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The aim of this prospective cohort study was to determine the prevalence of gut colonization with multidrug-resistant (MDR) bacteria, risk factors for colonization, infection risk, and outcomes among preterm neonates hospitalized at a tertiary-care center in Serbia. During the period from December 2017 to April 2018, 103 neonates were screened for rectal carriage at admission and on the seventh day of life. Characterization of MDR strains was done by conventional microbiology and molecular methods. Out of 61 (59.2%) colonized neonates,