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Defined criteria are essential for quality-assurance of rectal surveillance cultures.ESBL's were detected in 31.3% of soiled swabs vs. 23.7% of non-soiled swabs (P = .036).Enterobacteriaceae failed to grow on MacConkey agar from 36 (8.3%) of soiled swabs.Fecal soiling can serve as a simple QA criterion and prevent false-negative results.We aimed to validate quality assurance (QA) criteria for rectal surveillance cultures (RSCs) for extended-spectrum β-lactamase-producing Enterobacteriaceae. QA for RSCs were tested by observing the presence or absence of fecal soiling and by examining the growth of Enterobacteriaceae on MacConkey agar. Extended-spectrum β-lactamase-producing Enterobacteriaceae were detected in 136 out of 434 soiled swabs (31.3%) and in 61 out of 257 nonsoiled swabs (23.7%) (P = .036). Observation of fecal soiling on RSCs can serve as a simple QA criterion and prevent the reporting of false-negative results.