Since the 1970s, the spread of multidrug-resistant (MDR) Acinetobacter strains among critically ill, hospitalized patients, and subsequent epidemics, have become an increasing cause of concern. Reports of community-acquired Acinetobacter infections have also increased over the past decade. A recent manifestation of MDR Acinetobacter that has attracted public attention is its association with infections in severely injured soldiers. Here, we present an overview of the current knowledge of the genus Acinetobacter, with the emphasis on the clinically most important species, Acinetobacter baumannii.

Outbreaks of hospital infections caused by multidrug resistant Acinetobacter baumannii strains are of increasing concern worldwide. Although it has been reported that particular outbreak strains are geographically widespread, little is known about the diversity and phylogenetic relatedness of A. baumannii clonal groups. Sequencing of internal portions of seven housekeeping genes (total 2,976 nt) was performed in 154 A. baumannii strains covering the breadth of known diversity and including representatives of previously recognized international clones, and in 19 representatives of other Acinetobacter species. Restricted amounts of diversity and a star-like phylogeny reveal that A. baumannii is a genetically compact species that suffered a severe bottleneck in the recent past, possibly linked to a restricted ecological niche. A. baumannii is neatly demarcated from its closest relative (genomic species 13TU) and other Acinetobacter species. Multilocus sequence typing analysis demonstrated that the previously recognized international clones I to III correspond to three clonal complexes, each made of a central, predominant genotype and few single locus variants, a hallmark of recent clonal expansion. Whereas antimicrobial resistance was almost universal among isolates of these and a novel international clone (ST15), isolates of the other genotypes were mostly susceptible. This dichotomy indicates that antimicrobial resistance is a major selective advantage that drives the ongoing rapid clonal expansion of these highly problematic agents of nosocomial infections.

For bacterial typing to be useful, the development, validation and appropriate application of typing methods must follow unified criteria. Over a decade ago, ESGEM, the ESCMID (Europen Society for Clinical Microbiology and Infectious Diseases) Study Group on Epidemiological Markers, produced guidelines for optimal use and quality assessment of the then most frequently used typing procedures. We present here an update of these guidelines, taking into account the spectacular increase in the number and quality of typing methods made available over the past decade. Newer and older, phenotypic and genotypic methods for typing of all clinically relevant bacterial species are described according to their principles, advantages and disadvantages. Criteria for their evaluation and application and the interpretation of their results are proposed. Finally, the issues of reporting, standardisation, quality assessment and international networks are discussed. It must be emphasised that typing results can never stand alone and need to be interpreted in the context of all available epidemiological, clinical and demographical data relating to the infectious disease under investigation. A strategic effort on the part of all workers in the field is thus mandatory to combat emerging infectious diseases, as is financial support from national and international granting bodies and health authorities.

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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The species Acinetobacter calcoaceticus, A. baumannii, genomic species 3, and genomic species 13TU included in the Acinetobacter calcoaceticus-Acinetobacter baumannii complex are genetically highly related and difficult to distinguish phenotypically. Except for A. calcoaceticus, they are all important nosocomial species. In the present study, the usefulness of the 16S-23S rRNA gene intergenic spacer (ITS) sequence for the differentiation of (genomic) species in the A. calcoaceticus-A. baumannii complex was evaluated. The ITSs of 11 reference strains of the complex and 17 strains of other (genomic) species of Acinetobacter were sequenced. The ITS lengths (607 to 638 bp) and sequences were highly conserved for strains within the A. calcoaceticus-A. baumannii complex. Intraspecies ITS sequence similarities ranged from 0.99 to 1.0, whereas interspecies similarities varied from 0.86 to 0.92. By using these criteria, 79 clinical isolates identified as A. calcoaceticus (18 isolates) or A. baumannii (61 isolates) with the API 20 NE system (bioMérieux Vitek, Marcy l'Etoile, France) were identified as A. baumannii (46 isolates), genomic species 3 (19 isolates), and genomic species 13TU (11 isolates) by ITS sequencing. An identification rate of 96.2% (76 of 79 isolates) was obtained by using ITS sequence analysis for identification of isolates in the A. calcoaceticus-A. baumannii complex, and the accuracy of the method was confirmed for a subset of strains by amplified rRNA gene restriction analysis and genomic DNA analysis by AFLP analysis by using libraries of profiles of reference strains. In conclusion, ITS sequence-based identification is reliable and provides a promising tool for elucidation of the clinical significance of the different species of the A. calcoaceticus-A. baumannii complex.Since the 1970s, bacteria of the genus Acinetobacter have increasingly been recognized as important nosocomial pathogens that give rise to severe infections and episodes of epidemic spread among critically ill hospitalized patients (3,13,31,32). In a recent European study, Acinetobacter spp. appeared to be the eighth most common cause of nosocomial pneumonia (17). Nosocomial acinetobacters are notorious for their resistance to antibiotics, and strains resistant to most or all clinically important antibiotics, including expended-spectrum ␤-lactams and carbapenems, have now been identified worldwide (1,5,11,26,41).The genus Acinetobacter currently contains up to 33 described named and unnamed (genomic) species (9, 35). Of these, Acinetobacter calcoaceticus, A. baumannii, and genomic species 3 and 13TU are genetically and phenotypically very similar (21), which has led to the proposal to lump these and two closely related genomic species (genomic species close to 13TU and genomic species between 1 and 3) into the A. calcoaceticus-A. baumannii complex (20). The lumping of these species is unsatisfactory for clinical reasons because it obscures possible differences in the biology and pathology of the individual species. For example, A. c...

