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In this study presence of virulence genes in multidrug resistant Escherichia coli isolated from Mula-Mutha river, Pune, India was undertaken. The objective was to understand whether the isolates were of diarrhoeagenic or of environmental origin. This was essential since the river flows through urban and rural parts of Pune and its water is used not only for industrial and agricultural purposes but also for domestic usage. One hundred and two multidrug E. coli isolates were selected from our previous study which detected genes coding for antibiotic resistance as well as identified integrons associated with multidrug resistance. Isolates were subjected to multiplex PCR to detect presence of virulence genes, set1A, set1B, sen astA, aggA, aafA, pet, stx1 and stx. Sequencing was performed to confirm the amplified PCR product. Seven of the 102 E. coli isolates showed gene set1A alone identifying them as Enteroaggregative E. coli. Thus, the findings revealed that majority of drug resistant E. coli were environmental in origin. The presence of antibiotic resistant genes, integrons in the environment as well as diarrhoeagenic E. coli isolates is a warning and calls for efficient public health measures to ensure that untreated sewage and industrial waste does not enter the Mula-Mutha river. [ABSTRACT FROM AUTHOR]