International Journal of Systematic Bacteriology (1999), 49, 635-638 Printed in Great Britain

Facklamia sourekii sp. nov., isolated f rom NOTE 1 human sources Matthew D. Collins,' Roger A. Hutson,' Enevold Falsen2 and Berit Sj6den2

Author for correspondence : Matthew D. Collins. Tel : + 44 1 18 935 7226. Fax : + 44 1 18 935 7222. e-mail : [email protected]

1 Department of Food Two strains of a Gram-positive catalase-negative, facultatively anaerobic Science and Technology, coccus originating from human sources were characterized by phenotypic and University of Reading, Whiteknights, molecular taxonomic methods. The strains were found to be identical to each Reading RG6 6AP, other based on 165 rRNA gene sequencing and constitute a new subline within UK the genus . The unknown bacterium was readily distinguished from * Culture Collection, Facklamis hominis and by biochemicaltests and Department of Clinical electrophoretic analysis of whole-cell proteins. Based on phylogenetic and Bacteriology, University of Goteborg, Sweden phenotypic evidence it is proposed that the unknown bacterium be classified as sp. nov., the type strain of which is CCUG 28783AT.

Keywords : Facklamia sourekii, , phylogeny, 16s rRNA

The Gram-positive catalase-negative cocci constitute a Gram-positive coccus-shaped organisms, which con- phenotypically heterogeneous group of organisms stitute a third of the genus Facklamia, Fack- which belong to the Clostridium branch of the Gram- lamia sourekii sp. nov. This report adds to the many positive . This broad group of organisms new taxa and the diversity of Gram-positive catalase- includes many human and animal pathogens (e.g. negative cocci associated with human clinical materials enterococci, streptococci) and the taxonomy of these described in the past few years. organisms has undergone considerable development in Strain CCUG 3 1976 was received as a presumptive ' a- recent years. Much of this change has been due to an haemolytic-streptococcus' from the Public Health increased interest in the clinical importance of these Laboratory Service in Jonkoping (Sweden) in 1993. organisms in combination with the application of The strain was isolated from a haemoculture from a improved molecular identification techniques such as 72-year-old man. Initially, the strain could not be 16s rRNA gene sequencing. The most pronounced identified, but its biochemical profile matched that of a development in the taxonomy of the Gram-positive mislabelled strain, CCUG 276 18, received from Dr catalase-negative cocci has been the recognition of a Pavla Kuzemenska in Prague, Czech Republic, as plethora of new genera and species, which in the main CNCTC Str 2/84 ('SS 1019'). To eliminate any error, have been isolated from human sources [e.g. Abio- the curator of the Czechoslovak National Collection trophia elegans (Roggenkamp et al., 1998), Alloio- of Type Cultures (CNCTC), Dr Jiri Sourek was asked coccus (Aguirre & Collins, 1992), Dolosigranulum to deposit Str 2/84 again (= CCUG 28783AT). Ex- (Aguirre et al., 1993) Gemella bergeriae (Collins et al., tended phenotyping confirmed that CCUG 31976 and 1998a), Globicatella (Collins et al., 1992) and Helco- CCUG 28783AT possessed the same biochemical coccus et al., Facklamia horninis, (Collins 1993)]. features. Strain Str 2/84 (CNCTC + Rotta + Fack- another such example, was proposed to accommodate lam t Roguinsky + de Moore) originated from C. E. some unknown catalase-negative cocci from clinical de Moore (National Public Health Laboratory, specimens which could not be assigned to any es- Utrecht, The Netherlands) but no clinical information tablished taxon (Collins et al., 1997). A second species was available on the organism. The unidentified of the genus Facklamia, Facklamia ignava has recently organisms were cultured on Columbia agar (Difco) been characterized for some organisms originating supplemented with 5% horse blood at 37 "C, in air from blood specimens (Collins et al., 1998b). In this plus 5 % CO,. The strains were biochemically charac- article we describe the characteristics of two unknown terized by using the API Rapid ID32 Strep and API ZYM systems according to the manufacturer's in- The GenBank accession number for the 165 rRNA gene sequence of CCUG structions (API bioMerieux). PAGE analysis of whole- 28783T is Y17312. cell proteins was performed as described by Pot et al.

