coli DH5α was employed as a negative control in the virulence ass

coli DH5α was employed as a negative control in the virulence assays. A well-characterized collection of APEC, fecal E. coli isolated from the feces of healthy birds (avian fecal E. coli), human UPEC, and human NMEC were used for gene prevalence studies. Strains were grouped phylogenetically using multiplex PCR [16]. Cells were routinely grown at 37°C in Luria Bertani broth (LB) supplemented with an appropriate antibiotic: kanamycin (Km; 50 mg ml-1), Idasanutlin chloramphenicol (Cm; 25 mg ml-1), or ampicillin (Amp; 100 mg ml-1),

unless otherwise specified. Table 1 Bacterial strains and plasmids used in this study Strain Description Reference APEC O1 O1:K1:H7; fyuA, sitA, chuA, irp2, iroN, ireA, tsh, iucD, fimC, iss, ompA, vat, GSK2118436 selleck screening library traT; contains four plasmids, including pAPEC-O1-ColBM [14] BJ502 E. coli K12, ΔtktA

[15] DH5α E. coli K12   APEC O1-M tkt1 APEC O1 derivative, Δtkt1 this study APEC O1-M tktA APEC O1 derivative, ΔtktA this study S17λpir recA thi pro hsdR – M + RP4::2-Tc::Mu::Km Tn7 lysogenized with λpir phage [12] S17pGP tkt1 S17λpir with plasmid pGP704 tkt1 this study APEC O1-C tkt1 APEC O1 M tkt1 with plasmid pGP tkt1 inserted into bacterial chromosome this study APEC O1-P1 APEC O1 M tkt1 with plasmid pBAD tkt1 this study BJ502-P1 BJ502 with plasmid pBAD24 this study BJ502-P2 BJ502 with plasmid pBAD tkt1 this study BJ502-P3 BJ502 with plasmid pBAD tktA this study APEC collection 452 APEC strains isolated from lesions of birds clinically diagnosed with colibacillosis Etofibrate [17] Avian fecal E. coli 106 avian fecal E. coli strains were isolated from the feces of apparently healthy birds [17] UPEC collection 200 uropathogenic E. coli strains from from MeritCare Medical Center in Fargo, North Dakota [18] NMEC collection 91 human neonatal meningitis-causing E. coli strains from the cerebrospinal fluid of newborns in the Netherlands, isolated from 1989 through 1997 and from Dr. K. S. Kim at John Hopkins. [19] Plasmids     pGP704 Apr,

suicide plasmid [20] pBAD24 Apr, expression plasmid with arabinose-inducible promoter [21] pKD46 Apr; expresses λ red recombinase [22] pKD3 cat gene, template plasmid [22] pGP tkt1 pGP704 derivative harboring tkt1 gene this study pBAD tkt1 pBAD24 derivative, tkt1 gene under the control of PBAD this study pBAD tktA pBAD24 derivative, tktA gene under the control of PBAD this study PCR and multiplex PCR DNA templates were prepared by the rapid boiling-lysis method. Primer pairs used were tkt1- F 5′- cttacggcggtactttcctg-3′and tkt 1-R 5′-gtacgccgcatcctgattat-3′; genomic island left junction primer pair piaL-F 5′-cgacatcatggattcgattg-3′and piaL-R 5′-ggatggtgctggatcgtact-3′; and genomic island right junction primer pair piaR-F 5′-gcgccactcttcttctgttc-3′ and piaR-R 5′-tcagctaattgctcggcttt-3′ PCR was accomplished under the following reaction conditions: 4 mM magnesium chloride, 0.25 mM deoxynucleotide triphosphates 0.3 uM each primer, and 1 Unit Taq DNA polymerase.

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