Molecular typing of Legionella pneumophila serogroup 1 by pulsed-field gel electrophoresis with SfiI and comparison of this method with restriction fragment-length polymorphism analysis.
A panel of 131 well-characterised Legionella pneumophila serogroup 1 strains, previously used to evaluate a restriction fragment-length polymorphisms (RFLP) typing scheme, was examined by pulsed-field gel electrophoresis (PFGE) with the restriction endonuclease SfiI. The data obtained show that PFGE with SfiI is a highly discriminatory method yielding an index of discrimination (IOD) of 0.992 and 0.975, with 100% and 90% similarity thresholds respectively, compared with an IOD of 0.909 for the RFLP typing method. Reproducibility of PFGE profiles within gels was excellent and it was possible to compare the profiles visually. However, the reproducibility of the technique between gels was poor and visual comparison of the patterns was extremely difficult. Computer-aided analysis assisted the assessment of inter-gel reproducibility. Of 11 duplicates examined only four pairs showed 100% similarity, although 9 of 11 showed > or =90% similarity. In an attempt to determine if the PFGE banding patterns were sufficiently unambiguous to allow the method to be used as a definitive typing method, 20 coded strains were examined. At a 90% similarity level, 16 of these were placed in the correct PFGE type and four were not allocated to a type. Partial digestion of DNA by SfiI was noted despite careful control of DNA and enzyme concentrations, suggesting that an alternative enzyme might give more reproducible results.
DE Zoysa
A S
AS
Harrison
T G
TG
D000911:Antibodies, Monoclonal; D015373:Bacterial Typing Techniques; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D016521:Electrophoresis, Gel, Pulsed-Field; D006801:Humans; D016952:Legionella pneumophila / Q000145:classification* / Q000235:genetics; D012150:Polymorphism, Restriction Fragment Length; D015203:Reproducibility of Results; D012703:Serotyping
D000911:Antibodies, Monoclonal; C041749:endodeoxyribonuclease SfiI; D015252:Deoxyribonucleases, Type II Site-Specific