The agn43 primers (5′-CGTGGATGATGGCGGAAC-3′

and 5′-CACCGTTAATGGCTTCAACC-3′) amplify a 920 bp fragment spanning the regions that encode the α43 and β43 subunits (position 3492898..3493817 in Genbank NC_004431). The presence of putative pCTX-like plasmids was investigated employing primers designed to target consensus sequences displayed in the GenBank sequences AF550415 (pCTX-M3 plasmid from C. freundii), EU938349 (pCTXM360 plasmid from K. pneumoniae) and AY422214 (pEL60 plasmid from Erwinia amylovora). On basis of these sequences, the traJ primers (5′-AATACCGCTATCCAGCTAAGAG-3′ buy Acalabrutinib and 5′CCCACTTGCTGTAATCAACG-3′) generate an amplicon with 517 bp in length (position 35550..36312 in the sequence AF550415). Primers tra were designed based on the conserved sequences of the traA family genes. In relation to the prototype F pilus (Genbank: K01147), the forward primer (5′-AAGTGTTCAGGGTGCTTCTG-3′) target the traA signal sequence (position: 1940..1959) while the reverse primer (5′-TATTCTCGTCTCCCGACATC-3′) recognize the beginning of the traL gene (position: 2305..2324). traA primers detect the subtypes I (encoded by ATM Kinase Inhibitor ic50 ColVBtrp

and F plasmids), IIa (ColB2), IIb (R124), III (R1) and IV (R100) of the traA genes harbored by IncF plasmids [42, 43]. Cycling conditions for PCR were as follows: 30 cycles of 94°C for 60 s, 60°C for 60 s, and 72°C for 90 s. Specific EAEC molecular Gilteritinib markers as well as virulence factors for other E. coli pathotypes were detected using the primers listed in table 1[5, 9, 14, 44–48]. Supernatants derived from bacterial suspensions treated by boiling were used as the source of DNA. HeLa cells and infection assays HeLa cells were cultured

in DMEM (Dulbecco’s modified Eagle’s Calpain medium; Gibco BRL) with 10% fetal bovine serum (FBS) and antibiotics (ampicillin [120 μg/mL] and streptomycin [100 μg/mL]) under atmosphere with CO2 (4%) at 37°C [49]. For qualitative mixed infection assays, HeLa cells (0.6 × 105 cells/mL) were cultured on glass coverslips (10 × 10 mm) using 24-well culture plates (600 μL/well) (Costar). Cells were grown to 50%-70% confluence, and the medium was changed to DMEM supplemented with 1.4% mannose (DMEM-mannose) without FBS. For quantitative mixed infection assays, HeLa cells (0.8 × 105 cells/mL) were cultured in similar way using 12-well culture plates without glass coverslips. In order to carry out the adhesion assays, HeLa cells were infected with 150 μL of an overnight bacterial culture for three hours at 37°C. After infection, the coverslips were washed five times with Dulbecco’s PBS (D-PBS), and the cells were fixed with methanol, stained with May-Grünwald and Giemsa stains, and analyzed using light microscopy. EAEC prototype strain 042 was used as the positive control for the aggregative phenotype. Qualitative mixed infection assays were performed with two infection steps. Initially, C.