Molecular Characterization of a Multidrug
Abstract 
Escherichia coli sequence type 131 ( E. coli ST131 ) is a recently emerged and globally disseminated multidrug resistant clone associated with urinary tract and bloodstream infections . 
Plasmids represent a major vehicle for the carriage of antibiotic resistance genes in E. coli ST131 . 
In this study , we determined the complete sequence and performed a comprehensive annotation of pEC958 , an IncF plasmid from the E. coli ST131 reference strain EC958 . 
Plasmid pEC958 is 135.6 kb in size , harbours two replicons ( RepFIA and RepFII ) and contains 12 antibiotic resistance genes ( including the blaCTX-M-15 gene ) . 
We also carried out hyper-saturated transposon mutagenesis and multiplexed transposon directed in-sertion-site sequencing ( TraDIS ) to investigate the biology of pEC958 . 
TraDIS data showed that while only the RepFII replicon was required for pEC958 replication , the RepFIA replicon contains genes essential for its partitioning . 
Thus , our data provides direct evidence that the RepFIA and RepFII replicons in pEC958 cooperate to ensure their stable inheritance . 
The gene encoding the antitoxin component ( ccdA ) of the post-segregational killing system 
CcdAB was also protected from mutagenesis , demonstrating this system is active . 
Sequence comparison with a global collection of ST131 strains suggest that IncF represents the most common type of plasmid in this clone , and underscores the need to understand its evolution and contribution to the spread of antibiotic resistance genes in E. coli ST131 . 
Introduction
Escherichia coli sequence type 131 ( E. coli ST131 ) is a recently emerged and globally disseminated multidrug resistant clone associated with urinary tract and bloodstream infections [ 1 , 2 ] . 
E. coli ST131 was originally identified in 2008 as a major clone linked to the spread of the CTX-M-15 ex-tended-spectrum β-lactamase ( ESBL ) - resistance gene [ 3 -- 5 ] . 
Since then , E. coli ST131 has also been strongly associated with fluoroquinolone resistance , as well as co-resistance to aminoglycosides and trimethoprim-sulfamethoxazole [ 6 , 7 ] . 
Recent analyses of the global epidemiology of E. coli ST131 using whole genome sequencing has revealed the CTX-M-15 allele is highly prevalent within a fluoroquinolone resistant-FimH30 ( H30 ) ST131 sublineage [ 8 ] and demonstrated a significant role for recombination in the evolution of this E. coli lineage [ 9 ] . 
As observed for most other multidrug resistant Enterobacteriaciae pathogens , plasmids are the major vehicles for carriage of antibiotic resistance genes in E. coli ST131 . 
Multiple plasmids from a range of incompatibility groups and containing various combinations of antibiotic resistance genes , conjugative transfer genes and other cargo genes have been described in E. coli ST131 strains [ 2 ] . 
This includes the IncF plasmids pEK499 , pEK516 [ 10 ] , pGUE-NDM [ 11 ] , pC15-1a [ 12 ] , pJJ1886-5 [ 13 ] , pEC_B24 , pEC_L8 , pEC_L46 [ 14 ] , pJIE186-2 [ 15 ] , as well as the IncN plasmid pECN580 [ 16 ] , the IncX plasmid pJIE143 [ 17 ] and the IncI plasmid pEK204 [ 10 ] . 
E. coli EC958 represents one of the best-characterised genome-sequenced E. coli ST131 strains [ 18 ] . 
E. coli EC958 is a phylogenetic group B2 , CTX-M-15 positive , fluoroquinolone resistant , H30 E. coli ST131 strain [ 19 ] . 
The strain belongs to the pulse field gel electrophoresis defined United Kingdom ( UK ) epidemic strain A [ 20 ] , and the recently defined ST131 Clade C2/H30-Rx sublineage [ 8 , 9 ] . 
E. coli EC958 contains multiple genes associated with the virulence of extra-intestinal E. coli , including type 1 fimbriae which are required for adherence to and invasion of human bladder cells , the formation of intracellular bacterial communities , and colonization of the mouse bladder [ 19 , 21 ] . 
