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ek5
1 National Institute of Public Health, 
robárova 48, 100 42 Prague, Czech Republic
2 Department of Infectious Diseases, Leiden University Medical Center C5-P, P.O. Box 9600, 2300 RC Leiden, The Netherlands
3 BCCM/LMG Bacteria Collection, University of Ghent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
4 Department of Clinical Chemistry, Microbiology and Immunology, University Hospital, Blok A, B-9000 Gent, Belgium
5 Department of Clinical Microbiology, U nemocnice 85, P
íbram, Czech Republic
Correspondence
Alexandr Nemec
anemec{at}szu.cz
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Published online ahead of print on 20 June 2003 as DOI 10.1099/ijs.0.02631-0.
The EMBL accession numbers for the 16S rRNA gene sequences of Acinetobacter parvus LMG 21765T and LMG 21766 are AJ293691 and AJ293690, respectively.
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; Tjernberg & Ursing, 1989; Bouvet & Jeanjean, 1989; Gerner-Smidt & Tjernberg, 1993; Vaneechoutte et al., 1999; Nemec et al., 2001) and strains of these species usually form colonies of 1·0–2·0 mm in diameter after 24 h incubation under optimum growth conditions (Bouvet & Grimont, 1986; Nemec et al., 2001). In a taxonomic study of Acinetobacter clinical isolates (Nemec et al., 2000), two strains were found which formed notably small colonies on routine agar media and could not be identified as any known (genomic) species. These strains were glucose-non-acidifying, non-proteolytic, did not utilize any of the 14 carbon sources of the identification scheme of Bouvet & Grimont (1987) and had highly similar amplified rDNA restriction analysis (ARDRA) profiles. Later, five strains similar to the two strains were found among archive strains in our collections. The aim of the present study was to define the taxonomic status of these strains by a polyphasic analysis.
The seven strains used in this study are listed in Table 1. All had the properties of the genus Acinetobacter (Juni, 1984), i.e. they were Gram-negative, strictly aerobic, oxidase-negative, non-motile coccobacilli, and were positive in the transformation assay of Juni (1972). The methods for genotypic characterization included ARDRA, AFLP fingerprinting and comparative 16S rDNA sequence analysis. Phenotypic characterization was done essentially according to Bouvet & Grimont (1987) and Gerner-Smidt et al. (1991), with some modifications. Details of the methods and their interpretative criteria have been given by Dijkshoorn et al. (1998) and Nemec et al. (2000, 2001). The assimilation tests were performed in tubes containing the fluid medium of Cruze et al. (1979) supplemented with 0·1 % (w/v) carbon source. Results were read after 2, 6 and 10 days incubation at 30 °C.
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, with minor modifications. Strains Acinetobacter haemolyticus LMG 996T, Acinetobacter baumannii LMG 1041T, Acinetobacter calcoaceticus LMG 1046T and ‘Acinetobacter venetianus’ LMG 19082, which produced large amounts of exopolysaccharides, were subjected to a mild alkaline hydrolysis step before cell lysis, as described by Willems et al. (2001). The G+C content of the DNA was determined by HPLC according to the method of Mesbah et al. (1989). Non-methylated phage 
DNA (Sigma) was used as the calibration reference. DNA–DNA hybridizations were performed using a modification of the microplate method described by Ezaki et al. (1989) and Goris et al. (1998). Hybridizations were performed at 37 °C in a hybridization solution [2xSSC, 5xDenhardt's solution, 50 % (v/v) formamide, 2·5 % (w/v) dextran sulfate, low-molecular-mass denatured salmon sperm DNA to a final concentration of 100 µg ml-1, 1·25 µg biotinylated probe DNA ml-1]. The DNA–DNA relatedness percentages presented are means based on at least two hybridization experiments. Reciprocal reactions (e.g. AxB and BxA) were performed and the variation between them was within the limit of this method (Goris et al., 1998).
Colonies of all strains grown on TSA or Nutrient Agar (NA; Oxoid) were circular, convex, smooth and slightly opaque with entire margins. These colonies were notably smaller than those of the other described Acinetobacter species (Fig. 1). On NA, the colonies were 0·1–0·4 mm and 0·3–0·9 mm in diameter after 24 and 48 h incubation at 30 °C, respectively, while on TSA, the colonies were 0·3–0·7 mm and 1·0–1·4 mm in diameter after 24 and 48 h of incubation at 30 °C, respectively. The use of other agar media including chocolate and blood agar did not significantly affect colony size as compared with TSA.
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-alanine, L-histidine, D-malate, malonate, histamine, L-phenylalanine, phenylacetate, levulinate, citraconate, 4-hydroxybenzoate, L-tartrate, L-ornithine, L-leucine, L-arabinose and 2,3-butanediol.
The result of the comparative analysis of AFLP patterns of the seven strains and type and reference strains of all described Acinetobacter (genomic) species is shown in Fig. 2. The seven strains grouped at 63 %, which is well above the 50 % level seen in previous studies for the delineation of Acinetobacter species (Nemec et al., 2001). They were clearly separated from the other Acinetobacter (genomic) species (each species represented by one strain) at 34 %.
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; Nemec et al., 2001). Structural homogeneity of 16S rDNA was confirmed by ARDRA. All strains had identical or almost identical restriction patterns: CfoI 1 (LMG 21765T, LUH 3067, LMG 21766 and RUH 2714) or CfoI 1+5 (RUH 2008, LUH 4619 and LUH 7036), AluI 2, MboI 1, RsaI 2 and MspI 3.
