pathogens
1 School of Chemistry & Molecular Biosciences , the University of Queensland , Brisbane , Queensland 4072 , Australia ; E-Mails : n.benzakour@uq.edu.au ( N.L.B.Z. ) ; m.phan1@uq.edu.au ( M.-D.P. ) ; b.forde@uq.edu.au ( B.M.F. ) ; m.stantoncook@uq.edu.au ( M.S.-C . ) 
2 Australian Infectious Diseases Research Centre , the University of Queensland , Brisbane , Queensland 4072 , Australia 
1. Introduction
Uropathogenic Escherichia coli ( UPEC ) are a major cause of urinary tract infections ( UTI ) , causing ~ 80 % of all cases [ 1 ] . 
Over the last few decades , several pandemic clones of UPEC , some of which are associated with multidrug resistant infections , have disseminated worldwide . 
This includes UPEC clones belonging to several multi-locus sequence types , including sequence type 131 ( ST131 ) , ST69 , ST73 and ST95 [ 2,3 ] . 
E. coli ST131 was originally identified in 2008 as a major clone linked to the spread of the CTX-M-15 extended-spectrum β-lactamase ( ESBL ) - resistance gene [ 4 -- 6 ] , the most widespread CTX-M ESBL enzyme worldwide [ 7,8 ] . 
ST131 strains have now been identified in both hospital and community settings from virtually all parts of the globe [ 9 -- 12 ] . 
ST131 causes a variety of extra-intestinal infections , most commonly UTI and bacteremia . 
Many ST131 strains exhibit resistance to multiple antibiotics , and therefore these infections are often associated with limited treatment options and frequent recurrences . 
The largest sub-clonal lineage of E. coli ST131 is resistant to fluoroquinolones and contains the type 1 fimbriae fimH30 ( H30 ) allele [ 13 ] . 
Three complete ST131 genome sequences have been generated . 
This includes SE15 [ 14 ] , EC958 [ 15 ] and JJ1886 [ 16 ] . 
Another ST131 strain , NA114 , while listed among the completely sequenced genomes on the NCBI database , remains in draft format [ 15,17 ] . 
This review will present an overview of our recent genomic analysis of ST131 and provide an update on the molecular characterization of the ST131 reference strain EC958 . 
2. Global Epidemiology of ST131
ST131 belongs to the E. coli phylogenetic group B2 , which encompasses the largest group of E. coli associated with extra-intestinal infections . 
Based on phylogenetic analyses , the ST131 strains EC958 , NA114 and JJ1886 cluster together in a clade discrete from SE15 , and separate from representative strains from other E. coli phylogroups ( Figure 1 ) . 
Two recent studies have independently examined the global epidemiology of ST131 using genome sequence-based methods [ 18,19 ] . 
These studies identified a globally dominant fluoroquinolone resistant-FimH30 sub-lineage defined as H30 [ 18 ] or clade C [ 19 ] . 
All strains within this sub-lineage possessed the fluoroquinolone resistance alleles gyrA1AB and parC1aAB . 
Further analysis also revealed that ST131 strains containing the blaCTX-M-15 allele comprised a smaller subset of strains within this sub-lineage and were referred to as H30-Rx [ 18 ] or clade C2 [ 19 ] . 
Strikingly , the data from both studies supports the recent emergence and global dissemination of this sub-lineage from a single progenitor , provoking intriguing questions with respect to ST131 transmission , colonization and virulence . 
In addition to the dominant clade C that comprised 79 % of our sequenced ST131 strains , our analysis also identified two other well-supported ST131 clades referred to as A and B [ 19 ] . 
Clade A , represented by the reference strain SE15 , was the most divergent and comprised strains that contained the fimH41 allele . 
In contrast , strains from clade B were very similar to those from clade C and characterised by possession of the fimH22 allele . 
The prevalence of these fimH alleles , including the dominant H30 allele , is consistent with that reported previously from a large and extensive collection of ST131 strains [ 13 ] 
Our own detailed genomic analysis focused on the major defining features of the three ST131 clades [ 19 ] . 
