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Shiga toxin–producing Escherichia coli (STEC) is an important public health concern pathogen, as it produces two toxins, Stx1 and Stx2, with cytotoxic capacity. In addition, STEC strains are frequently involved in food outbreaks worldwide, leading to public health challenges and economic losses. In this context, the occurrence and antimicrobial resistance profile of the STEC isolated from fresh beef produced in Mato Grosso, Brazil, were estimated. One hundred seven retail beef under vacuum-packaged produced by 13 different slaughterhouses were submitted to microbiological, molecular, and antimicrobial resistance analyses. STEC occurrence in beef was of 4.67%, and five strains presented the stx2 gene. The O111 serogroup, reported in several outbreak cases worldwide, was detected, and other serotypes (O8:H20, O22:H16, and O141:H49) were also isolated. All isolated strains displayed sensitivity …