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1 Laboratorium voor Microbiologie, Universiteit Gent, Gent, Belgium
2 BCCM/LMG Bacteria Collection, Universiteit Gent, Gent, Belgium
3 CCUG Culture Collection, University of Göteborg, Göteborg, Sweden
Correspondence
Tom Coenye
Tom.Coenye{at}ugent.be
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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A picture of the protein profiles of Achromobacter spanius and Achromobacter insolitus strains and tables showing their fatty acid compositions and MIC values are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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and originally contained a single species, Achromobacter xylosoxidans. Following a polyphasic taxonomic study, Yabuuchi et al. (1998) transferred the species Alcaligenes ruhlandii and Alcaligenes piechaudii to the genus Achromobacter. The same authors also transferred Alcaligenes denitrificans to the genus Achromobacter as Achromobacter xylosoxidans subsp. denitrificans. However, the reclassification of Alcaligenes denitrificans as a subspecies of Achromobacter xylosoxidans contradicts previous work that showed that there is sufficient evidence to consider both taxa as distinct species (Vandamme et al., 1996). We recently proposed to formally reclassify Alcaligenes denitrificans as Achromobacter denitrificans (Coenye et al., 2003). Achromobacter xylosoxidans is widespread in aquatic habitats, but has also been involved in opportunistic infection of humans (Kersters & De Ley, 1984). For example, Achromobacter xylosoxidans is capable of causing persistent respiratory tract infection in cystic fibrosis patients (Liu et al., 2002). Achromobacter piechaudii can be isolated from soil and human clinical samples, including blood (Kiredjian et al., 1986), whereas Achromobacter ruhlandii is a soil commensal that is not known to be pathogenic to humans (Kersters & De Ley, 1984). We performed a polyphasic taxonomic study to elucidate the taxonomic positions of 10 isolates that were recovered from diverse environments, including human clinical samples. These isolates resembled Alcaligenes faecalis phenotypically, but whole-cell protein and fatty acid analyses suggested that they belonged to the genus Achromobacter. We show that these isolates belong to two novel Achromobacter species, for which we propose the names Achromobacter insolitus sp. nov. and Achromobacter spanius sp. nov.
Strains used in this study are listed in Table 1. Reference strains of other taxa have been described previously (Vandamme et al., 1995, 1996; Foss et al., 1998; Yabuuchi et al., 1998; Coenye et al., 2003). All strains were grown aerobically on trypticase soy agar (TSA; BBL) at 37 °C unless indicated otherwise. For SDS-PAGE of whole-cell proteins, strains were grown on TSA for 48 h at 37 °C. Preparation of whole-cell proteins and SDS-PAGE were performed as described previously (Pot et al., 1994). Densitometric analysis, normalization and interpolation of protein profiles and numerical analysis with the Pearson product–moment correlation coefficient were performed by using GelCompar 4.2 software (Applied Maths). Sequences of the 16S rRNA genes of strains LMG 6003T and LMG 5911T were determined as described previously (Coenye et al., 1999). Phylogenetic and bootstrap analyses (1000 replicates) were performed by using Kodon software (Applied Maths); a phylogenetic tree was constructed by using the neighbour-joining method (Saitou & Nei, 1987). Preparation of high-molecular-mass DNA for DNA–DNA hybridization experiments and determination of the degree of DNA–DNA binding by the initial renaturation rate method were performed as described previously (De Ley et al., 1970; Vandamme et al., 1992). Each value is the mean of at least two hybridization experiments. Total DNA concentration was 65 µg ml-1 and the optimal renaturation temperature in 2x SSC (1x SSC: 0·15 M NaCl, 0·015 M sodium citrate, pH 7·0) was 79 °C. Alternatively, high-molecular-mass DNA was prepared as described by Pitcher et al. (1989) and DNA–DNA hybridization was performed with photobiotin-labelled probes in microplate wells as described by Ezaki et al. (1989), using an HTS 7000 BioAssay Reader (PerkinElmer) for fluorescence measurements. Hybridization temperature was 50 °C. Reciprocal experiments were performed for every pair of strains. For determination of DNA base composition, DNA (prepared as described above) was degraded enzymically into nucleosides as described by Mesbah et al. (1989). The nucleoside mixture was then separated by HPLC, using a Waters SymmetryShield C8 column thermostatted at 37 °C. The solvent was 0·02 M NH4H2PO4 (pH 4·0) with 1·5 % acetonitrile. Non-methylated 
-phage DNA (Sigma) was used as the calibration reference. After 24 h incubation at 35 °C, a loopful of well-grown cells was harvested; fatty acid methyl esters were prepared as described previously (Vandamme et al., 1992) and separated and identified by using the Sherlock Microbial Identification system (version 3.0; MIDI). API galleries (API 50CH, API 50AO and API 50AA; bioMérieux) were used to determine assimilation of 147 organic compounds as sole carbon sources, as described previously (Kersters et al., 1984). Classical phenotypic tests were performed as described previously (Vandamme et al., 1993). API 20NE and API ZYM tests were performed according to the recommendations of the manufacturer (bioMérieux). MIC values towards levofloxacin, ciprofloxacin, ofloxacin, sparfloxacin, erythromycin, roxithromycin, clarithromycin, azithromycin, cefotaxime, cefpirome and rifampicin were determined for all strains listed in Table 1 by using the agar dilution method, conforming to NCCLS (1995) guidelines. Strains were grown on Mueller–Hinton agar (BRL) for 16–20 h at 35 °C.