While sequence analysis did not reveal any significant association with geographic origin , the majority of the single nucleotide polymorphisms that defined each clade were strongly associated with recombination . 
In total , 137 regions were defined as recombinant within our ST131 strain set , with the majority of large recombinant regions located adjacent to insertion sites for prophages and mobile genetic elements . 
Other recombination regions within the ST131 strain set were also identified , some of which encompassed virulence genes including fimH , the fliC flagella major subunit gene , and genes involved in capsule and O antigen biosynthesis . 
One other notable recombination region encompassed the fimB recombinase gene that contributes to the regulation of type 1 fimbriae expression . 
Most ST131 strains from clade C have a 1,895 bp insertion element within the fimB gene ( fimB : : ISEc55 ) , suggestin they may possess an altered type 1 fimbriae expression profile . 
Indeed , the fimB : : ISEc55 insertion has been associated with a slower `` off '' - to - `` on '' type 1 fimbriae switching phenotype in ST131 [ 20,21 ] . 
We are currently investing the impact of this insertion on ST131 virulence . 
3. Molecular Characterisation of the ST131 Reference Strain EC958
EC958 is an O25b : H4 serotype strain isolated in 2005 from the urine of an 8-year old girl presenting with a community-acquired UTI in the United Kingdom [ 21 ] . 
The complete genome sequence of EC958 has been determined [ 15 ] . 
EC958 contains multiple genes associated with UPEC virulence , including genes encoding adhesins ( e.g. , type 1 fimbriae , curli and the afimbrial adhesin ) , autotransporter proteins ( e.g. , Ag43 , UpaG , UpaH and PicU ) and the biosynthesis of several siderophores ( enterobactin , aerobactin and yersiniabactin ) . 
Both EC958 and JJ1886 belong to the globally dominant CTX-M-15 positive , fluoroquinolone resistant , H30 clade C ST131 sub-lineage . 
The two strains display a high level of synteny at the core genome level , with major differences due to the number , content and location of genomic islands ( GIs ) and other mobile elements ( Figure 1 ) . 
For example , GI-selC is present in EC958 but not JJ1886 , while the Phi8 prophage is only present in JJ1886 . 
The two strains cluster distinct from the ST131 clade A SE15 strain . 
Based on whole-genome BLASTn comparisons , the major structural differences between EC958/JJ1886 and SE15 are the presence of seven prophage loci ( Phi1-Phi7 ) and four genomic islands ( GI-thrW , GI-pheV , GI-selC , and GI-leuX ) ( Figure 2 ) . 
Future examination of complete genomes of ST131 strains from different origins will be required to determine the extent of divergence of prophage , genomic islands and other mobile genetic elements in the ST131 clonal group . 
4. Virulence of E. coli ST131
EC958 has been characterised extensively with respect to several virulence characteristics . 
The strain possesses the fimB : : ISEc55 insertion but can express type 1 fimbriae after several rounds of static subculture . 
The expression of type 1 fimbriae by EC958 is required for adherence to and invasion of human T24 bladder epithelial cells , and colonization of the mouse bladder [ 21 ] . 
In mice , E. coli EC958 causes acute and chronic UTI [ 22 ] . 
EC958 bladder infection involves the formation of intracellular bacterial communities ( IBCs ) in superficial epithelial cells and the subsequent release of rod-shaped and filamentous bacteria into the bladder lumen [ 22 ] . 
EC958 also causes impairment of rat uterine contractility [ 23 ] . 
The ability of EC958 to resist the bactericidal action of human serum has been extensively interrogated using hyper-saturated transposon mutagenesis in combination with transposon directed insertion-site sequencing ( TraDIS ) [ 24 ] . 
TraDIS is a high-throughput functional genomics method that enables a pool of transposon mutants to be characterized by direct sequencing of DNA flanking transposon insertion sites [ 25 ] . 
In total , 56 genes were defined by TraDIS to comprise the EC958 serum resistome , of which 46 genes were validated by the generation and testing of specific mutants . 
The majority of these genes encode outer membrane proteins , or were associated with the biosynthesis of lipopolysaccharide ( LPS ) , the enterobacterial common antigen or colonic acid . 
