PCR products were subjected to capillary electrophoresis on an ABI-310 Genetic Analyzer (Applied Biosystems). Each peak was identified according to colour and size and the allele number was assigned based on fragment sizes, as described by Lindstedt et al. (2007). Alleles for which amplicons were absent were designated an allele number of ‘0’. The allele numbers
were entered into bionumerics (Applied Maths) as character values and a dendogram was LDK378 constructed using categorical coefficients and the Ward algorithm. Nucleotide sequencing of the arcA gene (aerobic respiratory control protein A) was performed using the primers and conditions described previously (Leomil et al., 2005). Internal arcA sequences of 513 bp were used for analysis. The sequences were analysed using lasergene software (DNASTAR, Madison, WI) and accelrys gene v2.5 software (Accelrys Ltd, Cambridge, UK). Motility indicating flagellar antigens was found in 36 (58.1%) of the strains. Veliparib research buy Serotyping of H-antigens revealed the presence of the H32 antigen in six and the H11 antigen in 30 strains. The 26 (41.9%) nonmotile
E. coli O26 strains were shown to carry the fliCH11 gene. Fermentation of rhamnose and dulcitol (RDF+) was found with 18 O26:NM strains and with four O26:H32 strains. Thirty O26:H11 and seven O26:NM strains were negative for fermentation of rhamnose and dulcitol (RDF−). Two O26:H32 and one O26:NM strain were positive for fermentation of rhamnose but negative for dulcitol (Table Cyclic nucleotide phosphodiesterase 1). Twenty-three (37.1%)
of the O26 strains produced cytotoxins on Vero cells and were positive for Stx1 (n=15), Stx2 (n=5) or Stx1 and Stx2 (n=3) as tested by enzyme-linked immunosorbent assay. Subtyping of stx genes revealed stx1 in 18 strains, stx2 in seven strains and the mucus activatable stx2d gene in one strain (D618/98). All 56 O26:H11 and O26:NM strains carried an intimin (eae-β) gene. Thus, 33 isolates were identified as EPEC and 23 isolates as EHEC. The six O26:H32 strains were negative for stx- and eae-genes. Production of haemolysins was detected in 51 strains. The enterohaemolytic phenotype (Beutin et al., 2004) and the underlying e-hlyA gene was found with 27 O26:H11 and six O26:NM strains (53.2%). An α-haemolytic phenotype and the α-hlyA gene were present in all 18 RDF+ O26:NM strains (29.0%). The O26:H32 strains were negative for haemolysins and for e-hlyA and α-hlyA genes (Table 1). All O26 strains were tested for additional virulence genes associated with other E. coli pathotypes, STIa, STIb, LTI, ipaH, aggR, bfpB, saa, nleB, stcE, stcE-O103, cdt, and subA. One O26:H32 strain from a dog (C 4050) was positive for STIa and identified as enterotoxigenic E. coli (ETEC).