In animal models , E. coli EC958 causes acute and chronic urinary tract infection ( UTI ) [ 21 ] and impairment of uterine contractility [ 22 ] . 
E. coli EC958 is also resistant to the bactericidal action of human serum , and the complement of genes that define this phenotype have been comprehensively defined [ 23 ] . 
E. coli EC958 contains a large IncF plasmid ( pEC958 -- HG941719 ) containing multiple antibiotic resistance genes . 
Here we describe the full annotation of pEC958 , and demonstrate that genes encoded on pEC958 are common among other Clade C2/H30-Rx ST131 strains . 
Plasmid pEC958 contains two replicons , and we show that both replicons contribute to its maintenance in E. coli EC958 . 
Materials and Methods
Bacterial strains and growth conditions
E. coli EC958 is a UTI strain originally isolated in the UK in 2005 [ 19 ] . 
E. coli TOP10 has been described previously [ 24 ] . 
E. coli strains were stored in 15 % glycerol at -80 °C and routinely cultured at 37 °C on solid or in liquid Lysogeny Broth ( LB ) medium . 
Antimicrobial susceptibility testing
The minimal inhibitory concentrations ( MICs ) were determined by Etest ( bioMérieux Austra-lia ) on Mueller-Hinton agar at 37 °C . 
The procedure and interpretation of MIC were performed as recommended by the manufacturer using CLSI breakpoints [ 25 ] . 
Molecular methods
Plasmid DNA purification was performed using the PureLink HiPure Plasmid Filter Midiprep Kit ( Life Technologies ) . 
E. coli TOP10 electro-competent cells were prepared as previously described [ 23 ] and pEC958 plasmid DNA was transformed into TOP10 in a 2 mm cuvette using a BioRad GenePulser set to 2.5 kV , 25 mF and 200 O. Cells were resuspended in 1 mL SOC me-dium and incubated at 37 °C for 2 hours , then selected on LB agar plates supplemented with ampicillin 100 μg / mL . 
The FAB formula for IncF plasmids ( IncF RST scheme [ 26 ] ) was identified in silico using the online pMLST tool ( http://cge.cbs.dtu.dk/services/pMLST/ ) [ 27 ] . 
The pEC958 information was uploaded to the pMLST database ( http://pubmlst.org/ ) . 
Annotation of pEC958
The sequence of plasmid pEC958 ( emb | HG941719 ) [ 18 ] was manually curated in Artemis [ 28 ] using BLAST and literature searches . 
Antibiotic resistance genes were named in accordance with ResFinder 1.4 [ 29 ] and confirmed manually by BLAST and literature searches . 
TraDIS analyses
The TraDIS sequence data used in this work was generated from a previously published study that examined essential genes in EC958 ( BioProject number PRJNA189704 ; input A and B samples ) [ 23 ] . 
The short reads were mapped to the pEC958 sequence using Maq version 0.7.1 [ 30 ] . 
Counts of insertion per gene and insertion index were calculated as previously described [ 23 ] . 
Phylogenetic tree building
The maximum-likelihood phylogenetic tree of EC958_A0140 homologs was built using the PhyML v3 .0 online tool [ 31 ] . 
The tree used the WAG model for amino acid substitution and branch supports were calculated using approximate likelihood-ratio test ( aLRT ) [ 32 ] . 
Visualization
The read counts and insertion sites from TraDIS were visualized using Artemis version 15.0 [ 28 ] . 
The circular genome diagram was generated by DNAplotter [ 33 ] and linear genetic diagrams were constructed using Easyfig version 2.1 [ 34 ] . 
Circos [ 35 ] and Circoletto [ 36 ] were used to generate the sequence comparison figure . 
Sequence comparisons of pEC958 against ST131 strains were generated using BLAST Ring Image Generator ( BRIG ) [ 37 ] . 
