International Journal of Systematic Bacteriology (1999), 49, 1523-1 526 Printed in Great Britain

Characterization of a Gemella-like organism from the oral cavity of a dog: description of Gemella palaticanis sp. nov.

Matthew D. Collins,' Mar Rodriguez Jovita,' Geoffrey Foster,* Berit Sjsden3 and Enevold Falsen3

Author for correspondence: Matthew D. Collins. Tel: +44 118 935 7000. Fax: +44 118 926 7917. e-mail : [email protected]

Department of Food A hitherto unknown Gram-positive, -negative, facultatively anaerobic Science and Technology, coccus isolated from a vesicle on the gum of a dog was characterized by University of Reading, Reading RG6 6AP, phenotypic and molecular taxonomic methods. Comparative 16s rRNA gene UK sequencing studies demonstrated that the isolate represents a new subline

2 SAC Veterinary Science within the genus Gemella. The unknown bacterium was readily distinguished Division, Drummond Hill, from all currently described members of this genus, Gemella haemolysans, Inverness, UK Gemella bergeri, and , by biochemical 3 Culture Collection, tests and electrophoretic analysis of whole-cell proteins. On the basis of Department of Clinical phylogenetic and phenotypic evidence, it is proposed that the unknown Bacteriogy, University of Goteborg, Gbteborg, bacterium be classified as Gemella palaticanis sp. nov. The type strain of Sweden Gemella palaticanis is CCUG 3948gT.

1 Keywords: Gemella palaticanis sp. nov., , phylogeny, 16s rRNA

Species of the genus Gemella consist of catalase- mucous membranes of humans and of certain warm- negative, facultatively anaerobic, Gram-posi tive, blooded animals and are recognized opportunistic coccoid-shaped organisms that are either arranged in pathogens. Although the habitats of G. bergeri and G. pairs, often with adjacent sides flattened, or arranged sanguinis are unknown, it seems probable that these in tetrads, short-chains or small, irregular clusters. species also form part of the normal human micro- Until recently, only two species of the genus were biota. In this article, we report the characteristics of a recognized, Gemella haemolysans and Gemella mor- novel Gemella-like organism isolated from the oral billorum (Berger, 1992). G. haemolysans, the type cavity of a dog. On the basis of the results of a species of the genus, was originally classified as a polyphasic taxonomic study, we describe another new representative of the Gram-negative genus Neisseria species of the genus, Gemella palaticanis sp. nov. (Thjotta & Boe, 1938). However, numerous studies showed that the organism was biochemically incom- Strain M663-98-lT was recovered from a swab of a patible with the genus Neisseria, and a new genus, vesicle on the gum of a l-year-old male Labrador dog Gemella, was created (Berger, 1961). G. morbillorum, that was diagnosed as suffering from a long-standing the second species to be assigned to the genus, was mild inflammatory lesion. The strain was isolated originally classified as morbillorum along with Pasteurella spp. on Columbia sheep-blood (Prkvot, 1933) and subsequently transferred to Pepto- agar at 37 "C and has been deposited in the Culture streptococcus and then to Streptococcus before its Collection of the University of Goteborg, Sweden, placement in the genus Gemella (Kilpper-Balz & under accession number CCUG 39489T. The isolate Schleifer, 1988). Recently, two additional species of was biochemically characterized by using the API the genus have been described, Gemella bergeri (Collins Rapid ID32S and API ZYM systems according to the et al., 1998a) and Gemella sanguinis (Collins et al., manufacturer's instructions (API bioMerieux). PAGE 1998b), from human clinical specimens. Both G. analysis of whole-cell proteins was performed as haemolysans and G. morbillorum are residents of the described by Pot et al. (1 994). The GELCOMPARGCW 3.0 software package (Applied Maths) was used for densitometric analysis, normalization and interpret- The CenBank accession number for the 165 rRNA gene sequence of strain ation of protein patterns. The G + C content of DNA CCUC 3948gT is Y 17280. of strain CCUG 39489T was determined as described

