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1 Laboratory of Microbiology, Department of Biochemistry, Physiology and Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium
2 Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow G4 OBA, UK
Correspondence
Elke De Clerck
elke.declerck{at}Ugent.be
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Published online ahead of print on 9 January 2004 as DOI 10.1099/ijs.0.02960-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains LMG 21881T (=DSM 15866T) and LMG 21880T (=DSM 15865T) are AJ551330 and AJ551329, respectively.
Supplementary phylogenetic trees and fatty acid tables are available in IJSEM Online.
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) and, moreover, can survive extreme pH and temperature conditions during manufacturing, drying and ultra-high temperature (UHT) treatment, which should guarantee microbial sterility of the end product (De Clerck et al., 2004, unpublished results). We isolated and screened bacterial contaminants from gelatin batches of several production plants. Bacterial contamination of these gelatin batches was suspected, based on the quality control of the production plant. It is within the framework of this study that we characterized a group of five isolates (group 1) from a French production plant and a group of 66 isolates (group 2) from a Belgian production plant; we attribute each group of strains to a novel species in the genera Anoxybacillus and Bacillus, respectively, for which we propose the names Anoxybacillus contaminans sp. nov. and Bacillus gelatini sp. nov.
The genus Anoxybacillus was first described by Pikuta et al. (2000); at the time of writing, the genus comprises the three species Anoxybacillus pushchinoensis (Pikuta et al., 2000), Anoxybacillus flavithermus (Pikuta et al., 2000) and Anoxybacillus gonensis (Belduz et al., 2003). The genus Bacillus has a long taxonomic history. Since it was described by Cohn in 1872, its systematics have undergone major revisions [as reviewed by Stackebrandt & Swiderski (2002)].
Strains were isolated by enrichment of 30 g gelatin sample in 70 ml trypticase soy broth (Oxoid) at 45 and 55 °C for 24 h and plating on several media. Strains of group 1 were isolated on nutrient agar (Oxoid) that was supplemented with 1·2 % gelatin at 45 and 55 °C. Strains of group 2 were isolated on trypticase soy agar (TSA; Oxoid), brain heart infusion agar (BBL) that was supplemented with 1 mg vitamin B12 l–1 and nutrient agar that was supplemented with 1·2 % gelatin, at 45 and 55 °C. For repetitive extragenic palindromic DNA (rep)-PCR and 16S rDNA sequencing, template DNA was prepared by using a slight modification of the method of Pitcher et al. (1989), as described by Heyndrickx et al. (1996). Rep-PCR genomic fingerprinting was performed with the (GTG)5 primer (Versalovic et al., 1994), using PCR conditions that were described by Versalovic et al. (1994). The resulting fingerprints were analysed with the BioNumerics version 2.5 software package (Applied Maths). Sequencing of the 16S rDNA was performed as described by Heyrman & Swings (2001). Phylogenetic analysis was performed by using CLUSTAL W (Thompson et al., 1994) and TREECON (Van de Peer & De Wachter, 1994) software. Large-scale DNA extraction for determination of G+C content and DNA–DNA hybridization was based on the method described by Gevers et al. (2001). Cells were collected from 500 ml culture in the mid-exponential phase by centrifugation (3000 g, 10 min, 4 °C) and frozen for at least 10 min at –20 °C. The thawed pellet was washed in 15 ml RS buffer (0·15 M NaCl, 0·01 M EDTA, pH 8·0) and resuspended in a lysis buffer that contained 2250 µl STET buffer [8 % sucrose, 5 % Triton X-100, 50 mM Tris/HCl (pH 8·0), 50 mM EDTA], 412·5 µl TES buffer [6·7 % sucrose, 50 mM Tris/HCl (pH 8·0), 1 mM EDTA], 22·5 mg lysozyme, 200 µl RNase (10 mg ml–1) and 150 µl mutanolysine (5000 U ml–1). The suspension was incubated at 37 °C for 1 h. After addition of 500 µl 20 % SDS in TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8·0) and glass beads, cells were vortexed for 30 s and incubated at 60 °C for 10 min. Following the addition of 6 ml 5 M NaCl and vigorous shaking, the lysate was extracted with 10 ml chloroform/isoamyl alcohol (24 : 1) by centrifugation (15 000 g, 20 min, 4 °C). The aqueous phase was mixed carefully with 15 ml 2-propanol and precipitated DNA was twisted on a glass rod, washed in an ethanol gradient (70, 80 and 90 %), air-dried and dissolved in 5 ml 0·1x SSC (1·5 mM trisodium citrate, 15 mM NaCl). After an additional RNase treatment (50 µl, 10 mg RNase ml–1), 625 µl acetate/EDTA (3 M ammonium acetate, 1 mM EDTA, pH 7·5) was added. After a second round of chloroform/isoamyl alcohol extraction and DNA precipitation as stated above, DNA was dissolved in 0·5 ml 0·1x SSC. Determination of the DNA G+C content was performed as described previously (Logan et al., 2000). DNA–DNA hybridizations were performed by using a modification of the microplate method described by Ezaki et al. (1989), as described by Willems et al. (2001). A hybridization temperature of 37 °C was used. After growth of the cells on TSA for 24 h at 52 °C, fatty acid methyl esters (FAMEs) were prepared and separated as described by Yang et al. (1993). All strains were characterized phenotypically by the methods of Logan & Berkeley (1984), but the API tests (bioMérieux) were incubated at 50 °C and read after 24 h. Salt tolerance (5, 10 and 15 % NaCl) was tested in nutrient broth. Catalase and oxidase tests were performed according to the methods described by Smibert & Krieg (1994). Tests for other characteristics were done as described by Logan et al. (2000), but with the exception of growth-temperature ranges, all tests were incubated at 40 °C.
