International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1229–1236
DOI 10.1099/ijs.0.058081-0
Aerococcus vaginalis sp. nov., isolated from the vaginal mucosa of a beef cow, and emended descriptions of Aerococcus suis, Aerococcus viridans, Aerococcus urinaeequi, Aerococcus urinaehominis, Aerococcus urinae, Aerococcus christensenii and Aerococcus sanguinicola Masanori Tohno,1 Maki Kitahara,2 Shuichi Matsuyama,1 Koji Kimura,1 Moriya Ohkuma2 and Kiyoshi Tajima3 Correspondence Masanori Tohno [email protected]
1
National Agriculture and Food Research Organization, National Institute of Livestock and Grassland Science, Nasushiobara, Tochigi 329-2793, Japan
2
Japan Collection of Microorganisms, RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan
3
National Agriculture and Food Research Organization, National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki 305-0901, Japan
A gram-stain-positive, facultatively anaerobic, non-spore-forming, catalase-negative, coccoidshaped bacterial strain, designated BV2T, was isolated from the vaginal mucosa of a beef cow in Japan. Phylogenetic analysis showed that the isolate shared high 16S rRNA gene sequence similarity (92.9 %) with Aerococcus suis 1821/02T and low similarity (,92.7 %) with any other recognized species of the genus Aerococcus. The DNA G+C content was 44.7 mol%, which is within the range observed among species of the genus Aerococcus (37.5–48.4 mol%). The major cellular fatty acid was C18 : 1v9c, similar to other type strains of species of the genus Aerococcus. The results of genotypic, phenotypic and chemotaxonomic analyses as well as the low degree of DNA–DNA relatedness with all recognized members of the genus Aerococcus indicate that strain BV2T represents a novel species of the genus Aerococcus, for which the name Aerococcus vaginalis sp. nov. is proposed. The type strain is BV2T (5JCM 19163T5DSM 27293T). Emended descriptions of Aerococcus suis, Aerococcus viridans, Aerococcus urinaeequi, Aerococcus urinaehominis, Aerococcus urinae, Aerococcus christensenii and Aerococcus sanguinicola are also presented.
In bovines, the vaginal flora has a significant influence on the health and disease state of the host. Some bovine uterine pathogens, including Escherichia coli, Fusobacterium necrophorum, Arcanobacterium pyogenes and Prevotella melaninogenica, induce severe urogenital inflammation, which is associated with infertility and consequent economic loss (Sheldon & Dobson, 2004; Sheldon et al., 2002, 2006; Williams et al., 2005). Although lactobacilli are not predominantly found in the vaginal mucosa of cows, in contrast to humans, mice and monkeys (Herthelius et al., 1989; Otero et al., 2000; Redondo-Lopez et al., 1990; Vintin˜i et al., 2004), The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain BV2T is AB818493. One supplementary table and one supplementary figure are available with the online version of this paper.
058081 G 2014 IUMS
Printed in Great Britain
it is well known that lactic acid bacteria belonging to the genera Lactobacillus, Leuconostoc, Pediococcus and Weissella are naturally present and that certain strains isolated from the bovine vagina produce bacteriocin (Wang et al., 2013). During the course of isolating bacteria from the bovine reproductive tract, with particular interest in lactic acid bacteria, a bacterial strain (designated BV2T) capable of growing in de Man Rogosa Sharpe (MRS) medium (Difco) was isolated from a vaginal swab sample. The purpose of the present study was to establish the taxonomic position of this novel Aerococcus-like strain. The isolation of vaginal mucosa-associated bacterial strains was conducted basically as described by Otero et al. (2000). Vaginal swab samples were obtained from three healthy crossbred cows (Japanese Black6Holstein-Friesian) maintained at the National Institute of Livestock and Grassland 1229
M. Tohno and others
Science in Nasushiobara, Japan. Sampling was carried out on day 7 of the oestrus cycle (oestrus5day 0). Strain BV2T was isolated from one of the three cows, which had the following basic characteristics: age, 10.3 years; body weight, 750 kg; voluntary waiting period, 1048 days; parity, 4. After a sterile vaginal speculum was gently inserted into the vagina and opened, a long-handled sterile cotton swab was introduced to obtain a mucosal sample from the vaginal fornix. Samples serially diluted 10-fold with sterile PBS were plated on MRS agar and incubated for 4 days at 30 uC in an ANX-1 TE-HER Hard Anaerobox (Hirosawa) with an O2-free atmosphere of N2/H2/CO2 (80 : 10 : 10 %) in order to obtain bacterial isolates. The taxonomic position of strain BV2T was first determined by 16S rRNA gene sequence analysis, according to previous reports (Tohno et al., 2012a, b, 2013a, b). Briefly, the 16S rRNA gene sequences of strain BV2T and the closest recognized relatives were aligned using CLUSTAL X (version 2.0.12) (Larkin et al., 2007), and a distance matrix was obtained using the Kimura two-parameter model (Kimura, 1980). A phylogenetic tree was reconstructed according to the neighbour-joining method (Saitou & Nei, 1987). Maximum-likelihood and maximum-parsimony methods were also applied to the reconstruction of phylogenetic trees using the software package MEGA (version 5.0) (Tamura et al., 2011). The statistical reliability of the topology of the phylogenetic trees was evaluated by bootstrap analysis (1000 replications). T
Strain BV2 had the highest sequence similarity to the type strains of Aerococcus suis (92.9 %), Aerococcus viridans (92.7 %), Aerococcus urinaeequi (92.7 %), Aerococcus urinaehominis (92.5 %) and other members of the genus Aerococcus (,92 %). Neighbour-joining tree analysis showed that strain BV2T represents a distinct phylogenetic lineage close to the genus Aerococcus (Fig. 1). Maximumlikelihood and maximum-parsimony analyses confirmed this with similar topologies. In conjunction with additional data described below, the low degree of 16S rRNA gene similarity as compared with any other species of the genus Aerococcus indicate that strain BV2T represents a novel species of the genus Aerococcus. Therefore, strain BV2T and the reference strains Aerococcus christensenii JCM 18985T, Aerococcus sanguinicola JCM 11549T, Aerococcus suis JCM 18035T, Aerococcus urinae JCM 18986T, Aerococcus urinaeequi JCM 12519T, Aerococcus urinaehominis JCM 18987T and Aerococcus viridans JCM 20461T were maintained in MRS broth at 30 uC for 3–4 days and used for further analyses under identical conditions.
overnight, and the results are expressed as the mean±SD of three independent experiments. An automated RiboPrinter microbial characterization system (Qualicon) was used for ribotyping analysis (Kitahara et al., 2010). Because DNA G+C content measurements have not been conducted in some species of the genus Aerococcus with validly published names, we analysed not only strain BV2T but also all type strains of species of the genus Aerococcus with validly published names. The DNA G+C content of the reference strains was as follows: Aerococcus christensenii JCM 18985T (39.8 mol%), Aerococcus sanguinicola JCM 11549T (48.4 mol%), Aerococcus suis JCM 18035T (37.5 mol%), Aerococcus urinae JCM 18986T (44.1 mol%), Aerococcus urinaeequi JCM 12519T (40.0 mol%), Aerococcus urinaehominis JCM 18987T (43.5 mol%) and Aerococcus viridans JCM 20461T (40.2 mol%), indicating that the DNA G+C content of species of the genus Aerococcus ranges from 37.5 to 48.4 mol%. The DNA G+C content of strain BV2T was 44.7 mol%, which was consistent with the range observed in species of the genus Aerococcus. The DNA–DNA relatedness of strain BV2T with the other Aerococcus type strains was below 7.0 % (both fixed DNA and photobiotin-labelled results), confirming that strain BV2T represents a novel species in the genus Aerococcus (Table S1, available in the online Supplementary Material). The unique taxonomic position of strain BV2T within the genus Aerococcus was also confirmed through analysis of its riboprint band pattern (Fig. S1). For characterization of strain BV2T, both the cellular and colony morphologies were observed after growth on MRS agar plates at 30 uC for 72 h under anaerobic conditions or growth on Columbia blood agar supplemented with 5 % defibrinated horse blood (Oxoid) at 37 uC for 72 h under anaerobic conditions. Gram staining, motility, gas production from glucose, catalase production, spore formation, dextran production from sucrose, and the configuration of lactic acid enantiomers were determined as previously described (Tohno et al., 2013d). MRS broth supplemented with 2 % oxgall was used to test for bile tolerance. Nitrate reduction was analysed using the API rapid ID 32A system. The effects of temperature (4, 10, 15, 25, 30, 37, 45 and 50 uC), pH (3.0, 3.5, 4.0, 5.0, 6.0, 7.0 and 8.0) and NaCl concentration (4, 6.5, 8 and 10 %, w/v) on growth were investigated using cultures grown in MRS broth for 7 days (Tohno et al., 2013b).
