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Background Carbapenem-resistant Pseudomonas aeruginosa (CRPA) colonizes the gastrointestinal tract of intensive care unit (ICU) patients, and CRPA colonization puts patients at increased risk of CRPA infection. Prior studies have not examined relationships between the microbiota, medications, and CRPA colonization acquisition. Methods Data and perirectal swabs were obtained from a cohort of ICU patients at the University of Maryland Medical Center. Patients (N = 109) were classified into 3 groups by CRPA colonization-acquisition status and antimicrobial exposure. We conducted 16S ribosomal RNA gene sequencing of an ICU admission swab and ≥1 additional swab and evaluated associations between patient characteristics, medications, the gastrointestinal microbiota, and CRPA colonization acquisition. Results ICU patients had low levels of diversity and high relative abundances of pathobionts. Piperacillin-tazobactam was prescribed more frequently to patients with CRPA colonization acquisition than those without. Piperacillin-tazobactam was associated with low abundance of potentially protective taxa (eg, Lactobacillus and Clostridiales) and increased risk of Enterococcus domination (odds ratio [OR], 5.50; 95% confidence interval [CI], 2.03–14.92). Opioids were associated with dysbiosis in patients who did not receive antibiotics; potentially protective Blautia and Lactobacillus were higher in patients who did not receive opioids. Several correlated taxa, identified at ICU admission, were associated with lower risk of CRPA colonization acquisition (OR, 0.58; 95% CI,.38–.87). Conclusions Antibiotics differed in their impact on the microbiota, with piperacillin-tazobactam being particularly damaging. Certain bacterial taxa (eg, Clostridiales) were negatively associated with CRPA colonization acquisition. These taxa may be markers of risk for CRPA colonization acquisition and/or serve a protective role. [ABSTRACT FROM AUTHOR]