Rep-PCR screening of gelatin isolates allows determination of their genotypic diversity. Rep-PCR is known to discriminate at the subspecies level (Versalovic et al., 1994). The five strains of group 1 represent two rep-PCR fingerprint types (Fig. 1a). The 66 isolates of group 2 represent four different rep-PCR profiles. Cluster analysis based on representative strains for this group is shown in Fig. 1b.
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) showed best matches with Bacillus or Anoxybacillus species, but with sequence similarities of around or below 97 %. Cluster analysis revealed that these isolates grouped tightly as a separate rank within the genus Anoxybacillus (a phylogenetic tree with other Anoxybacillus species and the type species of related genera is available as Supplementary Fig. A in IJSEM Online). DNA–DNA relatedness was verified between representatives of both rep-PCR fingerprint types. A reciprocal DNA–DNA relatedness of 97 % between strains LMG 21881T and R-16223 assigns these strains to a single species, according to the recommendations for species designation (Wayne et al., 1987; Stackebrandt et al., 2002). The DNA G+C contents of strains LMG 21881T and R-16223 are 44·3 and 44·4 mol%, respectively.
The almost-complete 16S rRNA genes of strains R-13476 (1503 bp) and LMG 21880T (1485 bp) were sequenced, as well as the hypervariable part of strains R-13975 (354 bp) and R-13565 (477 bp), representing the four fingerprint types that are delineated in group 2. These sequences were 100 % similar in the most variable part of the gene. FASTA searches (Pearson & Lipman, 1988) showed best matches with Bacillus species, but with similarities of <94 %. Although 16S rDNA sequence similarities were low, the isolates cluster among Bacillus species (a phylogenetic tree is available as Supplementary Fig. B in IJSEM Online). These observations support separate species rank for the isolates within the genus Bacillus. As sequence similarities to entries in the GenBank/EMBL database were well below 97 %, the level below which strains are generally attributed to separate taxa (Stackebrandt & Goebel, 1994), DNA–DNA relatedness was verified between representatives of the most dissimilar rep-PCR fingerprint types. A reciprocal DNA–DNA relatedness of 95 % was found between strains R-13975 and R-13565, 94 % between strains LMG 21880T and R-13565 and 98 % between strains LMG 21880T and R-13975. According to the 70 % level for species delineation (Wayne et al., 1987; Stackebrandt et al., 2002), all strains may be assigned to a single species. The DNA G+C contents of strains R-13565, LMG 21880T and R-13975 are 41·2, 41·5 and 41·3 mol%, respectively.
The fatty acid profiles of four representative isolates of group 1, on the one hand, and eight representative isolates of group 2, on the other, are very homogeneous and can be used to calculate a mean fatty acid pattern, which is given in the species descriptions. Detailed FAME data are available as Supplementary Tables A and B in IJSEM Online.
Results of physiological characterization are given in the species descriptions. All strains had similar patterns of results in API and other routine phenotypic tests; no correlation of patterns of results with rep-PCR groups was seen. Table 1 shows some characters that distinguish strains of group 1 from other Anoxybacillus species. For group 2 isolates, production of acid from carbohydrates in the API 50 CHB gallery was usually weak, but strains R-13562 and R-13565 were more reactive and produced acid from a wider range of substrates. All strains liquefied gelatin within 24 h. Table 2 shows some characters that distinguish these strains from species of the genus Bacillus that react weakly in API tests. We considered an API 50 CHB reaction to be weak when phenol red turned from red to orange, instead of yellow.
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Description of Anoxybacillus contaminans sp. nov.
Anoxybacillus contaminans (con.ta'mi.nans. L. part. adj. contaminans contaminating).
