lnternational Journal of Systematic Bacteriology (1 999), 49,97-101 Printed in Great Britain

Ignavigranum ruoffiae sp. nov., isolated from human clinical specimens

Matthew D. Collins,’ Paul A. Lawson,’ Rafael Monasterio,’ Enevold Faken,’ Berit Sjod6n2 and Richard R. Facklam3

Author for correspondence: Richard R. Facklam. Tel: + 1 404 639 1379. Fax: + 1404 639 3123. e-mail : [email protected]

1 Department of Two strains of a hitherto undescribed Gram-positive catalase-negative, Microbiology, BBSRC facultatively anaerobic coccus isolated from human sources were characterized Institute of Food Research, Reading Laboratory, by phenotypic and molecular taxonomic methods. Comparative 16s rRNA gene Reading RG6 6BZ, UK sequencing studies demonstrated the unknown strains were genealogically

2 Culture Collection, identical, and constitute a new line close to, but distinct from, the genera Department of Clinical Facklamia and Globicatella. The unknown bacterium was readily distinguished Bacteriology, University of from Facklamia and Globicatella sanguinus by biochemical tests and GBteborg, Sweden electrophoretic analysis of whole-cell proteins. Based on phylogenetic and 3 Centers for Disease Control phenotypic evidence it is proposed that the unknown bacterium be classified and Prevention, 1600 Clifton Road, N.E., Atlanta, as /gnawigranum ruoffiae gen. nov., sp. nov. The type strain of rgnavigranum GA 30333, USA ruoffiae is CCUG 37658T.

Keywords : ruofiae, , phylogeny, 16s r RNA

During the past decade, knowledge of the taxonomic Abiotrophia-like bacterium from human clinical interrelationships of the Gram-positive catalase-nega- specimens. Based on the results of a polyphasic tive cocci has improved markedly. Much of this taxonomic study, a new genus and species, improvement has resulted from using a range of Ignavigranum ruofiae, is described. phenotypic methods (e.g. miniaturized biochemical Two strains (1607-97 and 3955-95) from human testing, protein profiling) in concert with molecular sources were referred to the Centers for Disease genetic approaches, notably comparative 16s rRNA Control and Prevention (Atlanta, GA, USA) for gene sequencing. 16s rRNA gene sequence analysis identification. Strain 1607-97 was recovered from a has not only facilitated new insights into the phylo- wound infection and strain 3955-95 was isolated from genetic interrelationships of the Gram-positive an ear abscess. Both strains have been deposited in the catalase-negative cocci but has provided systematists Culture Collection of the University of Goteborg with an immensely powerful means for characterizing (CCUG), Sweden, under accession numbers CCUG new diversity. Indeed primarily as a result of an 3765ST and CCUG 37841, respectively. Strains were increasing recognition by clinical microbiologists of cultured on Columbia agar (Difco) supplemented with the possible role of these organisms as opportunistic 5% (v/v) horse blood at 37 “C, in air plus 5% (v/v) human pathogens, in conjunction with the use of this CO,. The strains were biochemically characterized by molecular taxonomic tool, a plethora of new species using the API Rapid ID32 Strep, API CORYNE and and genera of Gram-positive catalase-negative cocci API ZYM systems according to the manufacturer’s [e.g. Aerococcus urinae (Aguirre & Collins, 1992b), instructions (API bioMirieux). The strains were also ‘ Abiotrophia elegans’ (Roggenkamp et al., 1998), examined using conventional biochemical tests as Alloiococcus (Aguirre & Collins, 1992a), Dolosi- described by Facklam & Elliot (1 995). PAGE of whole- granulum (Aguirre et al., 1993), Facklamia (Collins et cell proteins was performed as described by Pot et al. al., 1997), Globicatella (Collins et al., 1992) and (1994). The Pharmacia-LKB UltroScan XL densi- Helcococcus (Collins et al., 1993)] have been discovered tometer, with its software, was used for capture of gel and described in recent years. In this study we have data. For normalization and interpretation of protein used 16s rRNA gene sequencing to phylogenetically patterns the GelCompar GCW 3.0 software package characterize two strains of a hitherto unknown (Applied Maths) was used. The cell-wall murein structure and mol YOG + C content of DNA of strain The GenBank accession number for the 165 rRNA sequence of CCUG 37658T CCUG 3765STwere determined by methods described is Y 16426. by Schleifer & Kandler (1972) and Garvie (1978),

