Tjernberg, I. & Ursing. J. Clinical strains of ,lcinewhartrr classified by DNA-DNA hybridization. APMIS A collection of .-lcincrohn~tcr strains consisting of I68 consecutive clinical strains and 30 type and reference strains was studied by DNA-DNA hybridization and a few phenotypic tests. The field strains could be allotted to 13 DNA groups. By means of reference strains ten of these could be identified with groups described by Boiriw & Grirnonr (1986). while three groups were new: they were given the numbers 13-1 5. The type strain of .4. ruclior~~.~i.s,m.~recently described by Ni. rhimuru et al. ( 1988) was shown to be a member of DNA group 12. which comprised 3 1 clinical isolates. Of the 19 strains of A . ,jrinii. eight showed hemolytic activity on sheep and human blood agar and an additional four strains on human blood agar only.Strains of this species have previously been regarded as non-hemolytic. Reciprocal DNA pairing data for the reference strains of the DNA groups were treated by UPGMA clustering. The reference strains for .4. c~~~l~~o u r c~~i c~i r .~..4. haiiriiannii and for DNA groups 3 and 13 formed a cluster with about 70% relatedness within the cluster. Other DNA groups joined at levels below 6OYn.

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hydrolysis, and assimilation of 14 carbon sources. Of the strains tested, 181 represented 12 DNA groups in the matrix; at a probability level of .0.95, 78% of them were correctly identified, 2.2% were misidentified, and 19.8% were not identified. Seventeen strains represented two DNA groups not included in the matrix; nine of them were incorrectly assigned to a DNA group by these phenotypic tests. Because of problems of separating strains belonging to DNA groups 1, 2, 3, and 13 by using the phenotypic tests proposed by Bouvet and Grimont (Ann. Inst. Pasteur/Microbiol.), we suggest that these groups should be referred to as the Acinetobacter calcoaceticus-A. baumannii complex.

At least 19 genomic species are recognized as constituting the genus Acinetobacter. However, little is known about the natural reservoirs of the various members of the genus. An epidemiological study was therefore performed to investigate the colonization with Acinetobacter spp. of the skin and mucous membranes of 40 patients hospitalized in a cardiology ward and 40 healthy controls. Single samples were obtained once from each of nine different body sites, i.e., forehead, ear, nose, throat, axilla, hand, groin, perineum, and toe web. Identification of Acinetobacter isolates was achieved by using phenotypic properties and was compared to identification by amplified ribosomal DNA restriction analysis. Selected isolates were further investigated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ribotyping, and DNA-DNA hybridization. Plasmid profile analysis was used for epidemiological typing. Thirty patients (75%) and 17 controls (42.5%) were found to be colonized with Acinetobacter spp., and the colonization rates of patients increased during their hospital stay. The most frequently isolated species were Acinetobacter lwoffii (47%), A. johnsonii (21%), A. radioresistens (12%), and DNA group 3 (11%). In contrast, A. baumannii and DNA group 13TU, the most important nosocomial Acinetobacter spp., were found only rarely on human skin (0.5 and 1%, respectively) and their natural habitat remains to be defined. A good correlation between phenotypic and genotypic methods for identification of Acinetobacter spp. was observed, and only two isolates could not be assigned to any of the known DNA groups.

A total of 53 field and reference strains, including the type strains of the seven named species (nomenspecies) and belonging to the 18 described genomic species (DNA groups) of the genus Acinetobacter, were studied by amplified ribosomal DNA restriction analysis (ARDRA). Restriction analysis with the enzymes AluI, CfoI, MboI, RsaI, and MspI of the enzymatically amplified 16S rRNA genes allowed us to identify all species except the genomic species 4 (Acinetobacter haemolyticus) and 7 (A. johnsonii), 5 (A. junii) and 17, and 10 and 11, which clustered pairwise in three respective groups. Further analysis with the enzyme HaeIII, HinfI, NciI, ScrFI, or TaqI did not allow us to differentiate the species within these three clusters. However, use of a few additional simple phenotypic tests (hemolysis, growth at 37؇C, production of acid from glucose, and gelatin hydrolysis) can be used to differentiate between the species within these clusters. ARDRA proved to be a rapid and reliable method for the identification of most of the Acinetobacter genomic species, including the closely related DNA groups 1 (A. calcoaceticus), 2 (A. baumannii), 3, and 13. The results of this study suggest that ARDRA can be used for the identification of Acinetobacter species and as such may help to elucidate the ecology and clinical significance of the different species of this genus. Since ARDRA uses universal 16S rRNA gene primers, it is expected to be applicable to the identification of most bacterial species. Furthermore, ARDRA is less prone to contamination problems than PCR for detection, since the use of cultured organisms results in a large initial quantity of target DNA.

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Genotypic and phenotypic analyses were carried out to clarify the taxonomic position of the naturally transformable Acinetobacter sp. strain ADP1. Transfer tDNA-PCR fingerprinting, 16S rRNA gene sequence analysis, and selective restriction fragment amplification (amplified fragment length polymorphism analysis) indicate that strain ADP1 and a second transformable strain, designated 93A2, are members of the newly described species Acinetobacter baylyi. Transformation assays demonstrate that the A. baylyi type strain B2(T) and two other originally identified members of the species (C5 and A7) also have the ability to undergo natural transformation at high frequencies, confirming that these five strains belong to a separate species of the genus Acinetobacter, characterized by the high transformability of its strains that have been cultured thus far.

The Acinetobacter calcoaceticus‐Acinetobacter baumannii complex consists of four closely related “genospecies” or DNA groups: DNA group 1 (A. calcoaceticus), DNA group 2 (A. baumannii), DNA 3, and Tjernberg & Ursing‘s DNA group 13. Strains in this complex are so similar phenotypically that it is often impossible to identify them to the DNA group level by the use of biochemical tests. Twenty‐three Danish clinical strains from 23 patients phenotypically identified to the A. calcoaceticus‐A. baumannii complex were studied by ribotyping, plasmid profiling, and DNA/DNA hybridization. Multiple isolates were recovered from four patients. These were identical in each patient as judged by phenotype, ribotype and plasmic profile. Seventeen different ribotypes were observed among the 23 strains, and by using this method 19 out of the 23 strains could be identified to the DNA group level. Five strains were allocated to DNA group 2 (A. baumannii), eight to DNA group 3, and six to DNA group 13. These findings were confirmed by DNA/DNA hybridization. Two of the four unidentified strains were genotypically most closely related to but different from DNA groups 1 and 3. The last two strains were most closely related to DNA group 13. These four strains represent two new DNA groups within the A. calcoaceticus‐A. baumannii‐complex. One to four plasmids in the size range 2.1 kb‐ > 100 kb were detected in 13 of the strains. Nine plasmid profiles were seen, indicating the usefulness of this typing method if the strains contain plasmids. The study also indicates that ribotyping is useful both for typing and for identification purposes, and that the genetic relationship in this area are more diverse than hitherto perceived. Taxonomic reconsiderations are warranted.