The recent emergence of multidrug resistance (MDR) in Acinetobacter baumannii has raised concern in health care settings worldwide. In order to understand the repertoire of resistance determinants and their organization and origins, we compared the genome sequences of three MDR and three drug-susceptible A. baumannii isolates. The entire MDR phenotype can be explained by the acquisition of discrete resistance determinants distributed throughout the genome. A comparison of closely related MDR and drug-susceptible isolates suggests that drug efflux may be a less significant contributor to resistance to certain classes of antibiotics than inactivation enzymes are. A resistance island with a variable composition of resistance determinants interspersed with transposons, integrons, and other mobile genetic elements is a significant but not universal contributor to the MDR phenotype. Four hundred seventy-five genes are shared among all six clinical isolates but absent from the related environmental species Acinetobacter baylyi ADP1. These genes are enriched for transcription factors and transporters and suggest physiological features of A. baumannii that are related to adaptation for growth in association with humans.Among gram-negative pathogens that are reported as "multidrug resistant" (MDR), Acinetobacter baumannii is rapidly becoming a focus of significant attention (1,10,35,56). A. baumannii, a pleomorphic, gram-negative coccobacillus, is currently recognized by the Infectious Diseases Society of America as one of the most important pathogens threatening our health care delivery system (48). Global surveillance programs conducted over the last decade show an unparalleled increase in resistance rates among clinical Acinetobacter isolates (26). Acinetobacter spp. are now the third leading cause of respiratory tract infections among patients in intensive care units, and A. baumannii is responsible for up to 10% of hospital-acquired infections (26). These nosocomial infections are typically found in immunocompromised patients and are associated with an increased length of stay and excess morbidity (13,25,38,47 (19,40). Molecular typing of isolates from this outbreak revealed eight major clone types, and about 60% of the isolates were related to three pan-European types (5, 52), suggesting multiple independent origins (40). Examination of specific resistance determinants in the WRAMC isolates demonstrated considerable variability in the composition of resistance genes within each clone type and similar patterns across certain divergent clone types. Thus, genetic relatedness was a poor predictor of the MDR phenotype. This led to the hypothesis that there exist multiple independent genetic mechanisms leading to MDR in A. baumannii. A large cluster of antibiotic resistance genes and mobile genetic elements is present as an 86-kb "resistance island" (RI) in the A. baumannii AYE genome (15). The RI is not present in the genomes of the drug-susceptible A. baumannii isolates ATCC 17978 (43) and SDF (51), suggesting that it i...