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