DNA–DNA relatedness was determined between LMG 21765T, LMG 21766 and the type strains of the nomenspecies that had shown highest similarity (>96·5 %) of 16S rDNA sequences with the two strains (Table 2). The level of DNA–DNA binding between LMG 21765T and LMG 21766 was 82 %. DNA–DNA binding values between these strains and the type strains of Acinetobacter junii, A. haemolyticus, A. baumannii, Acinetobacter johnsonii, A. calcoaceticus and the reference strain of ‘A. venetianus’ were not higher than 35 %. The DNA G+C content of LMG 21765T and LMG 21766 was 41·8 and 41·5 %, respectively.
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A. parvus can be differentiated from other Acinetobacter (genomic) species by its negative results in biochemical tests suggested by Bouvet & Grimont (1987), in particular by the inability to oxidize D-glucose, to hydrolyse gelatin and to utilize DL-lactate, DL-4-aminobutyrate, citrate (Simmons), azelate, -alanine and L-histidine (Bouvet & Grimont, 1987; Bouvet & Jeanjean, 1989; Gerner-Smidt et al., 1991; Vaneechoutte et al., 1999; Nemec et al., 2001). The acetate utilization test which is positive in A. parvus is necessary to differentiate prototrophic A. parvus strains from auxotrophic strains of other Acinetobacter (genomic) species. Notably, its typical colony size is an important feature to recognize A. parvus amidst colonies of other species and genera, and to differentiate it from biochemically inactive strains of other Acinetobacter (genomic) species.
ARDRA allowed for differentiation of A. parvus from all described (genomic) species of Acinetobacter, except A. junii and proteolytic genomic species 17 (Dijkshoorn et al., 1998; Vaneechoutte et al., 1999; Nemec et al., 2001). Four A. parvus strains had the same ARDRA combination pattern (CfoI 1, AluI 2, MboI 1, RsaI 2, MspI 3) as the latter two (genomic) species. However, A. parvus strains can easily be distinguished from genomic species 17 and A. junii strains sharing this ARDRA profile by their small colonies and the inability to lyse sheep erythrocytes.
The A. parvus strains were isolated from human and animal non-sterile body sites, except for RUH 2008, which originated from the blood of a human. Isolation of this strain was followed by other isolates with similar characteristics from intravenous catheters, which indicates that the strain was involved in a catheter-related blood-stream infection. Strain LUH 7036 was isolated from the ear of a dog with refractory otitis media.
During this study, an additional strain (LUH 4826) that was phenotypically indistinguishable from the A. parvus strains was isolated from a human clinical specimen. However, LUH 4826 had AluI and RsaI ARDRA patterns different from those of the A. parvus strains and AFLP fingerprinting showed no significant similarity between this strain and any of the described Acinetobacter (genomic) species including A. parvus (not shown). Therefore, LUH 4826 may represent an as-yet-undescribed species of the genus Acinetobacter that is phenotypically similar to A. parvus. This finding demonstrates that, as is the case with most Acinetobacter (genomic) species, definitive species identification requires the use of genotypic methods.
Description of Acinetobacter parvus sp. nov.
Acinetobacter parvus (par'vus. L. masc. adj. parvus small, referring to the fact that its colonies on agar media are significantly smaller than those of the other known Acinetobacter species).
Characteristics correspond to those of the genus (Juni, 1984). The description is based on the characterization of seven strains of different origin. Colonies on TSA after 24 h incubation at 30 °C are approximately 0·3–0·7 mm in diameter, circular, convex, smooth and slightly opaque with entire margins. Growth occurs at 35 °C but not at 41 °C. Growth at 37 °C usually occurs but may be reduced. Good growth on ethanol and acetate as sole sources of carbon and energy. Negative results in the following tests: acid production from D-glucose, haemolysis of sheep blood, gelatinase production and the utilization of DL-lactate, DL-4-aminobutyrate, trans-aconitate, citrate (Simmons), glutarate, L-aspartate, azelate, -alanine, L-histidine, D-malate, malonate, histamine, L-phenylalanine, phenylacetate, levulinate, citraconate, 4-hydroxybenzoate, L-tartrate, L-ornithine, L-leucine, L-arabinose and 2,3-butanediol.
The type strain is LMG 21765T (=LUH 4616T=NIPH 384T=CCM 7030T). Isolated from the ear of an outpatient. This strain grows well at 37 °C and has the following restriction patterns of amplified 16S rDNA: CfoI 1, AluI 2, MboI 1, RsaI 2, MspI 3. Its DNA G+C content is 41·8 %.
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Acknowledgements |
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Note added in proof
Since the study was completed, seven additional strains with typical A. parvus colonies have been studied in our laboratories. All of them were isolated from human clinical specimens (blood, ear pus, vaginal swab) and showed AFLP fingerprints, ARDRA profiles and biochemical properties typical of A. parvus. The only exception was the ability of three of these strains to grow on L-ornithine. Since this article was accepted for publication, seven new species of Acinetobacter have been described (Carr et al., 2003). Comparison of published 16S rDNA sequences and phenotypic characteristics did not show the identity of A. parvus with any of these species.
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Bouvet, P. J. M. & Grimont, P. A. D. (1986). Taxonomy of the genus Acinetobacter with the recognition of Acinetobacter baumannii sp. nov., Acinetobacter haemolyticus sp. nov., Acinetobacter johnsonii sp. nov., and Acinetobacter junii sp. nov. and emended descriptions of Acinetobacter calcoaceticus and Acinetobacter lwoffii. Int J Syst Bacteriol 36, 228–240.
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