For DNA G+C content, DNA–DNA relatedness and Riboprint pattern analyses, genomic DNA of the novel strain and the closely related type strains was extracted and purified, as previously described (Tohno et al., 2013e). The DNA G+C content was determined using HPLC (Kitahara et al., 2010). DNA–DNA relatedness was assessed using the fluorometric microplate method (Ezaki et al., 1989; Tohno et al., 2013c). Hybridization was conducted at 43 uC
To compare the fatty acid profiles between strain BV2T and the reference type strains, cellular fatty acid methyl esters were prepared from cells grown in MRS broth at 30 uC for 3 days and analysed using the Microbial Identification System (Microbial ID; MIDI) (Sakamoto et al., 2002). All fatty acid data in the present study were obtained under identical conditions. The isolate and the reference type strains of the genus Aerococcus were characterized using the API 50 CH (bioMe´rieux), API ID 32 Strep (bioMe´rieux), API 20 Strep (bioMe´rieux) and API ZYM (bioMe´rieux) test strips in duplicate, according to the manufacturer’s instructions. The API 50 CH strips were incubated for 7 days.
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International Journal of Systematic and Evolutionary Microbiology 64
Aerococcus vaginalis sp. nov.
Lactococcus lactis subsp. lactis NCDO 604T (AB100803) Facklamia hominis ATCC 700628T (Y10772)
-/-/-
Dolosicoccus paucivorans 2992-95T (AJ012666)
-/-/74/-/-
Ignavigranum ruoffiae CCUG 37658T (Y16426)
87/75/-
Globicatella sanguinis LMG 18987T (S50214)
67/65/-
Eremococcus coleocola M1832/95/2T (Y17780) Granulicatella elegans ATCC 700633T (AF016390) Abiotrophia defectiva GIFU 12707T (D50541)
95/55/58
Aerococcus vaginalis BV2T (AB818493) Aerococcus urinaehominis CCUG 42038 bT (AJ278341) 100/99/98 59/-/-
-/-/-
Aerococcus suis 1821/02T (AM230658) 54/68/56
Aerococcus urinaeequi IFO 12173 (D87677)
T 100/100/100Aerococcus viridans ATCC 11563 (M58797)
0.02 88/74/68
Aerococcus urinae NCFB 2893T (M77819) 74/92/87 Aerococcus christensenii CCUG 28831T (Y17005) Aerococcus sanguinicola CCUG 43001T (AJ276512)
Fig. 1. 16S rRNA gene sequence-based neighbour-joining tree displaying the phylogenetic relationship of BV2T (bold type) among all species of the genus Aerococcus with validly published names and related taxa. GenBank/EMBL/DDBJ accession numbers are given in parentheses. Lactococcus lactis subsp. lactis NCDO 604T (AB100803) is the outgroup. Bootstrap values greater than 50 % (based on 1000 replications) from neighbour-joining/maximum-likelihood/minimum-evolution analyses are shown at branch points. Bar, 0.02 substitutions per nucleotide position.
The physiological, biochemical and morphological characteristics of strain BV2T and those that differentiate it from closely related type strains of species of the genus Aerococcus are summarized in the species description and Table 1. The phenotypic profile of strain BV2T differed from that of other type strains in the genus Aerococcus, and the ability to ferment D-tagatose and a positive result for N-acetyl-b-glucosaminidase activity uniquely differentiated strain BV2T from the other strains. Fatty acid profile analysis revealed that C18 : 1v9c was the major cellular fatty acid, together with smaller amounts of C16 : 0, C18 : 0 and summed feature 10 (C18 : 1v11c/9t/6t and/or unknown ECL 17.834), similar to other members of the genus Aerococcus (Table 2). The relatively high level of C20 : 1v9c fatty acid is a useful characteristic for differentiating the isolate from the other type strains. Some of the present phenotypic data for the recognized species of the genus Aerococcus grown in MRS broth differed from data reported in species descriptions of cells grown on blood agar at 37 uC. For example, with the type strains of Aerococcus urinaehominis, Aerococcus christensenii and Aerococcus sanguinicola, variable acetoin production results were observed, in contrast http://ijs.sgmjournals.org
to previous reports (Collins et al., 1999; Lawson et al., 2001a, b). These discrepancies can be explained by the fact that some culture conditions, such as medium type and incubation temperature, were different. Although a large divergence from other species was observed with respect to the 16S rRNA gene sequence of strain BV2T (.7.1 %), this novel isolate was not considered to represent a novel genus but was recognized as representing a novel species within the genus Aerococcus on the basis of the similar fatty acid profiles. Thus, BV2T is a novel member of the genus Aerococcus, and the name Aerococcus vaginalis sp. nov. is proposed to accommodate this strain. The descriptions of the recognized species of the genus Aerococcus must be emended in order to incorporate details regarding fatty acid composition and other characteristics when cells are grown in MRS broth at 30 uC. Emended description of Aerococcus viridans Williams et al. 1953 The species description is based on that of Williams et al. (1953), with the following amendments made for the type 1231
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Table 1. Differential phenotypic characteristics of Aerococcus vaginalis sp. nov. and other known species of the genus Aerococcus Strains: 1, BV2T; 2, Aerococcus suis JCM 18035T; 3, Aerococcus viridans JCM 20461T; 4, Aerococcus urinaeequi JCM 12519T; 5, Aerococcus urinaehominis JCM 18987T; 6, Aerococcus urinae JCM 18986T; 7, Aerococcus christensenii JCM 18985T; 8, Aerococcus sanguinicola JCM 11549T. All tested characteristics were investigated under identical conditions in the present study. Using the API 50 CH system, all strains produced acid from D-glucose and D-fructose but failed to produce acid from erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, D-adonitol, methyl b-D-xylopyranoside, L-rhamnose, dulcitol, inositol, methyl aD-mannopyranoside, melibiose, inulin, melezitose, starch, glycogen, D-lyxose, D-fucose, L-fucose, L-arabitol, 2-ketogluconate and 5-ketogluconate. Using the API ID 32 Strep system, all strains were negative for b-mannosidase. Using the API ZYM system, all strains were positive for naphthol-AS-BI-phosphohydrolase but negative for C14 lipase, valine aminopeptidase, trypsin, chymotrypsin, a-galactosidase, a-glucosidase, a-mannosidase and a-fucosidase. +, Positive; 2, negative. Characteristic API 20 strep Arginine Hippurate Aesculin Acetoin Pyrrolidonyl arylamidase API 50 CH Glycerol D-Ribose D-Galactose D-Mannose L-Sorbose D-Mannitol D-Sorbitol Methyl a-D-glucopyranoside N-Acetylglucosamine Amygdalin Arbutin Salicin Cellobiose Maltose Lactose Sucrose Trehalose Raffinose Xylitol Gentiobiose Turanose D-Tagatose D-Arabitol Gluconate API ZYM Alkaline phosphatase C4 esterase C8 esterase Leucine aminopeptidase Cystine aminopeptidase
1232
1
2
3
4
5
6
7
8
2 + 2 2 2
+ 2 2 2 +
2 + + 2 2
2 + 2 2 2
2 + + + 2
2 + 2 + 2
2 + 2 + 2
2 + + + +
2 + + + 2 2 2 2 + 2 2 2 2 + 2 2 2 2 2 2 2 + 2 2
2 2 + 2 2 2 2 2 2 2 2 2 2 + 2 2 2 2 2 2 2 2 2 2
+ + + + 2 + 2 + + 2 2 2 + + + + 2 2 2 2 + 2 2 2
+ + + + 2 + 2 2 + + + + + + + + + + 2 + 2 2 2 +
+ + 2 2 2 2 2 2 2 2 + + + + 2 + + 2 2 2 2 2 2 2
2 + 2 2 2 + + 2 + 2 2 2 2 2 2 + 2 2 + 2 2 2 + 2
2 2 2 2 + 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
+ 2 2 + 2 2 2 2 + 2 2 2 2 + 2 + + 2 2 2 2 2 2 2
+ + 2 + +
2 + + 2 2
+ + + 2 2
+ 2 2 2 2
+ + 2 2 2
+ + 2 + 2
2 + + + 2
2 + + + +
Table 1. cont. Characteristic
1
2
3
4
5
6
7
8
Acid phosphatase b-Galactosidase b-Glucuronidase b-Glucosidase N-Acetyl-b-glucosaminidase API ID 32 strep Alanine-phenylalanine-proline arylamidase Pyroglutamic acid arylamidase Glycine-tryptophan arylamidase Urease
+ + 2 2 +
+ + 2 2 2
2 2 2 2 2
+ 2 + + 2
+ 2 + + 2
+ 2 + 2 2
+ 2 2 2 2
+ 2 + 2 2
+ 2 2 2 2 + + 2 2 2 + 2 2 2 2 + + 2 2 2 2 + + 2 2 2 2 2 + 2 2 2
strain when grown at 30 uC on MRS medium. In the API 50 CH system (incubated for 7 days at 30 uC), acids are produced from glycerol, D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, methyl a-D-glucopyranoside, N-acetylglucosamine, cellobiose, maltose, lactose, sucrose and turanose, but acids are not produced from erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, Dadonitol, methyl b-D-xylopyranoside, L-sorbose, L-rhamnose, dulcitol, inositol, D-sorbitol, methyl a-D-mannopyranoside, amygdalin, arbutin, aesculin ferric citrate, salicin, melibiose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, D-lyxose, D-tagatose, Dfucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate and 5-ketogluconate. Using the commercial API ID 32 Strep, API 20 Strep and API ZYM systems, cells are positive for alkaline phosphatase, C4 esterase, C8 esterase and naphthol-AS-BI-phosphohydrolase activity. Cells are negative for C14 lipase, leucine aminopeptidase, valine aminopeptidase, cystine aminopeptidase, trypsin, chymotrypsin, acid phosphatase, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-bglucosaminidase, a-mannosidase, a-fucosidase, pyrrolidonyl arylamidase, alanine-phenylalanine-proline arylamidase, glycine-tryptophan arylamidase, b-mannosidase and urease activity. Hippurate and aesculin are hydrolysed but arginine is not. The major cellular fatty acid, as determined after growth in MRS broth at 30 uC for 3 days, is C18 : 1v9c. Emended description of Aerococcus urinae Aguirre and Collins 1992 The species description is based on that of Aguirre & Collins (1992), with the following amendments made for the type strain when grown at 30 uC on MRS medium. In the API 50 CH system (incubated for 7 days at 30 uC), acids are produced from D-ribose, D-glucose, D-fructose, D-mannitol, D-sorbitol, N-acetylglucosamine, sucrose, xylitol and D-arabitol, but acids are not produced from glycerol, erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, D-adonitol, methyl b-D-xylopyranoside, D-galactose, D-mannose, L-sorbose, L-rhamnose, dulcitol, inositol, methyl a-D-mannopyranoside, methyl a-D-glucopyranoside, amygdalin, arbutin, International Journal of Systematic and Evolutionary Microbiology 64
Aerococcus vaginalis sp. nov.
