Characterization of Community-acquired Methicillin-resistant Staphylococcus aureus by Pulsed-field Gel Electrophoresis, Multilocus Sequence Typing, and Staphylococcal Protein A Sequencing: Establishing a Strain Typing Database
Degree Granting Department
Andrew Cannons, Ph.D.
S. aureus, PFGE, MLST, Spa typing, Genotyping
Staphylococcus aureus has long been recognized as a leading cause of nosocomial infection. However, several recent publications have demonstrated this pathogen as the cause of community-acquired severe wound infections and necrotizing pneumonia in otherwise healthy individuals. These highly virulent endemic clones have been reported in several locations in the United States and Canada. The rapid spread of the organism, the ability of certain clones to cause serious infection, and the antibiotic resistance of the endemic clones, illustrates the importance of infection control measures. In this study we examined three S. aureus typing techniques; pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and Staphylococcal protein A (spa) sequencing for subspeciation of community-acquired methicillin-resistant S. aureus (CA-MRSA). It is hypothesized that PFGE will result in a higher level of discrimination among the strains, while MLST and spa typing will result in highly portable data that lacks the discriminatory power of PFGE.
Thirty CA-MRSA isolates that were obtained from Florida and Washington State were characterized by molecular typing methods. Whole genome restriction analysis was performed by PFGE using the SmaI enzyme. Sequence-based typing analyses, MLST and spa typing, were performed by polymerase chain reaction (PCR) followed by sequencing. PFGE data was analyzed using the BioNumericsÂ® software package and sequence-based data was analyzed using DNAstarÂ®. MLST Alleles were assigned using the online MLST database (www.mlst.net) and spa types were assigned using the Ridom SpaServer (www.ridom.de/spaserver). Molecular characterization of the 30 isolates resulted in 21 pulsotypes, four MLST sequence types (STs), and six spa types. Combining data from both MLST and spa typing resulted in only seven strain categories, many of which grouped isolates that are not epidemiologically linked.
These data demonstrate that techniques such as MLST and spa typing are not well suited for tracking isolates with limited evolutionary diversity such as the CA-MRSA epidemic clones.
Scholar Commons Citation
Roberts, Jill Carolyne, "Characterization of Community-acquired Methicillin-resistant Staphylococcus aureus by Pulsed-field Gel Electrophoresis, Multilocus Sequence Typing, and Staphylococcal Protein A Sequencing: Establishing a Strain Typing Database" (2006). Graduate Theses and Dissertations.