Abstract

While microbiome studies have focused on diversity on the species or higher level, bacterial species in microbiomes are represented by different, often multiple strains. These strains could be clonally and phenotypically very different, making assessment of strain content vital to a full understanding of microbiome function. This is especially important with respect to antibiotic resistant strains, the clonal spread of which may be dependent on competition between them and susceptible strains from the same species. The pandemic, multi-drug resistant, and highly pathogenic E. coli subclone ST131-H30 (H30) is of special interest, as it has already been found persisting in the gut and bladder of healthy people. In order to rapidly assess E. coli clonal diversity, we developed a novel method based on deep sequencing of two loci used for sequence typing, along with an algorithm for analysis of resulting data. Using this method, we assessed fecal and urinary samples from healthy women carrying H30, and were able to uncover considerable diversity, including strains with frequencies at <1% of the E. coli population. We also found that even in the absence of antibiotic use, H30 could complete dominate the gut and, especially, urine of healthy carriers. Our study offers a novel tool for assessing a species clonal diversity (clonobiome) within the microbiome, that could be useful in studying population structure and dynamics of multi-drug resistant and/or highly pathogenic strains in their natural environments.\n\nIMPORTANCEBacterial species in the microbiome are often represented by multiple genetically and phenotypically different strains, making insight into subspecies diversity critical to a full understanding of the microbiome, especially with respect to opportunistic pathogens. However, methods allowing efficient high-throughput clonal typing are not currently available. This study combines a conventional E. coli typing method with deep amplicon sequencing to allow analysis of many samples concurrently. While our method was developed for E. coli, it may be adapted for other species, allowing for microbiome researchers to assess clonal strain diversity in natural samples. Since assessment of subspecies diversity is particularly important for understanding the spread of antibiotic resistance, we applied our method to study of a pandemic multidrug-resistant E. coli clone. The results we present suggest that this clone could be highly competitive in healthy carriers, and that the mechanisms of colonization by such clones need to be studied.