Optimization of pulsed-field gel electrophoresis for Legionella pneumophila subtyping.

A total of 32 strains of Legionella pneumophila were used to optimize pulsed-field gel electrophoresis (PFGE) for subtyping of L. pneumophila. Twenty-six isolates of L. pneumophila with various origins and 11 isolates from five different water systems were used as the panels. For optimization of electrophoretic parameters (EPs) of SfiI PFGE, 26 isolates were analyzed with SfiI digestion, using four EPs yielding the same D value. The EP of a switch time of 5 to 50 s for 21 h had the smallest similarity coefficients and was declared the optimal EP for SfiI PFGE of L. pneumophila. By software analysis and pilot study, AscI was chosen as another PFGE enzyme. AscI PFGE could cluster the isolates from each water system into the same or very similar patterns and had a high degree of typing concordance with other molecular methods. In evaluating the discriminatory power of AscI with the panel of 26 isolates, AscI PFGE gave one single pattern and a D value of 100%. AscI PFGE had a high discriminatory power and a high degree of consistency with epidemiological data and other molecular typing methods for L. pneumophila subtyping, and hence, AscI could be used as a restriction enzyme in PFGE subtyping of L. pneumophila.