coli, the basis of any host specificity of those EHEC strains may be related to the production of specific colonization factors, although such adhesins of EHEC strains have not yet been identified (Bardiau et al., 2009). The aim of this study was (1) to explore the genomic differences, using suppressive subtractive hybridization (SSH), between two EHEC strains of serogroup O26, one isolated from a young calf and the other isolated from a human with diarrhea, to identify specific sequences of the bovine strain; (2) to analyze the bovine strain-specific sequences

regarding their potential implication in adherence to epithelial cells; and (3) to study the prevalence of these strain-specific sequences in a collection of human and bovine EHEC and EPEC strains. Subtractive suppressive Fostamatinib hybridization (SSH) was performed between the bovine EHEC strain

4276 of serogroup O26 isolated in Ireland from a diarrheic calf (Kerr et al., 1999) and the human EHEC strain 11368 of serogroup O26 isolated in Japan from a human suffering from diarrhea (Ogura et al., 2009). The distribution of the specific sequences was investigated in additional Compound Library in vitro EHEC (n = 44) and EPEC (n = 27) strains of serogroup O26 isolated from humans (n = 27) and from cattle (n = 44). Most of the strains have been described previously (Szalo et al., 2004; Bardiau et al., 2009), and their characteristics are described in the supplemental Table S1. PFGE was performed as already described (Cobbaut et al., 2009; Ooka et al., 2009) on most of the tested strains. In brief, bacterial cells were embedded in 1.8% Certified Low Melt Agarose (Bio-Rad Laboratories, Inc., Tokyo, Japan), lysed

with a buffer containing 0.2% sodium deoxycholate, 0.5% N-lauroylsarcosine, and 0.5% Brij-58, and treated with 100 μg mL−1 proteinase K. XbaI-digested genomic DNA was separated using CHEF MAPPER (Bio-Rad Laboratories, Inc.) with 1% Pulsed Field Certified Agarose (Bio-Rad Laboratories, Inc.) at 6.0 V cm−1 for 22 h and 18 min with pulsed times ranging from 47 to 44.69 s. Size of each DNA band was estimated Idelalisib datasheet by Biogene (Vilber Lourmat, France). The banding patterns were analyzed using the Dice coefficient, with an optimization and position tolerance of 1%. Dendrograms were prepared by the unweighted-pair group method using arithmetic average algorithm (UPGMA). Genomic DNA was extracted from E. coli strain 4276 and E. coli strain 11368 using the cetyltrimethylammonium bromide procedure described by Ausubel et al. (1994). Subtractive hybridization was carried out using the PCR-Select Bacterial Genome Subtractive kit (Clontech) as recommended by the manufacturer. The bovine EHEC strain 4276 was the tester, and the human EHEC strain 11368 was the driver. The PCR products obtained were cloned into the pGEM-T Easy Vector System (Promega) and transformed into E. coli JM109.