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Diarrheagenic Escherichia coli (DEC) cause acute and persistent diarrhoea worldwide, but little is known about their epidemiology in Mexico. We determined the prevalence of bacterial enteropathogens in 831 children with acute diarrhoea over a four-year period in Yucatan, Mexico. Six DEC supplementary virulence genes (SVG), mainly associated with enteroaggregative E. coli (EAEC), were sought in 3100 E. coli isolates. DEC was the most common bacterial enteropathogen (28%), surpassing Salmonella (12%) and Shigella (9%). Predominant DEC groups were diffusely adherent E. coli (DAEC) (35%), EAEC (24%), and enteropathogenic E. coli (EPEC) (19%). Among children with DEC infections, 14% had severe illness mainly caused by EPEC (26%) and DAEC (18%); 30% had moderate diarrhoea mainly caused by DAEC (36%), mixed DEC infections (33%) and EAEC (32%). DAEC was most prevalent during spring, while ETEC, EAEC and EPEC predominated in summer. EAEC was more frequent in children 6–24 months old than in those younger than 6 months of age (P = 0.008, OR = 4.2, 95% CI, 1.3–13.9). The presence of SVG dispersin, (aatA), dispersin-translocator (aatA), enteroaggregative heat-stable toxin 1 (astA), plasmid encoded toxin (pet), cytolethal distending toxin (cdt) was higher in DEC than non-DEC strains, (36% vs 26%, P <0.0001, OR = 1.5, 95% CI, 1.3–1.8). 98% of EAEC-infected children harboured strains with SVG; 85% carried the aap-aatA gene combination, and 33% of these also carried astA. 28% of both EPEC and ETEC, and 6% of DAEC patients had strains with SVG. 54% of EPEC patients carried pet-positive strains alone or in combination with astA; only this DEC group harboured cdt-positive isolates. All ETEC patients carried astA- or astA-aap-positive strains. astA and aap were the most common SVG in DAEC (3% and 2%) and non-DEC strains (21% and 13%). DEC carrying SVG are an important cause of moderate to severe bacterial diarrhoea in Mexican children. Author Summary: Diarrhoea is an important cause of illness and death among young children in low- and middle-income countries. Nonetheless, very few epidemiological studies of diarrhoea have been conducted in Mexico during the last two decades. In recent years, a group of bacteria known as diarrheagenic Escherichia coli (DEC) have been recognized as a major cause of diarrheal illness worldwide. This group cannot be identified by the conventional biochemical methods used for other diarrhoeal pathogens such as Salmonella or Shigella, which limits its identification. We conducted a study in Yucatan, Mexico, to identify the bacterial causes of acute diarrhoea in children less than five years of age who required hospitalization. All DEC strains were further characterized for the presence of six E. coli supplementary virulence genes (SVG). Of the 831 children with acute diarrhoea, a bacterial pathogen was found in 56%. DEC was the most prevalent (28%) pathogen, surpassing Salmonella and Shigella. Among children with DEC diarrhoea, 44% were moderately or severely ill. Thirty-six percent of the DEC strains had one or more SVG, which were more common in older children. Our results strongly suggest that DEC carrying SVG are an important cause of moderate to severe bacterial diarrhoeal disease in Mexico. [ABSTRACT FROM AUTHOR]