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Journal of Clinical Microbiology, January 2000, p. 351-353, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Simplified Protocol for Pulsed-Field Gel Electrophoresis Analysis of Streptococcus pneumoniae

M. Catherine McEllistrem,1,2,* Janet E. Stout,3 and Lee H. Harrison1,2

Public Health Infectious Diseases Laboratory, University of Pittsburgh Graduate School of Public Health,1 Department of Medicine, University of Pittsburgh School of Medicine,2 and Veterans Affairs Medical Center,3 Pittsburgh, Pennsylvania

Received 23 June 1999/Returned for modification 19 August 1999/Accepted 8 October 1999

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°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.

* Corresponding author. Mailing address: Public Health Infectious Diseases Laboratory, University of Pittsburgh Graduate School of Public Health, A521 Crabtree Hall, 130 DeSoto St., Pittsburgh, PA 15261. Phone: (412) 648-6401. Fax: (412) 648-6399. E-mail: McEllistremC{at}msx.dept-med.pitt.edu.

Journal of Clinical Microbiology, January 2000, p. 351-353, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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