> Pneumococcal Clone Identification
Pneumococcal Clone Identification: Using PFGE and CDC PFGE/MLST database
For the past several years we have used pulsed field gel electrophoresis (PFGE) together with multilocus sequence typing (MLST) to genotype pneumococcal isolates. PFGE offered advantages because it was less expensive for us to perform than MLST. For PFGE protocols see McEllistrem, M. C., J. E. Stout, and L. H. Harrison. 2000. Simplified protocol for pulsed-field gel electrophoresis analysis of Streptococcus pneumoniae . J. Clin. Microbiol. 38: 351-353 [Abstract/ Free Full Text]) (exit site). We prefer MLST for tracking clonal trends among invasive isolates in our surveillance (see http://www.cdc.gov/abcs/) because this data is very straightforward to analyze, and it provides an unambiguous, easily communicable digital identifier (see www.mlst.net (exit site) for relevant information, references, and protocols).
We have found that isolates of the same serotype with PFGE patterns sharing > 80% relatedness on an UPGMA- generated dendrogram nearly always share sequence identity among 5 or more of the 7 MLST targets. The situation is sometimes not as clear when comparing PFGE profiles between divergent serotypes, due to chance similarities in banding patterns. Therefore we only use the strategy of assigning MLST-based identifiers based upon isolates sharing PFGE profile relatedness ( > 80%) with isolates of the same serotype that have been subjected to MLST.
We have generated a large database of PFGE patterns from isolates of known serotypes with known multilocus sequence types (STs). Additionally our database contains patterns from the current recognized clones of the Pneumococcal Molecular Epidemiology network (http://www.sph.emory.edu/PMEN/) (exit site). All of our PFGE information is stored and analyzed within our Bionumerics (Applied Maths, Belgium) database.
Identifying Pneumococcal Clones using the CDC database
It is important to note that in order to supply you clone identification information that has the highest likelihood of being accurate, it is critical that you provide the serotype of the isolate.
Run PFGE of pneumococcal Sma I digests using your favorite protocol. You must co-run Sma I digests of our standard pneumococcal strain (we will send it to you upon request). At least every 8 th lane must contain the standard digest. Ideally, these 14 indicated standard strain bands should be discernible on your gel:
Simply run your gel with your isolates and standard (indicate the serotypes of your isolates) and email the digital tiff image to BBEALL@CDC.GOV . After analysis, we will respond back to you with our observations. Although we predict that we will usually be able to provide an accurate genotype result for a properly run digest, sometimes the PFGE profile may not match anything in our extensive database.