International Journal of Systematic and Evolutionary Microbiology (2000), 50, 1755–1760 Printed in Great Britain

Atopobacter phocae gen. nov., sp. nov., a novel NOTE bacterium isolated from common seals

Paul A. Lawson,1 Geoffrey Foster,2 Enevold Falsen,3 Maria Ohle! n3 and Matthew D. Collins1

Author for correspondence: Matthew D. Collins. Tel: j44 118 935 7000. Fax: j44 118 926 7917. e-mail: m.d.collins!reading.ac.uk

1 Department of Food Two strains of a Gram-positive, catalase-negative, facultatively anaerobic, rod- Science and Technology, shaped bacterium isolated from common seals were characterized using University of Reading, Reading RG6 6AP, UK phenotypic and molecular taxonomic methods. The two strains closely resembled each other based on their biochemical characteristics, and PAGE 2 SAC Veterinary Services, Inverness, UK analysis of whole-cell protein patterns confirmed their close phenotypic affinity. 16S rRNA gene sequencing showed that the two strains were 3 Culture Collection, Department of Clinical genetically highly related (998% sequence similarity) and that they constitute Bacteriology, University of a new line of descent within the lactic acid group of bacteria. The nearest Go$ teborg, Go$ teborg, phylogenetic neighbours of the unknown bacterium were Granulicatella spp., Sweden with related taxa such as enterococci, carnobacteria, Desemzia incerta, Lactosphaera pasteurii, Melissococcus plutonius, tetragenococci and vagococci more distantly related. Based on phylogenetic and phenotypic evidence it is proposed that the unknown bacterium from seals be classified in a new genus as Atopobacter phocae gen. nov., sp. nov. The type strain of Atopobacter phocae is CCUG 42358T (l CIP 106392T).

Keywords: Atopobacter phocae, 16S rRNA, taxonomy, phylogeny

The lactic acid bacteria constitute a phylogenetically organisms, numerous studies have shown these taxa heterogeneous assortment of Gram-positive, faculta- are only remotely related to lactobacilli, and in fact tively anaerobic, mostly catalase-negative, rod- or possess a closer phylogenetic affinity with several coccus-shaped organisms which belong to the Clos- catalase-negative coccus-shaped organisms such as tridium branch of the Gram-positive bacteria. The Granulicatella spp., enterococci, vagococci, tetrageno- genus Lactobacillus constitutes the largest group of cocci, Melissococcus plutonius and Lactosphaera pas- rod-shaped organisms within the lactic acid bacteria teurii (e.g. Collins & Lawson, 2000; Lawson et al., and currently over 50 species are recognized (Hammes 1999a,b; Stackebrandt et al., 1999). et al., 1992). Other rod-shaped genera within the lactic acid group of bacteria include Carnobacterium and During the course of an investigation into the Gram- Desemzia. The genus Carnobacterium was described to positive flora of aquatic mammals, we have isolated a accommodate the species Lactobacillus divergens and hitherto unknown rod-shaped bacterium from two Lactobacillus piscicola and some atypical lactobacilli dead seals which displays a phylogenetic association which were isolated from refrigerated meats (Collins et with Carnobacterium, Desemzia and the aforemen- al., 1987). The genus Carnobacterium originally con- tioned catalase-negative, coccus-shaped organisms. tained four species but two further species from The unknown bacterium was readily distinguished Antarctic lakes have subsequently been added to the from other described Gram-positive, catalase-negative genus (Franzmann et al., 1991). Recently, the genus organisms by biochemical tests and electrophoretic Desemzia was described for the species previously analysis of whole-cell proteins. Based on phylogenetic designated Brevibacterium incertum (Stackebrandt and phenotypic evidence it is proposed that the et al., 1999). Although both carnobacteria and De- unidentified bacterium be classified as Atopobacter semzia incerta consist of Gram-positive, rod-shaped phocae gen. nov., sp. nov. T ...... Strain M1590\94\2 was isolated following a post- The GenBank accession number for the 16S rRNA gene sequence of strain mortem examination of a common seal pup. The CCUG 42358T is Y16546. animal had general lymphadenopathy and acutely

