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ABSTRACT DiarrheagenicEscherichia coli, collectively known as DEC, is a leading cause of diarrhea, particularly in children in low- and middle-income countries. Diagnosing infections caused by different DEC pathotypes traditionally relies on the cultivation and identification of virulence genes, a resource-intensive and error-prone process. Here, we compared culture-based DEC identification with shotgun metagenomic sequencing of whole stool using 35 randomly drawn samples from a cohort of diarrhea-afflicted patients. Metagenomic sequencing detected the cultured isolates in 97% of samples, revealing, overall, reliable detection by this approach. Genome binning yielded high-qualityE. colimetagenome-assembled genomes (MAGs) for 13 samples, and we observed that the MAG did not carry the diagnostic DEC virulence genes of the corresponding isolate in 60% of these samples. Specifically, two distinct scenarios were observed: diffusely adherentE. coli(DAEC) isolates without corresponding DAEC MAGs appeared to be relatively rare members of the microbiome, which was further corroborated by quantitative PCR (qPCR), and thus unlikely to represent the etiological agent in 3 of the 13 samples (~23%). In contrast, ETEC virulence genes were located on plasmids and largely escaped binning in associated MAGs despite being prevalent in the sample (5/13 samples or ~38%), revealing limitations of the metagenomic approach. These results provide important insights for diagnosing DEC infections and demonstrate how metagenomic methods can complement isolation efforts and PCR for pathogen identification and population abundance. IMPORTANCEDiagnosing enteric infections based on traditional methods involving isolation and PCR can be erroneous due to isolation and other biases, e.g., the most abundant pathogen may not be recovered on isolation media. By employing shotgun metagenomics together with traditional methods on the same stool samples, we show that mixed infections caused by multiple pathogens are much more frequent than traditional methods indicate in the case of acute diarrhea. Further, in at least 8.5% of the total samples examined, the metagenomic approach reliably identified a different pathogen than the traditional approach. Therefore, our results provide a methodology to complement existing methods for enteric infection diagnostics with cutting-edge, culture-independent metagenomic techniques, and highlight the strengths and limitations of each approach. 

Abstract Heterotrophic activity, primarily driven by sulfate-reducing prokaryotes, has traditionally been linked to nitrogen fixation in the root zone of coastal marine plants, leaving the role of chemolithoautotrophy in this process unexplored. Here, we show that sulfur oxidation coupled to nitrogen fixation is a previously overlooked process providing nitrogen to coastal marine macrophytes. In this study, we recovered 239 metagenome-assembled genomes from a salt marsh dominated by the foundation plantSpartina alterniflora, including diazotrophic sulfate-reducing and sulfur-oxidizing bacteria. Abundant sulfur-oxidizing bacteria encode and highly express genes for carbon fixation (RuBisCO), nitrogen fixation (nifHDK) and sulfur oxidation (oxidative-dsrAB), especially in roots stressed by sulfidic and reduced sediment conditions. Stressed roots exhibited the highest rates of nitrogen fixation and expression level of sulfur oxidation and sulfate reduction genes. Close relatives of marine symbionts from theCandidatusThiodiazotropha genus contributed ~30% and ~20% of all sulfur-oxidizingdsrAand nitrogen-fixingnifKtranscripts in stressed roots, respectively. Based on these findings, we propose that the symbiosis betweenS. alternifloraand sulfur-oxidizing bacteria is key to ecosystem functioning of coastal salt marshes.