The prevalence of the currently known Acinetobacter species and related trends of antimicrobial resistance in a Dutch university hospital were studied. Between 1999 and 2006, Acinetobacter isolates from clinical samples were collected prospectively. Isolates were analyzed by amplified fragment length polymorphism fingerprinting. For species identification, a profile similarity cutoff level of 50% was used, and for strain identification, a cutoff level of 90% was used. Susceptibility for antimicrobial agents was tested by disk diffusion by following the CLSI guideline.The incidences of Acinetobacter isolates ranged from 1.7 to 3.7 per 10,000 patients per year, without a trend of increase, during the study years. Twenty different species were distinguished. Acinetobacter baumannii (27%) and Acinetobacter genomic species (gen. sp.) 3 (26%) were the most prevalent. Other species seen relatively frequently were Acinetobacter lwoffii (11%), Acinetobacter ursingii (4%), Acinetobacter johnsonii (4%), and Acinetobacter junii (3%). One large cluster of A. baumannii, involving 31 patients, and 16 smaller clusters of various species, involving in total 39 patients, with at most 5 patients in 1 cluster, occurred. Overall, 37% of the A. baumannii isolates were fully susceptible to the tested antibiotics. There was a borderline significant (P ؍ 0.059) trend of decreasing susceptibility. A. baumannii was the Acinetobacter species causing the largest burden of multiple-antibiotic resistance and transmissions in the hospital.More than 30 named and unnamed species of Acinetobacter have been described (14), some of which are of clinical importance, including A. baumannii, Acinetobacter gen. sp. 3, and Acinetobacter gen. sp. 13TU, while other species, like A. junii, A. johnsonii, A. ursingii, and Acinetobacter schindleri, can also incidentally be associated with infections (8). Much attention has been paid to outbreaks caused by acinetobacters (28), which in most cases are caused by A. baumannii (15,23). Notably, in diagnostic microbiology, isolates identified as A. baumannii may also include the closely related species Acinetobacter gen. sp. 3 or Acinetobacter gen. sp. 13TU. Bacteria belonging to other Acinetobacter species are frequently not further identified as or designated Acinetobacter species, as this would require genotypic methods that are usually not available in clinical diagnostic microbiology. These difficulties in identification explain why, overall, not much is known about the occurrences of the different Acinetobacter species in the hospital.The aim of the present study was to determine the prevalences of the currently known Acinetobacter species and related trends of antimicrobial resistance in our hospital through the years. To this aim, we identified all available Acinetobacter isolates obtained from our hospital in the period between 1999 and 2006 to the species level by amplified fragment length polymorphism (AFLP) analysis, a well-validated method for Acinetobacter species identification (7,8). Furthermore, ...