Results
Characteristics of plasmid pEC958
The plasmid pEC958 is a 135,600 bp circular DNA molecule containing 142 coding sequences ( CDSs ) and 10 pseudogenes ( Fig 1 ) . 
The most closely related plasmid to pEC958 is pEK499 ( 99 % identity covering 85 % of pEC958 ; pEK499 lacks the second transfer region present in pEC958 , which accounts for the remaining 15 % of pEC958 ) ( Fig 2 ) . 
In silico replicon sequence typing identified pEC958 as a hybrid plasmid containing both IncFII and IncFIA replicons ( FAB formula F2 : A1 : B - ) . 
The RepFIA replicon
The 6,509 bp RepFIA replicon in pEC958 is 99 % identical to the corresponding region on the F-plasmid ( nt 45922 to 52516 , accession no . 
NC_002483 .1 ) and 100 % identical to two other plasmids isolated from E. coli ST131 strains , pEK499 ( NC_013122 .1 [ 10 ] ) and pJJ1886_5 ( NC_022651 .1 [ 13 ] ) ( Fig 2 ) . 
As observed in many other RepFIA sequences , this region does not contain the repC gene ( replication initiation ) found on the F-plasmid . 
The first region of RepFIA in pEC958 contains two rfsF sites ( the target sequences of the site-specific resolvase ResD [ 38 ] ) , followed by the oriV-1 origin of replication , ccdAB genes ( post-segregational killing ) , and resD ( multimer resolution ) . 
The second region of RepFIA in pEC958 contains the replication repE gene ( RepFIA ) with its upstream sequences ssiA ( single strand initiation ) and oriV-2 ( including the DnaA boxes , A/T rich region and four iterons ) , and the downstream incC iterons ( incompatibility and copy-number control ) . 
The third region of RepFIA in pEC958 contains the sopAB partition genes and their target centromere-like sopC sequence . 
This is the only partition system found on pEC958 . 
Although this RepFIA replicon contains two origins of replication ( oriV-1 and oriV-2 ) , replication is predicted to start unidirectionally from oriV-2 because the bidirectional replication from oriV-1 is known to require the missing RepC protein [ 39 , 40 ] . 
The RepFII replicon
The second replicon in pEC958 , RepFII ( 4,068 bp ) , is 99 % identical to the IncFII replicon in the Shigella flexneri 2b plasmid R100 ( accession no . 
NC_002134 .1 , [ 41 ] ) and 100 % identical to the RepFII replicon in the E. coli ST131 plasmid pEK499 ( Fig 1 ) . 
This replicon encodes the essential gene for its replication , repA1 , which is regulated by the negative regulator RepA2 ( CopB ) , the non-coding RNA copA and the regulatory leading peptide RepA6 [ 42 -- 44 ] . 
The pEC958 RepFII origin of replication ( ori ) is located between repA1 and repA4 , consistent with previous descriptions for the initiation of DNA replication from this replicon [ 42 , 45 -- 47 ] . 
The repA4 region is important for plasmid stability and contains the ter sites for replication termination [ 48 ] . 
The pEC958 RepII replicon contains the tir ( transfer inhibition protein [ 49 ] ) and the type II toxin-antitoxin system pemI/pemK [ 50 , 51 ] downstream of repA4 . 
The transfer region of pEC958 is not functional
The transfer ( tra ) region of pEC958 is disrupted by a composite mobile element flanked by IS26a and IS26b , carrying blaTEM-1 gene ( Fig 1 ) . 
The first half of this tra region is 100 % identical to the corresponding region on pEK499 ( F2 : A1 : B - ) , and 99 % identical to the corresponding region of several other IncF plasmids including pJJ1886_5 ( F2 : A1 : B - ) , pEC_L46 ( F2 : A1 : B - ) , pEC_L8 ( F2 : A1 : B - ) , pEFC36a ( F2 : A - : B - ) and pChi7122-2 ( F11 : A - : B - ) . 
