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Residents in long-term care facilities (LTCFs) are frequently colonized by multidrug-resistant Gram-negative bacteria, putting them at risk for subsequent infections. We aimed to evaluate the effect of the multispecies probiotic Omnibiotic10AAD&reg, on the intestinal and inguinal skin colonization of patients by multidrug-resistant Gram-negative bacteria in LTCFs. Patients colonized by multidrug-resistant Gram-negative bacteria received a 12 week oral course of Omnibiotic10AAD&reg, Inguinal swabs and stool samples were collected during and after treatment for microbiological and microbiome analysis. The median age of patients was 76 years. Twelve patients completed the pilot study. Intestinal colonization was reduced to 42% of patients 8 weeks after the end of treatment, but increased to 66% 24 weeks after the end of probiotic treatment. Colonization of inguinal skin was lowest during probiotic treatment and increased thereafter. Fecal microbiome analysis revealed statistically significant increases of the genus Enterococcus comparing start and end of probiotic treatment. In conclusion, a 12 week course of a multispecies probiotic led to a transient reduction of intestinal colonization 8 weeks after the end of treatment. The findings of our pilot study warrant further research in the area of probiotics and intestinal colonization by multidrug-resistant bacteria.