Escherichia coli Clonobiome: Assessing the Strain Diversity in Feces and Urine by Deep Amplicon Sequencing.

While microbiome studies have focused on diversity at the species level or higher, 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, multidrug-resistant, and highly pathogenic Escherichia coli subclone ST131-H30 (H30) is of special interest, as it has already been found persisting in the gut and bladder in 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 the 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 completely 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, which could be useful in studying the population structure and dynamics of multidrug-resistant and/or highly pathogenic strains in their natural environments.IMPORTANCE Bacterial 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 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 the 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.