In contrast , the second half of the pEC958 tra region is 100 % identical to pC15-1a ( F2 : A - : B - ) , R100 ( F2 : A - : B - ) , pHN3A11 ( F2 : A - : B - ) , pFOS-HK151325 ( F2 : A - : B - ) , pXZ ( F2 : A - : B - ) , pHK23a ( F2 : A - : B - ) , pHK01 ( F2 : A - : B - ) and pEG356 ( F2 : A - : B - ) . 
However , the pEC958 conjugation system is missing three genes , namely trbI , traW and traU . 
TrbI is an inner membrane protein that affects pilus retraction [ 52 ] ; TraW is required for F-pilus assembly [ 52 ] ; and mutations in traU significantly reduce plasmid transfer proficiency [ 53 ] . 
Despite repeated attempts , we were unable to demonstrate conjugative transfer of pEC958 to recipient strains , supporting the bioinformatic prediction that its conjugation system is non-functional ( data not shown ) . 
Toxin-antitoxin systems
The pEC958 plasmid encodes four toxin-antitoxin ( TA ) systems : the hok/sok system , the ccdAB system encoded within RepFIA , the pemIK system encoded within RepFII and the vagDC system . 
The hok/sok locus encodes a type I TA system including a `` host killing '' ( hok ) transmembrane protein that damages the cell membrane , a `` modulation of killing '' ( mok ) and a `` suppression of killing '' ( sok ) antisense RNA that inhibits translation of mok [ 54 ] . 
Both ccdAB and pemIK belong to type II TA system where the toxin protein is inactivated by direct interaction with the antitoxin protein . 
The ccdB gene encodes for a gyrase inhibitor toxin [ 55 ] that kills the cell in the absence of the CcdA anti-toxin , which is unstable and degraded by the Lon protease [ 56 ] . 
PemK is a sequence-specific endoribonuclease that cleaves mRNAs to inhibit protein synthesis [ 50 ] whereas PemI blocks the endoribonuclease activity and is also subjected to Lon proteolysis [ 57 ] . 
There are two identical copies of the vagDC genes in pEC958 . 
Sequence analysis of VagD revealed a PIN_VapC-FitB ( cd09881 ) domain found in toxins of many bacterial TA systems . 
VagC contains an antitoxin-MazE ( pfam04014 ) domain . 
The vagDC genes have been shown to be involved in plasmid stability in Salmonella Dublin , where VagD inhibits cell division and VagC modulates the activity of VagD [ 58 ] . 
Mobile genetic elements and antibiotic resistance genes
The majority of mobile genetic elements and antibiotic resistant genes in pEC958 cluster in two regions : an 8-kb region in the middle of the tra region , and a 41-kb region located immediately downstream of the RepFII replicon ( Fig 1 ) . 
Plasmid pEC958 contains eight IS26 elements ( named IS26a-IS26h ) , two IS1 elements , one ISEc23 element and one group II intron ( E.c.I11 , found outside of the two regions ) ( Fig 3 ) . 
IS26a and IS26b are located at the two ends of the 8-kb region , flanking ISEcp1 , a remnant of Tn3 , which includes the blaTEM-1 gene , and a partial sequence of Tn21 . 
The beginning of the 41-kb region contains a partial sequence of Tn5403 followed by IS26c . 
The region between IS26c and IS26d contains a cluster of 6 genes ( EC958_A0096 to EC958_A0101 ) predicted to encode a series of ABC transporters and an iron permease . 
Downstream of IS26d is a class I integron In54 [ 59 ] with gene cassettes consisting of dfrA17 , aadA5 and sulI , encoding trimethoprim , streptomycin and sulfonamide resistance , respectively . 
The mphR-mrx-mph ( A ) operon encoding resistance to macrolides is located between IS6100 and IS26e . 
Immediately after IS26e is the blaCTX-M-15 gene encoding cefotaxime resistance . 
Located between IS26f and IS26g are catB4Δ ( non-functional ; disrupted by IS26f ) , blaOXA-1 ( beta-lactam resistance ) and aac ( 6 ' ) - Ib-cr ( fluoroquinolone and aminoglycoside resistance ) . 
