirmed by examination of raw reads), immediately upstream of the yfaA gene encoding a hypothetical protein, is A in pESBL-12. In pJIE174, pESBL-12, and also a cloned fragment of pJIE242, a region carrying blaCTX-M-15 and apparently derived from Tn2 is inserted in the same position, but the ISEcp1blaCTX-M-15-orf477Δ transposition unit is truncated at both ends by IS26 (Fig. 3A). One copy of IS26 truncates ISEcp1, leaving 388 bp of ISEcp1 in pJIE174/pJIE242 and 399 bp in pESBL-12. The IS26-ΔISEcp1-blaCTX-M-15 boundary in pJIE174 was also seen in plasmids from river sediment isolates from the UK (KF155155) (Amos et al., 2014), while the boundary in pESBL-12 was also reported in an E. coli from a bovine mastitis isolate from the UK (KC778404) (Timofte et al., 2014). The IRR end of this copy of IS26 is separated from a 38 bp IR matching that of Tn2 by a single C residue, which suggests that it corresponds to IRtnp of Tn2. The second copy of IS26 truncates orf477Δ and is followed by a region containing aac(3)-IId (Partridge, 2011), ISCfr1 and part of Tn2, including blaTEM-1b (but not its promoter region) and IRTEM. A related region is found in the IncL/M plasmid pCTX-M3 (AF550415) (Gołe˛biewski et al., 2007) except that the armA, mph(E) and msr(E) genes and a class 1 integron are found between the two IS26 elements (Tn1548; Galimand et al., 2005) instead of the ΔISEcp1-blaCTX-M-15-orf477Δ region (Fig. 3B). These two different structures could result from insertion of different IS26mediated “translocatable units” adjacent to the same copy of IS26 (Harmer et al., 2014), with an additional IS26mediated deletion possibly explaining the loss of most of the tnpA end of Tn2 compared with pCTX-M3. pCTX-M3 may have arisen from a plasmid like pCTX-M360 (EU938349), which carries an intact copy of Tn2 inserted in the same position (Zhu et al., 2009) (Fig. 3C). The inserts in pJIE174/242, pESBL-12 and pCTX-M3 and pCTX-M360 are flanked by 5-bp DR, suggesting insertion of an intact copy of Tn2 followed by modification. These DR are the reverse complements of one another in the IncI1 and IncL/M plasmids but the sequences immediately beyond these 5 bp are different and an uninterrupted version of each flanking sequence is available. Both pJIE174 and pESBL-12 have an additional insertion (positions 34432-81 in pJIE174) in the same location as Tn10 in R621a, flanked by the same 9-bp DR (TACCAG GTG). This insertion is a 50 nucleotide remnant of Tn10, consisting of IRL and IRR of IS10 separated by 6 bp of IS10. Such “nearly precise” excision of Tn10 was identified some time ago (Foster et al., 1981) and suggests that pJIE174/ESBL-12 type plasmids are derived from a plasmid carrying the complete Tn10.



7

Fig. 3. blaCTX-M-15 context in pJIE174/242 and pESBL-12. Components are generally shown as described in the legend of Fig. 1. Gene cassettes are shown as narrow open boxes with small filled boxes representing the attC sites. Δ indicates the 388-bp (pJIE174/242) or 399-bp (pESBL-12) remnant of ISEcp1 truncated by IS26. The single C residue separating the IRR end of this IS26 from a 38-bp IR suggests that this IR is IRtnp. A 135-bp fragment of the IRTEM end of Tn2 separates IRL of IS26 from the aac(3)-IId region. This structure is compared with the pCTX-M3 (GenBank accession no. AF550415) and pCTX-M360 (EU938349) resistance regions.