01033 0 1999 IUMS 1523 M. D. Collins and others

40 50 60 70 80 90 100 I I I I I Pediococcus pentosaceus CCUG 36942 Pediococcus pentosaceus CCUG 32205T Pediococcus acidilactici CCUG 35190 Pediococcus acidilactici CCUG 32T35’ Vagococcus fluv!al/sCCUG 32704 Vagococcus fluv/al/sCCUG 38909 Facklamia hominis CCUG 28830 Facklamia hominis CCUG 3681 3: Aerococcus urinae CCUG 29291 Aerococcus urinae CCUG 34223T Facklamia sourekii CCUG 28783A Facklamia sourekii CCUG 31 97y Alloiococcus otitis CCUG 32997 Aerococcus viridans CCUG 431 1 Aerococcus viridans CCUG 1 1676 Gemella morbillorum CCUG 3881 6 Gemella morbillorum CCUG 18164; Gemella morbillorum CCUG 15561 _. Gemella haemol sans CCUG 3798!jT - Gemella haernorsans CCUG 281 34 Gemella haerno6sans CCUG 41 1 Vagococcus salmoninarum CCUG 33394T Abiotrophia adiacens CCUG 276379 - I Abiotrophia adlacens CCUG 27809 Carnobacterium divergens CCUG 30094T Carnobacteriumgallinarum CCUG 30795’ Dolosigranulumpigrum CCUG 33392 Dolosigranulumpigrum CCUG 35446 Globicatella sanguinis CCUG 365639 Globicafella sanguinis CCUG 32999 Gemella pa/aricanissp. nov. CCUG 3948gT IHelcococcus kunzii CCUG 31742- Helcococcus kunzii CCUG 32213T Gemella bergeri CCUG 3781 8 Gemella bergeri CCUG 37817T ...... - ...... Abiotrophia elegans CCUG 38676 Abiotrophia elegans CCUG 38949’ Fig, 7. Similarity dendrogram based on Gemella sanguinis CCUG 37820T whole-cell protein pattern of Gemella Gemella sanguinis CCUG 33602 Abiotrophla defectiva CCUG 2763gT palaticanis sp. nov. and related species. Abiotrophia defecfiva CCUG 369Q7 Levels of correlation are expressed as Leuconostoc lactis CCUG 30064 Leuconostoc lactis CCUG 38156 percentages of similarity for convenience. by Garvie (1978). The 16s rRNA gene(s) of the isolate Activities for alanine-phenylalanine-proline aryl- was amplified by PCR and directly sequenced using a amidase and glycyl-tryptophan arylamidase were de- Taq Dye-Deoxy Terminator cycle sequencing kit tected but no activities were detected for alkaline (Applied Biosystems) and an automatic DNA se- phosphatase, arginine dihydrolase, N-acetyl-P-gluco- quencer (model 373A; Applied Biosystems). The saminidase, a-galactosidase, P-galactosidase, P- closest known relatives of the new isolate was de- mannosidase, pyroglutamic acid arylamidase or termined by performing database searches. These urease. The isolate did not produce acetoin and did not sequences and those of other known related strains hydrolyse hippurate. Based on the aforementioned were retrieved from the GenBank or the Ribosomal characteristics, the isolate showed some resemblance Database Project (RDP) databases and aligned with to members of the genus Gemella. The results of PAGE the newly determined sequence using the program analysis of whole-cell proteins depicted in Fig. 1, PILEUP (Devereux et ul., 1984). The resulting multiple however, revealed that the isolate was distinct from the sequence alignment was corrected manually and a four currently recognized species of this genus and distance matrix was calculated using the programs all reference Gram-positive, catalase-negative cocci PRETTY and DNADIST (using Kimura’s two-parameter examined. To investigate the phylogenetic position of correction) (Felsenstein, 1989). A phylogenetic tree the unknown isolate, comparative 16s rRNA gene was constructed according to the neighbour-joining sequence analysis was performed. The almost complete method with the program NEIGHBOR and the stability gene sequence (> 1400 nucleotides) of the dog isolate of the groupings was estimated by bootstrap analysis was determined and sequence searches of GenBank (200 replications) using the programs DNABOOT, and RDP databases revealed that the unknown isolate DNADIST, NEIGHBOR and CONSENSE (Felsenstein, 1989). was phylogenetically most closely associated with species of the genus Gemella. A tree constructed by the The isolate from the gum of a dog consisted of Gram- neighbour-joining method depicting the phylogenetic positive, ovoid-shaped cells that were catalase- and affinity of the unknown coccus is shown in Fig. 2 and oxidase-negative. On blood-agar plates after 72 h, the clearly shows that the bacterium represents a distinct strain formed small pin-head colonies. Using com- subline within the genus Gemella. mercial API systems, the isolate produced acid from , lactose, maltose, sucrose (weak reaction) and The bacterium, which originated from a vesicle on the trehalose. The organism failed to produce acid from D- gum of a dog, exhibited >4% 16s rRNA sequence arabitol, L-arabinose, cyclodextrin, glycogen, man- divergence from currently known Gemella species and nitol, melibiose, melezitose, methyl P-D-glucopyranos- clearly represents a new species of this genus. Bio- ide, pullulan, raffinose, ribose, tagatose or sorbitol. chemically, the unknown coccus does not correspond

1524 International Journal of Svstematic Bacterioloav 49 Gemella palaticanis sp. nov.

98 Aerococcus viridans ATCC 1 1563T (M58797) Aerococcus urinae NCFB 2893T (M77819)

...... , ...... , ...... , , , . .. . . ,. . .. , , , , , .. . , . .. . , ., , . Fig- 2. Unrooted tree showing the phylogenetic relationships of Gemella palaticanis sp. nov. and some other low- G + C-content, Gram-positive . The tree, constructed using the neighbour- joining method, was based on a comparison of approximately 1320 nucleotides. Boot- strap values, expressed as a percentage of 200 replications, are given at branching points. Bar, 1 % sequence divergence.

