Methicillin-resistant Staphylococcus aureus (MRSA) infections pose a major 
challenge in health care, yet the limited heterogeneity within this group 
hinders molecular investigations of related outbreaks. Pulsed-field gel 
electrophoresis (PFGE) has been the gold standard approach but is impractical 
for many clinical laboratories and is often replaced with PCR-based methods. 
Regardless, both approaches can prove problematic for identifying subclonal 
outbreaks. Here, we explore the use of whole-genome sequencing for clinical 
laboratory investigations of MRSA molecular epidemiology. We examine the 
relationships of 44 MRSA isolates collected over a period of 3 years by using 
whole-genome sequencing and two PCR-based methods, multilocus variable-number 
tandem-repeat analysis (MLVA) and spa typing. We find that MLVA offers higher 
resolution than spa typing, as it resolved 17 versus 12 discrete isolate groups, 
respectively. In contrast, whole-genome sequencing reproducibly cataloged 
genomic variants (131,424 different single nucleotide polymorphisms and indels 
across the strain collection) that uniquely identified each MRSA clone, 
recapitulating those groups but enabling higher-resolution phylogenetic 
inferences of the epidemiological relationships. Importantly, whole-genome 
sequencing detected a significant number of variants, thereby distinguishing 
between groups that were considered identical by both spa typing (minimum, 1,124 
polymorphisms) and MLVA (minimum, 193 polymorphisms); this suggests that these 
more conventional approaches can lead to false-positive identification of 
outbreaks due to inappropriate grouping of genetically distinct strains. An 
analysis of the distribution of variants across the MRSA genome reveals 47 
mutational hot spots (comprising ∼ 2.5% of the genome) that account for 23.5% of 
the observed polymorphisms, and the use of this selected data set successfully 
recapitulates most epidemiological relationships in this pathogen group.