00964 0 1999 lUM5 635 M. D. Collins and others

40 50 60 70 80 90 100 1 I I I I I I positive, non-spore-forming, catalase-negative, oxi- Vagococcus fluvialis CCUG 32704T Vagococcus fluvialis CCUG 38909 dase-negative facultative anaerobes. Using commer- Fadclamia horninis CCUG 36813T Facklarnia horninis urine CCUG 28572 cial API systems the isolates produced acid from D- Aerococcus urinae CCUG 34223T Aeroroccus urinae CCUG 29552 arabitol, maltose, mannitol, sorbitol, sucrose and Facklamia soumkii CCUG 27618 Facklamia soumkil CCUG 20703Ar trehalose. Acid was not produced from L-arabinose, Facklamia SOurekN CCUG 319?6 Alloiococcus ofifisCCUG 32997 cyclodextrin, glycogen, lactose, melibiose, melezitose, Aemcoccus v!r!dans CCUG 15548, Aemcoccus vrrtdans CCUG 431 1 Pedimusurinaeeaui CCUG 28094T pullulan, raffinose, ribose, taga tose or met hyl-P-D- Gemellabergerii CCdG 37969 <-Gemella bergerii CCUG 37817T glucopyranoside. Both isolates displayed pyroglutamic Helcococcus kunzii CCUG 32213’ Helcoroccus kunzii CCUG 31 742 acid arylamidase, leucine arylamidase and es terase C- Facklamiaignava CCUG 37659 FacklamiaMnava CCUG 3741gT 4 (weak reaction) activity. No activity was detected for fiTerracoccui luteus CCUG 391 86 Globcatella sanguinrs CCUG 3299gT arginine dihydrolase, alanine-phenylalanine-proline Globrcatella sanguinrs CCUG 33367 Lactococcus garvfeaeCCUG 3220ET arylamidase, a-galactosidase, /?-galactosidase, /?-gal- Lactococcus garvreae CCUG 38280 Enterococcus faecas CCUG 1991ST Entemccus faecalis CCUG 34289 acturonidase, a-glucosidase, P-glucosidase, /?-gluc- Dolosrgranulumpigrum CCUG 33081A Dolosrgranulumpigrum CCUG 33392T uronidase, lipase C 14, a-fucosidase, a-mannosidase, lgnavvranum ruoffrae CCUG 378413 lgnavigranumruoffrae CCUG 37658 N-acetylglucosaminidase, glycyl-trytophan arylamid- Pedioco~uspentosaceus CCUG 32205’ Pedrococcuspentosaceus CCUG 36942 ase, chymotrypsin, trypsin, valine arylamidase or Pedmmsacndrlacfrcr CCUG 351 90 Pedrococcus acndilacicr CCUG 32235T urease. They hydrolysed hippurate and were Voges- IIFGemella rnorbillorurn CCUG 18164T Gemella morbrllorurn CCUG 38816 Proskauer-negative. The close phenotypic affinity be- Gemella haemolysans CCUG 281 34 Gemella haemolysans CCUG 3798ST Vagococcus salmoninarumCCUG 33394T tween the two unknown isolates was confirmed by Carnobacferrumdrvergens CCUG 30094T Carnobacteriumgallinarum CCUG 30095’ PAGE analysis of whole-cell proteins in which the two Abrotrophia el6gans CCUG 3894gT Abrofrophraelegans CCUG 38676 organisms formed a robust and tight cluster which was Gemella sangurnrs CCUG 33602 Gemella sangurnis CCUG 37820T quite separate from all other Gram-positive catalase- Abrofrophia adracens CCUG 2780gT Abrotrophia adracens CCUG 37320 negative reference organisms examined, including Abrofrophia defecfiva CCUG 36937 Abrotrophradefecfrva CCUG 2763gT Abiotrophia spp., Aerococcus spp., Facklamia spp. and Leuconosfoc lactrs CCUG 37937 Leuconostoc lactrs CCUG 30064T Globicatella sanguinis (Fig. 1). Indeed on the basis of PAGE analysis the unknown isolates clustered, albeit Figrn 7# Similarity dendogram based on whole-cell protein loosely, with Alloiococcus otitis. Aerococcus viridans patterns of F. sourekii sp. nov. and related species. Levels of and Pediococcus urinaeequi were the next closest correlation are expressed as percentages of similarity for relatives. By contrast, F. horninis and F. ignuva formed convenience. distinct clusters which were far removed from the unknown bacterium (Fig. 1). (1994). For densitometric analysis, normalization and To assess the phylogenetic affinity between the two interpretation of protein patterns the GelCompar isolates and their relationship with other Gram- GCW 3.0 software package (Applied Maths) was used. positive catalase-negative taxa, comparative 16s The mol % G + C content of DNA of strain CCUG rRNA gene sequence analyses were performed. 28783AT was determined as described by Garvie Approximately 1400 nucleotides of the 16s rRNA (1978). The 16s rRNA genes of the isolates were gene of each of the strains was determined and pairwise amplified by PCR and directly sequenced using a Taq analysis revealed no base differences (i.e. 100% simi- Dye-Deoxy terminator cycle sequencing kit (Applied larity). Sequence searches of GenBank and RDP Biosystems) and an automatic DNA sequencer (model databases revealed the unknown bacterium was phylo- 373A; Applied Biosystems). The closest known rela- genetically most closely associated with a group of tives of the new isolates were determined by per- catalase-negative coccus-forming taxa which included forming database searches. These sequences and those A bio t r oph ia, A e r oco ccus, Fucklamia, Igna vigran urn of other known related strains were retrieved from the and Globicatella (data not shown). A tree constructed GenBank or Ribosomal Database Project (RDP) by neighbour-joining depicting the phylogenetic (Maidak et al., 1997) databases and aligned with the affinity of the unknown coccus as exemplified by strain newly determined sequences using the program PILEUP CCUG 28783AT is shown in Fig. 2. From the (Devereux et al., 1984). The resulting multiple se- branching pattern of the tree the nearest relative of the quence alignment was corrected manually and a unknown coccus was F. ignava. The clustering together distance matrix was calculated using the programs of these organisms occurred in 84% of 500 tree PRETTY and DNADIST (using the Kimura-two correction replications. F. horninis formed a somewhat deeper parameter) (Felsenstein, 1989). A phylogenetic tree subline branching from the unknown coccus/ F. ignava was constructed according to the neighbour-joining line (supported by 86% bootstrap value). The next method with the program NEIGHBOR (Felsenstein, nearest relatives of the aforementioned taxa corre- 1989). The stability of the groupings was estimated by sponded to Ignavigranum ruoflae and Globicatella bootstrap analysis (500 replications) using the pro- sanguinis. Parsimony analysis was also performed and grams DNABOOT,DNADIST, NEIGHBOR and CONSENSE the grouping of the unknown coccus with F. ignava (Felsenstein, 19 89). and I;. horninis confirmed. The two isolates were ovoid in shape and formed single Biochemical profiling and PAGE analysis of whole- cells, pairs or short chains. The strains were Gram- cell proteins demonstrated the two unidentified coccus- - 636 InternationalJournal of Systematic Bacteriology 49 Facklamia sourekii sp. nov.

