coli, E. fergusonii and E. albertii were concatenated in the order adk, fumC, gyrB, icd, mdh, purA and recA and aligned. Based on 3,423 bp of the concatenated sequences, a neighbor-joining tree was constructed by using MEGA 4 software. Serotyping and phylogenetic grouping To characterize the CTEC strains further, their serotype and phylogenetic Selleckchem MEK inhibitor groups were determined
(Table 2). The 81 cattle isolates were grouped into 12 different O serogroups and 31 O:H serotypes. Two cdt-I gene-positive E. coli (CTEC-I) isolates were identified as O112ac:H20 (phylogenetic group B1) and OUT:H26 (D), respectively. Three cdt-III gene-positive E. coli (CTEC-III) isolates were identified as O2:HUT (B2), 16 as OUT (B1) and 1 OUT (D), whereas one each of the 5 CTEC-III isolates belonged to serotype O2:NM (B2), O7:H6 (B1), O88:H2 (B1), O88:H4 (B1), and O88:H6 (B1), respectively. One cdt-IV gene-positive find protocol E. coli (CTEC-IV) isolate was identified as O169:H10 (B2). www.selleckchem.com/products/Vorinostat-saha.html The CTEC-V isolates belonged to divergent serotypes and phylogenetic groups, including O2:H10 (B2), O8:HUT (B1), O22:H8 (B1), O22:HUT (B1), O113:H21 (B1), O113:NM (B1), O118:NM (B1), O154:H34 (B1), O156:HUT (B1), O163:HUT (B1) and OUT (30 B1 and 2 D strains), as shown in Table 2. One isolate which was positive for both cdt-III and cdt-V genes was identified as O2:HUT (B2). Five and one CTEC-V isolates from swine were identified as O98:H10 (B1) and OUT:HUT
(B1), respectively. Interestingly, the E. albertii strain Sw-9 showed cross reaction with the E. coli O84 antiserum. heptaminol Virulence gene profile To analyze the virulence gene profile of the CTEC and E. albertii strains isolated in this study, genes for DEC, NTEC and putative adhesins reported in STEC
(see details in Material and Methods section) were investigated by colony hybridization assays (Table 2). In agreement with the previous report , all the CTEC-III strains possessed the cnf2 gene, indicating that cdt-III of these strains could be located on pVir-like plasmid. Surprisingly, 7 of the CTEC-V strains also possessed cnf2. The eaeA gene that encodes an outer membrane protein called intimin, which is necessary for intimate attachment of EPEC and EHEC strains to epithelial cells, was detected in the E. albertii strain Sw-9 from swine and all of the 3 CTEC-V O156:HUT (B1) strains from cattle (Table 2). The intimin subtype of three CTEC-V O156 strains was determined as θ/γ2 by PCR-RFLP, but the amplicon was not obtained in E. albertii strain Sw-9. Sixteen CTEC-V isolates (6 O22, 10 OUT) were positive for the stx1 and stx2 genes, while 6 CTEC-V strains (5 O113, 1 OUT) were positive for only stx2. Cytotoxicity assay using Vero and CHO cells, which are susceptible and unsusceptible to Stx intoxication, respectively, indicated that all the stx gene-positive CTEC strains produced functional Stx (titer ranging from 16 to 128<) and CDT (1 to 64) (Figure 3).