Table 2. Cellular fatty acid compositions of Aerococcus vaginalis sp. nov. and other species of the genus Aerococcus Strains: 1, BV2T; 2, Aerococcus suis JCM 18035T; 3, Aerococcus viridans JCM 20461T; 4, Aerococcus urinaeequi JCM 12519T; 5, Aerococcus urinaehominis JCM 18987T; 6, Aerococcus urinae JCM 18986T; 7, Aerococcus christensenii JCM 18985T; 8, Aerococcus sanguinicola JCM 11549T. Each value shown is expressed as a percentage of the total fatty acids. TR, trace (,0.5 %); –, not detected; DMA, dimethylacetal; ECL, equivalent chainlength, Un, unknown. All data were obtained under identical conditions in the present study. Fatty acid Saturated C10 : 0 C12 : 0 C14 : 0 C16 : 0 C16 : 0 DMA C18 : 0 C18 : 0 DMA Unsaturated C16 : 1v9c C18 : 1v9c C18 : 2v6,9c C20 : 1v9c C18 : 1v7c DMA Cyclopropane C19 : 1 cyclo 9,10 Summed features* 10 12 Un (ECL 18.199) and/or C18 : 0 anteiso DMA
1
2
3
4
5
6
7
– – – 2.9 – 4.2 –
– – – 3.9 – 3.3 –
– – – 3.1 – 1.1 –
– – – 2.5 – 1.4 –
–
– – 1.0 3.9
TR
–
– – 1.7 – 2.7 –
TR
– 73.2 – 16.3 –
– 89.8 – – –
– 89.8 – – –
– 93.6 – – –
4.0 80.5 0.5 – 0.9
2.7 82.3 0.7
–
–
4.3
–
3.4 – –
3.0 – –
1.7 – –
1.8 – 0.7
TR
2.5 4.6 – 2.2 –
TR
2.3 0.5
8
3.3 13.4 TR
3.9 TR
– 82.4
8.0 62.2
TR
TR
1.6
– 1.5
2.4 0.7
–
–
–
–
2.0 0.9 1.6
2.1 0.6 1.7
8.4 1.0 1.6
3.6 0.5 0.9
TR
*Summed feature 10: C18 : 111c/9t/6t and/or Un (ECL 17.834). Summed feature 12: Un (ECL 18.622) and/or iso-C19 : 0.
aesculin ferric citrate, salicin, cellobiose, maltose, lactose, melibiose, trehalose, inulin, melezitose, raffinose, starch, glycogen, gentiobiose, turanose, D-lyxose, D-tagatose, Dfucose, L-fucose, L-arabitol, gluconate, 2-ketogluconate and 5-ketogluconate. Using the commercial API ID 32 Strep, API 20 Strep and API ZYM systems, cells are positive for alkaline phosphatase, C4 esterase, leucine aminopeptidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, bglucuronidase, alanine-phenylalanine-proline arylamidase and glycine-tryptophan arylamidase activity. Cells are negative for C8 esterase, C14 lipase, valine aminopeptidase, cystine aminopeptidase, trypsin, chymotrypsin, a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, Nacetyl-b-glucosaminidase, a-mannosidase, a-fucosidase, pyrrolidonyl arylamidase, pyroglutamic acid arylamidase, b-mannosidase and urease activity. Acetoin is produced. The major cellular fatty acid, as determined after growth in MRS broth at 30 uC for 3 days, is C18 : 1v9c. Emended description of Aerococcus christensenii Collins et al. 1999 The species description is based on that of Collins et al. (1999), with the following amendments made for the type strain when grown at 30 uC on MRS medium. In the API 50 CH system (incubated for 7 days at 30 uC), acids are http://ijs.sgmjournals.org
produced from D-glucose, D-fructose and L-sorbose, but acids are not produced from glycerol, erythritol, Darabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-adonitol, methyl b-D-xylopyranoside, D-galactose, Dmannose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl a-D-mannopyranoside, methyl a-Dglucopyranoside, N-acetylglucosamine, amygdalin, arbutin, aesculin ferric citrate, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate and 5-ketogluconate. Using the commercial API ID 32 Strep, API 20 Strep and API ZYM systems, cells are positive for C4 esterase, C8 esterase, leucine aminopeptidase, acid phosphatase, naphthol-ASBI-phosphohydrolase, alanine-phenylalanine-proline arylamidase and glycine-tryptophan arylamidase activity. Cells are negative for alkaline phosphatase, C14 lipase, valine aminopeptidase, cystine aminopeptidase, trypsin, chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fucosidase, pyrrolidonyl arylamidase, pyroglutamic acid arylamidase, b-mannosidase and urease activity. Acetoin is produced. The major cellular fatty acid, as determined after growth in MRS broth at 30 uC for 3 days, is C18 : 1v9c. 1233
M. Tohno and others
Emended description of Aerococcus sanguinicola corrig. Lawson et al. 2001 The species description is based on that of Lawson et al. (2001b), with the following amendments made for the type strain when grown at 30 uC on MRS medium. In the API 50 CH system (incubated for 7 days at 30 uC), acids are produced from glycerol, D-glucose, D-fructose, D-mannose, N-acetylglucosamine, aesculin ferric citrate, maltose, sucrose and trehalose, but acids are not produced from erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, Lxylose, D-adonitol, methyl b-D-xylopyranoside, D-galactose, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl a-D-mannopyranoside, methyl a-Dglucopyranoside, amygdalin, arbutin, salicin, cellobiose, lactose, melibiose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate and 5-ketogluconate. Using the commercial API ID 32 Strep, API 20 Strep and API ZYM systems, cells are positive for C4 esterase, C8 esterase, leucine aminopeptidase, cystine aminopeptidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, b-glucuronidase, pyrrolidonyl arylamidase and pyroglutamic acid arylamidase activity. Cells are negative for alkaline phosphatase, C14 lipase, valine aminopeptidase, trypsin, chymotrypsin, agalactosidase, b-galactosidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fucosidase, alanine-phenylalanine-proline arylamidase, glycine-tryptophan arylamidase, b-mannosidase and urease activity. Acetoin is produced. The major cellular fatty acids, as determined after growth in MRS broth at 30 uC for 3 days, are C18 : 1v9c and C16 : 0. The DNA G+C content of the type strain is 48.4 mol%. Emended description of Aerococcus urinaehominis Lawson et al. 2001 The species description is based on that of Lawson et al. (2001a), with the following amendments made for the type strain when grown at 30 uC on MRS medium. In the API 50 CH system (incubated for 7 days at 30 uC), acids are produced from glycerol, D-ribose, D-glucose, D-fructose, arbutin, aesculin ferric citrate, salicin, cellobiose, maltose, sucrose and trehalose, but acids are not produced from erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, Dadonitol, methyl b-D-xylopyranoside, D-galactose, D-mannose, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl a-D-mannopyranoside, methyl a-Dglucopyranoside, N-acetylglucosamine, amygdalin, lactose, melibiose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, turanose, D-lyxose, D-tagatose, Dfucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate and 5-ketogluconate. Using the commercial API ID 32 Strep, API 20 Strep and API ZYM systems, cells are positive for alkaline phosphatase, C4 esterase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, b-glucuronidase, b-glucosidase and urease activity. Cells are 1234
negative for C8 esterase, C14 lipase, leucine aminopeptidase, valine aminopeptidase, cystine aminopeptidase, trypsin, chymotrypsin, a-galactosidase, b-galactosidase, aglucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fucosidase, pyrrolidonyl arylamidase, alanine-phenylalanine-proline arylamidase, pyroglutamic acid arylamidase, glycine-tryptophan arylamidase and b-mannosidase activity. Acetoin is produced. The major cellular fatty acid, as determined after growth in MRS broth at 30 uC for 3 days, is C18 : 1v9c. The DNA G+C content of the type strain is 43.5 mol%. Emended description of Aerococcus suis Vela et al. 2007 The species description is based on that of Vela et al. (2007), with the following amendments made for the type strain when grown at 30 uC on MRS medium. In the API 50 CH system (incubated for 7 days at 30 uC), acids are produced from D-galactose, D-glucose, D-fructose and maltose, but acids are not produced from glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, Lxylose, D-adonitol, methyl b-D-xylopyranoside, D-mannose, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl a-D-mannopyranoside, methyl a-Dglucopyranoside, N-acetylglucosamine, amygdalin, arbutin, aesculin ferric citrate, salicin, cellobiose, lactose, melibiose, sucrose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate and 5-ketogluconate. Using the commercial API ID 32 Strep, API 20 Strep and API ZYM systems, cells are positive for C4 esterase, C8 esterase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, b-galactosidase and pyrrolidonyl arylamidase activity. Cells are negative for alkaline phosphatase, C14 lipase, leucine aminopeptidase, valine aminopeptidase, cystine aminopeptidase, trypsin, chymotrypsin, a-galactosidase, b-glucuronidase, a-glucosidase, bglucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, afucosidase, alanine-phenylalanine-proline arylamidase, pyroglutamic acid arylamidase, glycine-tryptophan arylamidase, b-mannosidase and urease activity. The major cellular fatty acid, as determined after growth in MRS broth at 30 uC for 3 days, is C18 : 1v9c. Emended description of Aerococcus urinaeequi (Garvie 1988) Felis et al. 2005 The species description is based on that of Felis et al. (2005), with the following amendments made for the type strain when grown at 30 uC on MRS medium. In the API 50 CH system (incubated for 7 days at 30 uC), acids are produced from glycerol, D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, N-acetylglucosamine, amygdalin, arbutin, aesculin ferric citrate, salicin, cellobiose, maltose, lactose, sucrose, trehalose, raffinose, gentiobiose and gluconate, but acids are not produced from erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, D-adonitol, International Journal of Systematic and Evolutionary Microbiology 64
Aerococcus vaginalis sp. nov.