After IS26g lies Tn1721 , which harbours tetR and tet ( A ) , encoding resistance to tetracycline . 
The end of the 41-kb region contains a partial sequence of Tn5403 and IS26h . 
Functional characterization of antibiotic resistance genes on pEC958 
To investigate the antibiotic resistance phenotypes conferred by plasmid pEC958 , we transformed the plasmid into E. coli TOP10 . 
Table 1 shows the resistance profile of wild-type EC958 ( which contains pEC958 ) compared to TOP10 ( pEC958 ) . 
EC958 is resistant to 11 of the 18 antibiotics tested , five of which are fully transferable via pEC958 . 
EC958 is resistant to the cepha-mycin cefoxitin and the three third-generation cephalosporins tested ( cefotaxime , ceftazidime and cefpodoxime ) . 
These phenotypes , however , were not fully transferred to TOP10 by pEC958 . 
TOP10 ( pEC958 ) had elevated MICs to cefoxitin , cefotaxime , ceftazidime and cefpodoxime ( MIC of 6 , 1.5 , 1.5 and 8.0 μg / mL , respectively ) compared to the background strain TOP10 ( MIC of 4 , 0.047 , 0.38 and 0.25 μg / mL , respectively ) , but these MICs were still 6 -- 10 fold lower than those of the EC958 wild-type strain . 
This suggests that blaCTX-M-15 on pEC958 plasmid does not mediate the full resistance against third-generation cephalosporins . 
This is consistent with previous reports of lower resistance to cephalosporins in strains where the blaCTX-M-15 is separated by IS26 from its promoter within the ISEcp1 element [ 60 -- 63 ] . 
The other resistance phenotypes not transferred were for quinolones and fluoroquinolones . 
Chromosomal mutations in gyrA ( S83L , D87N , A828S ) and parC ( S80I , E84V , A192V , A471G , D475E , Q481H ) genes are likely to mediate these phenotypes , even though the plasmid carries aac ( 6 ' ) - Ib-cr [ 64 -- 66 ] . 
Genes required for the stable maintenance of pEC958
In order to gain insights into molecular mechanisms of plasmid stability , we analyzed the Tra-DIS data from a saturated transposon mutant library of EC958 [ 23 ] against the complete sequence of pEC958 to identify genes required for plasmid stability . 
We used a total of 12 million transposon-tagged reads , of which 901,588 reads ( 7.4 % ) were mapped to plasmid pEC958 , identifying 27,317 unique insertion sites ( i.e. one insertion site every 4.96 bp ) . 
To devise a biological threshold for the identification of genes required for the stable maintenance of pEC958 , the insertion index ( number of mapped reads normalized by gene length ) of each plasmid gene was calculated and compared with the sopAB genes , which are known to be essential for plasmid partitioning ( Fig 4 ) . 
A total of 9 genetic elements were identified to be required for the stable maintenance of pEC958 . 
They are the ccdA , sopA and sopB genes in RepFIA ; the copA , repA6 , repA1 , repA4 genes and the oriV region in RepFII ; and the hypothetical gene EC958_A0140 . 
Our results indicate that replication of pEC958 is initiated at the oriV of RepFII and requires at least the copA , repA6 , repA1 , repA4 genes . 
While RepFIA is not essential for replication , it is required for partitioning ( sopAB ) of pEC958 into daughter cells . 
Our data also demonstrated that the ccdAB TA system located within RepFIA is functional . 
EC958_A0140 represents a novel gene associated with plasmid maintenance . 
We screened the NCBI complete plasmid sequence database and identified 17 other plasmids that also contain this gene ( Fig 5 ) . 
All of these plasmids were IncF type except for pECL_A ( non-typable ) , and several were also isolated from E. coli ST131 strains ( pJJ1886_5 , pEK499 , pEC_L8 and pEC_L46 ) . 
Bioinformatic analysis of EC958_A0140 did not yield any clues regarding is function , and thus further work is required to confirm its role in plasmid stability . 