4. Conclusions Comparison of the plasmids analysed here with the wellcharacterised 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 highly conserved across their whole length. As more IncI1 plasmids are sequenced, this classification system may be able to be refined further. The various insertions carrying blaCTX-M-15 in the plasmids characterised here all lie in a similar region of the plasmid backbone, in or near the yagA hypothetical gene (see Figs 1 and 3). The insertion in pJIE113 and other ST31 plasmids lies downstream of yagA, while the hybrid transposon in pJIE139 and the resistance region in pJIE174/pJIE242 are inserted at different points within yagA (Fig. 2). This region, which lies between the sites targeted by the pMLST IncI1 rep and ardA primers, appears to be a more variable part of the IncI1 backbone and a common target for insertions in IncI1 plasmids (Johnson et al., 2011). The detection of closely related IncI1 ST37 plasmids pJIE174 and pJIE242 in different E. coli ST suggests local spread of a plasmid between strains, but this plasmid type is also present in The Netherlands (Brouwer et al., 2014) and cattle in the UK (http://pubmlst.org/plasmid), suggesting a wider distribution. Related IncF plasmids carrying blaCTX-M-15 have also spread to different locations, including Canada

(Boyd et al., 2004), the UK (Woodford et al., 2009) and Australia (Partridge et al., 2011b), but in this case associated with a single E. coli clonal type, ST131. The detection of an IncI1 ST31 plasmid in an S. sonnei isolate in Spain possibly as early as 2001, from a patient with no history of travel abroad (Seral et al., 2012), in isolates from humans and animals in different locations from 2004 onwards (http://pubmlst.org/plasmid), and of pJIE113 (the earliest ST31 plasmid to be sequenced) in Australia in 2006 suggests that this type of plasmid may have been widespread before the 2011 European O104:H4 outbreak. IncI1 ST31 plasmids do not appear to have been reported in more recent isolates, but may have spread beyond the outbreak, as an O91:H14 E. coli isolated in 2011 in Germany, from a patient with probable exposure to outbreak isolates but no symptoms, appeared to have a similar plasmid (Arvand et al., 2015). However, this patient had also been recently hospitalised and had travelled to India and two contemporaneous O104:H4 isolates epidemiologically unrelated to outbreak isolates appeared not to have the IncI1 ST31 plasmid (Tietze et al., 2015). As in IncF plasmids, blaCTX-M-15 in IncI1 plasmids appears to be commonly associated with Tn2 and/or IS26 in addition to ISEcp1, which may free this gene from some of the limitations of its original genetic background (O’Brien, 2002). The IncF and pJIE174/242 configurations may allow subsequent movement by a recently demonstrated mechanism that involves a single copy of IS26 (Harmer et al., 2014) or possibly as part of different IS26-mediated composite transposons, in either case retaining the promoter provided by ISEcp1. Association with Tn2, acting as a mobile target



8

for homologous recombination, may also be important in the spread of blaCTX-M-15, especially as complete or partial copies of Tn2 appear common in MRR and on plasmids (Bailey et al., 2011). Fully sequenced IncI1 plasmids carrying blaCTX-M-15, plus other limited information, suggest that these plasmids carry fewer resistance genes than IncF plasmids with blaCTX-M-15. The pJIE174/pJIE242 structure is slightly more complex, but still only includes one additional resistance gene, aac(3)IId conferring gentamicin and tobramycin resistance. The presence of smaller resistance regions could possibly reflect limitations in the “load” that can be carried by IncI1 plasmids. The increasing availability of economical nextgeneration sequencing methods should enable comparative analysis to shed further light on how IncI1 plasmids have evolved and acquired resistance genes. 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 Grant Hill-Cawthorne for help with sequence assembly. This work made use of the original E. coli MLST website (http://mlst.ucc.ie/mlst/dbs/Ecoli) supported by a grant from the Science Foundation Ireland (05/FE1/B882), now at http://mlst.warwick.ac.uk/mlst/dbs/Ecoli, supported by the UK Biotechnology and Biological Sciences Research Council, and the plasmid MLST website, developed by Keith Jolley, sited at the University of Oxford and funded by the Wellcome Trust. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.plasmid.2015.04.007. References Ahmed, S.A., Awosika, J., Baldwin, C., Bishop-Lilly, K.A., Biswas, B., Broomall, S., et al., 2012. Genomic comparison of Escherichia coli O104:H4 isolates from 2009 and 2011 reveals plasmid, and prophage heterogeneity, including Shiga toxin encoding phage stx2. PLoS ONE 7, e48228. Amos, G.C., Hawkey, P.M., Gaze, W.H., Wellington, E.M., 2014. Waste water effluent contributes to the dissemination of CTX-M-15 in the natural environment. J. Antimicrob. Chemother. 69, 1785–1791. Arvand, M., Bettge-Weller, G., Fruth, A., Uphoff, H., Pfeifer, Y., 2015. Extended-spectrum beta-lactamase-producing Shiga toxin gene (stx1)-positive Escherichia coli O91:H14 carrying blaCTX-M-15 on an IncI1-ST31 plasmid isolated from a human patient in Germany. Int. J. Med. Microbiol. 305, 404–407. Bailey, J., Pinyon, J., Abnantham, S., Hall, R., 2011. Distribution of the blaTEM gene and blaTEM-containing transposons in commensal Escherichia coli. J. Antimicrob. Chemother. 66, 745–751. Bankevich, A., Nurk, S., Antipov, D., Gurevich, A.A., Dvorkin, M., Kulikov, A.S., et al., 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 19, 455–477.