methyl b-D-xylopyranoside, L-sorbose, L-rhamnose, dulcitol, inositol, D-sorbitol, methyl a-D-mannopyranoside, methyl a-D-glucopyranoside, melibiose, inulin, melezitose, starch, glycogen, xylitol, turanose, D-lyxose, D-tagatose, D-fucose, Lfucose, D-arabitol, L-arabitol, 2-ketogluconate and 5-ketogluconate. Using the commercial API ID 32 Strep, API 20 Strep and API ZYM systems, cells are positive for alkaline phosphatase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, b-glucuronidase and b-glucosidase activity. Cells are negative for C4 esterase, C8 esterase, C14 lipase, leucine aminopeptidase, valine aminopeptidase, cystine aminopeptidase, trypsin, chymotrypsin, a-galactosidase, b-galactosidase, a-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fucosidase, pyrrolidonyl arylamidase, alanine-phenylalanine-proline arylamidase, pyroglutamic acid arylamidase, glycine-tryptophan arylamidase, b-mannosidase and urease activity. Hippurate is hydrolysed but aesculin and arginine are not. Acetoin is not produced. The major cellular fatty acid, as determined after growth in MRS broth at 30 uC for 3 days, is C18 : 1v9c. Description of Aerococcus vaginalis sp. nov. Aerococcus vaginalis (va.gi.na9lis. L. n. vagina sheath, vagina; L. masc. suff. -alis suffix denoting pertaining to; N.L. masc. adj. vaginalis pertaining to vagina, of the vagina). Cells are gram-stain-positive, catalase-negative, non-sporeforming and facultatively anaerobic. Cells are coccoid (1.0– 1.5 mm in diameter) and found singly, in pairs, in tetrads or in small groups. After 72 h of cultivation on MRS agar plates at 30 uC, pinpoint colonies are circular, grey–white or white and ,0.5 mm in diameter. Colonies are nonpigmented, circular and ,1 mm in diameter after 72 h on Columbia blood agar at 37 uC, and cells produce an ahaemolytic reaction. Gas is not produced from glucose. Both D- and L-lactic acids are produced from glucose. Using the commercial API 50 CH system, the carbohydrates D-ribose, D-galactose, D-glucose, D-fructose, Dmannose, N-acetylglucosamine, maltose and D-tagatose are fermented. The following carbohydrates are not fermented: glycerol, erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, D-adonitol, methyl b-D-xylopyranoside, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, Dsorbitol, methyl a-D-mannopyranoside, methyl a-D-glucopyranoside, amygdalin, arbutin, aesculin ferric citrate, salicin, cellobiose, lactose, melibiose, sucrose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, turanose, D-lyxose, D-fucose, L-fucose, Darabitol, L-arabitol, gluconate, 2-ketogluconate and 5ketogluconate. Using the commercial API ID 32 Strep, API 20 Strep and API ZYM systems, cells are positive for alkaline phosphatase, C4 esterase, leucine aminopeptidase, cystine aminopeptidase, acid phosphatase, naphthol-ASBI-phosphohydrolase, b-galactosidase, N-acetyl-b-glucosaminidase, alanine-phenylalanine-proline arylamidase and glycine-tryptophan arylamidase activity. Negative reactions http://ijs.sgmjournals.org
are observed for C8 esterase, C14 lipase, valine aminopeptidase, trypsin, chymotrypsin, a-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, a-mannosidase, a-fucosidase, pyrrolidonyl arylamidase, pyroglutamic acid arylamidase, b-mannosidase and urease activity. Nitrate is not reduced to nitrite. Dextran is not produced from sucrose. Hippurate is hydrolysed but aesculin and arginine are not. Acetoin is not produced. Growth is inhibited by 2 % oxgall. Growth occurs at 30–45 uC, pH 6.0–8.0 and in broth supplemented with 4.0 % NaCl. The major cellular fatty acids are C18 : 1v9c and C20 : 1v9c. The type strain is BV2T (5JCM 19163T5DSM 27293T), isolated from the vaginal mucosa of a beef cow maintained in Nasushiobara, Tochigi, Japan. The DNA G+C content of the type strain is 44.7 mol%.
Acknowledgements We thank Ms M. Ezure for technical support and helpful discussions and Dr J. P. Euze´by for advice on the etymology of the new taxon. This study was supported in part by a Grant-in-Aid for Young Scientists (A) (no. 25712032) from Japan Society for the Promotion of Science.
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Syst Bacteriol 49, 1125–1128. Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric
deoxyribonucleic acid deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane-filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229. Felis, G. E., Torriani, S. & Dellaglio, F. (2005). Reclassification of
Pediococcus urinaeequi (ex Mees 1934) Garvie 1988 as Aerococcus urinaeequi comb. nov. Int J Syst Evol Microbiol 55, 1325–1327. Herthelius, M., Gorbach, S. L., Mo¨llby, R., Nord, C. E., Pettersson, L. & Winberg, J. (1989). Elimination of vaginal colonization with
Escherichia coli by administration of indigenous flora. Infect Immun 57, 2447–2451. Kimura, M. (1980). A simple method for estimating evolutionary rates
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sp. nov., isolated from fermented cane molasses. Int J Syst Evol Microbiol 60, 187–190. Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A. & other authors (2007). CLUSTAL W and CLUSTAL_X version 2.0. Bioinformatics
23, 2947–2948. Lawson, P. A., Falsen, E., Ohle´n, M. & Collins, M. D. (2001a).
Aerococcus urinaehominis sp. nov., isolated from human urine. Int J Syst Evol Microbiol 51, 683–686. Lawson, P. A., Falsen, E., Truberg-Jensen, K. & Collins, M. D. (2001b). Aerococcus sanguicola sp. nov., isolated from a human
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tions during the growth of healthy cows. Lett Appl Microbiol 31, 251– 254.
Tohno, M., Kitahara, M., Inoue, H., Uegaki, R., Irisawa, T., Ohkuma, M. & Tajima, K. (2013a). Weissella oryzae sp. nov., isolated from
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Redondo-Lopez, V., Cook, R. L. & Sobel, J. D. (1990). Emerging role
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of lactobacilli in the control and maintenance of the vaginal bacterial microflora. Rev Infect Dis 12, 856–872.
fermented rice grain (Oryza sativa L. subsp. japonica). Int J Syst Evol Microbiol 63, 2957–2962.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406– 425.
Tohno, M., Kitahara, M., Irisawa, T., Masuda, T., Uegaki, R., Ohkuma, M. & Tajima, K. (2013c). Description of Lactobacillus iwatensis sp.