The prevalence of pEC958-like plasmid sequences was assessed in a previously described global collection of 97 E. coli ST131 strains [ 9 ] . 
Fig 6 shows the overview of plasmid sequences from 97 ST131 strains plus four complete ST131 plasmids available on GenBank in comparison with the pEC958 sequence . 
There are 20 strains and 2 database plasmids ( pEK499 and pJJ1886_5 ) that contain more than 70 % of pEC958 gene content , all of which belong to the clade C subli-neage C2 ( 40 % ) ( Fig 6 and S1 Table ) . 
Twelve out of these 20 strains ( plus pEK499 ) also harbor all 9 pEC958 essential genes identified above . 
In silico replicon sequence typing of IncF plasmids was also performed on the 97 strains . 
Table 2 shows the 8 most common FAB types found within this collection . 
The FAB formula of pEC958 , F2 : A1 : B - , is also the most common replicon type , accounting for 20.6 % of all 97 E. coli ST131 strains , or 27.8 % of clade C strains , all of which also belong to subclade C2 . 
The second most common type is F1 : A2 : B20 , of which 17 are in subclade C1 and 1 is in clade A . 
In terms of individual replicons , FIB is present in 100 % of clade A and B strains , while FII is most common in clade C ( 87.5 % ; S1 Table ) . 
Based on our sequence analysis , 3/97 strains do not harbor an IncF plasmid . 
Discussion
Our study presents a full annotation of pEC958 , a multi-drug resistance plasmid in the well-characterized E. coli ST131 strain EC958 [ 18 , 19 , 23 ] . 
In addition , we identified genes required for the maintenance and stability of pEC958 . 
Although IncF plasmids are extremely successful in the E. coli ST131 clonal lineage [ 67 ] , this is the first study to examine the biology of an IncF plasmid in its native host using TraDIS [ 68 ] . 
The replication and stability of IncF plasmids ( Fplasmid , R1 , and R100 ) has been well documented [ 39 , 47 , 69 , 70 ] . 
Here we provide insights into the interplay between two replicons in order to achieve stable maintenance of the circular plasmid DNA on which they co-exist . 
The data analysis in this study used a straight cut-off based on the insertion index of the sopAB genes , which encode the partitioning system of pEC958 . 
Mutation of sopAB is known to cause destabilization of IncF plasmids and thus they represent characterised genes involved in plasmid stability [ 71 , 72 ] . 
This deviation from the model-based approach , in which the cut-off is defined as the intercept of two distribution models representing essential and non-essential genes [ 23 ] , was chosen because of two reasons : ( i ) the number of genes on plasmid is insufficient to build two distribution models ; and ( ii ) the cut-off previously defined using chromosomal data is not applicable because of the higher insertion frequency on the plasmid ( i.e. one insertion every 4.96 bp compared to every 9.92 bp on the chromosome ) . 
In the case of the well-characterised IncF system , use of a straight cut-off assumed that any gene with an insertion index lower than the sopAB genes would have a similar or stronger effect on plasmid stability . 
The stable maintenance of large plasmids such as pEC958 is achieved by the contribution of multiple factors , including systems involved in replication , partitioning and toxin-antitoxin production . 
Using the strategy outlined , we aimed to identify genes that when mutated caused destabilization of plasmid pEC958 -- thus they must play a role in plasmid stability . 
Our results showed that RepFII , particularly the copA , repA1 , repA4 genes and oriV region , is required for the replication of pEC958 . 
This is consistent with previous studies on the function of RepFII in the IncFII plasmid R100 [ 41 ] . 
In contrast to R100 , the RepFII region on pEC958 does not contain its own intrinsic partition system ( stb locus on R100 [ 73 , 74 ] ) . 
Furthermore , we could not identify any region that resembles a partition site ( centromere-like ) elsewhere on pEC958 other than within the RepFIA region . 
Thus , it is reasonable to assume that the sopAB genes in the RepFIA region [ 75 , 76 ] represent the only active partition system on pEC958 . 
