Simplified protocol for pulsed-field gel electrophoresis analysis of Streptococcus pneumoniae

Abstract

A variety of pulsed-field gel electrophoresis (PFGE) protocols for the molecular subtyping of Streptococcus pneumoniae have been reported; most are time-consuming and complex. We sought to modify reference PFGE protocols to reduce the time required while creating high-quality gels. Only protocol modifications that resulted in high-quality banding patterns were considered. The following protocol components were modified. Lysis enzymes (lysozyme, mutanolysin, and RNase A) were deleted in a stepwise fashion, and then the lysis buffer was deleted. Lysis and digestion were accomplished in a single step with EDTA and N-lauroyl sarcosine (ES; pH 8.5 to 9.3) incubation at 50 degrees C in the absence of proteinase K. All enzymes except the restriction enzyme were omitted. A minimum incubation time of 6 h was required to achieve high-quality gels. All of the reactions were performed within 9 h, and the total protocol time from lysis to gel completion was reduced from 3 days to only 36 h. Combining lysis and digestion into a single step resulted in a substantial reduction in the time required to perform PFGE for S. pneumoniae. The ES solution may have caused cell lysis by activating N-acetylmuramyl-L-alanine amidase, the pneumococcal autolysin.

FIG. 1

PFGE of invasive S. pneumoniae isolates. Even-numbered lanes, simplified protocol; odd-numbered lanes, standard protocol. Lane 1, lambda ladder; lanes 2 and 3, serotype 6A; lanes 4 and 5, serotype 9V; lanes 6 and 7, serotype 19F; lanes 8 and 9, serotype 23F (Spanish clone); lanes 10 and 11, serotype 19A; lanes 12 and 13, serotype 14; lanes 14 and 15, serotype 9V.