Cells are curved or frankly curled, round-ended, Gram-variable (cells from the same colony stained either Gram-positive or Gram-negative), facultatively anaerobic, catalase-positive, oxidase-negative and feebly motile rods that occur singly, in pairs or in short chains. Cell diameter is 0·7–1·0 µm; cell length is 4–10 µm. Endospores are produced sparsely; they are oval in shape, lie subterminally or terminally and may swell sporangia slightly. Colonies grown on TSA at 40 °C are circular with regular margins and raised centres and edges, opaque, glossy and cream-coloured. Colony diameter ranges from 1 to 2 mm. Maximum temperature for growth lies between 50 and 60 °C; optimum temperature is 50 °C. Optimum pH for growth is 7·0; minimum pH for growth lies between 4·0 and 5·0 and maximum lies between 9·0 and 10·0. Weak growth occurs in nutrient broth with 5 % NaCl added; no growth occurs at a salt concentration of 10 %. Casein is not hydrolysed. In the API 20E strip, gelatin is hydrolysed and nitrate is reduced to nitrite. All strains are negative for o-nitrophenyl 
-D-galactopyranoside hydrolysis, arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase reactions, citrate utilization, hydrogen sulphide production, urease, tryptophan deaminase, indole production and the Voges–Proskauer reaction. In the API 50 CH gallery using the CHB suspension medium, hydrolysis of aesculin is weak and small amounts of acid without gas are produced from the following carbohydrates: L-arabinose, D-fructose, D-galactose, D-glucose, glycerol, glycogen, maltose, D-mannose, D-melezitose, methyl D-glucoside, N-acetylglucosamine, D-raffinose, ribose, starch, sucrose, D-trehalose, D-turanose and D-xylose. Production of acid is negative for adonitol, amygdalin, D-arabinose, D- and L-arabitol, arbutin, D-cellobiose, dulcitol, erythritol, D- and L-fucose, gentiobiose, gluconate, inulin, 2- and 5-keto-D-gluconate, lactose, D-lyxose, mannitol, D-melibiose, meso-inositol, methyl D-mannoside, methyl xyloside, rhamnose, salicin, sorbitol, L-sorbose, D-tagatose, L-xylose and xylitol. Major cellular fatty acids are iso-C15 : 0, C16 : 0 and iso-C17 : 0 (respectively representing about 52, 11 and 12 % of total fatty acids). The following fatty acids are present in smaller amounts: C14 : 0, anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0 (respectively representing about 3, 7, 5 and 7 % of total fatty acids).
The type strain is LMG 21881T (=DSM 15866T); the DNA G+C content of this strain is 44·4 mol%.
Description of Bacillus gelatini sp. nov.
Bacillus gelatini (ge.la.ti'ni. N.L. neut. gen. n. gelatini from gelatin).
Cells are straight, round-ended, Gram-variable, strictly aerobic, catalase-positive, oxidase-negative and feebly motile rods that form long chains or occasionally appear singly. Cell diameter is 0·5–0·9 µm; cell length is 4–10 µm. Endospores are oval, lie paracentrally and subterminally and do not swell sporangia. Colonies on TSA incubated at 30 °C for 4 days are cream-coloured, darker in the centre, smooth, have slightly irregular borders and are waxy in appearance, with eggshell-textured (not glossy, not matte) surfaces. They are slightly convex, but older colonies are flatter with concave, transparent centres. Colony diameter ranges from 1 to 4 mm. Maximum temperature for growth lies between 58 and 60 °C and optimum temperature lies between 40 and 50 °C. Good growth occurs at pH 5–8; minimum pH for growth is 4–5 and maximum is 9–10. Good growth occurs in nutrient broth with 15 % NaCl added. Casein is hydrolysed. In the API 20E strip, hydrolysis of gelatin is positive. All strains are negative for o-nitrophenyl -D-galactopyranoside hydrolysis, arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase reactions, citrate utilization, hydrogen sulphide production, urease, tryptophan deaminase, indole production, Voges–Proskauer reaction and nitrate reduction. In the API 50 CH gallery using the CHB suspension medium, hydrolysis of aesculin is positive and acid without gas is produced, often weakly, from the following carbohydrates: D-fructose, D-glucose, glycerol, mannitol, D-mannose, D-trehalose and D-xylose. Most strains show very weak production of acid from N-acetylglucosamine, maltose and ribose. The more reactive strains may produce acid from D-cellobiose, D-galactose, 5-keto-D-gluconate, D-melezitose, meso-inositol, methyl D-glucoside and D-turanose. No acid is produced from adonitol, amygdalin, D- or L-arabinose, D- or L-arabitol, arbutin, dulcitol, erythritol, D- or L-fucose, gentiobiose, gluconate, glycogen, inulin, 2-keto-D-gluconate, lactose, D-lyxose, D-melibiose, methyl D-mannoside, methyl xyloside, D-raffinose, rhamnose, salicin, sorbitol, L-sorbose, starch, sucrose, D-tagatose, L-xylose or xylitol. Major cellular fatty acids are iso-C15 : 0, iso-C17 : 0 and anteiso-C17 : 0 (respectively representing about 60, 13 and 10 % of total fatty acids). The following fatty acids are present in smaller amounts: anteiso-C15 : 0, iso-C16 : 0 and C16 : 0 (respectively representing about 9, 4 and 2 % of total fatty acids).
The type strain is LMG 21880T (=DSM 15865T); the DNA G+C content of this strain is 41·5 mol%.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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