The DNAs of these control strains were also used as template to PCR amplify each of the virulence gene followed by preparation of DNA probes. The E. coli eaeA gene was PCR amplified using the eae-F and eae-R primer set and subtyped by PCR-RFLP GDC-0941 research buy with MspI as described previously [34]. Cytotoxicity assay Cytotoxicity assay was performed as described earlier [10]. Briefly, test strains were grown overnight in 3 mL of tryptic soy broth at 37°C overnight with shaking. Bacterial cells were lysed by sonication using an Astrason ultrasonic processor (Heat-System

7 Ultrasonics, Farmingdale, NY, USA) and each sonic lysate was passed through sterile disposable filter with 0.22-μm pore size and each filtrate was used for cytotoxicity assay. Vero and CHO cells were seeded at density of 1 × 104 cells in a 96 well plate (Asahi glass Co., Ltd., Tokyo, Japan) respectively, and 20 μL of 2-fold serially diluted each toxin solution was added to assay their cytotoxic effects. After 9 h of incubation, 100 μL of fresh medium was added per well and cytotoxic effect of each test sample, if any, was examined microscopically after 72 h of incubation. The toxin titer was expressed as the reciprocal of the highest dilution that caused

50% of the Vero and CHO cells in a well to be killed and distended, respectively. E. coli strains Sakai and GB1371 were always used as positive controls and as a negative

control we used E. coli strain C600. Vero and CHO cells were cultured in Minimum Essential Medium (MEM) and MEM-α (Life technologies), respectively, BIBW2992 clinical trial containing 10% fetal bovine serum (EuroClone S.p.A., Pero, Italy), and 1% antibiotic-antimycotic (100x) (Penicillin G sodium [10,000 U/mL], streptomycin sulfate [10,000 μg/mL], and 25 μg/mL amphotericin B in 0.85% saline [Life technologies]). Cells were cultured at 37°C under 5% CO2 Thymidylate synthase in air. Sequence analysis of cdt-III and cdt-V To determine the entire sequence of the cdt genes, the cdt gene-cluster including their flanking regions were PCR amplified followed by sequencing as previously described [10]. For the cdt-III genes, PCR product obtained by the pVir-u and pVir-d Ralimetinib order primers specific to the flanking region of cdt-III on the pVir plasmid was sequenced. For the cdt-V genes, PCR products obtained by the P2-A2 and CdtVC-D2 primers and the CdtIII/VB-F2 and P2-C3 primers were sequenced (Figure 1). Each PCR product was purified by the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) and the nucleotide sequence of the PCR product was determined as described above. Nucleotide and amino acid sequences were analyzed and compared with each subtype using the BLAST program through the DDBJ (DNA Data Bank of Japan), and the DNA Lasergene software package (DNASTAR).