Barton, B.M., Harding, G.P., Zuccarelli, A.J., 1995. A general method for detecting and sizing large plasmids. Anal. Biochem. 226, 235–240. Boetzer, M., Henkel, C.V., Jansen, H.J., Butler, D., Pirovano, W., 2011. Scaffolding pre-assembled contigs using SSPACE. Bioinformatics 27, 578–579. Boyd, D.A., Tyler, S., Christianson, S., McGeer, A., Muller, M.P., Willey, B.M., et al., 2004. Complete nucleotide sequence of a 92-kilobase plasmid harboring the CTX-M-15 extended-spectrum beta-lactamase involved in an outbreak in long-term-care facilities in Toronto, Canada. Antimicrob. Agents Chemother. 48, 3758–3764. Brolund, A., Edquist, P.J., Makitalo, B., Olsson-Liljequist, B., Soderblom, T., Wisell, K.T., et al., 2014. Epidemiology of extended-spectrum β-lactamase-producing Escherichia coli in Sweden 2007–2011. Clin. Microbiol. Infect. 20, O344–O352. Brouwer, M.S., Bossers, A., Harders, F., van Essen-Zandbergen, A., Mevius, D.J., Smith, H.E., 2014. Complete genome sequences of IncI1 plasmids carrying extended-spectrum β-lactamase genes. Genome Announc 2 (4), pii: e00859-14. Brouwer, M.S., Tagg, K.A., Mevius, D.J., Iredell, J.R., Bossers, A., Smith, H.E., et al., 2015. IncI shufflons: assembly issues in the next-generation sequencing era. Plasmid. In this issue. Chen, L., Hu, H., Chavda, K.D., Zhao, S., Liu, R., Liang, H., et al., 2014. Complete sequence of a KPC-producing IncN multidrug-resistant plasmid from an epidemic Escherichia coli sequence type 131 strain in China. Antimicrob. Agents Chemother. 58, 2422–2425. D’Andrea, M.M., Arena, F., Pallecchi, L., Rossolini, G.M., 2013. CTX-M-type β-lactamases: a successful story of antibiotic resistance. Int. J. Med. Microbiol. 303, 305–317. Foster, T.J., Lundblad, V., Hanley-Way, S., Halling, S.M., Kleckner, N., 1981. Three Tn10-associated excision events: relationship to transposition and role of direct and inverted repeats. Cell 23, 215–227. Galimand, M., Sabtcheva, S., Courvalin, P., Lambert, T., 2005. Worldwide disseminated armA aminoglycoside resistance methylase gene is borne by composite transposon Tn1548. Antimicrob. Agents Chemother. 49, 2949–2953. Garcia-Fernandez, A., Chiaretto, G., Bertini, A., Villa, L., Fortini, D., Ricci, A., et al., 2008. Multilocus sequence typing of IncI1 plasmids carrying extended-spectrum β-lactamases in Escherichia coli and Salmonella of human and animal origin. J. Antimicrob. Chemother. 