01409 # 2000 IUMS 1755 P. A. Lawson and others congested lungs with pulmonary haemorrhaging. The blood to the basal medium exhibiting the same colony rod-shaped bacterium was recovered from the small morphology as above. Both strains were facultatively intestine and from the mesenteric, external iliac, pre- anaerobic and catalase-negative. Growth was ob- scapular and pancreatic lymph nodes. Additionally it served at 25 but not at 42 mC. Both strains produced was co-isolated with Arcanobacterium phocae from the acid from cyclodextrin, glucose, glycogen, maltose, lungs (Pascual Ramos et al., 1997). Strain M955\98\1 pullulan and ribose, but failed to produce acid from - was isolated from an adult common seal that had died arabitol, -arabinose, mannitol, melibiose, melezitose, from gun-shot wounds. It was isolated from a mixed methyl β--glucopyranoside, raffinose, sorbitol, taga- culture with an unidentified Actinomyces sp. obtained tose and -xylose. The production of acid from lactose, from the liver, spleen and blood. All of the rod-shaped sucrose and trehalose was variable. Both isolates isolates recovered from these multiple sites of the showed acid phosphatase, alkaline phosphatase, ar- respective seals were found to possess identical cellular ginine dihydrolase, esterase C-4 (weak reaction), ester and colonial morphologies and biochemical profiles. lipase C-8 (weak reaction), pyroglutamic acid aryl- Strains M1590\94\2T and M955\98\1 have been amidase (weak reaction), pyrazinamidase (weak re- deposited in the Culture Collection of the University action) and pyrrolidonyl arylamidase activity. Activity of Go$ teborg (CCUG) under accession numbers 42358T of alanine phenylalanine proline arylamidase and and 42577, respectively. leucine arylamidase was either weak or negative. Neither of the strains however displayed activity for N- Both strains were cultured on Columbia agar sup- acetylglucosaminidase, chymotrypsin, cystine aryl- plemented with 5% defibrinated horse blood (Oxoid amidase, α-fucosidase, α-galactosidase, β-glucosidase, Unipath) and incubated in air plus 5% CO at 37 C. # m β-glucuronidase, glycine tryptophan arylamidase, α- The strains were biochemically characterized by using mannosidase, β-mannosidase lipase C-14, trypsin or the API rapid ID32S and API ZYM systems according valine arylamidase. Neither of the strains hydrolysed to the manufacturer’s instructions (API bioMe! rieux). aesculin, gelatin, hippurate or urease. They were PAGE analysis of whole-cell proteins was performed Voges–Proskauer-negative and did not reduce nitrate. as described by Pot et al. (1994). The GelCompar GCW 3.0 software package (Applied Maths) was used PAGE analysis of whole-cell proteins confirmed the for densitometric analysis, normalization and inter- close phenotypic resemblance of the two bacterial seal pretation of protein patterns. The cell wall murein isolates as they formed a tight cluster (approx. 78% structure of strain CCUG 42358T was determined by similarity), which was separate from all other Gram- the method of Schleifer & Kandler (1972) except that positive, catalase-negative reference organisms ascending TLC on cellulose sheets (Merck) was used. examined (see Fig. 1). Taxa recovered close to the The 16S rRNA genes of the isolates were amplified by unknown strains on PAGE analysis included Tetra- PCR and directly sequenced using a Taq DyeDeoxy genococcus halophilus, Aerococcus viridans and some terminator cycle sequencing kit (Applied Biosystems) Leuconostoc spp., although their association was not and an automatic DNA sequencer (Applied Bio- particularly close (joining the unknown bacterium systems; model 373A). The closest known relatives of cluster at less than 60% similarity). An examination of the new isolates were determined by performing the cell wall of strain CCUG 42358T revealed a murein database searches. These sequences and those of other based on -Orn--Asp (type A4β) (nomenclature of known related strains were retrieved from the Gen- Schleifer & Kandler, 1972). Within the Gram-positive, Bank or Ribosomal Database Project (RDP) data- catalase-negative, non-sporing, rod-shaped taxa this bases and aligned with the newly determined sequences murein type is relatively uncommon, occurring in using the program  (Devereux et al., 1984). The some bifidobacteria, Lactobacillus fermentum, Lacto- resulting multiple sequence alignment was corrected bacillus pontis, Lactobacillus vaginalis and Lacto- manually and a distance matrix was calculated using bacillus uli. Similarly, amongst coccus-shaped taxa this the programs  and  (using the Kimura murein type is to our knowledge found only in two-correctionparameter)(Felsenstein,1989).Aphylo- Granulicatella balaenopterae,‘Peptostreptococcus gly- genetic tree was constructed according to the cinophilus’, Sporosarcina halophila and Halobacillus neighbour-joining method with the program  spp. It is pertinent to note that carnobacteria and D. (Felsenstein, 1989). The stability of the groupings was incerta, the nearest rod-shaped phylogenetic relatives estimated by bootstrap analysis (500 replications) of the unknown seal bacterium, possess a directly using the programs , ,  and cross-linked wall based on meso-diaminopimelic acid  (Felsenstein, 1989). The GenBank accession (type A1γ) and a -Lysine--Glu-type murein, re- number for the 16S rRNA gene sequence of strain spectively (Collins et al., 1987; Stackebrandt et al., CCUG 42358T is Y16546. 1999). The two isolates were Gram-positive, irregular-rod- To investigate the genealogical affinity between the shaped cells which formed smooth, grey-coloured, two isolates and their relationship with other Gram- non-haemolytic, pin-sized colonies on Columbia agar positive, catalase-negative taxa, comparative 16S supplemented with 5% defibrinated horse blood when rRNA gene sequence analyses were performed. Pair- incubated at 37 mC in air plus 5% CO# for 24 h. wise analysis of the almost complete gene sequence Growth was also observed without the addition of (" 1400 nucleotides) of the two isolates from seals were

