High-throughput Targeted Amplicon Screening Tool for Characterizing Intrahost Diversity Instaphylococcus Aureusdirectly from Sample

A significant proportion of people are asymptomatic carriers of Staphylococcus aureus (SA), an important risk factor for the development of opportunistic infections. SA colonization is dynamic, appearing and disappearing, with strains evolving and potentially shifting in composition over time and between body sites. These changes make detection challenging, and the numerous potential sources of reintroduction from other people and even other body site reservoirs preclude efficient efforts to prevent transmission and spread. Identifying typical sources is therefore critical for mitigation. Whole-genome sequencing (WGS), ideally of multiple colonies from multiple body sites, is the gold standard for characterizing SA strains and confirming transmission. However, this is often too resource-intensive for initial assessments of transmission and not feasible for large-scale studies involving various body sites from multiple individuals over time. To address these challenges, we developed a low-cost, custom, species-specific amplicon sequencing (AmpSeq) assay optimized to provide high-resolution discrimination of SA genotypes directly from samples. We tested this approach on a subset of samples that were a part of a large-scale longitudinal study of SA carriage. Oral and nasal samples were collected from nine participants every 2 weeks for up to 18 weeks and qPCR positive samples were analysed using our AmpSeq assay directly from the sample without culturing. The longitudinal sampling strategy enabled us to characterize changes in SA colonization patterns over time, detect potential strain mixtures and identify rare variants that may serve as signatures of transmission between different body sites or among individuals. Without using WGS, we were able to rapidly eliminate the possibility of transmission between sampled residents. Participants who had positive oral and nasal samples had no fixed SNP differences between the two body sites, suggesting likely within-person spread. Analysis of rare variants segregating in the oral and nasal populations suggests that the nasal populations were the likely source of the spread because the nasal samples had higher diversity and most of the variants identified in the oral samples were shared with the nasal samples. While WGS can be used to provide higher resolution to colonization patterns and validate these findings, our AmpSeq approach offers a rapid, cost-effective, direct-from-sample method for species-specific screening intended for population-level characterization that allows researchers to strain type, identify or eliminate likely transmission cases and identify potential reservoirs before resorting to more expensive WGS methods.