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This study was undertaken to determine the virulence and antibiotic resistance profiles of diarrhoeagenic Escherichia coli (DEC) in environmental waters of Johannesburg, South Africa. Samples were collected and cultured on selective media. An 11-plex PCR assay was used to differentiate five DEC, namely: enteroaggregative (EAEC), enterohaemorrhagic (EHEC), enteroinvasive (EIEC), enteropathogenic (EPEC) and enterotoxigenic (ETEC). The antibiotic resistance profile of isolates was determined using the VITEK®-2 automated system. The virulence profiles of 170 E. coli tested showed that 40% (68/170) were commensals and 60% (102/170) were pathogenic. EPEC had a prevalence of 19.2% (32/170), followed by ETEC 11.4% (19/170), EAEC 6% (10/170) and EHEC 3% (5/170). Hybrid DEC carrying a combination of simultaneously two and three pathogenic types was detected in twenty-eight and nine isolates, respectively. The antibiotic susceptibility testing showed isolates with multidrug resistance, including cefuroxime (100%), ceftazidime (86%), cefotaxime (81%) and cefepime (79%). This study highlighted the widespread occurrence of DEC and antibiotic resistance strains in the aquatic ecosystem of Johannesburg. The presence of hybrid pathotypes detected in this study is alarming and might lead to more severe diseases. There is a necessity to enhance surveillance in reducing the propagation of pathogenic and antibiotic-resistant strains in this area.