1756 International Journal of Systematic and Evolutionary Microbiology 50 Atopobacter phocae gen. nov., sp. nov.

...... Fig. 1. Similarity dendrogram based on whole-cell protein patterns of Atopobacter phocae sp. nov. and related species. Levels of correlation are expressed as percentages of similarity for convenience. determined and revealed 99n8% 16S rRNA similarity. blance. Sequence searches of GenBank and RDP This close genealogical affinity between the isolates databases revealed the unknown isolates were phylo- was consistent with the results of whole-cell protein genetically most closely associated with the lactic acid PAGE analysis and their close biochemical resem- group of bacteria, particularly Granulicatella spp.,

International Journal of Systematic and Evolutionary Microbiology 50 1757 P. A. Lawson and others

...... Fig. 2. Unrooted tree showing the phylogenetic relationships of Atopobacter phocae sp. nov. and some other low GjC content, Gram-positive bacteria. The tree, constructed using the neighbour-joining method, was based on a comparison of approximately 1320 nucleotides. Bootstrap values, expressed as a percentage of 500 replications, are given at branching points.

Lactosphaera pasteurii, M. plutonius, enterococci, car- tetragenococci and vagococci, and the rod-shaped D. nobacteria, vagococci and tetragenococci (data not incerta and carnobacteria (Fig. 2). Based on tree shown). A tree constructed by the neighbour-joining topology considerations and sequence divergence method depicting the phylogenetic affinity of the values of 6% or greater with the aforementioned taxa, unknown rod as exemplified by strain CCUG 42358T the unknown bacterium merits classification as a new is shown in Fig. 2. The treeing analysis confirmed the genus. Chemotaxonomic data (whole-cell protein association of the unknown bacterium with the lactic patterns and wall murein composition) reinforce the acid group of bacteria and showed that it represents a separateness of the unknown rod from all currently previously unknown line, close to but distinct from, described Gram-positive, catalase-negative reference Granulicatella adiacens and related species. taxa. Therefore, based on both phenotypic and phylo- genetic findings, we consider the unknown catalase- It is evident from the findings of the investigation that negative, Gram-positive rod from seals merits classi- the two rod-shaped strains originating from seals fication in a new genus, for which the name Atopo- represent a hitherto unrecognized bacterium within bacter phocae gen. nov., sp. nov. is proposed. Charac- the low GjC content, Gram-positive, catalase-nega- teristics which are useful in distinguishing Atopobacter tive group of organisms. Phylogenetically, the rod- phocae from its closest phylogenetic relatives and the shaped bacterium shows a loose association with a rod-shaped carnobacteria and D. incerta are shown in cluster of coccus-shaped species which includes G. Table 1. adiacens, G. balaenopterae and Granulicatella elegans (approx. 93n0–93n7% similarity) although bootstrap Description of Atopobacter gen. nov. resampling showed this relationship is not statistically significant. Other taxa displaying a somewhat looser Atopobacter (A.to.po.bachter. Gr. adj. atopos having affinity with the unknown bacterium included the no place, strange; M.L. masc. n. bacter rod; M.L. coccus-shaped Lactosphaera pasteurii, M. plutonius, masc. n. Atopobacter, strange rod).

