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Lactobacillus Vaginalis Sp. Nov. from the Human Vagina T INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1989, p. 368-370 VOl. 39. No. 3 0020-7713/89/030368-03$02 .OO/O Copyright 0 1989, International Union of Microbiological Societies Lactobacillus vaginalis sp. nov. from the Human Vagina T. MARTIN EMBLEY,l* NAZIR FAQUIR,l WALTER BOSSART,2 AND MATTHEW D. COLLINS' Microbial Technology Research Unit, North East London Polytechnic, Stratford, London El5 4LZ, and Division of Microbiology, AFRC Institute of Food Research, Reading Laboratory, Shinfeld, Reading RG2 9AT,3 United Kingdom, and Vaccine Division, Solco Bade Ltd., CH 4127 Birsfelden, Switzerland2 Biochemical, chemical, and nucleic acid analyses were carried out on some lactobacilli isolated from the vaginas of patients suffering from trichomoniasis. Phenotypically the strains resembled Lactobacillus ferrnen- turn and L. reuteri. Nucleic acid studies, however, showed that the vaginal strains were distinct from these and all other lactobacilli examined and therefore warrant a new species. The name Lactobacillus vaginalis sp. nov. is proposed. The type strain of L. vaginalis is NCTC 12197. In the course of investigations aimed at developing a glucose fermentation were determined by gas chromatogra- vaccine (Solco Trichovac/Gynatren; Solco Basle Ltd., Birs- phy as described in the Anaerobe LLiboratory Manual (4). felden, Switzerland) against recurrent vaginitis, a number of The isomer of lactic acid produced by the strains in MRS lactobacilli were isolated from vaginal swabs from patients broth was determined with a commercial test kit (Boehringer with trichomoniasis. These strains showed similarities with Mannheim Biochemicals). Lactobacillus fermentum, a species commonly isolated from The guanine-plus-cytosine compositions of the five strains the vagina. L. fermentum, however, exhibits considerable of L. vaginalis varied between 38 and 41 mol%. The results phenotypic diversity (6), and the assignment of the vaginal of preliminary slot blot hybridizations indicated that they strains to this species is uncertain. In view of this, we were homogeneous and distinct from reference strains of L. investigated the relationship of the vaginal strains to repre- fermenturn and L. reuteri. The results of quantitative DNA- sentatives of L. fermenturn and other heterofermentative DNA hybridizations are shown in Table 1. The strains of L. lactobacilli by using biochemical, chemical, and nucleic acid vaginalis formed a single homology group that was 85 to 90% techniques. The results indicated that the vaginal strains related to the type strain L. vaginalis NCTC 12197. Strains form a new species, for which the name Lactobacillus of L. oris and L. reuteri were most closely related to L. vaginalis sp. nov. is proposed. vaginalis NCTC 12197T at 26 to 29% and 24 to 35% homol- Cultures. Details of the strains and their sources are given ogy, respectively. L.fermentum and the remaining reference in Table 1. strains were only distantly related (118%) to L. vaginalis. If DNA base composition and DNA-DNA hybridization. Cells 70% homology under optimum conditions is used as a for deoxyribonucleic acid (DNA) isolation were grown in guideline for species differentiation (9,the DNA data clearly MRS broth (CM361; Oxoid Ltd.) at 25 or 37°C to the late demonstrate that L. vaginalis is a distinct taxon worthy of exponential phase, harvested by centrifugation, and washed separate specific status. Biochemical features for the differ- with Tris-EDTA buffer (10 mM Tris, 1 mM EDTA [pH 8.01). entiation of from and DNA was prepared by a modification of the method of L. vaginalis L.fermentum, L. oris, L. Garvie (2). DNA base composition was estimated from the reuteri are given in Table 2. A full description of L. vaginalis midpoint of the DNA melting profile (T,) in 0.1 x SSC (1 x is given below. SSC is 0.15 M NaCl plus 0.015 M sodium citrate [pH 7.0]), Description of Lactobacillus vaginalis sp. nov. Lactobacillus with DNA from Escherichia coli K-12 as a standard. Prelim- vaginalis (vaginalis. L. gen. n. vaginalis, of the vagina). inary DNA hybridizations were done on Zeta-Probe nylon Cells are rod shaped, are of variable size (0.5 to 0.8 by 1.5 to membranes (Bio-Rad Laboratories Ltd.) with a slot-blot 25 pm), and occur singly or in pairs or short chains. Colonies apparatus (Bio-Rad) and probe DNA labeled with biotin- are white to grey, small to large (1 to 5 mm), and semirough, UTP by nick translation (Bethesda Research Laboratories often with raised areas. Cells are gram positive. Nonmotile. Ltd.). Hybridizations were carried out under semistringent Grows at 30 and 45°C but not at 22 or 48°C. Facultatively conditions (T,,, - 20°C). Quantitative data were generated anaerobic. Catalase negative. Heterofermentative, produc- with radiolabeled DNA and membrane filter hybridizations ing D- and L-lactic acid. Acid