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). One cluster contained Achromobacter spanius LMG 5896, LMG 14525, LMG 5911T and LMG 5909. Achromobacter insolitus strains LMG 5997, LMG 6000, LMG 5999, LMG 6003T, LMG 6010 and LMG 6001 formed the second cluster. The protein patterns of strains in both clusters were also clearly different from those of Bordetella and Kerstersia strains (data not shown). The 16S rDNA sequences of isolates LMG 5911T and LMG 6003T (representatives of the two protein-electrophoretic groups) were determined and compared with available 16S rDNA sequences of other 
-Proteobacteria (Fig. 2). The 16S rDNA sequences of LMG 5911T and LMG 6003T were very similar to each other (98·3 % similarity) and were related closely to those of Achromobacter piechaudii (98·6 and 98·0 % similarity, respectively), Achromobacter ruhlandii (98·0 and 98·5 %, respectively), Achromobacter xylosoxidans (98·1 and 98·4 %, respectively) and Achromobacter denitrificans (97·6 and 98·0 %, respectively). Bootstrap analysis indicated that strains LMG 5911T and LMG 6003T formed a stable phylogenetic group with other Achromobacter species (bootstrap value, 99·9 %).
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). DNA was prepared from isolates LMG 6003T, LMG 6001, LMG 6010, LMG 6000 and LMG 5911T and from reference strains of Achromobacter piechaudii, Achromobacter xylosoxidans, Achromobacter denitrificans, Achromobacter ruhlandii, Alcaligenes faecalis, Bordetella hinzii and Kerstersia gyiorum. DNA binding values are shown in Table 2; they confirm that strains from both electrophoretic groups formed two separate novel Achromobacter species. The DNA G+C content of isolates LMG 6003T, LMG 6001, LMG 6010, LMG 6000 and LMG 5911T was 64·9, 65·3, 64·9, 65·5 and 64·9 mol%, respectively.
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). Predominant fatty acids that were present in both novel Achromobacter species were C16 : 0, C17 : 0 cyclo, summed feature 2 (comprising C14 : 0 3-OH, iso-C16 : 1 I, an unidentified fatty acid with equivalent chain-length of 10·928 and/or C12 : 0 ALDE) and summed feature 3 (comprising C16 : 1
7c and/or iso-C15 : 0 2-OH). Detailed results of the fatty acid composition analysis are available as supplementary material in IJSEM Online.
All strains examined showed oxidase, catalase, C4-esterase and leucine arylamidase activities but no lysine decarboxylase, ornithine decarboxylase, arginine dihydrolase, gelatinase, amylase, -galactosidase or DNase activity. All strains were capable of growth between 28 and 37 °C and could grow at NaCl concentrations from 0 to 4·5 %. All strains reduced nitrate but none reduced nitrite or were capable of denitrification. No haemolysis, hydrolysis of aesculin or production of indole, H2S or acid on TSI agar was observed. Growth was never observed on 10 % lactose, Tween 80 or oxidation–fermentation (OF) medium supplemented with glucose, maltose, adonitol, fructose or xylose. C8-ester-lipase, C14-lipase, valine arylamidase, cysteine arylamidase, trypsin, chymotrypsin, phosphoamidase, 
-galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase activities were never observed. All strains assimilated the following substrates: gluconate, acetate, propionate, butyrate, isobutyrate, n-valerate, succinate, fumarate, adipate, pimelate, suberate, azelate, sebacate, DL-lactate, DL-3-hydroxybutyrate, D-malate, L-malate, meso-tartrate, pyruvate, citraconate, itaconate, citrate, phenylacetate, m-hydroxybenzoate, D--alanine, L--alanine, L-leucine, L-isoleucine, L-serine, L-threonine, L-phenylalanine, L-histidine, L-aspartate, L-glutamate, L-proline, DL-4-aminobutyrate and 2-aminobenzoate. None of the strains assimilated the following substrates: glycerol, erythritol, D-arabinose, L-arabinose, ribose, D-xylose, L-xylose, adonitol, methyl -D-xyloside, galactose, D-glucose, D-fructose, D-mannose, L-sorbose, L-rhamnose, dulcitol, inositol, mannitol, sorbitol, methyl -D-mannoside, methyl -D-glucoside, N-acetylglucosamine, amygdalin, arbutin, aesculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, inulin, D-melezitose, raffinose, starch, glycogen, xylitol, -gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, 2-ketogluconate, 5-ketogluconate, caprylate, oxalate, malonate, glycolate, levulinate, o-hydroxybenzoate, D-mandelate, L-mandelate, phthalate, isophthalate, terephthalate, trigonelline, L-arginine, betaine, creatine, DL-5-aminovalerate, 2-aminobenzoate, 3-aminobenzoate, 4-aminobenzoate, urea, acetamide, sarcosine, ethylamine, butylamine, amylamine, ethanolamine, benzylamine, spermine, histamine or glucosamine. Urease activity, growth on acetamide and assimilation of isovalerate, n-caproate, heptanoate, pelargonate, caprate, malate, glutarate, DL-glycerate, D-tartrate, L-tartrate, 2-oxoglutarate, mesaconate, aconitate, benzoate, p-hydroxybenzoate, glycine, L-norleucine, L-valine, DL-norvaline, DL-2-aminobutyrate, L-cysteine, L-methionine, L-tyrosine, D-tryptophan, L-tryptophan, L-ornithine, L-lysine, L-citrulline, DL-kynurenine, -alanine, DL-3-aminobutyrate, diaminobutane and tryptamine were strain-dependent characteristics.
