L NOTE Human Vagina Matthew D

international Journal of Systematic Bacteriology (1 999), 49, 1 125-1 128 Printed in Great Britain

Aerococcus christensenii sp. nov., f rom the L NOTE human vagina Matthew D. Collins,’ Mar Rodriguez Jovita,’ Roger A. Hutson,’ Maria Ohlen2and Enevold Faisen’

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

1 Department of Food Phenotypic and phylogenetic studies were performed on two strains of a Science and Technology, hitherto undescribed Aerococcus-like organism isolated from the human University of Reading, Reading RG6 6AP. UK vagina. Comparative 16s rRNA gene sequencing studies demonstratedthat the unknown strains constitute a new subline within the genus Aerococcus. The 2 Culture Collection, Department of Clinical unknown bacterium was readily distinguished from the two currently Bacteriology, University of recognized Aerococcus species, Aerococcus viridans and Aerococcus urinae, by Gsteborg, 5-41346 biochemical tests and electrophoretic analysis of whole-cell proteins. On the Gbteborg, Sweden basis of phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as Aerococcus christenseniisp. nov. The type strain of A. christenseniiis CCUG 2883IT.

Keywords: Aerococcus christensenii sp. nov., phylogeny, taxonomy, 16s rRNA

The genus Aerococcus until recently contained a single UW06T (= CCUG 2883 lT), were submitted to the species, Aerococcus viridans (Williams et al., 1953). Culture Collection of the University of Goteborg by This species is found in a wide range of environments L. K. Rabe and S. L. Hillier (University of Washing- and, although considered saprophytic, has been shown ton, Seattle, USA) for identification. Both strains were to cause disease in lobsters and to be associated, albeit isolated from vaginal sources and tentatively identified rarely, with human infections, e.g. subacute bacterial as Streptococcus acidorninimus. The organisms were endocarditis (Janosek et al., 1980; Parker & Ball, cultured on Columbia agar (Difco) supplemented with 1976), urinary tract infections (Colman, 1967) and 5% horse blood at 37 “C, in air plus 5 % CO,. The meningitis (Nathavitharana et al., 1983). Recently, strains were characterized biochemically by using the Christensen et al. (1 989, 1991) reported the isolation of API Rapid ID32S system according to the manu- some Aerococcus-like organisms from human clinical facturer’s instructions (API bioMirieux). PAGE specimens that were distinct from A. viridans. These analysis of whole-cell proteins was performed as Aerococcus-like organisms were assigned to the genus described by Pot et al. (1994). For densitometric Aerococcus, as Aerococcus urinae, by Aguirre & Collins analysis, normalization and interpretation of protein (1992). A. urinae has been isolated from urine speci- patterns, the GELCOMPARGCW 3.0 software package mens of elderly persons suffering from urinary tract (Applied Maths) was used. The cell wall murein infections and from blood cultures taken from patients structure and G + C content of DNA of strain CCUG with endocarditis and urosepticaemia (Christensen et 28831T was determined as described by Schleifer & al., 1989, 1991, 1995). In the course of a study of Kandler (1972) and Garvie (1978), respectively. The Aerococcus-like organisms from human sources, we 16s rRNA genes of the isolates were amplified by PCR have used 16s rRNA gene sequencing to characterize and directly sequenced by using a Taq Dye-Deoxy two strains of a hitherto unknown Aerococcus-like terminator cycle sequencing kit (Applied Biosystems) bacterium. On the basis of the results of a polyphasic and an automatic DNA sequencer (model 373A, taxonomic study, a new species, Aerococcus christen- Applied Biosystems). The closest known relatives of senii sp. nov., is described. the new isolates were determined by performing sequence database searches. These sequences and those Two strains, designated UWOl (= CCUG 28826) and of other known related strains were retrieved from the GenBank or Ribosomal Database Project libraries and aligned with the newly determined sequences by The GenBank accession numbers for the 165 rRNA gene sequences of using the program PILEUP (Devereux et al., 1984). The strains CCUG 28826 and CCUG 28831T are Y17318 and Y17005. resulting multiple sequence alignment was corrected

