Complete sequence of pEC14_114, a highly conserved IncFIB/FIIA plasmid associated with uropathogenic Escherichia coli cystitis strains
Introduction
Extraintestinal pathogenic Escherichia coli (ExPEC) strains have been implicated in a variety of diseases of humans, including urinary tract infection (UTI), pneumonia, neonatal meningitis, and septicemia (Johnson, 1991). Additionally, this pathotype has been associated with important diseases of production and domestic animals, including colibacillosis in poultry, bovine septicemia and hemorrhagic pneumonia, and UTI in canine and feline hosts (Breitschwerdt et al., 2005, DebRoy et al., 2008, De Rycke et al., 1999, Handt et al., 2003, Sura et al., 2007, Smith et al., 2007). Because of the vast diversity of ExPEC, subpathotypes have been described specific to each disease type, including uropathogenic E. coli (UPEC), neonatal meningitis-associated E. coli (NMEC), necrotoxigenic E. coli (NTEC), and avian pathogenic E. coli (APEC), all of which are considered to be ExPEC because they carry some common virulence factors that are distinct from other diarrheagenic E. coli (Kaper et al., 2004, Kaper, 2005). Of the ExPEC-associated diseases, UTIs are the most commonly recognized infection. During the establishment of UTI, UPEC are thought to colonize the urogenital tract, form biofilms, and subsequently infect other urothelial cells. UPEC then have the ability to lead to recurrent or persistent UTI, or to disseminate to the bladder and kidneys to cause cystitis and pyelonephritis, respectively (Johnson, 1991, Johnson and Russo, 2002). They may ultimately invade the bloodstream to cause septicemia.
UPEC carry a diverse array of virulence genes enabling them to cause disease, encoding traits such as adhesins, toxins, siderophores, capsule, iron-utilization factors, surface coatings, outer membrane proteins, and lipopolysaccharides (Johnson et al., 2008, Ewers et al., 2007, Donnenberg and Welch, 2005, Johnson and Russo, 2005, Rodriguez-Siek et al., 2005). The genomes of several human UPEC have been sequenced, including those isolated from cases of uncomplicated UTI (strain IAI 39) (Touchon et al., 2009), cystitis (strains UTI89, F11, and UMN026) (Touchon et al., 2009, Chen et al., 2006), and pyelonephritis (strains CFT073 and 536) (Brzuszkiewicz et al., 2006, Welch et al., 2002). Historically, it has been postulated that UPEC virulence factors are primarily found within pathogenicity-associated islands (PAIs) on the bacterial chromosome (Dobrindt, 2005). Indeed, the archetypic UPEC genomes have been characterized by their chromosomal PAI possession (Brzuszkiewicz et al., 2006, Welch et al., 2002, Dobrindt et al., 2002, Guyer et al., 1998, Rasko et al., 2001). A role for plasmid-encoded virulence in UPEC is not well established, despite the fact that ExPEC strains are known to carry large plasmids (Johnson et al., 2007, Johnson et al., 2008, Ewers et al., 2007, Rodriguez-Siek et al., 2005, Zhao et al., 2009, Sorsa et al., 2003). In contrast, several plasmids associated with APEC and NTEC virulence have been described, such as the ColV and ColBM plasmids of APEC and the Vir plasmid of NTEC-2 (De Rycke et al., 1999, Ewers et al., 2007, Mellata et al., 2009, Tivendale et al., 2009, Johnson et al., 2006a, Johnson et al., 2006b, Rodriguez-Siek et al., 2005, DebRoy and Maddox, 2001).
Recent genome sequencing efforts involving three cystitis-associated UPEC strains (Touchon et al., 2009, Chen et al., 2006, Rasko et al., 2008) identified a conserved IncFIB/FIIA plasmid as a component of their genome that includes a large number of uncharacterized coding regions. Population-based approaches have proven effective at identifying traits that are highly prevalent among ExPEC and, as such, potentially implicated in ExPEC pathogenesis (Johnson, 1991, Johnson et al., 2005, Johnson et al., 2008, Ewers et al., 2007, Johnson and Russo, 2005). Here, we describe the completed sequence of a plasmid from a UPEC strain and utilize comparative genomics and gene prevalence in an effort to understand the conservation and distribution of this plasmid type among ExPEC populations.
Section snippets
Bacterial strains and characterization
UPEC strain EC14 (O6:H31; ECRC# 89.0590), isolated from a human UTI case, was used for plasmid isolation. This strain was grown in Luria–Bertani broth medium (LB broth, Difco Laboratories, Detroit, MI) overnight at 37 °C with shaking. For determining prevalence of plasmid-associated genes among ExPEC strains, a total of 1456 ExPEC isolates were examined. These included E. coli isolated from UTI from humans (n = 300), dogs (n = 139), and cats (n = 90); neonatal meningitis from humans (n = 91); diarrhea
Characteristics of UPEC strain EC14
Escherichia coli strain EC14 (O6:H31; ECRC# 89.0590) was submitted to the E. coli Reference Center (Penn State University, University Park, PA) from a case of human UTI in Illinois, USA. Analysis of this isolate revealed that it harbored a large plasmid and it contained the following virulence-associated genes: fimH, sfaA, cnf1, fyuA, iroN, entC, iroB, sitA, marR, yhjX, emrA, glnA, traT, uspA, kII, kpsII, uidA, paI, and papG allele III. The strain was susceptible to all antimicrobials tested.
Analysis of pEC14_114
Conclusions
Plasmids pEC14_114, pUTI89, and p1ESCUM represent a highly homogeneous RepFIB/FIIA plasmid subset with a distribution among human ExPEC and a strong association with highly virulent ExPEC belonging to the B2 phylogenetic group. The prevalence of these plasmids among ExPEC populations, and the presence of a number of putative virulence genes within an accessory region of this plasmid suggest that it might make contributions towards ExPEC virulence and/or fitness. Circumstantial evidence exists
Acknowledgments
The authors thank James R. Johnson, Lodewijk Spanjaard, and Lisa K. Nolan for providing some of the isolates used in this study.
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