61, 1229–1233. Gerdes, K., Moller-Jensen, J., Bugge Jensen, R., 2000. Plasmid and chromosome partitioning: surprises from phylogeny. Mol. Microbiol. 37, 455–466. Golub, E.I., Low, K.B., 1985. Conjugative plasmids of enteric bacteria from many different incompatibility groups have similar genes for singlestranded DNA-binding proteins. J. Bacteriol. 162, 235–241. Gołe˛biewski, M., Kern-Zdanowicz, I., Zienkiewicz, M., Adamczyk, M., Z˙ylin`ska, J., Baraniak, A., et al., 2007. Complete nucleotide sequence of the pCTX-M3 plasmid and its involvement in spread of the extended-spectrum β-lactamase gene blaCTX-M-3. Antimicrob. Agents Chemother. 51, 3789–3795. Harmer, C.J., Moran, R.A., Hall, R.M., 2014. Movement of IS26-associated antibiotic resistance genes occurs via a translocatable unit that includes a single IS26 and preferentially inserts adjacent to another IS26. MBio 5, e1801–e1814. Hopkins, K.L., Liebana, E., Villa, L., Batchelor, M., Threlfall, E.J., Carattoli, A., 2006. Replicon typing of plasmids carrying CTX-M or CMY β-lactamases circulating among Salmonella and Escherichia coli isolates. Antimicrob. Agents Chemother. 50, 3203–3206. Howland, C.J., Rees, C.E., Barth, P.T., Wilkins, B.M., 1989. The ssb gene of plasmid ColIb-P9. J. Bacteriol. 171, 2466–2473. Johnson, T.J., Shepard, S.M., Rivet, B., Danzeisen, J.L., Carattoli, A., 2011. Comparative genomics and phylogeny of the IncI1 plasmids: a common plasmid type among porcine enterotoxigenic Escherichia coli. Plasmid 66, 144–151. Koonin, E.V., 1993. A superfamily of ATPases with diverse functions containing either classical or deviant ATP-binding motif. J. Mol. Biol. 229, 1165–1174. Magoc, T., Salzberg, S.L., 2011. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27, 2957–2963. Manges, A.R., Johnson, J.R., Foxman, B., O’Bryan, T.T., Fullerton, K.E., Riley, L.W., 2001. Widespread distribution of urinary tract infections caused by a multidrug-resistant Escherichia coli clonal group. N. Engl. J. Med. 345, 1007–1013. Mellmann, A., Harmsen, D., Cummings, C.A., Zentz, E.B., Leopold, S.R., Rico, A., et al., 2011. Prospective genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology. PLoS ONE 6, e22751. Naseer, U., Sundsfjord, A., 2011. The CTX-M conundrum: dissemination of plasmids and Escherichia coli clones. Microb. Drug Resist. 17, 83–97.