Sakamoto, M., Suzuki, M., Umeda, M., Ishikawa, I. & Benno, Y. (2002). Reclassification of Bacteroides forsythus (Tanner et al. 1986) as
nov., isolated from orchardgrass (Dactylis glomerata L.) silage, and Lactobacillus backii sp. nov. Int J Syst Evol Microbiol 63, 3854–3860. Tohno, M., Kitahara, M., Irisawa, T., Masuda, T., Uegaki, R., Ohkuma, M. & Tajima, K. (2013d). Lactobacillus silagei sp. nov., isolated from
Tannerella forsythensis corrig., gen. nov., comb. nov. Int J Syst Evol Microbiol 52, 841–849.
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cattle. Anim Reprod Sci 82-83, 295–306. Sheldon, I. M., Noakes, D. E., Rycroft, A. N., Pfeiffer, D. U. & Dobson, H. (2002). Influence of uterine bacterial contamination after par-
from subarctic timothy grass (Phleum pratense L.) silage. Int J Syst Evol Microbiol 63, 2526–2531.
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Vela, A. I., Garcı´a, N., Latre, M. V., Casamayor, A., Sa´nchez-Porro, C., Briones, V., Ventosa, A., Domı´nguez, L. & Ferna´ndez-Garayza´bal, J. F. (2007). Aerococcus suis sp. nov., isolated from clinical specimens
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Vintin˜i, E., Ocan˜a, V. & Elena Nader-Macı´as, M. (2004). Effect of
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maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739. Tohno, M., Kobayashi, H., Nomura, M., Kitahara, M., Ohkuma, M., Uegaki, R. & Cai, Y. (2012a). Genotypic and phenotypic character-
lactobacilli administration in the vaginal tract of mice: evaluation of side effects and local immune response by local administration of selected strains. Methods Mol Biol 268, 401–410. Wang, Y., Ametaj, B. N., Ambrose, D. J. & Ga¨nzle, M. G. (2013).
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International Journal of Systematic and Evolutionary Microbiology 64
DOI 10.1099/ijs.0.058081-0
Aerococcus vaginalis sp. nov., isolated from the vaginal mucosa of a beef cow, and emended descriptions of Aerococcus suis, Aerococcus viridans, Aerococcus urinaeequi, Aerococcus urinaehominis, Aerococcus urinae, Aerococcus christensenii and Aerococcus sanguinicola Masanori Tohno,1 Maki Kitahara,2 Shuichi Matsuyama,1 Koji Kimura,1 Moriya Ohkuma2 and Kiyoshi Tajima3 Correspondence Masanori Tohno [email protected]
1
National Agriculture and Food Research Organization, National Institute of Livestock and Grassland Science, Nasushiobara, Tochigi 329-2793, Japan
2
Japan Collection of Microorganisms, RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan
3
National Agriculture and Food Research Organization, National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki 305-0901, Japan
A gram-stain-positive, facultatively anaerobic, non-spore-forming, catalase-negative, coccoidshaped bacterial strain, designated BV2T, was isolated from the vaginal mucosa of a beef cow in Japan. Phylogenetic analysis showed that the isolate shared high 16S rRNA gene sequence similarity (92.9 %) with Aerococcus suis 1821/02T and low similarity (,92.7 %) with any other recognized species of the genus Aerococcus. The DNA G+C content was 44.7 mol%, which is within the range observed among species of the genus Aerococcus (37.5–48.4 mol%). The major cellular fatty acid was C18 : 1v9c, similar to other type strains of species of the genus Aerococcus. The results of genotypic, phenotypic and chemotaxonomic analyses as well as the low degree of DNA–DNA relatedness with all recognized members of the genus Aerococcus indicate that strain BV2T represents a novel species of the genus Aerococcus, for which the name Aerococcus vaginalis sp. nov. is proposed. The type strain is BV2T (5JCM 19163T5DSM 27293T). Emended descriptions of Aerococcus suis, Aerococcus viridans, Aerococcus urinaeequi, Aerococcus urinaehominis, Aerococcus urinae, Aerococcus christensenii and Aerococcus sanguinicola are also presented.
In bovines, the vaginal flora has a significant influence on the health and disease state of the host. Some bovine uterine pathogens, including Escherichia coli, Fusobacterium necrophorum, Arcanobacterium pyogenes and Prevotella melaninogenica, induce severe urogenital inflammation, which is associated with infertility and consequent economic loss (Sheldon & Dobson, 2004; Sheldon et al., 2002, 2006; Williams et al., 2005). Although lactobacilli are not predominantly found in the vaginal mucosa of cows, in contrast to humans, mice and monkeys (Herthelius et al., 1989; Otero et al., 2000; Redondo-Lopez et al., 1990; Vintin˜i et al., 2004), The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain BV2T is AB818493. One supplementary table and one supplementary figure are available with the online version of this paper.
058081 G 2014 IUMS
Printed in Great Britain
it is well known that lactic acid bacteria belonging to the genera Lactobacillus, Leuconostoc, Pediococcus and Weissella are naturally present and that certain strains isolated from the bovine vagina produce bacteriocin (Wang et al., 2013). During the course of isolating bacteria from the bovine reproductive tract, with particular interest in lactic acid bacteria, a bacterial strain (designated BV2T) capable of growing in de Man Rogosa Sharpe (MRS) medium (Difco) was isolated from a vaginal swab sample. The purpose of the present study was to establish the taxonomic position of this novel Aerococcus-like strain. The isolation of vaginal mucosa-associated bacterial strains was conducted basically as described by Otero et al. (2000). Vaginal swab samples were obtained from three healthy crossbred cows (Japanese Black6Holstein-Friesian) maintained at the National Institute of Livestock and Grassland 1229
M. Tohno and others
Science in Nasushiobara, Japan. Sampling was carried out on day 7 of the oestrus cycle (oestrus5day 0). Strain BV2T was isolated from one of the three cows, which had the following basic characteristics: age, 10.3 years; body weight, 750 kg; voluntary waiting period, 1048 days; parity, 4. After a sterile vaginal speculum was gently inserted into the vagina and opened, a long-handled sterile cotton swab was introduced to obtain a mucosal sample from the vaginal fornix. Samples serially diluted 10-fold with sterile PBS were plated on MRS agar and incubated for 4 days at 30 uC in an ANX-1 TE-HER Hard Anaerobox (Hirosawa) with an O2-free atmosphere of N2/H2/CO2 (80 : 10 : 10 %) in order to obtain bacterial isolates. The taxonomic position of strain BV2T was first determined by 16S rRNA gene sequence analysis, according to previous reports (Tohno et al., 2012a, b, 2013a, b). Briefly, the 16S rRNA gene sequences of strain BV2T and the closest recognized relatives were aligned using CLUSTAL X (version 2.0.12) (Larkin et al., 2007), and a distance matrix was obtained using the Kimura two-parameter model (Kimura, 1980). A phylogenetic tree was reconstructed according to the neighbour-joining method (Saitou & Nei, 1987). Maximum-likelihood and maximum-parsimony methods were also applied to the reconstruction of phylogenetic trees using the software package MEGA (version 5.0) (Tamura et al., 2011). The statistical reliability of the topology of the phylogenetic trees was evaluated by bootstrap analysis (1000 replications). T
Strain BV2 had the highest sequence similarity to the type strains of Aerococcus suis (92.9 %), Aerococcus viridans (92.7 %), Aerococcus urinaeequi (92.7 %), Aerococcus urinaehominis (92.5 %) and other members of the genus Aerococcus (,92 %). Neighbour-joining tree analysis showed that strain BV2T represents a distinct phylogenetic lineage close to the genus Aerococcus (Fig. 1). Maximumlikelihood and maximum-parsimony analyses confirmed this with similar topologies. In conjunction with additional data described below, the low degree of 16S rRNA gene similarity as compared with any other species of the genus Aerococcus indicate that strain BV2T represents a novel species of the genus Aerococcus. Therefore, strain BV2T and the reference strains Aerococcus christensenii JCM 18985T, Aerococcus sanguinicola JCM 11549T, Aerococcus suis JCM 18035T, Aerococcus urinae JCM 18986T, Aerococcus urinaeequi JCM 12519T, Aerococcus urinaehominis JCM 18987T and Aerococcus viridans JCM 20461T were maintained in MRS broth at 30 uC for 3–4 days and used for further analyses under identical conditions.
overnight, and the results are expressed as the mean±SD of three independent experiments. An automated RiboPrinter microbial characterization system (Qualicon) was used for ribotyping analysis (Kitahara et al., 2010). Because DNA G+C content measurements have not been conducted in some species of the genus Aerococcus with validly published names, we analysed not only strain BV2T but also all type strains of species of the genus Aerococcus with validly published names. The DNA G+C content of the reference strains was as follows: Aerococcus christensenii JCM 18985T (39.8 mol%), Aerococcus sanguinicola JCM 11549T (48.4 mol%), Aerococcus suis JCM 18035T (37.5 mol%), Aerococcus urinae JCM 18986T (44.1 mol%), Aerococcus urinaeequi JCM 12519T (40.0 mol%), Aerococcus urinaehominis JCM 18987T (43.5 mol%) and Aerococcus viridans JCM 20461T (40.2 mol%), indicating that the DNA G+C content of species of the genus Aerococcus ranges from 37.5 to 48.4 mol%. The DNA G+C content of strain BV2T was 44.7 mol%, which was consistent with the range observed in species of the genus Aerococcus. The DNA–DNA relatedness of strain BV2T with the other Aerococcus type strains was below 7.0 % (both fixed DNA and photobiotin-labelled results), confirming that strain BV2T represents a novel species in the genus Aerococcus (Table S1, available in the online Supplementary Material). The unique taxonomic position of strain BV2T within the genus Aerococcus was also confirmed through analysis of its riboprint band pattern (Fig. S1). For characterization of strain BV2T, both the cellular and colony morphologies were observed after growth on MRS agar plates at 30 uC for 72 h under anaerobic conditions or growth on Columbia blood agar supplemented with 5 % defibrinated horse blood (Oxoid) at 37 uC for 72 h under anaerobic conditions. Gram staining, motility, gas production from glucose, catalase production, spore formation, dextran production from sucrose, and the configuration of lactic acid enantiomers were determined as previously described (Tohno et al., 2013d). MRS broth supplemented with 2 % oxgall was used to test for bile tolerance. Nitrate reduction was analysed using the API rapid ID 32A system. The effects of temperature (4, 10, 15, 25, 30, 37, 45 and 50 uC), pH (3.0, 3.5, 4.0, 5.0, 6.0, 7.0 and 8.0) and NaCl concentration (4, 6.5, 8 and 10 %, w/v) on growth were investigated using cultures grown in MRS broth for 7 days (Tohno et al., 2013b).
