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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.