International Journal of Systematic and Evolutionary Microbiology (2002), 52, 207–210 Printed in Great Britain Isobaculum melis gen. nov., sp. nov., a NOTE Carnobacterium-like organism isolated from the intestine of a badger 1 School of Food Biosciences, Matthew D. Collins,1 Roger A. Hutson,1 Geoffrey Foster,2 Enevold Falsen3 Whiteknights, University 4 of Reading, Reading and Norbert Weiss RG6 6AP, UK 2 SAC Veterinary Science Author for correspondence: Matthew D. Collins. Tel: j44 118 935 7000. Fax: j44 118 926 7917. Division, Drummondhill, e-mail: m.d.collins!reading.ac.uk Inverness, UK 3 Culture Collection, Department of Clinical Phenotypic and phylogenetic studies were performed on a hitherto Bacteriology, University of undescribed facultatively anaerobic, catalase-negative, Gram-positive rod- Go$ teborg, Go$ teborg T Sweden shaped organism, strain M577-94 , isolated from the small intestine of a dead badger. It resembled carnobacteria in terms of its long-chain cellular fatty acid 4 Deutsche Sammlung von Mikroorganismen und composition, but differed markedly from the latter in possessing a cell-wall Zellkulturen GmbH, murein based on L-lysine (type L-Lys–L-Thr–Gly). Comparative 16S rRNA gene Braunschweig, Germany sequencing showed that the unknown bacterium represents a new line closely related to, albeit distinct from, the genera Carnobacterium and Desemzia.On the basis of phylogenetic and phenotypic evidence, it is proposed that strain M577-94T be classified as Isobaculum melis gen. nov., sp. nov. The type strain of Isobaculum melis is CCUG 37660T (l DSM 13760T). Keywords: Isobaculum melis, 16S rRNA, taxonomy, phylogeny The genus Carnobacterium was proposed as the genus known non-spore-forming rod-shaped bacterium, accommodating the species Lactobacillus divergens isolated from a badger, which somewhat resembles and Lactobacillus piscicola and a number of atypical carnobacteria. Here we report the results of a poly- lactobacilli which had been isolated from refrigerated phasic taxonomic study on the unknown rod, and meats (Collins et al., 1987). The genus originally propose that it be classified as a new genus and species, contained four species (Carnobacterium divergens, namely Isobaculum melis. Carnobacterium piscicola Carnobacterium gallinarum T T T , Strain M577-94 (l CCUG 37660 l DSM 13760 ) and Carnobacterium mobile). Three further species, was isolated from the small intestine of a dead badger Carnobacterium funditum and Carnobacterium and was cultured on Columbia agar supplemented alterfunditum , originating from Antarctic lakes with 5% defibrinated horse blood in air at 37 mC. The (Franzmann et al., 1993), and Carnobacterium inhi- strain was characterized by using conventional physio- bens, isolated from Atlantic salmon (Jo$ born et al., logical tests (Facklam & Elliot, 1995) and also by using 1999), have subsequently been assigned to the genus. the API rapid ID32Strep, API CORYNE and API Carnobacteria resemble lactobacilli in consisting of ZYM systems according to the manufacturer’s instruc- Gram-positive, catalase-negative, fermentative, as- tions (API bioMe! rieux). The cell-wall murein structure porogenous rod-shaped organisms. Molecular genetic of strain CCUG 37660T was determined by the studies, however, have revealed that carnobacteria are methods of Schleifer & Kandler (1972) except that only remotely related to lactobacilli and in fact show a ascending TLC on cellulose sheets was used. Long- closer phylogenetic affinity with several catalase-nega- chain cellular fatty acids were examined using the tive coccus-shaped organisms such as enterococci, MIDI (MIDI) system. The 16S rRNA gene of the vagococci and Lactosphaera pasteurii (e.g. Collins isolate was amplified by a PCR and directly sequenced et al., 1991, 1997, 1998; Stackebrandt et al., 1999). using a Taq dye-Deoxy terminator cycle sequencing kit During the course of an investigation of unusual (Applied Biosystems) and an automatic DNA se- Gram-positive catalase-negative organisms associated quencer (model 373A; Applied Biosystems). The with animals, we have characterized a hitherto un- closest known relatives of the new isolate were de- ................................................................................................................................................. termined by performing database searches. A phylo- The GenBank accession number for the 16S rRNA gene sequence of strain genetic tree was constructed according to the neigh- CCUG 37660T (l DSM 13760T) is AJ302648. bour-joining method with the program 01864 # 2002 IUMS 207 M. D. Collins and others ..................................................................................................... Fig. 1. Unrooted tree showing the phylogenetic relationships of Isobaculum