Since its discovery in the early 2000s, methicillin-resistant Staphylococcus 
aureus (MRSA) clonal complex 398 (CC398) has become a rapidly emerging cause of 
human infections, most often associated with livestock exposure. We applied 
whole-genome sequence typing to characterize a diverse collection of CC398 
isolates (n = 89), including MRSA and methicillin-susceptible S. aureus (MSSA) 
from animals and humans spanning 19 countries and four continents. We identified 
4,238 single nucleotide polymorphisms (SNPs) among the 89 core genomes. Minimal 
homoplasy (consistency index = 0.9591) was detected among parsimony-informative 
SNPs, allowing for the generation of a highly accurate phylogenetic 
reconstruction of the CC398 clonal lineage. Phylogenetic analyses revealed that 
MSSA from humans formed the most ancestral clades. The most derived lineages 
were composed predominantly of livestock-associated MRSA possessing three 
different staphylococcal cassette chromosome mec element (SCCmec) types (IV, V, 
and VII-like) including nine subtypes. The human-associated isolates from the 
basal clades carried phages encoding human innate immune modulators that were 
largely missing among the livestock-associated isolates. Our results strongly 
suggest that livestock-associated MRSA CC398 originated in humans as MSSA. The 
lineage appears to have undergone a rapid radiation in conjunction with the jump 
from humans to livestock, where it subsequently acquired tetracycline and 
methicillin resistance. Further analyses are required to estimate the number of 
independent genetic events leading to the methicillin-resistant sublineages, but 
the diversity of SCCmec subtypes is suggestive of strong and diverse 
antimicrobial selection associated with food animal production.
IMPORTANCE: Modern food animal production is characterized by densely 
concentrated animals and routine antibiotic use, which may facilitate the 
emergence of novel antibiotic-resistant zoonotic pathogens. Our findings 
strongly support the idea that livestock-associated MRSA CC398 originated as 
MSSA in humans. The jump of CC398 from humans to livestock was accompanied by 
the loss of phage-carried human virulence genes, which likely attenuated its 
zoonotic potential, but it was also accompanied by the acquisition of 
tetracycline and methicillin resistance. Our findings exemplify a bidirectional 
zoonotic exchange and underscore the potential public health risks of widespread 
antibiotic use in food animal production.