For DNA G+C content, DNA–DNA relatedness and Riboprint pattern analyses, genomic DNA of the novel strain and the closely related type strains was extracted and purified, as previously described (Tohno et al., 2013e). The DNA G+C content was determined using HPLC (Kitahara et al., 2010). DNA–DNA relatedness was assessed using the fluorometric microplate method (Ezaki et al., 1989; Tohno et al., 2013c). Hybridization was conducted at 43 uC
To compare the fatty acid profiles between strain BV2T and the reference type strains, cellular fatty acid methyl esters were prepared from cells grown in MRS broth at 30 uC for 3 days and analysed using the Microbial Identification System (Microbial ID; MIDI) (Sakamoto et al., 2002). All fatty acid data in the present study were obtained under identical conditions. The isolate and the reference type strains of the genus Aerococcus were characterized using the API 50 CH (bioMe´rieux), API ID 32 Strep (bioMe´rieux), API 20 Strep (bioMe´rieux) and API ZYM (bioMe´rieux) test strips in duplicate, according to the manufacturer’s instructions. The API 50 CH strips were incubated for 7 days.
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International Journal of Systematic and Evolutionary Microbiology 64
Aerococcus vaginalis sp. nov.
Lactococcus lactis subsp. lactis NCDO 604T (AB100803) Facklamia hominis ATCC 700628T (Y10772)
-/-/-
Dolosicoccus paucivorans 2992-95T (AJ012666)
-/-/74/-/-
Ignavigranum ruoffiae CCUG 37658T (Y16426)
87/75/-
Globicatella sanguinis LMG 18987T (S50214)
67/65/-
Eremococcus coleocola M1832/95/2T (Y17780) Granulicatella elegans ATCC 700633T (AF016390) Abiotrophia defectiva GIFU 12707T (D50541)
95/55/58
Aerococcus vaginalis BV2T (AB818493) Aerococcus urinaehominis CCUG 42038 bT (AJ278341) 100/99/98 59/-/-
-/-/-
Aerococcus suis 1821/02T (AM230658) 54/68/56
Aerococcus urinaeequi IFO 12173 (D87677)
T 100/100/100Aerococcus viridans ATCC 11563 (M58797)
0.02 88/74/68
Aerococcus urinae NCFB 2893T (M77819) 74/92/87 Aerococcus christensenii CCUG 28831T (Y17005) Aerococcus sanguinicola CCUG 43001T (AJ276512)
Fig. 1. 16S rRNA gene sequence-based neighbour-joining tree displaying the phylogenetic relationship of BV2T (bold type) among all species of the genus Aerococcus with validly published names and related taxa. GenBank/EMBL/DDBJ accession numbers are given in parentheses. Lactococcus lactis subsp. lactis NCDO 604T (AB100803) is the outgroup. Bootstrap values greater than 50 % (based on 1000 replications) from neighbour-joining/maximum-likelihood/minimum-evolution analyses are shown at branch points. Bar, 0.02 substitutions per nucleotide position.
The physiological, biochemical and morphological characteristics of strain BV2T and those that differentiate it from closely related type strains of species of the genus Aerococcus are summarized in the species description and Table 1. The phenotypic profile of strain BV2T differed from that of other type strains in the genus Aerococcus, and the ability to ferment D-tagatose and a positive result for N-acetyl-b-glucosaminidase activity uniquely differentiated strain BV2T from the other strains. Fatty acid profile analysis revealed that C18 : 1v9c was the major cellular fatty acid, together with smaller amounts of C16 : 0, C18 : 0 and summed feature 10 (C18 : 1v11c/9t/6t and/or unknown ECL 17.834), similar to other members of the genus Aerococcus (Table 2). The relatively high level of C20 : 1v9c fatty acid is a useful characteristic for differentiating the isolate from the other type strains. Some of the present phenotypic data for the recognized species of the genus Aerococcus grown in MRS broth differed from data reported in species descriptions of cells grown on blood agar at 37 uC. For example, with the type strains of Aerococcus urinaehominis, Aerococcus christensenii and Aerococcus sanguinicola, variable acetoin production results were observed, in contrast http://ijs.sgmjournals.org
to previous reports (Collins et al., 1999; Lawson et al., 2001a, b). These discrepancies can be explained by the fact that some culture conditions, such as medium type and incubation temperature, were different. Although a large divergence from other species was observed with respect to the 16S rRNA gene sequence of strain BV2T (.7.1 %), this novel isolate was not considered to represent a novel genus but was recognized as representing a novel species within the genus Aerococcus on the basis of the similar fatty acid profiles. Thus, BV2T is a novel member of the genus Aerococcus, and the name Aerococcus vaginalis sp. nov. is proposed to accommodate this strain. The descriptions of the recognized species of the genus Aerococcus must be emended in order to incorporate details regarding fatty acid composition and other characteristics when cells are grown in MRS broth at 30 uC. Emended description of Aerococcus viridans Williams et al. 1953 The species description is based on that of Williams et al. (1953), with the following amendments made for the type 1231
M. Tohno and others
Table 1. Differential phenotypic characteristics of Aerococcus vaginalis sp. nov. and other known species of the genus Aerococcus Strains: 1, BV2T; 2, Aerococcus suis JCM 18035T; 3, Aerococcus viridans JCM 20461T; 4, Aerococcus urinaeequi JCM 12519T; 5, Aerococcus urinaehominis JCM 18987T; 6, Aerococcus urinae JCM 18986T; 7, Aerococcus christensenii JCM 18985T; 8, Aerococcus sanguinicola JCM 11549T. All tested characteristics were investigated under identical conditions in the present study. Using the API 50 CH system, all strains produced acid from D-glucose and D-fructose but failed to produce acid from erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, D-adonitol, methyl b-D-xylopyranoside, L-rhamnose, dulcitol, inositol, methyl aD-mannopyranoside, melibiose, inulin, melezitose, starch, glycogen, D-lyxose, D-fucose, L-fucose, L-arabitol, 2-ketogluconate and 5-ketogluconate. Using the API ID 32 Strep system, all strains were negative for b-mannosidase. Using the API ZYM system, all strains were positive for naphthol-AS-BI-phosphohydrolase but negative for C14 lipase, valine aminopeptidase, trypsin, chymotrypsin, a-galactosidase, a-glucosidase, a-mannosidase and a-fucosidase. +, Positive; 2, negative. Characteristic API 20 strep Arginine Hippurate Aesculin Acetoin Pyrrolidonyl arylamidase API 50 CH Glycerol D-Ribose D-Galactose D-Mannose L-Sorbose D-Mannitol D-Sorbitol Methyl a-D-glucopyranoside N-Acetylglucosamine Amygdalin Arbutin Salicin Cellobiose Maltose Lactose Sucrose Trehalose Raffinose Xylitol Gentiobiose Turanose D-Tagatose D-Arabitol Gluconate API ZYM Alkaline phosphatase C4 esterase C8 esterase Leucine aminopeptidase Cystine aminopeptidase
1232
1
2
3
4
5
6
7
8
2 + 2 2 2
+ 2 2 2 +
2 + + 2 2
2 + 2 2 2
2 + + + 2
2 + 2 + 2
2 + 2 + 2
2 + + + +
2 + + + 2 2 2 2 + 2 2 2 2 + 2 2 2 2 2 2 2 + 2 2
2 2 + 2 2 2 2 2 2 2 2 2 2 + 2 2 2 2 2 2 2 2 2 2
+ + + + 2 + 2 + + 2 2 2 + + + + 2 2 2 2 + 2 2 2
+ + + + 2 + 2 2 + + + + + + + + + + 2 + 2 2 2 +
+ + 2 2 2 2 2 2 2 2 + + + + 2 + + 2 2 2 2 2 2 2
2 + 2 2 2 + + 2 + 2 2 2 2 2 2 + 2 2 + 2 2 2 + 2
2 2 2 2 + 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
+ 2 2 + 2 2 2 2 + 2 2 2 2 + 2 + + 2 2 2 2 2 2 2
+ + 2 + +
2 + + 2 2
+ + + 2 2
+ 2 2 2 2
+ + 2 2 2
+ + 2 + 2
2 + + + 2
2 + + + +
Table 1. cont. Characteristic
1
2
3
4
5
6
7
8
Acid phosphatase b-Galactosidase b-Glucuronidase b-Glucosidase N-Acetyl-b-glucosaminidase API ID 32 strep Alanine-phenylalanine-proline arylamidase Pyroglutamic acid arylamidase Glycine-tryptophan arylamidase Urease
+ + 2 2 +
+ + 2 2 2
2 2 2 2 2
+ 2 + + 2
+ 2 + + 2
+ 2 + 2 2
+ 2 2 2 2
+ 2 + 2 2
+ 2 2 2 2 + + 2 2 2 + 2 2 2 2 + + 2 2 2 2 + + 2 2 2 2 2 + 2 2 2
