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1 Institut für Zoo- und Wildtierforschung, Alfred-Kowalke-Str.17, D-10315 Berlin, Germany
2 Institut für Bakteriologie, Mykologie und Hygiene, Veterinärmedizinische Universität, Veterinärplatz 1, 1210 Wien, Austria
3 Mammal Research Institute PAS, ul. Waszkiewicza 1c, 17-230 Bialowieza, Poland
4 Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität, Heinrich Buff-Ring 26-32 (IFZ), D-35392 Giessen, Germany
Correspondence
Stephanie Speck
speck{at}izw-berlin.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains 1(W3/01)T and 2(W106/04)T are AJ879696 and AJ879697, respectively.
The polar lipid profile of strain 1(W3/01)T is available as a supplementary figure in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). The advanced stage of the disease is characterized by oedema of the skin surrounding the preputial orifice and accumulation of thick exudate and necrotic tissue within the preputial cavity (Jakob et al., 2000). In the course of a study concerning the aetiological agent of the balanoposthitis, several bacterial strains were isolated. Based on preliminary classification, these isolates were identified as members of the genera Arcanobacterium, Corynebacterium, Bacillus, Fusobacterium, Peptostreptococcus, Prevotella, Staphylococcus and Streptococcus and the family Enterobacteriaceae (A. Lehnen, unpublished results). Thirteen strains isolated from the prepuces of 13 European bison bulls suffering from balanoposthitis [strains 1(W3/01)T, 1(W10/02), 1(W89/03), 1(W91/04), 2(W106/04)T, 2(W4/01), 2(W34/02), 2(W76/03), 2(W78/03), 2(W87/03), 2(W101/04), 2(W103/04) and 2(W105/04)] were found to be morphologically similar to Arcanobacterium pyogenes, but could not be identified by standard diagnostic tests. Although these strains were isolated exclusively from bulls with balanoposthitis, their pathological significance is still unknown. However, the phylogenetic relationship to A. pyogenes underlines their possible role in the aetiology of balanoposthitis. In order to clarify the taxonomic status of this set of isolates, the strains were characterized by a combination of genomic, physiological and chemotaxonomic methods.
Genomic analyses
Based on almost identical genomic fingerprints generated after BOX-PCR according to the method described by Louws et al. (1994), the Arcanobacterium isolates were grouped into two clusters and each cluster was assumed to represent a distinct species. Strains 1(W3/01)T and 2(W106/04)T were selected as representative strains from each of the clusters. Cluster I, represented by strain 1(W3/01)T, exhibited four predominant bands, with sizes of approximately 450, 550, 750 and 1500 bp. Cluster II, represented by strain 2(W106/04)T, exhibited three predominant bands, with sizes of approximately 550, 850 and 1100 bp. A. pyogenes DSM 20630T exhibited a distinct BOX fingerprint (Fig. 1).
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. Amplified PCR products were sequenced bidirectionally using an ABI 3100 Genetic Analyzer (Applied Biosystems). The resulting 16S rRNA gene sequences of strains 1(W3/01)T and 2(W106/04)T consisted of 1481 [positions 36–1517, Escherichia coli numbering (Brosius et al., 1978)] and 1444 nucleotides (36–1480, E. coli numbering), respectively. In a FASTA search (Pearson & Lipman, 1988), the two isolates shared 97·2 % gene sequence similarity. Isolates 1(W3/01)T and 2(W106/04)T exhibited highest sequence similarities to A. pyogenes DSM 20630T (96·1 and 96·4 %, respectively) and Arcanobacterium bernardiae DSM 9152T (95·5 and 95·8 %, respectively). Sequence similarities with other species of Arcanobacterium were in the range 93·3–94·7 %. These results indicate that strains 1(W3/01)T and 2(W106/04)T are affiliated to the genus Arcanobacterium, but are not members of an established species. In order to display the phylogenetic relationships of the two representative strains with established species of the genus Arcanobacterium, a dendrogram was constructed after multiple alignment with publicly available 16S rRNA gene sequences using MEGA version 2.1 (Kumar et al., 2001). The results clearly demonstrated that the two isolates are closely related and that they are located on the Arcanobacterium lineage (Fig. 2), with A. pyogenes DSM 20630T and A. bernardiae DSM 9152T as the closest related species.
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; Kämpfer et al., 2003) between strains 1(W3/01)T and 2(W106/04)T revealed a DNA–DNA relatedness of only 9·4 % (reciprocal 13·2 %). This low value unambiguously demonstrated that strains 1(W3/01)T and 2(W106/04)T are representatives of two separate species.
