Metagenomic Profiling of Diarrheagenic Escherichia coli Pathotypes Reveals Predominance of Diffusely Adherent and Enteroaggregative Subtypes and Mixed Signatures Among Under-Fives in Tanzania

Diarrheagenic Escherichia coli (DEC) is a leading enteric pathogen in children under five in sub-Saharan Africa, yet conventional targeted assays fail to capture mixed virulence signatures or provide quantitative context relative to E. coli carriage. We applied a virulence-aware nanopore metagenomics workflow to characterise DEC pathotype distribution, mixed signatures, and virulence gene burden in 126 under-five stool metagenomes from six Tanzanian regions. Virulence support was quantified as aligned bases normalised to per-sample E. coli-aligned bases, termed GPMB (gene bases per million E. coli-aligned bases). At least one DEC virulence family was detected in 79/126 (62.7%) samples. afa/dra (diffusely adherent E. coli [DAEC] marker) was the most prevalent family (46/126, 36.5%); DAEC-containing pathotypes accounted for 46/79 (58.2%) of all assigned calls across all six regions. Mixed signatures occurred in 21/79 (26.6%) assigned samples, most commonly DAEC + enteroaggregative E. coli (EAEC) (n = 11). Enterotoxigenic E. coli (ETEC) was geographically concentrated in Mwanza. Age-stratified analysis revealed declining DAEC prevalence with age. Multivariable logistic regression found no significant independent associations between DEC positivity and age, sex, or rainfall. Unassigned samples had 6.8-fold lower E. coli carriage depth, implicating sequencing depth rather than pathotype absence as the primary non-detection driver. DAEC and EAEC should be elevated as priority DEC surveillance targets in Tanzania.