00866 0 1999 IUMS 97 M. D. Collins and others respectively. The 16s rRNA genes of the isolates were amplified by PCR and directly sequenced using a Taq Dye-Deoxy terminator cycle sequencing kit (Applied Biosystems) and an automatic DNA sequencer (model 373A; Applied Biosystems). The closest known relatives of the new isolates were determined by performing database searches. These sequences and - Gemella haemolysans CCUG 37985T those of other known related strains were retrieved Gemella haemolysans CCUG 281 34 Gemella haemolysans CCUG 41 1 from GenBank or Ribosomal Database Project (RDP) Abiotrophia adiacens CCUG 27637A Abiotrophia adiacens CCUG 2780gT databases and aligned with the newly determined Abiotrophia adiacens CCUG 3513fB Aerococcus viridans CCUG 431 1 sequences using the program PILEUP (Devereux et al., ANoiococcus otitis CCUG 32997T Dolosigranulum pigrum CCUG 33081 A 1984). The resulting multiple sequence alignment was Dolosigranulum pigrum CCUG 35446 DdosigranulumpigNm CCUG 3339ZT corrected manually and a distance matrix was calcu- Globicatellasanguinis CCUG 36563A Globkatella sanguinis CCUG 33367 lated using the programs PRETTY and DNADIST (using Globicatellasanguinis CCUG 3299gT Ignavigranum ruoffae CCUG 37841 the Kimura-2 correction parameter) (Felsenstein, lgnavigranum Noffiae CCUG 3765BT Facklamia ignava CCUG 37659 1989). A phylogenetic tree was constructed according IFacklamia ignava CCUG 3741gT Facklamia hominis CCUG 28830 to the neighbour-joining method with the program - Facklamia hominis CCUG 28572 Facklamia hominis CCUG 32738 NEIGHBOR (Felsenstein, 1989). The stability of the Fackiamia hominis CCUG 28829 Facklamia hominis CCUG 28827 groupings was estimated by bootstrap analysis (500 Facklamia hominis CCUG 3681 3T replications) using the programs DNABOOT,DNADIST, NEIGHBOR and CONSENSE (Felsenstein, 1989). In ad- dition a parsimony analysis (Felsenstein, 1989) was also performed on the same data set...... , ...... Fig, 1. Similarity dendogram based on whole-cell protein The two clinical isolates were ovoid in shape and pattern of sp. nov. and related species. Levels of correlation are expressed as percentages of similarity formed single cells, pairs or groups. The strains were for convenience. Gram-positive, non-spore-forming, catalase-negative, oxidase-negative facultative anaerobes. Both CCUG 3765tlT and CCUG 37841 grew in 6.5% NaCl and weakly at 45 “C (growth apparent only after 7 d hominis and Globicatella sanguinis (Fig. 1). An exam- incubation). The only positive conventional tests other ination of the cell wall of strain CCUG 37658Trevealed than growth at 6.5 % NaCl and at 45°C were reactions that the unknown coccus possessed a directly cross- for PYRase and LAPase by the disc method. Using the linked murein based on L-lysine [type A 1a, according API systems the isolates produced acid from glucose to nomenclature of Schleifer & Kandler (1972)l. This and were similar to each other in not producing acid murein structure is found in a number of Gram- from D-arabitol, L-arabinose, cyclodextrin, glycogen, positive catalase-negative cocci such as aerococci lactose, melibiose, melezitose, methyl-P-D-gluco- (Aguirre & Collins, 1992b),Alloiococcus otitis (Aguirre pyranoside, pullulan, raffinose, ribose, sorbitol, taga- & Collins, 1992a), Globicatella sanguinis (Collins et al., tose, trehalose or D-xylose. Strain CCUG 37841 1992) and Abiotrophia defectiva (Collins et al., 1997). showed weak acid production from mannitol and To assess the genealogical affinity between the two sucrose. Acid production from mannitol and sucrose isolates and their relationship with other Gram- was either negative or weak for strain CCUG 37658T. positive catalase-negative taxa, comparative 16s Using the API systems the two isolates showed rRNA gene sequence analyses were performed. The arginine dihydrolase, leucine arylamidase, pyro- almost complete gene sequences (> 1400 nucleotides) glutamic acid arylamidase and urease activity; no of the two clinical strains were determined and pairwise activity was detected for alkaline phosphatase, alanyl- analysis revealed no base differences (i.e. 100% simi- phenylalanine-proline arylamidase, N-acetyl-glucos- larity) thereby showing their high phylogenetic aminidase, cystine arylamidase, chymotrypsin, a- relatedness. Sequence searches of GenBank and RDP fucosidase, a-galactosidase, P-galactosidase, p- databases revealed that the unknown bacterium was galacuronidase, a-glucosidase, P-glucosidase, P- phylogenetically most closely associated with the lactic glucuronidase, glycyl-tryptophan arylamidase, lipase acid group of . A tree constructed by C 14, P-mannosidase, pyrazinamidase, trypsin or valine neighbour-joining depicting the phylogenetic affinity arylamidase. Neither of the isolates hydrolysed of the unknown coccus as exemplified by strain CCUG aesculin, hippurate or gelatin. They were Voges- 37658Tis shown in Fig. 2. From the branching pattern Proskauer-negative and did not reduce nitrate. The of the tree the nearest relative of the unknown coccus close phenotypic affinity between the clinical isolates was Facklamia hominis. The clustering together of was confirmed by PAGE analysis of whole-cell these organisms occurred in 84% of 500 tree repli- proteins in which the two strains formed a robust and cations. Globicatella sanguinis formed a somewhat tight cluster which was quite separate from all other deeper subline branching off from the unknown Gram-positive catalase-negative reference organisms coccuslFacklamia hominis line (supported by 83 % examined, including, Abiotrophia spp., Facklamia bootstrap value). The next nearest relative of the