...... I ...... I ...... Fig. 2. Unrooted tree showing the phylogenetic relationships of F. sourekii 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 approx. 1320 nucleotides. Bootstrap values, expressed as a percentage of 500 replications, are given at branching points.

Table I. Characteristics that differentiate F. sourekii sp. nov. from F. ignava and F. hornin is

I Character F. sourekii F. ignava F. hominis

Acid from : D-Arabitol Maltose Mannitol Sorbitol S ucr o se Trehalose + +" - Production of: Arginine dihydrolase Alanyl-phenylalanine-prolinearylamidase a-Galactosidase (API Rapid ID32 Strep) P-Galactosidase (API Rapid ID32 Strep shaped organisms were phenotypically similar. This also differs from F. ignava and F. hominis in being affinity between the isolates was confirmed by com- negative for alanyl-phenylalanine-proline arylamid- parative 16s rRNA gene sequencing. Phylogenetically ase, and from F. horninis in being negative for arginine the unidentified coccus represents a new subline within dihydrolase, a-galactosidase and P-galactosidase. the recently proposed genus Facklarnia. A 16s rRNA Thus based on the results of 16s rRNA gene sequence gene sequence divergence value of about 7 YObetween and electrophoretic whole-cell protein analyses, and the unidentified coccus and F. ignava and F. horninis, the distinctive biochemical characteristics of the un- however, unequivocally demonstrated that the bac- known coccus, we consider that this bacterium merits terium represents a hitherto unknown species. classification as a new species, for which the name Although the above divergence value indicates the Facklarnia sourekii is proposed. Tests that distinguish affinity between the unknown coccus and F. ignava/F. F. sourekii from F. ignava and F. horninis are horninis is not particularly close, based on currently summarized in Table 1. described taxa, the genus Facklamia is the most appropriate home for this bacterium. It is pertinent to Description of Facklamia sourekii sp. nov. point out that the novel bacterium can be readily distinguished from both F. ignava and F. horninis by Facklamia sourekii (Czech pronounciation producing acid from D-arabitol, maltose, mannitol, sch0u.rek.i.i. sourekii named after the late J. Sourek, sorbitol and sucrose, and additionally from F. horninis curator of the CNCTC, Czech Republic). in producing acid from trehalose. The unknown coccus Cells are Gram-positive, ovoid in shape occurring in