Biochemical characteristics
An investigation of A. bernardiae DSM 9152T, Arcanobacterium haemolyticum DSM 20595T, Arcanobacterium hippocoleae DSM 15539T, Arcanobacterium pluranimalium DSM 13483T, Arcanobacterium phocae DSM 10002T, A. pyogenes DSM 20630T and strains 1(W3/01)T and 2(W106/04)T using commercially available API Coryne, API CH50 and API ZYM tests (bioMérieux) revealed metabolic profiles that were distinct for each strain (Table 1). The other isolates which, based on BOX band patterns, had been grouped with strain 1(W3/01)T [strains 1(W10/02), 1(W89/03) and 1(W91/04)] or strain 2(W106/04)T [strains 2(W4/01), 2(W34/02), 2(W76/03), 2(W78/03), 2(W87/03), 2(W101/04), 2(W103/04) and 2(W105/04)] showed profiles which were almost indistinguishable from the selected representative of each cluster. The two similarity groups could be distinguished from one another by their biochemical profiles, confirming that each group represents a distinct species. Gram-staining (Quinn et al., 2000) of cells of the two novel isolates revealed Gram-positive behaviour. Isolates 1(W3/01)T and 2(W106/04)T were catalase- and oxidase-negative, showed no serolysis on Löffler serum agar and no growth on Gassner or MacConkey agar. The optimum growth temperature on sheep blood agar was 37 °C. No growth occurred at 42 °C. The best growth occurred under aerobic conditions. Growth in a CO2-enriched (CO2Gen) or anaerobic atmosphere (AnaeroGen) was slowed. Other characteristics are listed in the species descriptions and in Table 1.
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. The fatty acid profiles of the representatives of each BOX similarity group, strains 1(W3/01)T, 1(W10/02), 2(W106/04)T and 2(W34/02), as well as A. haemolyticum DSM 20595T and A. pyogenes DSM 20630T, consisted predominantly of unbranched acids (>98 %). The six strains shared the presence of the major components C16 : 0, C18 : 1
9c and C18 : 0 which was in good agreement with the profiles of A. pyogenes and A. haemolyticum as reported by Bernard et al. (1991). The fatty acid profiles distinguished the representatives of the two BOX similarity groups by revealing significant differences in the relative amounts of C14 : 0, C18 : 19c, C18 : 16c, C16 : 0 and/or C18 : 0 (Table 2) and also distinguished them from the two reference strains.
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. The polar lipid profiles of strains 1(W3/01)T (see Supplementary Fig. S1 in IJSEM Online) and 2(W106/04)T were rather complex. Both strains contained diphosphatidylglycerol, phosphatidylglycerol, three unknown phosphoglycolipids, an unknown aminolipid (AL2), two unknown phospholipids and an unknown aminophospholipid as predominant compounds, but they could be distinguished by the presence of two hydrophilic lipids in strain 1(W3/01)T; an unknown aminoglycolipid and an unknown aminophosphoglycolipid. Similar, but not identical, profiles were detected for A. pyogenes DSM 20630T and A. haemolyticum DSM 20595T (results not shown).
Analyses of quinones were performed as described previously (Altenburger et al., 1996). Strain 1(W3/01)T exhibited a quinone system with menaquinone MK-10(H4) as the predominant compound (98 %) and MK-9(H4) (2 %). A similar quinone system, with MK-10(H4) (91 %) and MK-9(H4) (9 %), was detected in strain 2(W106/04)T. This result was not in agreement with the previously determined quinone systems of A. haemolyticum, which was reported to contain MK-9(H4) as the predominant compound (Collins et al., 1982). Hence, we analysed the quinone system of A. haemolyticum DSM 20595T and A. pyogenes DSM 20630T. A. haemolyticum DSM 20595T was found to possess a quinone system with MK-9(H4) as the predominant compound (85 %) and minor amounts of MK-8(H4) (15 %). In contrast, A. pyogenes DSM 20630T, which is the closest phylogenetic relative of strains 1(W3/01)T and 2(W106/04)T, also contained a quinone system with the major compounds MK-10(H4) (85 %) and MK-9(H4) (15 %). The results from the quinone analyses reflect the phylogenetic relatedness of strains 1(W3/01)T and 2(W106/04)T to A. pyogenes DSM 20630T and clearly distinguish them from the type species of the genus, A. haemolyticum.
