Multidrug-resistant gram-negative bacteria (MDR-GNB) are one of the main challenges in intensive care units (ICUs), mainly extended-spectrum β-lactamase-producing Klebsiella pneumoniae (EKP), carbapenemase-producing Enterobacterales, carbapenem-resistant (CR) Pseudomonas aeruginosa, and CR Acinetobacter spp. The objectives were to determine the prevalence and incidence of colonization/infection by these bacteria in Spanish ICUs, their antimicrobial susceptibility, resistome, and phylogeny. All patients admitted to ICUs of seven Spanish hospitals were evaluated (15 February to 30 March 2023). Colonization was determined in rectal and oropharyngeal swabs upon admission and the first and second weeks after admission. Infections were studied for up to 60 days from admission. Antimicrobial susceptibility was determined by broth microdilution. Whole-genome sequencing was performed to analyze the resistome and phylogeny (core-genome Multi-Locus Sequence-Typing). In total, 767 patients were studied. The prevalence of MDR-GNB colonization on admission was 2.9% (23/767), and EKP was the most prevalent bacterium. The incidence of acquired colonization was 1.3 (22/1,694) cases per 100 ICU-admitted patients-day, with colonization by CR P. aeruginosa being the most frequent ( n = 14/22). Twenty-one patients had an infection (prevalence: 2.7%), mainly CR P. aeruginosa (12/21). The major resistance mechanisms and clones detected were some of the most frequent circulating clones in Spain: EKP-ST15/ bla _VIM-1+OXA-48 , EKP-ST307/ bla _CTXM-15 , and CR P. aeruginosa ST175/OprD porin deficiency. The antibiotics with the greatest activity against Enterobacterales ( n = 26) were colistin, cefiderocol, and meropenem-vaborbactam (100%); while in CR P. aeruginosa ( n = 20), they were colistin, cefiderocol, and imipenem-relebactam (95%–100%). The prevalence/incidence of colonization/infection by MDR-GNB in the ICUs of this study was low, but with important variations between hospitals. CR P. aeruginosa was the most common bacterium identified. IMPORTANCE: This comprehensive study integrates incidence/prevalence, clinical, microbiological, and genomic data to provide an up-to-date overview of multidrug-resistant gram-negative bacteria in Spanish intensive care units (ICUs). Its main strengths lie in its multidisciplinary approach and in the involvement of seven ICUs from major university hospitals across different regions in Spain. We believe that monitoring resistance epidemiology at the ICU level—especially through molecular surveillance of resistance mechanisms and clonal dissemination—is essential for optimizing antimicrobial use and guiding stewardship and infection control programs. Notably, carbapenem-resistant Pseudomonas aeruginosa was the most frequently identified organism in both colonization (first/second week) and infection cases, highlighting the need for its inclusion in routine hospital colonization screening.

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