InternationalJournal of Systematic Bacteriology 49 637 M. D. Collins and others pairs or short-chains. Cells are non-pigmented, a- tion of Dolosigranulum pigrum gen. nov., sp. nov. J Appl haemolytic and non-motile. Spores are not produced. Bacteriol75, 608-612. Facultatively anaerobic, oxidase-negative and Collins, M. D., Aguirre, M., Facklam, R. R., Shallcross, J. & catalase-negative. Grows in 5% NaCl. Acid is pro- Williams, A. M. (1992). Globicatella sanguis gen. nov., sp. nov., a duced from D-arabitol, glucose, maltose, mannitol, new Gram-positive catalase-negative bacterium from human sorbitol, sucrose and trehalase. Acid is not produced sources. J Appl Bacteriol73, 422-437. from L-ara bin0 se, cy clodextrin, glycogen, lactose, Collins, M. D., Facklam, R. R., Rodrigues, U. M. & Ruoff, K. L. melibiose, melezitose, pullulan, raffinose, ribose, taga- (1993). Phylogenetic analysis of some Aerococcus-like organisms tose or methyl-P-D-glucopyranoside. Pyroglutamic from clinical sources : description of Helcococcus kunzii gen. acid arylamidase and leucine arylamidase are pro- nov., sp. nov. Int J Syst Bacteriol43, 425429. duced. Weak activity for esterase C-4 detected. Acid Collins, M. D., Falsen, E., Lemozy, J., Akervall, E., SjadBn, B. & phosphatase, alkaline phosphatase and cystine aryl- Lawson, P. A. (1997). Phenotypic and phylogenetic charac- amidase activity may or may not be detected. Arginine terization of some Globicatella-like organisms from human alanine-phenylalanine-proline sources: description of gen. nov., sp. nov. Int dihydrolase, arylamid- J Syst Bacteriol47, 880-882. ase, a-galactosidase, P-galactosidase, P-galacturonid- ase, a-glucosidase, P-glucosidase, P-glucuronidase, li- Collins, M. D., Hutson, R. A,, Falsen, E., Sj&I&n, B. & Facklam, R. R. (1998a). Gemella bergeriae sp. nov., isolated from human pase C 14, a-fucosidase, a-mannosidase, N-acetyl- clinical specimens. J Clin Microbiol36, 1290-1 293. glucosaminidase, glycyl-trytophan arylamidase, chy- motrypsin, trypsin, valine arylamidase and urease are Collins, M. D., Lawson, P. A., Monasterio, R., Falsen, E., SjadBn, B. & Facklam, R. R. (1998b). Facklamia ignava sp. nov, isolated from not produced. Hippurate is hydrolysed. Acetoin is not human clinical specimens. J Clin Microbiol36, 2 146-2 148. produced. The G + C content of DNA is 413 mol %. The type strain of Facklamia sourekii is CCUG Devereux, J., Haeberli, P. & Smithies, 0. (1984). A comprehensive Facklamia sourekii set of sequence analysis programs for the VAX. Nucleic Acids 28783AT. The habitat of is not Res 12, 387-395. known. Felsenstein, 1. (1989). PHYLIP-phylogeny inference package (version 3.2). Cladistics 5, 164-166. Acknowledgements Garvie, E. 1. (1 978). Streptococcus rafinolactis (Orla-Jensen and Hansen) ; a group N streptococcus found in raw milk. Int J Syst We thank Dr S. Lofgren for one of the human isolates and Bacteriol28, 190-193. Dr R. R. Facklam for providing information on the second Maidak, B. L., Olsen, G. J., Larsen, N., Overbeek, R., McCaughey, strain. M. J. & Woese, C. R. (1997). The RDP (Ribosomal Database Project). Nucleic Acids Res 25, 109-1 11. References Pot, B., Vandamme, P. & Kersters, K. (1994). Analysis of electrophoretic whole-organism protein fingerprints. In Modern Aguirre, M. & Collins, M. D. (1992). Phylogenetic analysis of an Microbial Methods. Chemical Methods in Prokaryotic Sys- unknown bacterium from human middle ear fluid : description tematics, pp. 493-521. Edited by M. Goodfellow & A. G. of Alloiococcus otitis gen. nov., sp. nov. Int J Syst Bacteriol42, O’Donnell. Chichester : Wiley. 79-8 3. Roggenkamp, A., Abele-Horne, M., Trebesius, K. H., Tretter, U., Aguirre, M., Morrison, D., Cookson, B. D., Gay, F. W. & Collins, Autenreith, 1. B. & Belesemann, J. (1998). Abiotrophia elegans sp. M. D. (1 993). Phenotypic and phylogenetic characterization of nov., a possible pathogen in patients with culture-negative some Gemella-like organisms from human infections : descrip- endocarditis. J Clin Microbiol36, 100-104.

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