~ ~ 00963 0 1999 IUMS 1125 M. D. Collins and others

40 50 60 70 80 90 100

Gemella morbillorum CCUG 18164T Gemella morbillorum CCUG 38816 Gemella haemolysans CCUG 28134 Gemella haemolysans CCUG 37985' Vagococcus salmoninarum CCUG 333MT Carnobacterium divergens CCUG 30094T Carnobacterium gallinarum CCUG 30095T Abiotrophia elegans CCUG 3894gT Abiotrophia elegans CCUG 38676 Gemella sanguinis CCUG 33602 Gemella sanguinis CCUG 37820' Abiofrophia adiacens CCUG 2780gT Abiofrophia adiacens CCUG 37320 Aerococcus chrlstensenll CCUG 28826T Aerococcus christensenllCCUG 28831 Globicateilasanguinis CCUG 3299gT Globicatellasanguinis CCUG 33367 Alloiococcus ofrf/s CCUG 32997T Lactococcus garvieae CCUG 32208T Lactococcus garv!eae CCUG 38280 Dolosigranulum prgrum CCUG 33081A Dolosigranulum prgrum CCUG 33392T lgnavigranum ruoTae CCUG 37841# lgnavigranum ruoffiae CCUG 37658# Enterococcus faecalis CCUG 19916T Enterocaccus faecalis CCUG 34289 Aerococcus viridans CCUG 431 1 Aerococcus viridans CCUG 30285 Aerococcus viridans CCUG 15548 Pediococcus urinaeeaui CCUG 280MT Gemella bergeri CCUG 37969 Gemella bergeri CCUG 37817T Helcococcus kunzii CCUG 32213T Helcococcus kunzii CCUG 31742 Facklamia ignava CCUG 37659 Facklamia ignava CCUG 3741gT Terracoccus luteus CCUG 391 86 +- Vagococcus fluvialis CCUG 32704' Vagococcus f/uvialisCCUG 38909 Facklamia hominis CCUG 36813' Facklamia hominis CCUG 28572 Aerococcus urinae CCUG 29552 Aerococcus urinae CCUG 29554 Aeromccus urinae CCUG 35279A Aerococcus ucnae CCUG 29291T Aerococcus urrnae CCUG 34223T IAerocokus urinae CCUG 39330 Pediococcus penfosaceus CCUG 32205T II , Pediococcus Dentosaceus CCUG 36942 -- Pediococcus acidilactici CCUG 35190 Pediococcus acidilactici CCUG 32235' Leuconostoc Iactis CCUG 37937 Leuconostoc lactis CCUG 30064T Abiotrophia defectiva CCUG 36937 Abiotrophia defectiva CCUG 2763gT

...... I...... Fig, 7. Similarity dendrogram based on whole-cell protein patterns of A. christensenii sp. nov. and related species. Levels of correlation are expressed as percentages of similarity for convenience.

manually and a distance matrix was calculated by sidase, P-galacturonidase, P-glucosidase, P-glucuro- using the programs PRETTY and DNADIST (with nidase, glycyl-tryptophan arylamidase, pyroglutamic the Kimura two-correction parameter) (Felsenstein, acid arylamidase, P-mannosidase and urease. They 1989). A phylogenetic tree was constructed according were also Voges-Proskauer-negative. Indeed, the hy- to the neighbour-joining method with the program drolysis of hippurate was the only positive test ob- NEIGHBOR (Felsenstein, 1989). The stability of the served with the API Rapid ID32S system. An exam- groupings was estimated by bootstrap analysis (500 ination of the cell wall of strain CCUG 2883 lTrevealed replications) with the programs DNABOOT, DNADIST, a directly cross-linked murein based on L-lysine (type NEIGHBOR and CONSENSE (Felsenstein, 1989). Ala). This murein type is found in several Gram- positive, catalase-negative taxa such as the aerococci, Morphologically, the two isolates consisted of Gram- A lloiococcus otitis, A bio t roph ia defect iva and Globi- positive cocci that occurred in pairs, tetrads or small catella sanguinis. groups. They produced a-haemolytic reactions on blood agar and were non-motile. Utilizing the com- PAGE analysis of whole-cell proteins further dem- mercial API Rapid ID32S system, both strains were onstrated the close phenotypic relatedness of the two biochemically unreactive, failing to produce acid from isolates. A dendrogram depicting the relationships of D-arabitol, L-arabinose, cyclodextrin, glycogen, lac- the two isolates is shown in Fig. 1 and illustrates clearly tose, melibiose, mannitol, maltose, melezitose, methyl that they represent a taxon distinct from all other P-D-glucopyranoside, pullulan, raffinose, ribose, sor- reference Gram-positive, catalase-negative cocci ex- bitol, sucrose, tagatose or trehalose. Both strains tested amined to date. To determine the phylogenetic related- negative for alkaline phosphatase, alanine-phenyl- ness of the two isolates, their 16s rRNA genes were alanine-proline arylamidase, arginine dihydrolase, N- amplified by PCR and sequenced. Pairwise analysis acetyl P-glucosaminidase, P-galactosidase, a-galacto- showed that the isolates were genealogically identical

