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.

Inactivation of Transcriptional Regulators during Within-Household Evolution of Escherichia coli.

We analyzed the within-household evolution of two household-associated Escherichia coli strains from pandemic clonal group ST131-H30, using isolates recovered from five individuals within two families, each of which had a distinct strain. Family 1's strain was represented by a urine isolate from the index patient (older sister) with recurrent cystitis and a blood isolate from her younger sister with fatal urosepsis. Family 2's strain was represented by a urine isolate from the index patient (father) with pyelonephritis and renal abscesses, blood and kidney drainage isolates from the daughter with emphysematous pyelonephritis, and urine and fecal isolates from the mother with cystitis. Collectively, the several variants of each family's strain had accumulated a total of 8 (family 1) and 39 (family 2) point mutations; no two isolates were identical. Of the 47 total mutations, 36 resulted in amino acid changes or truncation of coded proteins. Fourteen such mutations (39%) targeted genes encoding transcriptional regulators, and 9 (25%) involved DNA-binding transcription factors (TFs), which significantly exceeded the relative contribution of TF genes to the isolates' genomes (∼6%). At least one-half of the transcriptional regulator mutations were inactivating, based on phenotypic and/or transcriptional analysis. In particular, inactivating mutations in the global regulator LrhA (repressor of type 1 fimbriae and flagella) occurred in the blood isolates from both households and increased the virulence of E. coli strains in a murine sepsis model. The results indicate that E. coli undergoes adaptive evolution between and/or within hosts, generating subpopulations with distinctive phenotypes and virulence potential.IMPORTANCE The clonal evolution of bacterial strains associated with interhost transmission is poorly understood. We characterized the genome sequences of clonal descendants of two Escherichia coli strains, recovered at different time points from multiple individuals within two households who had different types of urinary tract infection. We found evidence that the E. coli strains underwent extensive mutational diversification between and within these individuals, driven disproportionately by inactivation of transcriptional regulators. In urosepsis isolates, the mutations observed in the global regulator LrhA increased bacterial virulence in a murine sepsis model. Our findings help in understanding the adaptive dynamics and strategies of E. coli during short-term natural evolution.

A novel group of diverse Polinton-like viruses discovered by metagenome analysis.

BACKGROUND: The rapidly growing metagenomic databases provide increasing opportunities for computational discovery of new groups of organisms. Identification of new viruses is particularly straightforward given the comparatively small size of viral genomes, although fast evolution of viruses complicates the analysis of novel sequences. Here we report the metagenomic discovery of a distinct group of diverse viruses that are distantly related to the eukaryotic virus-like transposons of the Polinton superfamily. RESULTS: The sequence of the putative major capsid protein (MCP) of the unusual linear virophage associated with Phaeocystis globosa virus (PgVV) was used as a bait to identify potential related viruses in metagenomic databases. Assembly of the contigs encoding the PgVV MCP homologs followed by comprehensive sequence analysis of the proteins encoded in these contigs resulted in the identification of a large group of Polinton-like viruses (PLV) that resemble Polintons (polintoviruses) and virophages in genome size, and share with them a conserved minimal morphogenetic module that consists of major and minor capsid proteins and the packaging ATPase. With a single exception, the PLV lack the retrovirus-type integrase that is encoded in the genomes of all Polintons and the Mavirus group of virophages. However, some PLV encode a newly identified tyrosine recombinase-integrase that is common in bacteria and bacteriophages and is also found in the Organic Lake virophage group. Although several PLV genomes and individual genes are integrated into algal genomes, it appears likely that most of the PLV are viruses. Given the absence of protease and retrovirus-type integrase, the PLV could resemble the ancestral polintoviruses that evolved from bacterial tectiviruses. Apart from the conserved minimal morphogenetic module, the PLV widely differ in their genome complements but share a gene network with Polintons and virophages, suggestive of multiple gene exchanges within a shared gene pool. CONCLUSIONS: The discovery of PLV substantially expands the emerging class of eukaryotic viruses and transposons that also includes Polintons and virophages. This class of selfish elements is extremely widespread and might have been a hotbed of eukaryotic virus, transposon and plasmid evolution. New families of these elements are expected to be discovered.

Multiple layers of chimerism in a single-stranded DNA virus discovered by deep sequencing.

Viruses with single-stranded (ss) DNA genomes infect hosts in all three domains of life and include many medically, ecologically, and economically important pathogens. Recently, a new group of ssDNA viruses with chimeric genomes has been discovered through viral metagenomics. These chimeric viruses combine capsid protein genes and replicative protein genes that, respectively, appear to have been inherited from viruses with positive-strand RNA genomes, such as tombusviruses, and ssDNA genomes, such as circoviruses, nanoviruses or geminiviruses. Here, we describe the genome sequence of a new representative of this virus group and reveal an additional layer of chimerism among ssDNA viruses. We show that not only do these viruses encompass genes for capsid proteins and replicative proteins that have distinct evolutionary histories, but also the replicative genes themselves are chimeras of functional domains inherited from viruses of different families. Our results underscore the importance of horizontal gene transfer in the evolution of ssDNA viruses and the role of genetic recombination in the emergence of novel virus groups.