strain when grown at 30 uC on MRS medium. In the API 50 CH system (incubated for 7 days at 30 uC), acids are produced from glycerol, D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, methyl a-D-glucopyranoside, N-acetylglucosamine, cellobiose, maltose, lactose, sucrose and turanose, but acids are not produced from erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, Dadonitol, methyl b-D-xylopyranoside, L-sorbose, L-rhamnose, dulcitol, inositol, D-sorbitol, methyl a-D-mannopyranoside, amygdalin, arbutin, aesculin ferric citrate, salicin, melibiose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, D-lyxose, D-tagatose, Dfucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate and 5-ketogluconate. Using the commercial API ID 32 Strep, API 20 Strep and API ZYM systems, cells are positive for alkaline phosphatase, C4 esterase, C8 esterase and naphthol-AS-BI-phosphohydrolase activity. Cells are negative for C14 lipase, leucine aminopeptidase, valine aminopeptidase, cystine aminopeptidase, trypsin, chymotrypsin, acid phosphatase, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-bglucosaminidase, a-mannosidase, a-fucosidase, pyrrolidonyl arylamidase, alanine-phenylalanine-proline arylamidase, glycine-tryptophan arylamidase, b-mannosidase and urease activity. Hippurate and aesculin are hydrolysed but arginine is not. The major cellular fatty acid, as determined after growth in MRS broth at 30 uC for 3 days, is C18 : 1v9c. Emended description of Aerococcus urinae Aguirre and Collins 1992 The species description is based on that of Aguirre & Collins (1992), with the following amendments made for the type strain when grown at 30 uC on MRS medium. In the API 50 CH system (incubated for 7 days at 30 uC), acids are produced from D-ribose, D-glucose, D-fructose, D-mannitol, D-sorbitol, N-acetylglucosamine, sucrose, xylitol and D-arabitol, but acids are not produced from glycerol, erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, D-adonitol, methyl b-D-xylopyranoside, D-galactose, D-mannose, L-sorbose, L-rhamnose, dulcitol, inositol, methyl a-D-mannopyranoside, methyl a-D-glucopyranoside, amygdalin, arbutin, International Journal of Systematic and Evolutionary Microbiology 64
Aerococcus vaginalis sp. nov.
Table 2. Cellular fatty acid compositions of Aerococcus vaginalis sp. nov. and other species of the genus Aerococcus Strains: 1, BV2T; 2, Aerococcus suis JCM 18035T; 3, Aerococcus viridans JCM 20461T; 4, Aerococcus urinaeequi JCM 12519T; 5, Aerococcus urinaehominis JCM 18987T; 6, Aerococcus urinae JCM 18986T; 7, Aerococcus christensenii JCM 18985T; 8, Aerococcus sanguinicola JCM 11549T. Each value shown is expressed as a percentage of the total fatty acids. TR, trace (,0.5 %); –, not detected; DMA, dimethylacetal; ECL, equivalent chainlength, Un, unknown. All data were obtained under identical conditions in the present study. Fatty acid Saturated C10 : 0 C12 : 0 C14 : 0 C16 : 0 C16 : 0 DMA C18 : 0 C18 : 0 DMA Unsaturated C16 : 1v9c C18 : 1v9c C18 : 2v6,9c C20 : 1v9c C18 : 1v7c DMA Cyclopropane C19 : 1 cyclo 9,10 Summed features* 10 12 Un (ECL 18.199) and/or C18 : 0 anteiso DMA
1
2
3
4
5
6
7
– – – 2.9 – 4.2 –
– – – 3.9 – 3.3 –
– – – 3.1 – 1.1 –
– – – 2.5 – 1.4 –
–
– – 1.0 3.9
TR
–
– – 1.7 – 2.7 –
TR
– 73.2 – 16.3 –
– 89.8 – – –
– 89.8 – – –
– 93.6 – – –
4.0 80.5 0.5 – 0.9
2.7 82.3 0.7
–
–
4.3
–
3.4 – –
3.0 – –
1.7 – –
1.8 – 0.7
TR
2.5 4.6 – 2.2 –
TR
2.3 0.5
8
3.3 13.4 TR
3.9 TR
– 82.4
8.0 62.2
TR
TR
1.6
– 1.5
2.4 0.7
–
–
–
–
2.0 0.9 1.6
2.1 0.6 1.7
8.4 1.0 1.6
3.6 0.5 0.9
TR
*Summed feature 10: C18 : 111c/9t/6t and/or Un (ECL 17.834). Summed feature 12: Un (ECL 18.622) and/or iso-C19 : 0.
aesculin ferric citrate, salicin, cellobiose, maltose, lactose, melibiose, trehalose, inulin, melezitose, raffinose, starch, glycogen, gentiobiose, turanose, D-lyxose, D-tagatose, Dfucose, L-fucose, L-arabitol, gluconate, 2-ketogluconate and 5-ketogluconate. Using the commercial API ID 32 Strep, API 20 Strep and API ZYM systems, cells are positive for alkaline phosphatase, C4 esterase, leucine aminopeptidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, bglucuronidase, alanine-phenylalanine-proline arylamidase and glycine-tryptophan arylamidase activity. Cells are negative for C8 esterase, C14 lipase, valine aminopeptidase, cystine aminopeptidase, trypsin, chymotrypsin, a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, Nacetyl-b-glucosaminidase, a-mannosidase, a-fucosidase, pyrrolidonyl arylamidase, pyroglutamic acid arylamidase, b-mannosidase and urease activity. Acetoin is produced. The major cellular fatty acid, as determined after growth in MRS broth at 30 uC for 3 days, is C18 : 1v9c. Emended description of Aerococcus christensenii Collins et al. 1999 The species description is based on that of Collins et al. (1999), with the following amendments made for the type strain when grown at 30 uC on MRS medium. In the API 50 CH system (incubated for 7 days at 30 uC), acids are http://ijs.sgmjournals.org
produced from D-glucose, D-fructose and L-sorbose, but acids are not produced from glycerol, erythritol, Darabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-adonitol, methyl b-D-xylopyranoside, D-galactose, Dmannose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl a-D-mannopyranoside, methyl a-Dglucopyranoside, N-acetylglucosamine, amygdalin, arbutin, aesculin ferric citrate, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate and 5-ketogluconate. Using the commercial API ID 32 Strep, API 20 Strep and API ZYM systems, cells are positive for C4 esterase, C8 esterase, leucine aminopeptidase, acid phosphatase, naphthol-ASBI-phosphohydrolase, alanine-phenylalanine-proline arylamidase and glycine-tryptophan arylamidase activity. Cells are negative for alkaline phosphatase, C14 lipase, valine aminopeptidase, cystine aminopeptidase, trypsin, chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fucosidase, pyrrolidonyl arylamidase, pyroglutamic acid arylamidase, b-mannosidase and urease activity. Acetoin is produced. The major cellular fatty acid, as determined after growth in MRS broth at 30 uC for 3 days, is C18 : 1v9c. 1233
M. Tohno and others
Emended description of Aerococcus sanguinicola corrig. Lawson et al. 2001 The species description is based on that of Lawson et al. (2001b), with the following amendments made for the type strain when grown at 30 uC on MRS medium. In the API 50 CH system (incubated for 7 days at 30 uC), acids are produced from glycerol, D-glucose, D-fructose, D-mannose, N-acetylglucosamine, aesculin ferric citrate, maltose, sucrose and trehalose, but acids are not produced from erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, Lxylose, D-adonitol, methyl b-D-xylopyranoside, D-galactose, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl a-D-mannopyranoside, methyl a-Dglucopyranoside, amygdalin, arbutin, salicin, cellobiose, lactose, melibiose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate and 5-ketogluconate. Using the commercial API ID 32 Strep, API 20 Strep and API ZYM systems, cells are positive for C4 esterase, C8 esterase, leucine aminopeptidase, cystine aminopeptidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, b-glucuronidase, pyrrolidonyl arylamidase and pyroglutamic acid arylamidase activity. Cells are negative for alkaline phosphatase, C14 lipase, valine aminopeptidase, trypsin, chymotrypsin, agalactosidase, b-galactosidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fucosidase, alanine-phenylalanine-proline arylamidase, glycine-tryptophan arylamidase, b-mannosidase and urease activity. Acetoin is produced. The major cellular fatty acids, as determined after growth in MRS broth at 30 uC for 3 days, are C18 : 1v9c and C16 : 0. The DNA G+C content of the type strain is 48.4 mol%. Emended description of Aerococcus urinaehominis Lawson et al. 2001 The species description is based on that of Lawson et al. (2001a), with the following amendments made for the type strain when grown at 30 uC on MRS medium. In the API 50 CH system (incubated for 7 days at 30 uC), acids are produced from glycerol, D-ribose, D-glucose, D-fructose, arbutin, aesculin ferric citrate, salicin, cellobiose, maltose, sucrose and trehalose, but acids are not produced from erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, Dadonitol, methyl b-D-xylopyranoside, D-galactose, D-mannose, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl a-D-mannopyranoside, methyl a-Dglucopyranoside, N-acetylglucosamine, amygdalin, lactose, melibiose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, turanose, D-lyxose, D-tagatose, Dfucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate and 5-ketogluconate. Using the commercial API ID 32 Strep, API 20 Strep and API ZYM systems, cells are positive for alkaline phosphatase, C4 esterase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, b-glucuronidase, b-glucosidase and urease activity. Cells are 1234