98 In termtiona I Jou rna I of Systematic Bacteriology 49 Ignavigranum ruofiae sp. nov.

Carnobacterium divergens NCDO 2763 (X54270) ...... Carnobacterium gallrnarum NCFB 2766 (X54269) Fig. 2. Unrooted tree showing the Carnobacterium pisciwla NCDO 2762 (X54268) phylogenetic relationships lgnavigranum L?ctosph~era.pasteun’iDSM 2381 (X87150 of ‘Abrotrophla adlacens’ ATCC 491 75 (D50540\ ruofiiae sp. nov. and some other low G+C -_ Abiotrophia elegans DSM 11693 (AFO16390) content Gram-positive bacteria (all strains Facklamia hominis CCUG 36813 (Y 1 0772) are type strains). The tree, constructed using Ignavigranum ruofffae CCUG 37658 (Y16426) Globicatella sanguinis NCFB 2835 (S50214) the neighbour-joining method, was based Abiotrophia defectiva ATCC 491 76 (D50541) on a comparison of about 1320 nucleotides. Aerococcus urrnae NCFB 2893 (M77819) Bootstrap values, expressed as a percentage Aerococcus viridans ATCC 11563 (M58797) 500 Dolosigranulum i rum NCFB 2975 (X70907) of replications, are given at branching Alloiococcus otitis i8FB 2890 (X59765) points.