1758 International Journal of Systematic and Evolutionary Microbiology 50 Atopobacter phocae gen. nov., sp. nov.

Table 1. Characteristics differentiating Atopobacter phocae sp. nov. from its nearest phylogenetic relatives and the rod-shaped carnobacteria and D. incerta ...... Data from Janssen et al. (1995), Stackebrandt et al. (1999) and this study. Biochemical tests were determined using API rapid ID32S and ZYM systems. v, Variable; w, weak; , not determined.

Characteristic Atopobacter G. G. G. D. C. C. C. C. C. C. phocae adiacens balaenopterae elegans incerta alterfundium divergens funditum gallinarum mobile piscicola

Production of acid from: Cyclodextrin jk kkkkjkkkj Glycogen jk kkkkkkkkk Lactose v kkkjkkkjkk Pullulan jk jkkkkkkkk Ribose jk kkkkjkjkj Trahalose v kjkjjjw jjj Sucrose v v kjjw j w jjj Production of: Alkaline phosphatase jk kjkkkkkkk Arginine dihydrolase jk jjkkjkjjj β-Glucosidase k v kkjjjkjw k β-Galactosidase v kkkjkkkkkk β-Glucuronidase k v kkjjjjjjj Murein type A4β A3α A4β  A4α A1γ A1γ A1γ A1γ A1γ A1γ

Cells consist of Gram-positive, short, non-spore- or may not be produced. Activity for acid phosphatase, forming, irregular rods. Non-haemolytic, small pin- alkaline phosphatase, arginine dihydrolase, esterase sized, grey-coloured, smooth colonies are formed on C-4 (weak reaction), ester lipase C-8 (weak reaction), horse blood agar after incubation at 37 mC for 24 h. pyroglutamic acid arylamidase (weak reaction), pyra- Growth is observed without the addition of blood or zinamidase (weak reaction) and pyrrolidonyl aryl- serum to the basal medium. Facultatively anaerobic amidase is detected. No activity for N-acetylgluco- and catalase-negative. Growth is observed at 25 but samidase, chymotrypsin, cystine arylamidase, α-fuco- not at 42 mC. Acid but no gas is produced from - sidase, α-galactosidase, β-glucosidase, β-glucuroni- glucose. Acid phosphatase, arginine dihydrolase, al- dase, glycine tryptophan arylamidase, β-mannosidase, kaline phosphatase and pyrrolidonyl arylamidase are lipase C-14, trypsin or valine arylamidase is detected. produced. Aesculin, gelatin, hippurate and urea are Activity for β-galactosidase may or may not be not hydrolysed. Nitrate is not reduced to nitrite. detected and β-galacturonidase activity is either weak Voges–Proskauer-negative. The cell wall contains an or negative. Aesculin, gelatin, hippurate and urease are -Orn--Asp-type murein (A4β). Isolated from dead not hydrolysed. Acetoin is not produced. Nitrate is not common seals. Habitat is not known. The type species reduced to nitrite. The cell wall contains an -Orn-- of the genus is Atopobacter phocae. As determined by Asp-based murein (type A4β). Isolated from dead 16S rRNA gene sequence analysis, the genus Atopo- common seals. Habitat is not known. The type strain is T T bacter belongs to the lactic acid group of bacteria and CCUG 42358 (l CIP 106392 ). is phylogenetically most closely related to G. adiacens, G. balaenopterae and G. elegans. Acknowledgements