Table 1. Characteristics useful in differentiating Gemella palaticanis sp. nov. from other Gemella species

~ ~~~~ Test G.palaticanis G. bevgevi G. haemolysans G. morbillorum G. sanguinis

Acid from : Lactose + Mannitol - V + Sorbitol - ,/(+I + Sucrose + + + Trehalose + Production of: Alkaline phosphatase - + - + Alanine-phenylalanine-prolinearylamidase + - V V Glycyl-tryptophan arylamidase + V V - v, Variable; - /( +), a few strains tested positive. to any of the four recognized species and PAGE mannitol, melibiose, melezitose, methyl p-D-gluco- analysis of whole-cell proteins confirmed the pheno- pyranoside, pullulan, raffinose, ribose, tagatose or typic separateness of the bacterium. On the basis of sorbitol. Alanine-phenylalanine-proline arylamidase both phenotypic and phylogenetic evidence, we there- and glycyl-tryptophan arylamidase are produced. fore consider that the unknown bacterium should be Alkaline phosphatase, arginine dihydrolase, N-acetyl- classified as a new species of the genus Gemella, for P-glucosaminidase, a-galactosidase, P-galactosidase, which the name Gemella palaticanis sp. nov. is pro- P-galacturonidase, P-glucosidase, P-glucuronidase, p- posed. Tests that serve to distinguish the novel mannosidase, pyroglutamic acid arylamidase and bacterium from other Gemella species are given in urease are not produced. Hippurate is not hydrolysed. Table 1. Acetoin is not produced. The G + C content of DNA is 32 mol %. Isolated from a vesicle on the gum of a dog. Habitat is unknown. The type strain is M663-98-lT Description of Gemella palaticanis sp. nov. (= CCUG 39489T).

Gemella palaticanis (pa.la.ti.ca’nis. L. neut. n. palatum References gum; L. masc. n. canis dog; M.L. gen. masc. n. palaticanis of the gum of a dog). Berger, U. (1961). A proposed new genus of Gram-negative cocci: Gemella. Int Bull Bacteriol Nomencl Taxorz 11, 17-19. Cells are Gram-positive, non-spore-forming cocci that Berger, U. (1992). The genus Gemella. In The Prokaryotes, 2nd occur in pairs, in clusters or in short chains. Cocci edn, pp. 1643-1653. Edited by A. Balows, H.G. Truper, are sometimes elongate. Colonies on blood-agar M. Dworkin, W. Harder & K.-H. Schleifer. New York: plates after 72 h are small, circular, entire, low- Springer. convex, translucent to opaque and smooth. Non-pig- Collins, M. D., Hutson, R. A., Falsen, E., Sjijden, 6. 81 Facklam, mented and non-haemolytic. Facultatively anaerobic. R. R. (1998a). Gemella bergeriae sp. nov., isolated from human Catalase- and oxidase-negative. Acid is produced clinical specimens. J Clin Microbiol36, 1290-1293. from glucose, lactose, maltose, sucrose (weak Collins, M. D., Hutson, R. A., Falsen, E., SjadBn, B. 81 Facklam, reaction) and trehalose. Acid is not produced R. R. (199813). Description of Gemella sanguinis sp. nov., isolated from D-arabitol, L-arabinose, cyclodextrin, glycogen, from human clinical specimens. J Clin Microbiol36,3090-3093.

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Devereux, J., Haeberli, P. & Smithies, 0. (1984). A comprehensive Pot, B., Vandamme, P. 81 Kersters, K. (1994). Analysis of set of sequence analysis programs for the VAX. Nucleic Acids electrophoretic whole-organism protein fingerprints. In Modern Res 12, 387-395. Microbial Methods (Chemical Methods in Prokaryotic System- Felsenstein, 1. (1989). PHYLIP - phylogeny inference package atics Series), pp. 493-521. Edited by M. Goodfellow & A. G. (version 3.2). Cladistics 5, 164-166. O’Donnell. Chichester : Wiley. Garvie, E. I. (1 978). Streptococcus rafinolactis Orla-Jensen and PrBvot, A. R. (1933). Etudes de systematique bactirienne. I. Lois Hansen, a group N streptococcus found in raw milk. Int J Syst generals, 11. Cocci anaerobies. Ann Soc Nat Zoo1 Biol Anim 15, Bacteriol28, 190-193. 23-26. Kilpper-Balz, R. & Schleifer, K. H. (1988). Transfer of Strepto- Thjotta, T. & Bee, J. (1938). Neisseria haemolysans. A haemolytic coccus morbillorum to the genus Gemella as Gemella morbillorum species of Neisseria Trevisan. Acta Pathol Microbiol Scand comb. nov. Int J Syst Bacteriol38, 442-443. Suppl37, 727-731.

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