Table I. Characteristics that differentiate rgnavigranum ruoffiae sp. nov. from Facklamia hominis and Globicatella sanguinis 1 Character I. ruofiae F. hominis G. sanguinis I

Production of acid from: Glycogen + Lactose V Mannitol + Melibiose + Methy1-P-D-gluco p yranoside V Pullulan v Raffinose + Ribose + Sorbitol V Sucrose + Trehalose + Hydrolysis of: Hippurate + + Production of: Alanyl-phenylalanine-prolinearylamidase - + + Arginine dihydrolase + + a-Galactosidase - + + P-Galactosidase - + + Murein type: Ala A4a Ala

aforementioned taxa corresponded to Abiotrophia positive catalase-negative species within the Lactic defectiva (Fig. 2). Parsimony analysis was also per- acid group of bacteria. By contrast, 16s rRNA permits formed and the grouping of the unknown coccus with an assessment of more distant phylogenetic affinities Facklamia hominis and Globicatella sanguinis (as (e.g. interspecies relationships). From the 16s rRNA shown in Fig. 2) confirmed. All other significant sequence analysis it is evident that the coccus has a associations shown in the neighbour-joining tree were close phylogenetic relationship with Facklamia also reproduced in the parsimony analysis (data not hominis. The association between these taxa was shown). supported by a bootstrap value of 84 YO.A sequence divergence value of 8.2 YOsuggests this relationship is PAGE analysis of whole-cell proteins is an excellent that of two phylogenetically closely related, but never- tool for assessing close taxonomic relationships (e.g. theless separate, genera. Support for the separate species, subspecies). Using this approach it is clear that generic status of the unknown bacterium comes from the two unknown isolates from human clinical sources its possession of a Lys-directly cross-linked murein clearly belong to a hitherto unrecognized Gram- (type Ala). Facklamia hominis contains a cell wall

