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DNA‐based subtyping of verocytotoxin‐producing Escherichia coli (VTEC) O128ab:H2 strains from human and raw meat sources
Author(s) -
Domingue G.,
Willshaw G.A.,
Smith H.R.,
Perry N.,
Radford D.,
Cheasty T.
Publication year - 2003
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1046/j.1472-765x.2003.01424.x
Subject(s) - verocytotoxin , biology , pulsed field gel electrophoresis , vtec , subtyping , restriction fragment length polymorphism , intimin , escherichia coli , genetics , amplicon , genotype , plasmid , polymerase chain reaction , shiga like toxin , dna profiling , microbiology and biotechnology , gene , dna , enterobacteriaceae , computer science , programming language
Aims: To investigate subtyping methods for verocytotoxin‐producing Escherichia coli (VTEC) O128ab:H2. Methods and Results: Eleven human and food strains isolated over a 15‐year period were examined. All were intimin ( eae )‐negative, but all possessed enterohaemolysin and VT1‐encoding sequences which in nine strains were vtx 1c variant. Ten strains had VT2 genes which were all vtx 2d. Plasmid profiles and randomly amplified polymorphic DNA‐PCR were not discriminatory. Long‐PCR restriction fragment length polymorphism of amplicons bound by the p gene and the VT2A subunit had screening potential. Pulsed field gel electrophoresis (PFGE) using Xba I gave fine discrimination although VT2 sequences were located on a 220 kbp fragment conserved in nine strains and on a 200 kbp fragment in the 10th. Conclusions: As a result of apparent clonality, PFGE proved essential for differentiation. Long‐PCR has promise for screening but requires further evaluation of inter‐strain variable sequences. Significance and Impact of the Study: A combined phenotypic and genotypic screen, and PFGE for selected strains was effective.