The sample learn more was obtained from the Enteric Diseases Laboratory Branch, Center of Disease Control and Prevention (CDC, Atlanta, GA). Furthermore, 2 E. coli O104:H4 APO866 chemical structure strains 2050 and 2071, recovered from an outbreak in the Republic of Georgia, were also obtained from the CDC. Unless indicated, strains were grown overnight in Luria-Bertani (LB) medium at 37 °C, shaking at 225 rpm. The aerobactin transport iutA mutant CSS001 was constructed by PCR amplification and cloning of a fragment containing the iutA gene, disrupted with the cam cassette and cloned into the pCVD442 suicide vector. The mutagenesis approach was previously described [23]. The iutA mutant was confirmed

by PCR by using the oligonucleotides listed in Table 1, under the following conditions: 1 cycle at 94 °C for 3 min, and then 30 cycles at 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min. For the spatial-temporal location of E. coli O104:H4 in mice, the transformed RJC001 was constructed selleck kinase inhibitor by electroporation with 3 μg of pCM17 plasmid, containing the luxCDABE operon driven by the OmpC promoter (constitutive expression), which was previously used to visualize pathogenic E. coli[19]. The plasmid was generously donated by J.B. Kaper. Transformants were selected on LB agar plates supplemented with kanamycin (50 mg/ml), and BLI was confirmed by using the

IVIS Spectrum (Caliper Corp., Alameda, CA). Table 1 qRT-PCR primers used in this study Primer name Sequence Characteristics References 5RTRRSB 5’-TGCAAGTCGAACGGTAACAG-3’ qRT-PCR rrsb gene [40] 3RTRRSB 5’-AGTTATCCCCCTCCATCAGG-3’

rpoS Fw 5’-AGTCAGAATACGCTGAAAGTTCATG-3’ qRT-PCR BCKDHA rpoS gene [41] rpoS Rv 5’-AAGGTAAAGCTGAGTCGCGTC-3’ iutAFw 5’- GATCATAGTGTCTGCCAGCC-3’ qRT-PCR iutA gene This study iutARv 5’- GCTCTTTACCGCCCTGAATC-3’ iutAO104_F 5’-ATGGAGTTTGAGGCTGGCAC-3’ iutA mutant confirmation This study iutAO104_R 5’-GCTTACTGTCGCTGACGTTC-3’     Growth curves Cultures containing no antibiotics were grown overnight at 37 °C, 225 rpm. On the next day, 1:500 dilutions of overnight were inoculated into 30 mL of pre-warmed, sterile LB media. The growth of CSS001 was compared to the growth of wild-type E. coli O104:H4 strain C3493. Sampling was performed at approximately 1-h intervals during the first 9 h of the assay, and a final sample was analyzed 24 h from the start of the experiment. The growth of the E. coli wild- type and CSS001 strains was monitored by plating serial dilutions (log10 CFU/ml) from the time points on LB media with and without 2,2’-dipyridyl as well as by OD600 readings (Additional file 1: Figure S1). Mice Female ICR (CD-1) mice of 20 to 25 g were obtained from Charles River Laboratories and housed in the pathogen-free animal facility at UTMB upon arrival for 72 h prior to experiments. Animal studies were performed in accordance with the Animal Care and Use Committee’s guidelines at UTMB as recommended by the National Institute of Health.