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Vagococcus Lutrae Sp. Nov., Isolated from the Common Otter (Lutra Lutra)

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Vagococcus Lutrae Sp. Nov., Isolated from the Common Otter (Lutra Lutra) International Journal of Systematic Bacteriology (1 999), 49, 125 1-1 254 Printed in Great Britain Vagococcus lutrae sp. nov., isolated from the ~ NOTE common otter (Lutra lutra) Paul A. Lawson,’ Geoffrey Foster,’ Enevold Fal~en,~Maria Ohlen3 and Matthew D. Collins’ Author for correspondence: Matthew D. Collins. Tel: +44 118 935 7000. Fax: +44 118 926 7917. e-mail : david.collins@)bbsrc.ac.uk 1 Dep rtment of Fo d Phenotypic and phylogenetic studies were performed on an unknown Gram- Science and Technology, positive catalase-negative coccus isolated from a common otter (Lufra lutra). University of Reading, Reading, RG6 6AP, UK Comparative 16s rRNA gene sequencing demonstrated that the unknown bacterium represents a new subline within the genus Vagococcus, close to, but * SAC Veterinary Services, lnverness IV2 4JZ, UK distinct from, Vagococcus fluvialis and Vagococcus salmoninarum. The unknown bacterium was readily distinguished from the two currently 3 Culture Collection, Department of Clinical recognized Vagococcus species by biochemical tests and electrophoretic Bacteriology, University of analysis of whole-cell proteins. Based on phylogenetic and phenotypic Gbteborg, 5-41 346 evidence it is proposed that the unknown bacterium be classified as a new Gbteborg, Sweden species, Vagococcus lutrae, the type strain of which is CCUG 39187T. I Keywords: Vagococcus lutrae sp. nov., phylogeny, taxonomy, 16s rRNA The genus Vagococcus was described by Collins et al. An unknown coccus was isolated following a post- (1989) to accommodate some motile Lancefield group mortem examination of a common otter following a N Gram-positive, catalase-negative cocci, which were road traffic accident on the Isle of Mull, UK. The phylogenetically distinct from lactococci. Initially the coccus was recovered from the blood, liver, lungs and genus consisted of a single species, Vagococcusfluvialis, spleen specimens from the otter as the dominant which was isolated from chicken faeces and river water organism co-isolated with an unidentified oxidase- and first described by Hashimoto et al. (1974). V. positive, non-fermentative, non-motile, Gram-nega- fluvialis has subsequently been reported from a variety tive rod-shaped bacterium. All of the coccoid isolates of sources including human clinical specimens, viz. recovered from these multiple sites of the otter were blood, peritoneal fluid and wounds (Teixeira et al., found to possess identical cellular and colonial 1997) and domestic animals, viz. chickens, pigs, cattle, morphologies and biochemical profiles. The post- horses and cats (Pot et al., 1994a). A second species of mortem revealed the animal to be in good bodily the genus, Vagococcus salmoninarum, was described by condition prior to the road traffic accident and Wallbanks et al. (1990) for two strains isolated from therefore it was not possible to draw conclusions diseased rainbow trout in the USA. V. salmoninarum regarding the possible, if any, clinical significance of has subsequently been recovered from Atlantic the unknown isolate. Isolate M 1134/97/ lT was de- salmon, rainbow trout and brown trout with per- posited in the Culture Collection of the University of itonitis (Schmidtke & Carson, 1994). In the course of a Goteborg (CCUG) Sweden, as number CCUG study of Gram-positive catalase-negative cocci 39187T. The unidentified organism was cultured on associated with aquatic mammals, we have used 16s Columbia agar supplemented with 5 % defibrinated rRNA gene sequencing to phylogenetically charac- horse blood (Oxoid Unipath) at 37 “C,in air plus 5 % terize a hitherto-unknown Vagococcus-like bacterium CO,. The strain was biochemically characterized by from a common otter. Based on the results of a using the API Rapid ID32S and API ZYM systems polyphasic taxonomic study, we consider the unknown according to the manufacturer’s instructions (APT coccus represents a third species of the genus bioMirieux). PAGE analysis of whole-cell proteins Vagococcus, for which the name Vagococcus lutrae sp. was performed as described by Pot et al. (1994b). The nov., is proposed. GELCOMPAR GCW 3.0 software package (Applied Maths, Kortrijk, Belgium) was used for densitometric analysis, normalization and interpretation of protein The GenBank accession number for the 165 rRNA gene sequence of strain patterns. Similarity between pairs of traces was carried CCUG 39187T is Y17152. out using the unweighted pair group method with 01024 0 1999 IUMS 1251 P. A. Lawson and others Vagococcus fluvialis CCUG 32704 Vagococcus fluvialis CCUG 38909 Vagococcus fluvialis CCUG 33397 - Facklamia hominis CCUG 36813 I Facklamia hominis CCUG 28572 Pediococcus pentosaceus CCUG 3220!jT I Pediococcus pentosaceus CCUG 36942 I Gemella morbillorum CCUG 38816 Gemella haemol sans CCUG 28134 I Gemella haemo&ans CCUG 37985T Vagococcus salmoninarum CCUG 33394T - Abiotrophia elegans CC UG 3894gT Fig. 1. Similarity dendogram based on whole-cell protein patterns of Vagococcus lutrae sp. nov. and related species. Levels of correlation are expressed as percentages of similarity for convenience. averages (UPGMA) clustering method and expressed 1989). A phylogenetic tree was constructed according by the Pearson product-moment correlation to the neighbour-joining method with the program coefficient converted for convenience to a percentage NEIGHBOR (Felsenstein, 1989). The stability of the value (Pot et al., 1994b). The 16s rRNA genes of the groupings was estimated by bootstrap analysis (500 isolate were amplified by PCR and directly sequenced replications) using the programs DNABOOT, DNADIST, using a Taq Dye-Deoxy terminator cycle sequencing NEIGHBOR and CONSENSE (Felsenstein, 1989). kit and a model 373A automatic DNA sequencer (both from Applied Biosystems). The closest known relatives The unknown bacterium from the common otter of the new isolate were determined by performing consisted of Gram-positive coccus-shaped cells which database searches. These sequences and those of other occurred singly, in pairs or short chains, and which known related strains were retrieved from the were elongated in the direction of the chain. The GenBank or Ribosomal Database Project (RDP) isolate was catalase-negative, facultatively anaerobic databases and aligned with the newly determined and motile. It produced small (0-1-0-2 mm diameter) sequence using the program PILEUP (Devereux et al., smooth colonies on Columbia agar supplemented with 1984). The resulting multiple sequence alignment was 5 % defibrinated horse blood at 37 "C. The isolate corrected manually and a distance matrix was calcu- produced acid from glucose and some other carbo- lated using the programs PRETTY and DNADIST (using hydrates, and displayed acid phosphatase, N-acetyl- the Kimura two-correction parameter) (Felsenstein, glucosaminidase, chymotrypsin, ester lipase C-8, a- 1252 international Journal of Systematic Bacteriology 49 Vagucuccus lutrae sp. nov., from the common otter Streptococcus iniae NCDO 2772T(X58316) 100 Tetragenococcus halophilus IAM 1676T (D88668) 1Yo Tetragenococcusmuriaticus JCM 10006T (D88824) Enterococcus faecalis NCIMB 775T (Y18293) Enterococcus sulfureus NCDO 237gT(X55133'+ Enterococcus saccharolyticus NCDO 2594 (U30931) Vagococcus fluvialis CCUG 32704' (Y 18098) Vagococcus salmoninarum CCUG 33394' (Y18097) Vagococcus lutrae sp. nov. CCUG 391 87T (Y17152) Carnobacterium alterfunditum ATCC 49837T (L08623) Carnobacterium funditum DSM 5970T(S86170) Carnobacterium mobile NCFB 2765T(X54271) Carnobacterium divergens NCDO 2763T X54270) Carnobacterium gallinarum NCFB 2766! (X54269) L Carnobacterium piscicola NCDO 2762T (X54268) Lactosphaera pasteurii DSM 2381 (X87150) Abiotrophia balaenopterae CCUG 37380T (Y 16547) Abiotrophia adiacens ATCC 49175T (D50540) 11693T (AFOI 63904 mia hominis CCUG 36813 (Y 10772) bicatella sanguinis NCFB 2835T(S50214) hia defectiva ATCC 491 76T (D50541) us urinae NCFB 2893T M77819) ns ATCC 11 563 (T (M58797) m NCFB 2975T(X70907) 890T (X59765) Fig. 2. Unrooted tree showing the phylogenetic relationships of Vagococcus lutrae sp. nov. and some other low-G+C- 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. The bar represents a sequence divergence range of 1 %. detailed biochemical characteristics of the unknown coccus are presented in the species description of this Test V. Zutrae (n=l) V.fluvialis (n=6) organism. PAGE analysis of whole-cell proteins showed the unknown organism to be separate from all Acid from : other Gram-positive catalase-negative reference Cyclodextrin + species examined, including Vagococcus spp., Mannito1 - Abiotrophia spp., Aerococcus spp., Carnobacterium Sorbitol + spp., Facklamia hominis and Globicatella sunguinis Sucrose + (Fig. 1). On the basis of PAGE analysis the unknown D-Ri bose + coccus formed a loose association with Aerococcus Production of: urinae. By constrast V.fluvialis and V. salmoninarum or-Galactosidase + were far-removed from the unknown bacterium. To P-Galactosidase + assess the genealogical affinity between the unidentified organism and other Gram-positive catalase-negative taxa, comparative 16s rRNA gene sequence analyses were performed. The almost complete gene sequence from the treeing analysis that the nearest relatives of (>1470 nucleotides) of the unknown coccus was the unidentified coccus were V. Juvialis and V. determined. Sequence searches of GenBank and RDP salmoninarum.
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