Description of Atopobacter phocae sp. nov. We are grateful to Professor Hans Tru$ per (University of Atopobacter phocae (phohcae. N.L. gen. n. phocae of or Bonn, Germany) for help in coining the genus name. We also pertaining to the common seal Phoca vitulina, from thank Anthony Patterson and Robert Reid of SAC Vet- erinary Science Division, Inverness. This work is part of an which the organism was isolated). ongoing study of Scottish strandings and is conducted under On blood agar cells are short, irregular rods that form contract to the UK Department of the Environment, small, smooth, grey, pin-sized colonies after 24 h Transport and the Regions as part of its co-ordinated incubation at 37 mC. Haemolysis is not observed. Cells programme of research on the North Sea. stain Gram-positive. Facultatively anaerobic and catalase-negative. Growth is observed at 25 but not at 42 mC. Using commercial API systems, acid is pro- References duced from cyclodextrin, glucose, glycogen, maltose, Collins, M. D. & Lawson, P. A. (2000). The genus Abiotrophia pullulan and ribose, but not from -arabitol, - (Kawamura et al.) is not monophyletic: proposal of Granu- arabinose, mannitol, melibiose, melezitose, methyl β- licatella gen. nov., Granulicatella adiacens comb. nov., Granu- -glucopyranoside, raffinose, sorbitol, tagatose and - licatella elegans comb. nov. and Granulicatella balaenopterae xylose. Acid from lactose, sucrose and trehalose may comb. nov, Int J Syst Evol Microbiol 50, 365–369.

International Journal of Systematic and Evolutionary Microbiology 50 1759 P. A. Lawson and others

$ ! Collins, M. D., Farrow, J. A. E., Phillips, B. A., Ferusu, S. & Jones, Lawson, P. A., Foster, G., Falsen, E., Sjoden, B. & Collins, M. D. D. (1987). Classification of Lactobacillus divergens, Lactobacillus (1999a). Abiotrophia balaenopterae sp. nov., isolated from the piscicola, and some catalase-negative, asporogenous, rod- minke whale (Balaenoptera acutorostrata), Int J Syst Bacteriol shaped bacteria from poultry in a new genus, Carnobacterium, 49, 503–506. ! Int J Syst Bacteriol 37, 310–316. Lawson, P. A., Foster, G., Falsen, E., Ohlen, M. & Collins, M. D. Devereux, J., Haeberli, P. & Smithies, O. (1984). A comprehensive (1999b). Vagococcus lutrae sp. nov., isolated from the common set of sequence analysis programs for the VAX, Nucleic Acids otter (Lutra lutra), Int J Syst Bacteriol 49, 1251–1254. Res 12, 387–395. Pascual Ramos, C., Foster, G. & Collins, M. D. (1997). Phylogenetic Felsenstein, J. (1989).  – phylogeny inference package analysis of the genus Actinomyces based on 16S rRNA gene (version 3.2), Cladistics 5, 164–166. sequences: description of Arcanobacterium phocae sp. nov., $ Franzmann, P. D., Hopfl, P., Weiss, N. & Tindall, B. J. (1991). Arcanobacterium bernardiae comb. nov., and Arcanobacterium Psychrophilic, lactic acid-producing bacteria from anoxic pyogenes comb. nov, Int J Syst Bacteriol 47, 46–53. waters in Ace Lake, Antarctica; Carnobacterium funditum sp. Pot, B., Vandamme, P. & Kersters, K. (1994). Analysis of nov. and Carnobacterium alterfunditum sp. nov, Arch Microbiol electrophoretic whole-organism protein fingerprints. In Modern 156, 255–262. Microbial Methods: Chemical Methods in Prokaryotic System- Hammes, W. P., Weiss, N. & Holzapfel, W. (1992). The genera atics, pp. 493–521. Edited by M. Goodfellow & A. G. Lactobacillus and Carnobacterium.InThe Prokaryotes, 2nd O’Donnell. Chichester: Wiley. edn, pp. 1535–1594. Edited by A. Balows, H. G. Tru$ per, M. Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of Dworkin, W. Harder & K.-H. Schleifer. New York: Springer. bacterial cell walls and their taxonomic implications, Bacteriol Janssen, P. H., Evers, S., Rainey, F. A., Weiss, N., Ludwig, W., Rev 36, 407–477. Harfoot, C. G. & Schink, D. (1995). Lactosphaera gen. nov., a new Stackebrandt, E., Schumann, P., Swiderski, J. & Weiss, N. (1999). genus of lactic acid bacteria, and transfer of Ruminococcus Reclassification of Brevibacterium incertum (Breed 1953) as pasteurii Schink 1984 to Lactosphaera pasteurii comb. nov, Int Desemzia incerta gen. nov., comb. nov, Int J Syst Bacteriol 49, J Syst Bacteriol 45, 565–571. 185–188.

1760 International Journal of Systematic and Evolutionary Microbiology 50