O’Brien, T.F., 2002. Emergence, spread, and environmental effect of antimicrobial resistance: how use of an antimicrobial anywhere can increase resistance to any antimicrobial anywhere else. Clin. Infect. Dis. 34, S78–S84. Overbeek, R., Olson, R., Pusch, G.D., Olsen, G.J., Davis, J.J., Disz, T., et al., 2014. The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST). Nucleic Acids Res. 42, D206–D214. Partridge, S.R., 2011. Analysis of antibiotic resistance regions in Gramnegative bacteria. FEMS Microbiol. Rev. 35, 820–855. Partridge, S.R., Hall, R.M., 2005. Evolution of transposons containing blaTEM genes. Antimicrob. Agents Chemother. 49, 1267–1268. Partridge, S.R., Ellem, J.A., Tetu, S.G., Zong, Z., Paulsen, I.T., Iredell, J.R., 2011a. Complete sequence of pJIE143, a pir-type plasmid carrying ISEcp1-blaCTX-M-15 from an Escherichia coli ST131 isolate. Antimicrob. Agents Chemother. 55, 5933–5935. Partridge, S.R., Zong, Z., Iredell, J.R., 2011b. Recombination in IS26 and Tn2 in the evolution of multi-resistance regions carrying blaCTX-M-15 on conjugative IncF plasmids from Escherichia coli. Antimicrob. Agents Chemother. 55, 4971–4978. Platell, J.L., Cobbold, R.N., Johnson, J.R., Trott, D.J., 2010. Clonal group distribution of fluoroquinolone-resistant Escherichia coli among humans and companion animals in Australia. J. Antimicrob. Chemother. 65, 1936–1938. Poirel, L., Decousser, J.W., Nordmann, P., 2003. Insertion sequence ISEcp1B is involved in expression and mobilization of a blaCTX-M β-lactamase gene. Antimicrob. Agents Chemother. 47, 2938–2945. Poirel, L., Lartigue, M.F., Decousser, J.W., Nordmann, P., 2005. ISEcp1Bmediated transposition of blaCTX-M in Escherichia coli. Antimicrob. Agents Chemother. 49, 447–450. Rodríguez, M.M., Power, P., Radice, M., Vay, C., Famiglietti, A., Galleni, M., et al., 2004. Chromosome-encoded CTX-M-3 from Kluyvera ascorbata: a possible origin of plasmid-borne CTX-M-1-derived cefotaximases. Antimicrob. Agents Chemother. 48, 4895–4897. Rohde, H., Qin, J., Cui, Y., Li, D., Loman, N.J., Hentschke, M., et al., 2011. Open-source genomic analysis of Shiga-toxin-producing E. coli O104:H4. N. Engl. J. Med. 365, 718–724. Sakuma, T., Tazumi, S., Furuya, N., Komano, T., 2013. ExcA proteins of IncI1 plasmid R64 and IncIγ plasmid R621a recognize different segments of their cognate TraY proteins in entry exclusion. Plasmid 69, 138–145. Sampei, G., Furuya, N., Tachibana, K., Saitou, Y., Suzuki, T., Mizobuchi, K., et al., 2010. Complete genome sequence of the incompatibility group I1 plasmid R64. Plasmid 64, 92–103. Seral, C., Rojo-Bezares, B., Garrido, A., Gude, M.J., Saénz, Y., Castillo, F.J., 2012. [Characterisation of a CTX-M-15-producing Shigella sonnei in a Spanish