melis sp. nov. and some other low-GjC Gram-positive bacteria. The tree constructed using the neighbour-joining method was based on a comparison of approximately 1320 nucleotides. Bootstrap values, ex- pressed as percentages of 500 replications, are given at branching points. (Felsenstein, 1989). The stability of the groupings was unsaturated types with C14:0 (11%), C16:0 (32%), estimated by bootstrap analysis (500 replications) C18:0 (15%) and C18:1ω9c (30%) predominating. using the programs , , and The DNA base composition of the organism was 39 (Felsenstein, 1989). mol% GjC, and cell wall analysis revealed the T presence of an A3α murein (type -Lys–-Thr–Gly). The unknown strain CCUG 37660 originating from To ascertain the phylogenetic relationships of the the small intestine of a dead badger consisted of Gram- unidentified rod, its 16S rRNA gene sequence was positive asporogenous rods which were non-motile. determined and subjected to a comparative analysis. The organism was facultatively anaerobic and cata- Sequence searches showed that the rod-shaped or- lase- and oxidase-negative. It produced growth at ganism was a member of the Clostridium subphylum of 10 mC but not 45 mC and did not grow in broth the Gram-positive bacteria and displayed highest containing 6n5% NaCl. The isolate failed to produce sequence relatedness to the genus Carnobacterium gas in deMan, Rogosa and Sharpe broth, gave a (94–95% sequence similarity) and Desemzia incerta positive bile aesculin reaction, was pyrrolydonyl aryla- (94n5%). A tree showing the position of the rod- midase-positive and leucine aminopeptidase-negative, shaped bacterium in relation to its nearest phylogenetic and did not hydrolyse hippurate, starch or urea using relatives is shown in Fig. 1. The unknown rod formed conventional methods (Facklam & Elliot, 1995). It a distinct line branching near to the base of the genera produced acid from -glucose, glycerol, -ribose and Carnobacterium and Desemzia. trehalose but not from -arabinose, inulin, lactose, maltose, melezitose, melibiose, -raffinose, sorbitol, It is evident from the results of the polyphasic sorbose or sucrose in conventional heart infusion base taxonomic study that the unidentified asporogenous medium (Facklam & Elliot, 1995). The organism did rod-shaped organism isolated from a badger represents not utilize pyruvate and was Voges–Proskauer-nega- a hitherto unknown taxon. Phylogenetically, the bac- tive. When API kits were used, the isolate produced terium forms a distinct subline close to the genera acid from -glucose, trehalose and -ribose but not Carnobacterium and Desemzia. Bootstrap resampling from -arabinose, -arabitol, cyclodextrin, glycogen, showed that the unknown rod did not possess a lactose, melibiose, melezitose, maltose, mannitol, pul- statistically significant association with either of these lulan, -raffinose, sorbitol, sucrose, -tagatose, - taxa. The badger bacterium was also found to be xylose or methyl β--glucopyranoside. Activity was phenotypically very distinct. For example, the presence observed for acid phosphatase (weak reaction), ar- of large amounts of oleic acid meant that the unknown ginine dihydrolase, esterase C-4 (weak reaction), ester organism resembled the carnobacteria, whereas many lipase C-8 (weak reaction), β-glucosidase, pyrogluta- other catalase-negative, asporogenous rod-shaped mic acid arylamidase, pyrrolydonyl arylamidase, taxa such as lactobacilli and D. incerta contain cis- phosphoaminidase and β-mannosidase but not for vaccenic acid (Collins et al., 1987). However, the alanine phenylalanine proline arylamidase, chymo- presence of an A3α murein based on -lysine (type - trypsin, cystine arylamidase, α-fucosidase, α-galacto- Lys–-Thr–Gly) in the badger bacterium represents a sidase, β-galactosidase, α-glucosidase, β-glucuroni- marked difference with respect to the wall mureins of dase, glycine tryptophan arylamidase, α-mannosidase, members of the genus Carnobacterium, which are leucine arylamidase, lipase C-14, pyrazinamidase, invariably based on meso-diaminopimelic acid. Simi- trypsin, urease or valine arylamidase. Different results larly, the cell wall composition reinforces the difference were obtained for N-acetyl-β-glucosaminidase: acti- between the badger bacterium and D. incerta, as the vity was detected with the API rapid ID32Strep system latter contains the -lysine–-glutamic acid murein but not with the API ZYM or API CORYNE systems. type (Stackebrandt et al., 1999). The 16S rRNA tree The long-chain cellular fatty acids of the unidentified branching pattern together with these marked chemo- isolate consisted of straight-chain saturated and mono- taxonomic differences strongly support the assignment 208 International Journal of Systematic and Evolutionary Microbiology