DNA-based subtyping of verocytotoxin-producing Escherichia coli (VTEC) O128ab:H2 strains from human and raw meat sources.
OBJECTIVE
To investigate subtyping methods for verocytotoxin-producing Escherichia coli (VTEC) O128ab:H2.


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 vtx1c variant. Ten strains had VT2 genes which were all vtx2d. 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 XbaI 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.


CONCLUSIONS
A combined phenotypic and genotypic screen, and PFGE for selected strains was effective.
Domingue
G
G
Laboratory of Enteric Pathogens, Specialist and Reference Microbiology Division, Health Protection Agency, London, UK. g.dominique@ex.ac.uk
Willshaw
G A
GA
Smith
H R
HR
Perry
N
N
Radford
D
D
Cheasty
T
T
D018829:Adhesins, Bacterial / Q000032:analysis; D015373:Bacterial Typing Techniques / Q000379:methods*; D002352:Carrier Proteins / Q000032:analysis; D016172:DNA Fingerprinting / Q000379:methods; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods; D004926:Escherichia coli / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004927:Escherichia coli Infections / Q000453:epidemiology / Q000382:microbiology*; D029968:Escherichia coli Proteins / Q000032:analysis; D005516:Food Microbiology; D005838:Genotype; D006460:Hemolysin Proteins / Q000032:analysis; D006801:Humans; D008460:Meat / Q000382:microbiology*; D008461:Meat Products / Q000382:microbiology; D017720:Molecular Epidemiology / Q000379:methods; D010641:Phenotype; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D019105:Random Amplified Polymorphic DNA Technique / Q000379:methods; D012703:Serotyping; D022622:Shiga Toxin 1 / Q000032:analysis; D022641:Shiga Toxin 2 / Q000032:analysis
D018829:Adhesins, Bacterial; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; C066219:enterohemolysin; C073819:eaeA protein, E coli