9

patient who had not travelled abroad]. Enferm. Infecc. Microbiol. Clin. 30, 469–471. Smet, A., Van Nieuwerburgh, F., Vandekerckhove, T.T., Martel, A., Deforce, D., Butaye, P., et al., 2010. Complete nucleotide sequence of CTX-M-15plasmids from clinical Escherichia coli isolates: insertional events of transposons and insertion sequences. PLoS ONE 5, e11202. Tagg, K.A., Iredell, J.R., Partridge, S.R., 2014. Complete sequencing of IncI1 sequence type 2 plasmid pJIE512b indicates mobilization of blaCMY-2 from an IncA/C plasmid. Antimicrob. Agents Chemother. 58, 4949–4952. Takahashi, H., Shao, M., Furuya, N., Komano, T., 2011. The genome sequence of the incompatibility group Iγ plasmid R621a: evolution of IncI plasmids. Plasmid 66, 112–121. Tietze, E., Dabrowski, P.W., Prager, R., Radonic, A., Fruth, A., Aurass, P., et al., 2015. Comparative genomic analysis of two novel sporadic Shiga toxin-producing Escherichia coli O104:H4 strains isolated 2011 in Germany. PLoS ONE 10, e0122074. Timofte, D., Maciuca, I.E., Evans, N.J., Williams, H., Wattret, A., Fick, J.C., et al., 2014. Detection and molecular characterization of Escherichia coli CTX-M-15 and Klebsiella pneumoniae SHV-12 β-lactamases from bovine mastitis isolates in the United Kingdom. Antimicrob. Agents Chemother. 58, 789–794. Wirth, T., Falush, D., Lan, R., Colles, F., Mensa, P., Wieler, L.H., et al., 2006. Sex and virulence in Escherichia coli: an evolutionary perspective. Mol. Microbiol. 60, 1136–1151. Woodford, N., Carattoli, A., Karisik, E., Underwood, A., Ellington, M.J., Livermore, D.M., 2009. Complete nucleotide sequences of plasmids pEK204, pEK499, and pEK516, encoding CTX-M enzymes in three major Escherichia coli lineages from the United Kingdom, all belonging to the international O25:H4-ST131 clone. Antimicrob. Agents Chemother. 53, 4472–4482. Yamaichi, Y., Chao, M.C., Sasabe, J., Clark, L., Davis, B.M., Yamamoto, N., et al., 2015. High-resolution genetic analysis of the requirements for horizontal transmission of the ESBL plasmid from Escherichia coli O104:H4. Nucleic Acids Res. 43, 348–360. Zerbino, D.R., Birney, E., 2008. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18, 821–829. Zhu, W.H., Luo, L., Wang, J.Y., Zhuang, X.H., Zhong, L., Liao, K., et al., 2009. Complete nucleotide sequence of pCTX-M360, an intermediate plasmid between pEL60 and pCTX-M3, from a multidrug-resistant Klebsiella pneumoniae strain isolated in China. Antimicrob. Agents Chemother. 53, 5291–5293. Zong, Z., Partridge, S.R., Thomas, L., Iredell, J.R., 2008. Dominance of blaCTX-M within an Australian extended-spectrum β-lactamase gene pool. Antimicrob. Agents Chemother. 52, 4198–4202.


ST114 conjugative plasmids carrying blaCTX-M-8 in Escherichia coli isolated from poultry in Brazil.

Coverage of diarrhoea-associated Escherichia coli isolates from different origins with two types of phage cocktails.

Complex dissemination of the diversified mcr-1-harbouring plasmids in Escherichia coli of different sequence types.

AmpC Genes on IncI1 Plasmids in the Colombian Poultry Chain.

F' plasmids from HfrH and HfrC in recA- Escherichia coli.

Chloramphenicol resistance plasmids in Escherichia coli isolated from diseased piglets.

Analysis of K99 plasmids from enterotoxigenic Escherichia coli.

Microcin plasmids: a group of extrachromosomal elements coding for low-molecular-weight antibiotics in Escherichia coli.

Plasmids of Escherichia coli as cloning vectors.

DNA synthesis of plasmids in Escherichia coli.

High efficiency transformation of Escherichia coli with plasmids.

Antibiotic resistance and Hly plasmids in serotypes of Escherichia coli associated with porcine enteric disease.

Analogs of the dnaB gene of Escherichia coli K-12 associated with conjugative R plasmids.

-65 in Escherichia coli from chickens in China.

The different ecological niches of enterotoxigenic Escherichia coli.

Different lethal effects by enzyme-generated triplet indole-3-aldehyde in different Escherichia coli strains.

Sensitivity of Escherichia coli to cephaloridine at different growth rates.

Are the aerobic and anaerobic phosphofructokinases of Escherichia coli different?

Envelope-associated folded chromosomes for Escherichia coli: variations under different physiological conditions.

Identity elements of Escherichia coli tRNA(Ala).

Surveys of human enterotoxigenic Escherichia coli from three different geographical areas for possible colonization factors.

Escherichia coli E-39 ADPglucose synthetase has different activation kinetics from the wild-type allosteric enzyme.

Relationship of biofilm formation and different virulence genes in uropathogenic Escherichia coli isolates from Northwest Iran.

Characterization of Vaginal Escherichia coli Isolated from Pregnant Women in Two Different African Sites.