Our results suggest that CD14 and Toll-like receptor 4 play a key role in innate sensing of A. baumannii via the LPS moiety, resulting in effective elimination of the bacteria from the lung, whereas Toll-like receptor 2 signaling seems to counteract the robustness of innate responses during acute A. baumannii pneumonia.

In the past decade, various methods have been developed for the identification and typing of prokaryotic and eukaryotic organisms at the DNA level. These methods differ in their taxonomic range, discriminatory power, reproducibility, and ease of interpretation and standardization (62,67,86,87,101,106,110,116). The ideal genotyping method produces results that are invariable from laboratory to laboratory and allows unambiguous comparative analyses and the establishment of reliable databases.One of the newest and most promising methods is amplifiedfragment length polymorphism (AFLP) analysis (11,118,122), developed by Keygene BV, Wageningen, The Netherlands. This method combines universal applicability with high powers of discrimination and reproducibility (45). An increasing number of reports describe the use of AFLP analysis for plant and animal genetic mapping, medical diagnostics, phylogenetic studies, and microbial typing. This minireview describes the principles, advantages, and disadvantages of AFLP analysis and summarizes its applications in different fields. PRINCIPLE OF AFLPIn the nomenclature of Vaneechoutte (110), AFLP analysis belongs to the category of selective restriction fragment amplification techniques, which are based on the ligation of adapters (i.e., linkers and indexers) to genomic restriction fragments followed by a PCR-based amplification with adapterspecific primers. For AFLP analysis ( Fig. 1), only a small amount of purified genomic DNA is needed; this is digested with two restriction enzymes, one with an average cutting frequency (like EcoRI) and a second one with a higher cutting frequency (like MseI or TaqI). Double-stranded oligonucleotide adapters are designed in such a way that the initial restriction site is not restored after ligation, which allows simultaneous restriction and ligation, while religated fragments are cleaved again. An aliquot is then subjected to two subsequent PCR amplifications under highly stringent conditions with adapter-specific primers that have at their 3Ј ends an extension of one to three nucleotides running into the unknown chromosomal restriction fragment. An extension of one selective nucleotide amplifies 1 of 4 of the ligated fragments, whereas three selective nucleotides in both primers amplify 1 of 4,096 of the fragments. The PCR primer which spans the average-frequency restriction site is labeled. After polyacrylamide gel electrophoresis a highly informative pattern of 40 to 200 bands is obtained. The patterns obtained from different strains are polymorphic due to (i) mutations in the restriction sites, (ii) mutations in the sequences adjacent to the restriction sites and complementary to the selective primer extensions, and (iii) insertions or deletions within the amplified fragments.Since the original publication by Vos et al. in 1995 (118) several enzyme combinations have been used, such as EcoRI, PstI, HindIII, or ApaI combined with MseI or TaqI. For animal genomes EcoRI and TaqI appear to be the most suitable (2). Alternative AFLP typing proce...