Comparison of genomic diversity between single and pooled Staphylococcus aureus colonies isolated from human colonization cultures

The most common approach to sampling the bacterial populations within an infected or colonized host is to sequence genomes from a single colony obtained from a culture plate. However, it is recognized that this method does not capture the genetic diver-sity in the population. Sequencing a mixture of several colonies (pool- seq) is a better approach to detect population heterogene-ity, but it is more complex to analyse due to different types of heterogeneity, such as within -clone polymorphisms, multi-strain mixtures, multi-species mixtures and contamination. Here, we compared 8 single-colony isolates (singles) and pool- seq on a set of 2286 Staphylococcus aureus culture samples to identify features that can distinguish pure samples, samples undergoing intraclonal variation and mixed strain samples. The samples were obtained by swabbing 3 body sites on 85 human partici-pants quarterly for a year, who initially presented with a methicillin-resistant S. aureus skin and soft-tissue infection (SSTI). We compared parameters such as sequence quality, contamination, allele frequency, nucleotide diversity and pangenome diversity in each pool to those for the corresponding singles. Comparing singles from the same culture plate, we found that 18% of sample collections contained mixtures of multiple multilocus sequence types (MLSTs or STs). We showed that pool- seq data alone could predict the presence of multi -ST populations with 95% accuracy. We also showed that pool- seq could be used to estimate the number of intra-clonal polymorphic sites in the population. Additionally, we found that the pool may contain clini-cally relevant genes such as antimicrobial resistance markers that may be missed when only examining singles. These results highlight the potential advantage of analysing genome sequences of total populations obtained from clinical cultures rather than single colonies.