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To investigate the assumption that usage of novobiocin (20 mg l−1) in Shiga toxin-producing Escherichia coli (STEC) enrichment broths could achieve false-negative results.First, the minimum inhibitory concentration (MIC) of 74 E. coli O157:H7 and 55 non-O157:H7 STEC strains to novobiocin was determined. Second, to visualize the potential impact of novobiocin on the STEC growth during the enrichment step, the growth experiments were carried out in trypticase soy broth (TSB) with and without 20 mg l−1 of novobiocin. The MIC values varied from 32 to >64 mg l−1 for the 74 E. coli O157:H7 strains, and from 16 to >64 mg l−1 for the 55 non-O157:H7 STEC strains. The E. coli O157:H7 strains were significantly (P < 0·001) more resistant to novobiocin than the non-O157:H7 STEC strains. The present study shows that the addition of novobiocin into enrichment broths inhibits the growth of some non-O157:H7 STEC strains, and slows down the growth of some STEC strains.Enrichment broths supplemented by novobiocin could lead to false-negative results for detecting STEC from food.We strongly suggest that novobiocin should not be systematically added into enrichment broths for detecting STEC from food.