In ternationa I lo urnaI of Systematic Bacteriology 49 99 M. D. Collins and others murein type L-LYS-D-ASP(type A4a) (Collins et al., negative. Strains grow at 45 "C and 6.5 % NaC1. Acid 1997). The unknown bacterium displayed a slightly produced weakly from glucose. Acid is not produced higher 16s rRNA sequence similarity with Globicatella from L-arabinose, D-arabitol, cyclodextrin, glycogen, sanguinis (93.8 YO).A sequence divergence of > 6 YO lactose, D-mannose, melibiose, melezitose, pullulan, D- and the branching pattern of the tree, however, raffinose, D-ribose, sorbitol, tagatose, trehalose or D- indicates the relationship between these two taxa is xylose. Some strains produce acid from mannitol and that of two closely related, albeit different, genera. sucrose. Using commercial API systems arginine Additional evidence for the separateness of the un- dihydrolase, leucine arylamidase, pyroglutamic acid known coccus from the genus Globicatella comes from arylamidase and urease activity is detected. Alanyl- its very characteristic phenotype, in particular its phenylalanine-proline arylamidase, cy stine asaccharolytic nature. By contrast, Globicatella arylamidase, chymotrypsin, a-fucosidase, alkaline sanguinis is a highly saccharolytic organism capable of phosphatase, a-galactosidase, P-galactosidase, P- utilizing a broad range of carbohydrates (Collins et al., galacturonidase, P-glucuronidase, glycyl-tryptophan 1992). It is pertinent to note that no other taxon arylamidase, lipase C 14, P-mannosidase, trypsin and examined possessed less than 8 YO16s rRNA sequence pyrazinamidase activity is not detected. Aesculin, divergence with the unknown bacterium. Examples of hippurate, gelatin and starch are not hydrolysed. characteristics which serve to distinguish the unknown Arginine dihydrolase and urease are negative in human bacterium from Facklarnia horninis and conventional tests. Voges-Proskauer negative. Nitrate Globicatella sanguinis are outlined in Table 1. Based on is not reduced. The G + C content of DNA is 40 mol %. 16s rRNA sequence considerations and the distinctive The cell-wall murein type is L-lysine-direct (A1 a). phenotypic traits of the unknown coccus, we consider Isolated from clinical sources. Habitat not known. The this bacterium merits classification as a new genus, for type strain is CCUG 37658T. Strain CCUG 37658T which the name Zgnavigranurn ruofiae gen. nov., sp. was isolated from a wound infection. nov. is proposed. Acknowledgements Description of rgnavigranum gen. nov. This work was supported in part by a grant from the Zgnavigranurn gen. nov. (1g.na.vi.gra'num. L. adj. European Union (pI02-CT94-3098).The excellent technical ignuvus lazy, non-reacting; L. neut. n. granurn grain, assistance of Eva Akervall is acknowledged. kernel ; Ignavigranurn lazy grain). Cells are Gram-positive, non-spore-forming, non- References motile, cocci occurring as single cells, in pairs or Aguirre, M. & Collins, M. D. (1992a). Phylogenetic analysis of an groups. Facultatively anaerobic and catalase-negative. unknown bacterium from human middle ear fluid : description Growth at 45 "C and in 6.5% NaCl. Weak acid of Alloiococcus otitis gen. nov., sp. nov. Int J Syst Bacteriol42, production but no gas from glucose. Other carbo- 79-8 3. hydrates generally not acidified. Hippurate, aesculin, Aguirre, M. & Collins, M. D. (1992b). Phylogenetic analysis of gelatin and starch are not hydrolysed. Arginine some Aerococcus-like organisms from urinary tract infections : dihydrolase, leucine arylamidase and urease are description of Aerococcus urinae sp. nov. J Gen Microbioll38, produced according to the API system. Voges- 401405. Proskauer negative. Nitrate is not reduced. The murein Aguirre, M., Morrison, D., Cookson, B. D., Gay, F. W. & Collins, type is L-lysine-direct (Ala). The G+C content of M. D. (1993). Phenotypic and phylogenetic characterization of DNA is 40 mol YO(Tm). The type species of the genus some Gemella-like organisms from human infections : descrip- is Zgnavigranum ruojiae. As determined by 16s rRNA tion of Dolosigranulum pigrum gen. nov., sp. nov. J Appl gene sequence analysis, the genus Zgnavigranurn Bacteriol75, 608-612. belongs to the Lactic acid group of bacteria with low Collins, M. D., Aguirre, M., Facklam, R. R., Shallcross, J. & DNA G + C contents, and is phylogenetically closely Williams, A. M. (1992). Globicatella sanguis gen. nov., sp. nov., a related to Facklamia horninis and Globicatella new gram-positive catalase-negative bacterium from human sanguinis, but may be distinguished from the latter two sources. J Appl BacterioE73, 422-437. taxa using traits shown in Table 1. Collins, M. D., Facklam, R. R., Rodrigues, U. M. & Ruoff, K. L. (1 993). Phylogenetic analysis of some Aerococcus-likeorganisms from clinical sources : description of Helcococcus kunzii gen. Description of lgnavigranum ruoffiae sp. nov. nov., sp. nov. Int J Syst Bacteriol43, 425-429. Zgnavigranurn ruojiae (ru.ofYiae. Named after Collins, M. D., Falsen, E., Lemozy, J., Akervall, E., SjMCn, B. & Kathryn L. Ruoff an American microbiologist, in Lawson, P. A. (1997). Phenotypic and phylogenetic charac- terization of some Globicatella-like organisms from human recognition of her contributions to the microbiology of sources: description of Facklamia horninis gen. nov., sp. nov. Int Gram-positive cocci). J Syst Bacteriol47, 880-882. Cells are Gram-positive, ovoid in shape, occurring as Devereux, J., Haeberli, P. & Smithies, 0. (1984). A comprehensive single cells, in pairs or groups. Cells are non-spore- set of sequence analysis programs for the VAX. Nucleic Acids forming and non-motile. Some strains show satelliting Res 12, 387-395. enhancements. Facultatively anaerobic and catalase- Facklam, R. R. & Elliot, 1. A. (1995). Identification, classification,

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