Taxonomic considerations
16S rRNA gene sequence analyses and the chemotaxonomic characteristics of the isolates suggest the assignment of strains 1(W3/01)T and 2(W106/04)T to the genus Arcanobacterium. Unique physiological profiles and genomic fingerprints and characteristic fatty acid and polar lipid profiles demonstrate that strains 1(W3/01)T and 2(W106/04)T each represent a separate species within the genus.
In general, the quinone system is a rather conserved characteristic. The finding of MK-10(H4) in one lineage within the genus Arcanobacterium, represented by our isolates and A. pyogenes, is rather exciting because the representative of the second lineage, A. haemolyticum, is characterized by the MK-9(H4) quinone system. Hence, both quinone system and phylogeny indicate a subdivision within the genus Arcanobacterium, and a restriction of the genus to the species A. haemolyticum, A. phocae, A. pluranimalium and A. hippocoleae might be desirable. This would lead to A. pyogenes, A. bernardiae and the two novel species described in this study being reclassified as a novel genus. In a reclassified genus Arcanobacterium, the presence of a MK-9(H4) quinone system might be a genus-specific trait, while MK-10(H4) might characterize the neighbouring genus. However, the suitability of the quinone system as the basis for such differentiation would have to be substantiated by examination of all species of Arcanobacterium.
Since the presence of MK-9(H4) is listed as a characteristic trait of the genus Arcanobacterium (Collins et al., 1982), the description of the genus has to be emended to include the presence of MK-10(H4).
Emended description of the genus Arcanobacterium Collins et al. 1983
In contrast to the original description of the genus, some species do not contain a quinone system with menaquinone MK-9(H4) as the major compound; MK-10(H4) can also predominate. Other characteristics of the genus are as given by Collins et al. (1982).
Description of Arcanobacterium bialowiezense sp. nov.
Arcanobacterium bialowiezense (bi.a.lo.wi.e.zen'se. N.L. neut. adj. bialowiezense pertaining to Bialowieza, Poland, where the type strain was isolated).
Cells are short pleomorphic rods that stain Gram-positive. After 48 h of growth under aerobic conditions on sheep blood agar, colonies are translucent, convex, approx. 0·5 mm in diameter and surrounded by a narrow zone of 
-haemolysis. Cells are non-motile (motility agar), facultatively anaerobic and are catalase- and oxidase-negative. Best growth occurs at 37 °C; no growth at 42 °C. No growth is observed on Gassner or MacConkey agar. Other physiological and biochemical characteristics are summarized in Table 1
. The quinone system contains MK-10(H4) as the predominant compound. Diphosphatidylglycerol is predominant in the polar lipid profile. Phosphatidylglycerol, three unknown phosphoglycolipids, two unknown aminolipids, two unknown phospholipids, an unknown aminophospholipid, an unknown aminoglycolipid and an unknown aminophosphoglycolipid are present in moderate to minor amounts. The fatty acid profile consists predominantly of unbranched acids with the major compounds C16 : 0, C18 : 19c, C18 : 0 and C14 : 0. Complete fatty acid profiles are listed in Table 2.
The type strain, strain 1(W3/01)T (=DSM 17162T=NCTC 13354T), was isolated from the prepuce of a European bison.
Description of Arcanobacterium bonasi sp. nov.
Arcanobacterium bonasi [bo.na'si. L. gen. n. bonasi of the European bison (Bison bonasus) from which the type strain was isolated].
Cells are short pleomorph rods that stain Gram-positive. After 48 h of growth under aerobic conditions on sheep blood agar, colonies are translucent, convex, approx. 0·5 mm in diameter and surrounded by a narrow zone of -haemolysis. Cells are non-motile (motility agar), facultatively anaerobic and catalase- and oxidase-negative. Best growth occurs at 37 °C; no growth at 42 °C. No growth is observed on Gassner or MacConkey agar. Other physiological and biochemical characteristics are summarized in Table 1. The quinone system contains MK-10(H4) as the predominant compound. Diphosphatidylglycerol is predominant in the polar lipid profile. Phosphatidylglycerol, three unknown phosphoglycolipids, an unknown aminolipid, two unknown phospholipids and an unknown aminophospholipid are present in moderate to minor amounts. The fatty acid profile consists predominantly of unbranched acids with the major compounds C16 : 0, C18 : 19c and C18 : 0. Complete fatty acid profiles are listed in Table 2.
The type strain, strain 2(W106/04)T (=DSM 17163T=NCTC 13355T), was isolated from the prepuce of a European bison.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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