negative for C8 esterase, C14 lipase, leucine aminopeptidase, valine aminopeptidase, cystine aminopeptidase, trypsin, chymotrypsin, a-galactosidase, b-galactosidase, aglucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fucosidase, pyrrolidonyl arylamidase, alanine-phenylalanine-proline arylamidase, pyroglutamic acid arylamidase, glycine-tryptophan arylamidase and b-mannosidase activity. Acetoin is produced. The major cellular fatty acid, as determined after growth in MRS broth at 30 uC for 3 days, is C18 : 1v9c. The DNA G+C content of the type strain is 43.5 mol%. Emended description of Aerococcus suis Vela et al. 2007 The species description is based on that of Vela et al. (2007), with the following amendments made for the type strain when grown at 30 uC on MRS medium. In the API 50 CH system (incubated for 7 days at 30 uC), acids are produced from D-galactose, D-glucose, D-fructose and maltose, but acids are not produced from glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, Lxylose, D-adonitol, methyl b-D-xylopyranoside, D-mannose, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl a-D-mannopyranoside, methyl a-Dglucopyranoside, N-acetylglucosamine, amygdalin, arbutin, aesculin ferric citrate, salicin, cellobiose, lactose, melibiose, sucrose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate and 5-ketogluconate. Using the commercial API ID 32 Strep, API 20 Strep and API ZYM systems, cells are positive for C4 esterase, C8 esterase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, b-galactosidase and pyrrolidonyl arylamidase activity. Cells are negative for alkaline phosphatase, C14 lipase, leucine aminopeptidase, valine aminopeptidase, cystine aminopeptidase, trypsin, chymotrypsin, a-galactosidase, b-glucuronidase, a-glucosidase, bglucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, afucosidase, alanine-phenylalanine-proline arylamidase, pyroglutamic acid arylamidase, glycine-tryptophan arylamidase, b-mannosidase and urease activity. The major cellular fatty acid, as determined after growth in MRS broth at 30 uC for 3 days, is C18 : 1v9c. Emended description of Aerococcus urinaeequi (Garvie 1988) Felis et al. 2005 The species description is based on that of Felis et al. (2005), with the following amendments made for the type strain when grown at 30 uC on MRS medium. In the API 50 CH system (incubated for 7 days at 30 uC), acids are produced from glycerol, D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, N-acetylglucosamine, amygdalin, arbutin, aesculin ferric citrate, salicin, cellobiose, maltose, lactose, sucrose, trehalose, raffinose, gentiobiose and gluconate, but acids are not produced from erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, D-adonitol, International Journal of Systematic and Evolutionary Microbiology 64
Aerococcus vaginalis sp. nov.
methyl b-D-xylopyranoside, L-sorbose, L-rhamnose, dulcitol, inositol, D-sorbitol, methyl a-D-mannopyranoside, methyl a-D-glucopyranoside, melibiose, inulin, melezitose, starch, glycogen, xylitol, turanose, D-lyxose, D-tagatose, D-fucose, Lfucose, D-arabitol, L-arabitol, 2-ketogluconate and 5-ketogluconate. Using the commercial API ID 32 Strep, API 20 Strep and API ZYM systems, cells are positive for alkaline phosphatase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, b-glucuronidase and b-glucosidase activity. Cells are negative for C4 esterase, C8 esterase, C14 lipase, leucine aminopeptidase, valine aminopeptidase, cystine aminopeptidase, trypsin, chymotrypsin, a-galactosidase, b-galactosidase, a-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fucosidase, pyrrolidonyl arylamidase, alanine-phenylalanine-proline arylamidase, pyroglutamic acid arylamidase, glycine-tryptophan arylamidase, b-mannosidase and urease activity. Hippurate is hydrolysed but aesculin and arginine are not. Acetoin is not produced. The major cellular fatty acid, as determined after growth in MRS broth at 30 uC for 3 days, is C18 : 1v9c. Description of Aerococcus vaginalis sp. nov. Aerococcus vaginalis (va.gi.na9lis. L. n. vagina sheath, vagina; L. masc. suff. -alis suffix denoting pertaining to; N.L. masc. adj. vaginalis pertaining to vagina, of the vagina). Cells are gram-stain-positive, catalase-negative, non-sporeforming and facultatively anaerobic. Cells are coccoid (1.0– 1.5 mm in diameter) and found singly, in pairs, in tetrads or in small groups. After 72 h of cultivation on MRS agar plates at 30 uC, pinpoint colonies are circular, grey–white or white and ,0.5 mm in diameter. Colonies are nonpigmented, circular and ,1 mm in diameter after 72 h on Columbia blood agar at 37 uC, and cells produce an ahaemolytic reaction. Gas is not produced from glucose. Both D- and L-lactic acids are produced from glucose. Using the commercial API 50 CH system, the carbohydrates D-ribose, D-galactose, D-glucose, D-fructose, Dmannose, N-acetylglucosamine, maltose and D-tagatose are fermented. The following carbohydrates are not fermented: glycerol, erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, D-adonitol, methyl b-D-xylopyranoside, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, Dsorbitol, methyl a-D-mannopyranoside, methyl a-D-glucopyranoside, amygdalin, arbutin, aesculin ferric citrate, salicin, cellobiose, lactose, melibiose, sucrose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, turanose, D-lyxose, D-fucose, L-fucose, Darabitol, L-arabitol, gluconate, 2-ketogluconate and 5ketogluconate. Using the commercial API ID 32 Strep, API 20 Strep and API ZYM systems, cells are positive for alkaline phosphatase, C4 esterase, leucine aminopeptidase, cystine aminopeptidase, acid phosphatase, naphthol-ASBI-phosphohydrolase, b-galactosidase, N-acetyl-b-glucosaminidase, alanine-phenylalanine-proline arylamidase and glycine-tryptophan arylamidase activity. Negative reactions http://ijs.sgmjournals.org
are observed for C8 esterase, C14 lipase, valine aminopeptidase, trypsin, chymotrypsin, a-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, a-mannosidase, a-fucosidase, pyrrolidonyl arylamidase, pyroglutamic acid arylamidase, b-mannosidase and urease activity. Nitrate is not reduced to nitrite. Dextran is not produced from sucrose. Hippurate is hydrolysed but aesculin and arginine are not. Acetoin is not produced. Growth is inhibited by 2 % oxgall. Growth occurs at 30–45 uC, pH 6.0–8.0 and in broth supplemented with 4.0 % NaCl. The major cellular fatty acids are C18 : 1v9c and C20 : 1v9c. The type strain is BV2T (5JCM 19163T5DSM 27293T), isolated from the vaginal mucosa of a beef cow maintained in Nasushiobara, Tochigi, Japan. The DNA G+C content of the type strain is 44.7 mol%.
Acknowledgements We thank Ms M. Ezure for technical support and helpful discussions and Dr J. P. Euze´by for advice on the etymology of the new taxon. This study was supported in part by a Grant-in-Aid for Young Scientists (A) (no. 25712032) from Japan Society for the Promotion of Science.
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Pediococcus urinaeequi (ex Mees 1934) Garvie 1988 as Aerococcus urinaeequi comb. nov. Int J Syst Evol Microbiol 55, 1325–1327. Herthelius, M., Gorbach, S. L., Mo¨llby, R., Nord, C. E., Pettersson, L. & Winberg, J. (1989). Elimination of vaginal colonization with
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sp. nov., isolated from fermented cane molasses. Int J Syst Evol Microbiol 60, 187–190. Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A. & other authors (2007). CLUSTAL W and CLUSTAL_X version 2.0. Bioinformatics
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Aerococcus urinaehominis sp. nov., isolated from human urine. Int J Syst Evol Microbiol 51, 683–686. Lawson, P. A., Falsen, E., Truberg-Jensen, K. & Collins, M. D. (2001b). Aerococcus sanguicola sp. nov., isolated from a human
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