The range of MIC values and MIC50 and MIC90 of the strains are available as supplementary data in IJSEM Online.
Previous studies have indicated that SDS-PAGE of whole-cell proteins is a valuable tool for identification of members of the family Alcaligenaceae (Vancanneyt et al., 1995; Vandamme et al., 1995, 1996; Coenye et al., 2003). By using whole-cell protein analysis, both Achromobacter insolitus and Achromobacter spanius could be differentiated easily from each other, from known Achromobacter species and from members of the genera Alcaligenes, Bordetella and Kerstersia (Fig. 1
and data not shown). Both quantitative and qualitative differences occur in the fatty acid compositions of different Achromobacter species; however, these differences are small and it seems questionable whether they will suffice to identify strains at the species level. Biochemically, both novel Achromobacter species are difficult to separate from each other, from other Achromobacter species and from Alcaligenes faecalis. Biochemical characteristics that are useful for the differentiation of Achromobacter insolitus and Achromobacter spanius from each other and from other Achromobacter species are given in Table 3. In addition, differentiation of Achromobacter insolitus and Achromobacter spanius from Alcaligenes faecalis is possible due to the absence of assimilation of azelate, D-malate and adipate in the latter.
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Gram-negative, small (1–2 µm long) and coccoid cells that occur as a single unit, in pairs or in short chains. Motility is strain-dependent. On nutrient agar, colonies are flat or slightly convex with smooth margins and range from white to light brown in colour. Oxidase- and catalase-positive, but no urease, -galactosidase or DNase activity is present. Nitrate reduction is present. Other biochemical characteristics are listed in the Results section above. In addition, Achromobacter insolitus grows on acetamide and assimilates isovalerate, malate, glutarate, DL-glycerate, mesaconate, aconitate, L-norleucine, L-valine, L-cysteine, L-tryptophan and tryptamine, but does not assimilate D-tartrate or diaminobutane. The following fatty acid components are present in significant amounts: C12 : 0, C12 : 0 2-OH, C14 : 0, C16 : 0, C17 : 0 cyclo, C18 : 0, C18 : 17c and summed features 2 and 3. G+C content of the DNA is 64·9–65·5 mol%. Isolated from various human clinical samples.
The type strain, LMG 6003T (=API 201-3-84T=CCUG 47057T), was isolated from a leg wound. Characteristics of the type strain are the same as those for the species. In addition, the type strain assimilates n-caproate, pelargonate, caprate, L-tartrate, 2-ketoglutarate, benzoate, DL-norvaline, DL-2-aminobutyrate, L-tyrosine, L-lysine, -alanine and DL-3-aminobutyrate. DNA G+C content of the type strain is 64·9 mol%.
Description of Achromobacter spanius sp. nov.
Achromobacter spanius (spa'ni.us. N.L. masc. adj. spanius from Gr. adj. spanios rare, scarce, referring to the fact that, so far, only a few strains have been found).
Gram-negative, small (1–2 µm long), coccoid cells that occur as a single unit, in pairs or in short chains. Motility is strain-dependent. On nutrient agar, colonies are flat or slightly convex with smooth margins and range from white to light brown in colour. Oxidase- and catalase-positive, but no urease, -galactosidase or DNase activity is present. Nitrate reduction is present. Other biochemical characteristics are listed in the Results section above. In addition, Achromobacter spanius does not grow on acetamide, assimilates 2-ketoglutarate, L-tyrosine, -alanine, DL-3-aminobutyrate, diaminobutane and tryptamine and does not assimilate mesaconate, aconitate, n-caproate, heptanoate, pelargonate, caprate, benzoate, p-hydroxybenzoate, glycine, L-methionine, L-ornithine, L-lysine, L-citrulline or DL-kynurenine. The following fatty acid components are present in significant amounts: C12 : 0 2-OH, C14 : 0, C16 : 0, C16 : 0 2-OH, C17 : 0 cyclo, C18 : 0, C18 : 17c and summed features 2 and 3. G+C content of the DNA is 64·9 mol%.
The type strain, LMG 5911T (=API 198-2-84T=CCUG 47062T), was isolated from blood. Characteristics of the type strain are the same as those for the species. In addition, the type strain utilizes glutarate, DL-glycerate and L-tryptophan. DNA G+C content of the type strain is 64·9 mol%.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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