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Background: Pyomyositis is primarily a disease primarily of children in tropical regions. 90% of cases are caused by Staphylococcus aureus. Only small case series have been reported, and the bacterial genetic factors underlying this disease are incompletely understood. We present a genome wide association study of S. aureus isolated from pyomyositis and asymptomatic carriage from the same human paediatric population. Material/methods: Bacterial isolates were identified from 102 children, attending Angkor Hospital for Children in Siem Reap, Cambodia over a six-year period 2007-12, who had a clinical diagnosis of pyomyositis and S. aureus cultured from the abscess samples. 418 S. aureus nasal carriers were identified from two studies of S. aureus carriage in the same population in 2008 (n=222) and 2012 (n=196). A single isolate was cultured from each clinical sample and underwent whole-genome sequencing on the Illumina HiSeq platform, yielding 520 high quality sequences. Single nucleotide polymorphisms (SNPs) were identified after mapping reads against a reference genome (MRSA252) and de novo assembly was performed using Velvet. We tested for loci associated with pyomyositis by performing association testing for all SNPs and short sequences (kmers) using Gemma, which controls for bacterial population structure. We examined strains associated with pyomyositis using Bugwas, which identifies bacterial lineages and tests them for association with phenotype. Multi-locus sequence type was determined in silico using blast. Results: The phylogeny of S. aureus isolated from asymptomatic carriage and pyomyositis shows that 87/102 (85%) of pyomyositis isolates are found within a single clade (Figure) (p=1.6x10-30). These isolates belong to Clonal Complex (CC) 121. In contrast, carriage isolates include a global diversity of global lineages, including CC121, as well as CC1, CC5, CC15, CC45. Strikingly, 41 carriage isolates form a sub-clade within CC121 and no cases of pyomyositis are observed in this sub-clade. This lineage is strongly associated with carriage rather than pyomyositis (p=4x10-14). Both CC121 lineages were found as asymptomatic carriers in 2008 and 2012. In addition to lineage effects, we find a strong association between the presence of Panton-Valentine leucocidin (PVL) and pyomyositis, even after controlling for population structure (p=5x10-20). Kmers mapping to PVL were found in 99/102 (97%) of pyomyositis isolates, compared with 84/418 (20%) of carriage isolates. PVL is a known S. aureus virulence factor, and is often carried by CC121, but is absent from the protective carriage sub-clade. PVL was not limited to CC121 isolates in this study, but is found in multiple lineages (Figure). Conclusions: We have discovered strong evidence for bacterial genetic determinants for this relatively neglected, serious bacterial disease using a non-selective method with no prior assumptions. We find both lineage and gene specific effects, with an overwhelming association between PVL and pyomyositis in Cambodian children.



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Staphylococcus aureus, pyomyositis, PVL