1126 International Journal of Systematic Bacteriology 49 Aerococcus christensenii sp. nov.

Fig. 2. Unrooted tree showing the phylogenetic relationships of A. christensenii sp. nov. and some other low-G+C- content, Gram-positive bacteria. With the exception of A. christensenii, all taxa are represented by their type strains. The tree, constructed by 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. Bar represents 1 % sequence divergence.

Table 1. Tests useful for differentiating A. christensenii sp. nov. from A. urinae and A. viridans

I Test A. christensenii A. urinae A. viridans

Production of acid from: Lactose - Maltose - Mannitol - Ribose - Sucrose - Trehalose - Production of: P-Glucuronidase - Pyroglutamic acid arylamidase - v, Variable.

(100 Yo sequence identity). Sequence database searches member of the genus Aerococcus. Sequence divergence showed that the unknown bacterium was phylo- values of 46% from A. urinae and A. viridans, genetically most closely related to the aerococci however, demonstrate unequivocally that the un- (approx. 94-96 % identity). Considerably lower levels known bacterium represents a hitherto undescribed of relatedness (<92 % identity) were shown to other species of the genus. It is evident from both sequence Gram-positive taxa (data not shown). A tree con- divergence values and tree topology considerations structed by using the neighbour-joining method and that the bacterium is more closely related phylo- showing the phylogenetic position of the unknown genetically to A. urinae than to A. viridans. Christensen bacterium within the ‘lactic acid’ group of bacteria is et al. (1997) have shown that A. urinae as presently illustrated in Fig. 2. Treeing analysis showed that the defined embraces two biotypes, which share high DNA unknown bacterium was a member of the Aerococcus relatedness. The two isolates reported here can be clade and was most closely related to A. urinae (approx. readily distinguished from both A. urinae biotypes by 4 % 16s rRNA sequence divergence). This association their failure to produce acid from mannitol and with A. urinae was supported in 100 % of bootstrapped sucrose. Using conventional testing, Christensen et al. trees. The next nearest relative corresponded to A. (1 997) reported that all 22 strains of A. urinae examined viridans, which displayed about 6% 16s rRNA se- by them produced acid from these substrates. Using quence divergence from the unknown coccus. the commercial API Rapid ID32S system, we examined 38 strains of A. urinae and found that all It is clear from the comparative 16s rRNA gene fermented mannitol and 36 of 38 also fermented sequence analysis that the unidentified coccus is a sucrose. Similarly, by using the API Rapid ID32S