is produced from D-galactose, done under optimum conditions (T, - 25°C) (2). D-glUCOSe, lactose, D-levulose, D-mannose, maltose, melibi- Fatty acid analyses. Dried biomass (30 mg) of cells grown ose, D-raffinose, and sucrose. Most strains produce acid in yeast-glucose-phosphate broth (3) was degraded by over- from ribose. Acid is not produced from N-acetylglucos- night methanolysis at 55°C. Long-chain fatty acid methyl amine, adonitol, D-arabinose, L-arabitol, arbutin, cellobiose, esters were purified and analyzed by capillary gas chroma- dulcitol, erythritol, D- and L-fucose, glycerol, glycogen, tography as described previously (1). Capillary gas chroma- inositol, inulin, 2- and 5-ketogluconate, D-lyxose, mannitol, tography-mass spectrometry of picolinyl esters of fatty acids melezitose, a-methy1-D-mannoside, rhamnose, sorbitol, L- was done as described by Wait and Hudson (7). sorbose, starch, D-tagatose, trehalose, D-turanose, xylitol, Biochemical tests. The API 50CH system was used in and D- and L-xylose. Esculin is hydrolyzed slowly by most accordance with the instructions of the manufacturer (API- strains. Produces straight-chain and monounsaturated long- BioMerieux Ltd.).Tests were incubated at 30 and 37°C and chain fatty acids of the vaccenic series and cis-11,12-meth- read after 24 and 48 h and after 7 days. The products of ylene-octadecenoic (lactobacillic) acid. The guanine-plus- cytosine content of the DNA ranges from 38 to 41 mol% (T,). Isolated from the vagina of patients suffering from * Corresponding author. trichomoniasis. 368 VOL. 39, 1989 NOTES 369 TABLE 1. Strain histories, DNA base compositions, and DNA TABLE 1.-Continued homologies of L. vaginafis and other lactobacilli % Relative % Relative binding with Gze- binding with "::F"- Species and Source or other 3H-labeled 'H-labeled strain description DNA from cytosine Species and strain Sourcedescription Or Other DNA from cytosine L. vaginalis 'Ontent L. vuginulis 'Ontent NCTC 12197T (mol%' NCTC 12197' (mol%) L. reuteri L. vaginalis NCDO 2589T ATCC 23272T, DSM NCTC 12197T Human vagina 100 39 20016T; adult human 4 Human vagina 86 38 intestine 6 Human vagina 85 38 NCDO 1089 M. E. Sharpe 33 38 8 Human vagina 90 39 NCDO 219 M. E. Sharpe 24 38 10 Human vagina 99 41 NCDO 230 M. E. Sharpe 27 39 L. acidophilus ATCC 4356T, DSM 16 34 NCDO 2655 Rat 28 40 NCDO 174gT 20079T L. rhamnosus ATCC 11977, NCIB 9 ND L. agilis NCDO DSM 20509T, NCIB 13 41 NCDO 86 76, NCTC 76 2744T 117XT; municipal L. viridescens sewage NCDO 1655T ATCC 1270fiT, DSM 15 43 L. brevis 20410T NCDO 391 ATCC 4356, DSM 16 34 NCDO 403 C. F. Niven 14 43 ~ 20079 ND, Not determined. NCDO 476 12 44 L. buchneri ATCC 4005T, DSM 11 45 NCDO llOT 20057T; tomato TABLE 2. Differential features of L. vaginalis, L. fermenturn, Pulp L. oris, and L. reUteria L. casei NCDO ATCC 393T, DSM 10 ND 161T 20011T No. of the following strains positivec: L. collinoides ATCC 27612T, DSM 8 47 Acid produced NCDO 2746T 20515T, NCIB fromb: L. vaginalis L.fermenturn L. oris L. reuteri (n = 6) (n = 13) (n = 5) (n = 8) 10925T L. confusus Human 12 ND Am ygdalin - + - 1635-85 L- Arabinose - + 7 L. curvatus NCDO DSM 20019T; milk 13 ND ~-Arabitol - + - 2739T Esculin 4D 3 1 L. falsus NCDO Originally L. casei 8 ND D-Fructose + + - 151 D-Furanose - 3 - L. farciminis ATCC 29644T, DSM 11 36 Gentiobiose - + - NCDO 2330T 20184T; sausage Gluconate - 4 5 L. fermenturn D-Mannose + 1 - NCDO 217 M. E. Sharpe 17 ND a-Methylglucoside - 4 2 (strain F3) a- Methylxyloside - 3D - NCDO 223 M. E. Sharpe 17 ND Salicin - 3 - (strain F4) Sucrose + + + L. gasseri NCDO ATCC 33323, DSM 10 34 2233 20243, NCIB " The guanine-plus-cytosine contents for L. vaginalis, L. fermenturn, L. 11718 oris, and L. reuteri were 38 to 41, 42 to 53, 49 to 51, and 37 to 41 mol%, respectively. L. helveticus Jll M. E. Sharpe 13 ND All L. vaginalis strains produced acid from ribose (after 7 days), D- L. hifgardii NCDO ATCC 8290T, DSM 11 40 galactose, D-glucose, lactose, maltose, melibiose, D-raffinose, and saccha- 264T 20176T; wine rose; all L. voginulis strains failed to produce acid from N-acetylglucosamine, L. intestinalis HE1 10 37 adonitol, D-arabinose, L-arabitol, arbutin, cellobiose, dulcitol, erythritol, D- NCDO 2176 and L-fucose, glycerol, glycogen, inositol, inulin, 2- and 5-ketogluconate, L. ke$r NCDO Kefir 12 41 D-lyxose, mannitol, melezitose, a-methyl-D-mannoside, rhamnose, sorbitol, 2132 L-sorbose, starch, D-tagatose, trehalose, D-turanose, xylitol, and D- and 20177, 13 41 L-xylose. L. malefermentans DSM JCM +, All strains positive; -, all strains negative; D, delayed reaction. NCDO 2344 1189 L. minor NCDO 0. Kandler 11 38 1973 The type strain is NCTC 12197. In most respects the L. oris description of the type strain resembles the species descrip- NCDO 2160T NCIB 883 lT; human 29 49 tion. The type strain does not produce acid from ribose and saliva does not hydrolyze esculin.
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