A standard procedure for pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments of Acinetobacter baumannii was set up and validated for its interlaboratory reproducibility and its potential for use in the construction of an Internet-based database for international monitoring of epidemic strains. The PFGE fingerprints of strains were generated at three different laboratories with ApaI as the restriction enzyme and by a rigorously standardized procedure. The results were analyzed at the respective laboratories and also centrally at a national reference institute. In the first phase of the study, 20 A. baumannii strains, including 3 isolates each from three well-characterized hospital outbreaks and 11 sporadic strains, were distributed blindly to the participating laboratories. The local groupings of the isolates in each participating laboratory were identical and allowed the identification of the epidemiologically related isolates as belonging to three clusters and identified all unrelated strains as distinct. Central pattern analysis by using the band-based Dice coefficient and the unweighted pair group method with mathematical averaging as the clustering algorithm showed 95% matching of the outbreak strains processed at each local laboratory and 87% matching of the corresponding strains if they were processed at different laboratories. In the second phase of the study, 30 A. baumannii isolates representing 10 hospital outbreaks from different parts of Europe (3 isolates per outbreak) were blindly distributed to the three laboratories, so that each laboratory investigated 10 epidemiologically independent outbreak isolates. Central computer-assisted cluster analysis correctly identified the isolates according to their corresponding outbreak at an 87% clustering threshold. In conclusion, the standard procedure enabled us to generate PFGE fingerprints of epidemiologically related A. baumannii strains at different locations with sufficient interlaboratory reproducibility to set up an electronic database to monitor the geographic spread of epidemic strains.Acinetobacter baumannii is a well-recognized opportunistic pathogen that gives rise to nosocomial infections and outbreaks, in particular, in the intensive care unit setting (1). The increasing rates of resistance of A. baumannii to the major antimicrobial drugs make early identification and control of hospital outbreaks mandatory. Recent data indicate that several successful epidemic A. baumannii strains (clones) circulate in Europe, and a better understanding of the diversity within the species and the emergence of epidemic clones is urgently needed (19,25,29). Molecular typing plays an important role in the study of the epidemiology of A. baumannii and in coping with its epidemic spread.Various genotypic methods have been developed for the typing of acinetobacters, including ribotyping (11), macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) (21), randomly amplified polymorphic DNA (RAPD) analysis (13), and total genomic fingerpr...

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In the last few years, phenotypically carbapenem resistant Acinetobacter strains have been identified throughout the world, including in many of the hospitals and intensive care units (ICUs) of Australia. Genotyping of Australian ICU outbreak-associated isolates by pulsed-field gel electrophoresis of whole genomic DNA indicated that different strains were cocirculating within one hospital. The carbapenem-resistant phenotype of these and other Australian isolates was found to be due to carbapenem-hydrolyzing activity associated with the presence of the bla OXA-23 gene. In all resistant strains examined, the bla OXA-23 gene was adjacent to the insertion sequence ISAba1 in a structure that has been found in Acinetobacter baumannii strains of a similar phenotype from around the world; bla OXA-51 -like genes were also found in all A. baumannii strains but were not consistently associated with ISAba1, which is believed to provide the promoter required for expression of linked antibiotic resistance genes. Most isolates were also found to contain additional antibiotic resistance genes within the cassette arrays of class 1 integrons. The same cassette arrays, in addition to the ISAba1-bla OXA-23 structure, were found within unrelated strains, but no common plasmid carrying these accessory genetic elements could be identified. It therefore appears that antibiotic resistance genes are readily exchanged between cocirculating strains in epidemics of phenotypically indistinguishable organisms. Epidemiological investigation of major outbreaks should include whole-genome typing as well as analysis of potentially transmissible resistance genes and their vehicles.Acinetobacter spp. are emerging opportunistic nosocomial pathogens, with increasing prevalence worldwide. Their epidemiology is complex, and genotypic methods or a combination of genotypic and phenotypic methods are required for species identification (28). Differentiation of Acinetobacter baumannii and Acinetobacter genomic species 3 and 13TU (the three most clinically important members of the genus) and the environmental species A. calcoaceticus is impractical in the routine laboratory, and these four species are generally grouped phenotypically as the A. calcoaceticus-A. baumannii complex. DNA fingerprinting by ApaI digestion of total DNA and pulsed-field gel electrophoresis (PFGE) is regarded as the typing method of choice for outbreak investigation and can be used to compare outbreaks in different locations when the methodology is uniform (41).Studies at the species level have shown that the problems of epidemic spread of antibiotic-resistant Acinetobacter strains are mostly due to bacteria belonging to the A. calcoaceticus-A. baumannii complex (3). The potential for acquisition of transferable carbapenem resistance has long been recognized (37a), and the adjusted mortality risk for intensive-care patients infected by carbapenem resistant Acinetobacter may be increased more than threefold (33).Carbapenem resistance in strains of the A. calcoaceticus-A.baumannii comp...

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