Indeed , our transposon mutagenesis revealed a very low insertion index for both sopA and sopB , confirming the requirement of these two genes for pEC958 partitioning and allowing us to use these genes as a reference threshold to identify biologically significant genes required for plasmid maintenance . 
Using TraDIS , we were able to demonstrate that none of the known replication genes in RepFIA are required for pEC958 replication . 
This included the oriV-1 of RepFIA , which was not expected to be functional due to the absence of the repC gene [ 40 ] . 
The oriV-2 and its associated genes in RepFIA appear to be intact yet dispensable in pEC958 . 
Similar behavior has been reported in the dual-replicon plasmid pCG86 , which contains an active RepFII replicon and an inactive ( but intact ) RepFIB replicon [ 77 ] . 
This is consistent with a previously proposed model for plasmid speciation , in which the existence of co-integrate plasmids ( such as pEC958 ) allows one replicon to be relaxed and free to accumulate mutations whilst the other replicon is constrained by evolutionary pressure to maintain its replication function [ 78 ] . 
The RepFIA also carries one toxin-antitoxin system ccdAB in which the antitoxin CcdA is protected from transposon mutagenesis , indicating that the system is active in pEC958 . 
There are three other TA systems in pEC958 , none of which were required for plasmid stability under the conditions tested in this study . 
Others have suggested that TA systems are more than just plasmid maintenance systems ; they can also function as a stress-response system [ 79 , 80 ] , as a programmed cell-death network [ 81 ] , or as a reversible bacteriostasis system ( i.e. induction of dormancy or persistence ) [ 82 , 83 ] . 
It is conceivable that the redundancy of TA systems on pEC958 is linked to other functions that provide a fitness advantage to its host . 
Plasmids of several different incompatibility types have been identified in E. coli ST131 , including IncF , IncI1 , IncN , IncA/C and pir-type [ 2 ] . 
Our data demonstrates that IncF plasmids are the most common plasmid type in E. coli ST131 , and is in accordance with previous studies [ 2 , 4 ] . 
To investigate the prevalence of pEC958 sequences in our strain collection , we used ge-nome sequence data to evaluate the prevalence of pEC958 genes and to perform in silico IncF replicon sequence typing . 
We identified 20 strains ( including EC958 ) that contained more than 70 % of the genes identified on pEC958 , suggesting that many ST131 strains carry very similar plasmids . 
We also identified 20 strains that possess the F2 : A1 : B - plasmid replicon formula , 17 of which contain > 70 % of pEC958 genes . 
Taken together , our data demonstrate that pEC958 belongs to the most common group of IncF plasmids found in E. coli ST131 . 
The overall success of IncFII plasmids extends beyond the carriage of blaCTX-M-15 in E. coli ST131 . 
IncFII plasmids that have acquired the blaNDM-1 gene ( thus conferring carbapenem resistance ) have been described in the ST131 lineage [ 11 , 84 ] , but strain EC958 was isolated prior to the discovery of NDM determinants and we did not find any NDM determinants in the 97 ST131 strain collection . 
The IncFIIk plasmid , a replicon type originally found in Klebsiella [ 26 ] , has also been found in KPC-producing ST131 strains in the USA and China [ 85 , 86 ] . 
The evolution and continual gain of new antimicrobial resistance determinants in IncFII plasmids represents a major challenge for our understanding of plasmid biology and the spread of antibiotic resistance genes . 
Here , we shed novel insight into our knowledge of plasmid replication by providing direct evidence that the RepFIA and RepFII replicons in pEC958 cooperate to ensure their stable inheritance . 
The combination of replication from RepFII and partition from RepFIA may represent a co-evolutionary adaptation for this common plasmid type . 
Supporting Information
We thank Majed Alghoribi for technical assistance.
Author Contributions
Conceived and designed the experiments : MDP MU SAB MAS . 
Performed the experiments : MDP KMP SS SH MU . 
Analyzed the data : MDP BMF MSC NLBZ MU . 
Contributed reagents / materials/analysis tools : MSC MU SAB . 
Wrote the paper : MDP MU SAB MAS .