Overall , the murein lipoprotein Lpp and two lipidA-core biosynthesis enzymes ( WaaP and WaaG ) were most strongly associated with serum resistance . 
The hyxR gene , which has previously been shown to contribute to th nitrosative stress response and intramacrophage survival of UPEC [ 26 ] , was also identified as a minor regulator of O-antigen chain length . 
5. Plasmids of ST131
Plasmids represent a major vehicle for the carriage of antibiotic resistance genes . 
Among the Enterobacteriaceae , plasmids from a range of incompatibility ( Inc ) groups have been characterised that contain various combinations of resistance , conjugative transfer and other cargo genes . 
The diversity of plasmid types in ST131 has been examined , with 50 % of the most frequent gamma-proteobacterial plasmid groups identified within the ST131 lineage [ 28 ] . 
Our own analysis revealed that the majority of ST131 strains harbor an IncF plasmid , many of which are associated with the carriage of antibiotic resistance genes [ 29 ] . 
Indeed , complete genome sequencing of EC958 demonstrated it contains a large 135.6 kb plasmid that harbors two replicons ( RepFIA and RepFII ) and 12 antibiotic resistance genes ( including blaCTX-M-15 ) . 
The most closely related plasmid to pEC958 is pEK499 ( 99 % identity covering 85 % of pEC958 ; Figure 3 ) , which was also isolated from an ST131 strain in the United Kingdom [ 30 ] . 
Interestingly , despite the presence of the blaCTX-M-15 gene on pEC958 , we have shown that this is not the major determinant responsible for EC958 resistance to second and third generation cephalosporins . 
Instead , EC958 contains a chromosomally-located blaCMY-23 gene that drives this resistance phenotype [ 31 ] 
We employed TraDIS as a novel approach to investigate the biology of pEC958 [ 29 ] . 
Analysis of TraDIS data from our saturated transposon mutant library of EC958 identified 27,317 reads that mapped to unique insertion sites in plasmid pEC958 ( i.e. , one insertion site every 4.96 bp ) . 
Genetic elements required for pEC958 stability were identified in both the RepFIA and RepFII replicons ; the ccdA , sopA and sopB genes in RepFIA , and the copA , repA6 , repA1 , repA4 genes as well as the oriV region in RepFII . 
Interestingly , this data suggests a model where both replicons contain features that ensure their stable inheritance : replication in RepFII and partition as well as post-segregational killing in RepFIA . 
Our analysis also identified EC958_A0140 as a novel gene of unknown function that is associated with pEC958 stability . 
Screening of the NCBI complete plasmid sequence database revealed EC958_A0140 is present in 17 other plasmids , all of which are IncF type except for pECL_A ( non-typable ) . 
However , bioinformatic analysis of EC958_A0140 did not yield any clues regarding its function and thus this remains an area of ongoing study . 
6. Conclusions
Our current understanding of ST131 epidemiology supports its divergence into three discrete sub-lineages sometime before the year 2000 , with acquisition of multiple mobile genetic elements , associated recombination events and point-mutations jointly responsible for the emergence of the most prevalent clade C/H30 strains . 
Several studies have now reported the identification of ST131 strains resistant to last-line carbapenem antibiotics [ 32 -- 35 ] , highlighting the alarming scenario of pan-resistance in a UPEC clone that has already demonstrated its capacity to disseminate rapidly across the globe . 
Future work will explore the continued evolution of the globally dominant clade C/H30 group , and address important questions that relate to ST131 resistance , transmission , colonization and virulence . 
Acknowledgments
M.A.S. and S.A.B. would like to thank other members of their research teams who have contributed to this ongoing area of research . 
In addition , we thank our many national and international collaborators for their valuable contributions . 
This work was supported by a grant from the National Health an 
Medical Research Council ( NHMRC ) of Australia ( APP1067455 ) . 
M.A.S. is supported by an Australian Research Council Future Fellowship ( FT100100662 ) and S.A.B. is supported by an NHMRC Career Development Fellowship ( APP1090456 ) . 
Conflicts of Interest
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