International Journal of Systematic Bacteriology 49 1127 M. D. Collins and others system, neither of the two human isolates produced References P-glucuronidase. However, 37 of 38 strains of A.urinae Aguirre, M. & Collins, M. D. (1992). Phylogenetic analysis of produced this enzyme. The unknown bacterium could some Aerococcus-like organisms from urinary tract infections : also be distinguished readily from A. viridans by using description of Aerococcus urinae sp. nov. J Gen Microbioll38, the API Rapid ID32S system. A. viridans tests positive 401-405. for pyroglutamic acid arylamidase and produces acid Christensen, 1. J., Korner, 6. & Kjaergaard, H. (1989). Aerococcus- from lactose, maltose, sucrose and trehalose. By like organism - an unnoticed urinary tract pathogen. APMIS contrast, strains CCUG 28826 and CCUG 28831T 97, 539-546. were negative for these tests (Table 1). Thus, on the Christensen, 1. J., Gutschik, E., Friis-Moller, A. & Korner, B. (1991). basis of phylogenetic evidence and their distinctive Urosepticemia and fatal endocarditis caused by Aerococcus-like biochemical characteristics, we consider that the two organisms. Scand J Infect Dis 23, 7 17-72 1. human vaginal isolates merit classification as a new Christensen, 1. J., Jensen, 1. P., Faerk, J., Kristensen, B., Skov, R. & species of the genus Aerococcus, for which the name Korner, B. (1995). Bacteremia/septicemia due to Aerococcus-like Aerococcus christensenii sp. nov. is proposed. organisms : report of seventeen cases. The Danish ALO Study Group. Clin Infect Dis 21, 943-947. Christensen, J. J., Whitney, A. M., Teixeira, L. M., Steigerwalt, Description of Aerococcus christensenii sp. nov. A. G., Facklam, R. R., Korner, B. & Brenner, D. 1. (1997). Aerococcus urinae : intraspecies genetic and phenotypic Aerococcus christensenii (christ.en.sen’i.i. M.L. gen. n. relatedness. Int J Syst Bacteriol47, 28-32. christensenii named after the Danish microbiologist, Colman, G. (1967). Aerococcus-like organisms isolated from Jens J. Christensen). human infections. J Clin Pathol20, 294-297. Devereux, J., Haeberli, P. & Smithies, 0. (1984). A comprehensive Cells are Gram-positive, non-spore-forming cocci that set of sequence analysis programs for the VAX. Nucleic Acids occur in pairs, tetrads or small groups. Non-pigmented Res 12, 387-395. and non-motile. Grows in 6.5% NaCl and on blood Felsenstein, J. (1989). PHYLIP - phylogeny inference package agar producing an a-haemolytic reaction. Faculta- (version 3.2). Cladistics 5, 164-166. tively anaerobic. Catalase- and oxidase-negative. Garvie, E. 1. (1 978). Streptococcus raflnolactis (Orla-Jensen and Using the commercial API Rapid ID32S system, acid Hansen) ; a group N streptococcus found in raw milk. Int J Syst is not produced from D-arabitol, L-arabinose, cyclo- Bacteriol28, 190-1 93. dextrin, glycogen, lactose, melibiose, mannitol, malt- Janosek, J., Eckert, J. & Hudac, A. (1980). Aerococcus viridans as ose, melezitose, methyl /?-D-glucopyranoside,pullulan, a causative agent of infectious endocarditis. J Hyg Epidemiol raffinose, ribose, sorbitol, sucrose, tagatose or tre- Microbiol Immunol24, 92-96. halose. Alkaline phosphatase, alanine-phenylalanine- Nathavitharana, K. A., Arseculeratne, 5. N., Aponso, H. A., proline arylamidase, arginine dihydrolase, N-acetyl Vijeratnam, R., Jayasena, L. & Navaratman, C. (1983). Acute P-glucosaminidase, glycyl-tryptophan arylamidase, meningitis in early childhood caused by Aerococcus viridans. Br a-galactosidase, P-galactosidase, P-galacturonidase, Med J 286, 1248. P-glucosidase, P-glucuronidase, pyroglutamic acid Parker, M. T. & Ball, L. C. (1976). Streptococci and aerococci arylamidase, P-mannosidase and urease activity are associated with systemic infection in man. J Med Microbiol9, not detected. Acetoin is not produced. Hippurate is 275-302. hydrolysed. The cell wall murein is of the L-lysine Pot, B., Vandamme, P. & Kersters, K. (1994). Analysis of directly cross-linked type. The G + C content of the electrophoretic whole-organism protein fingerprints. In Modern DNA is 38-5 mol% (Tm). Isolated from the human Microbial Methods. Chemical Methods in Prokaryotic System- vagina. The type strain is CCUG 28831T. atics, pp. 493-521. Edited by M. Goodfellow & A. G. O’Donnell. Chichester : Wiley. Schleifer, K. H. & Kandler, 0. (1972). Peptidoglycan types of Acknowledgements bacterial cell walls and their taxonomic implications. Bacteriol Rev 36,407-477. We are grateful to Lorna K. Rabe and Sharon L. Hillier for Williams, R. E. O., Hirch, A. & Cowan, 5. T. (1953). Aerococcus, a the gift of strains. new bacterial genus. J Gen Microbiol8, 475-480.

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