1 The Diversity of the Genus Acinetobacter

The genus Acinetobacter comprises 17 validly named and 14 unnamed (genomic) species. Some unrelated (genomic) species have common designations, while some other species seem to be congruent but have different names. The knowledge of the biology or ecology of acinetobacters at species level is limited. This is due to the fact that identification of acinetobacters at species level is difficult. A phenotypic species identification system comprising c. 20 tests has been described (Bouvet and Grimont, 1986) but is not widely used and some closely related species cannot be separated well with this system. A variety of genotypic methods has been explored and applied to investigate the diversity or phylogeny in the genus. These methods include high resolution fingerprinting with AFLP, PCR-RFLP with digestion of PCR amplified sequences, and analysis of various DNA sequences. Of these, AFLP analysis and amplified 16SrDNA ribosomal DNA restriction analysis have been validated with large numbers of strains of all described species. Nucleotide sequence based methods are expected to be the standard for identification in the near future, but a prerequisite for their successful application is the availability of libraries of sequences of strains of all described genomic species. For each species, the sequences should cover the intra-species diversity. Sequence comparisons will also provide a valuable tool to study the phylogenetic relatedness of species. Introduction Bacteria that belong to the genus Acinetobacter constitute a heterogeneous group of organisms. Yet, it was not before the mid 1980s that different species could be distinguished within the genus. Since, up to 33 genomic groups have been identified by DNA–DNA hybridization, 17 of which have been given species names (Table 1.1). Unfortunately, in applied microbiology the knowledge on the existence of the many named and unnamed species and the availability of methods for species identification are lagging behind. Hence, relatively little is still known about the biology of the different (genomic) species except for the clinically relevant species Acinetobacter baumannii. Progress in the field of the diversity of the genus Acinetobacter has largely been made by the use of molecular methods including DNA-based methods. It is of note that different methods may provide different groupings. The challenge of present-day taxonomy or Dijkshoorn and Nemec | epidemiology is to find the grouping which reflects the natural diversity and evolutionary history of the bacteria of study. It is the aim of the present chapter to discuss the diversity of the genus Acinetobacter as currently known. We will start with an overview of the taxonomy of the genus from a historic perspective. This section is followed by a paragraph on the problems of the current taxonomy of Acinetobacter. For those not familiar with bacterial systematics, an introduction to the basic concepts of taxonomy is included. This may help to understand the particular problems of the classification and nomenclature of acinetobacters. The body of the chapter provides an overview of the diversity of the genus as assessed by different methods and the use of these methods in classification and identification. The taxonomy of the genus Acinetobacter from a historic perspective It was at the beginning of the 20th century that Martinus Willem Beijerinck, a Dutch microbiologist and father of the Delft School of Microbiology (la Riviere, 1997), described an organism that was isolated from soil by enrichment cultivation on a calciumacetate-mineral medium (Beijerinck, M.W., 1911). This organism was named Micrococcus calco-aceticus. In the following decades similar organisms were described independently and assigned different genus and species names like e.g. Herellea vaginicola, Mima polymorpha, Bacterium anitratum and Moraxella lwoffii (Henriksen, 1973). By the late 1960s a comprehensive study was published in which strains from these different taxa were compared for a large number of physiological characters (Baumann et al., 1968). The conclusion was that the organisms represented a single genus, but no clear-cut criteria for subdivision of the genus into species could be given. Thus, it was proposed to name the genus Acinetobacter and to consider only one species, named Acinetobacter calco-aceticus. The Beijerinck strain described in 1911, being the oldest living and documented Acinetobacter strain, was presented as the type strain for both the genus and the species. Interestingly, the report of that study (Baumann et al., 1968) included unpublished evidence from M. Mandel on DNA composition and from J. Johnson on DNA homologies indicating that multiple species could be recognized. A first published DNA hybridization study assigned strains to five DNA groups with four strains remaining ungrouped ( Johnson et al., 1970). Twelve DNA–DNA hybridization groups—then named “genospecies”—were described in 1986, seven of which were given species names while the others were provisionally given numbers (Bouvet and Grimont, 1986) (Table 1.1). In a next report, five additional DNA–DNA hybridization groups—we will use the term genomic species or gen. sp. for these groups—with the designations 13–17 were described (Bouvet and Jeanjean, 1989). Independently, a concurrent report also described three new genomic species numbered 13–15, where gen. sp. 13 of this paper (Tjernberg and Ursing, 1989) appeared to correspond to genomic species 14 of the other paper (Bouvet and Jeanjean, 1989). It was also shown by DNA hybridization that the previously described species Acinetobacter radioresistens (Nishimura et al., 1988) corresponds to gen. sp. 12 (Bouvet and Grimont, 1986), and that A. lwoffii (gen. sp. 8) and gen. sp. 9 belong to one hybridization group (Tjernberg and Ursing, 1989). Likewise, recent results indicate that A. grimontii and A. junii represent one species making A. grimontii a junior synonym (Vaneechoutte, unpublished data). Furthermore, A. calcoaceticus, A. baumannii, The Diversity of the Genus Acinetobacter | and gen. sp. 3 and 13TU were found so closely related (~65%) that lumping them into one species—to be named A. calcoaceticus—was considered but not formally proposed. Ten additional species have been validly described over the past five years. These include three species of possible clinical relevance, A. ursingii, A. schindleri (Nemec et al., 2001) and the small colony forming A. parvus (Nemec et al., 2003), and seven species from activated sludge, A. baylyi, A. bouvetii, A. grimontii, A. tjernbergiae, A. towneri, A. tandoii, and A. gerneri (Carr et al., 2003). It is of note that the well studied mutant strain ADP1—originally described as BD413 and derived from the soil isolate BD4 ( Juni and Janik, 1969)—has recently been found to belong to one of these species, A. baylyi (Young et al., 2005; Vaneechoutte et al., 2006). DNA–DNA hybridization studies have also revealed the existence of two additional genomic species which have not yet been validly described. These were designated “between 1 and 3” and “close to 13TU” indicating their position based on DNA homology as relatively close to A. calcoaceticus and gen. sp 3, and to gen. sp. 13, respectively (Gerner-Smidt and Tjernberg, 1993). Another species, “Acinetobacter venetianus” which includes the biochemically important, oil-degrading strain RAG-1 has not yet been validly described either, although it fulfils the criteria for validation (Vaneechoutte et al., 1999). In conclusion, considering that the respective pairs of designations A. lwoffii and gen. sp. 9, 13BJ and 14TU, and A. junii and A. grimontii are synonyms, the actual number of published (genomic) species is 30 (Table 1.1). Current problems related to the taxonomy of Acinetobacter In the preceding paragraph we have seen that considerable progress has been made to elucidate the diversity of the genus Acinetobacter at species level. Nevertheless, there are still several problems and inconsistencies (Box 1.1). For example, only 17 described DNA–DNA hybridization groups (genomic species) have valid names, while the remaining groups have numbers or other designations that do not contribute to their familiarity. The reasons for not giving species names to these DNA hybridization groups were that the groups were considered too small (< 10 strains) to describe their characteristics as a group properly, and that criteria for phenotypic differentiation were insufficient (Bouvet Table 1.1 Hitherto described genomic species of the genus Acinetobacter