INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1986, p. 8-12 Vol. 36, No. 1 0020-7713/86/010008-05$02 .OO/O Copyright 0 1986, International Union of Microbiological Societies

Enterococcus mundtii sp. nov.

MATTHEW D. COLLINS,l* JOHN A. E. FARROW,l AND DOROTHY JONES2 Department of Food Microbiology, Food Research Institute, Shinfield, Reading RG2 and Department of Microbiology, School of Medicine and Biological Sciences, University of Leicester, Leicester LEI 7RH,2 United Kingdom

Deoxyribonucleic acid base composition, deoxyribonucleic acid-deoxyribonucleic acid hybridization, and biochemical studies were performed on some problematic enterococci of unknown taxonomic position. Our results indicate that some motile, nonpigmented strains are members of gallinarum, whereas motile, yellow-pigmented strains belong to Enterococcus casselijlavus. Four nonmotile, yellow-pigmented strains were biochemically and genetically distinct from all previously described Enterococcus species and constitute a new species, for which the name Enterococcus mundtii is proposed. The type strain of Enterococcus mundtii is strain NCDO 2375.

The use of chemotaxonomical techniques has done much Garvie (7). DNA base composition was estimated by thermal to clarify the classification of Streptococcus faecalis, Strep- denaturation in standard saline citrate, as described by tococcus faecium, and related streptococci belonging to Garvie (8), using DNAs from Leuconostoc mesenteroides Lancefield serological group D. On the basis of nucleic acid NCDO 768 and Escherichia coli K-12 NCDO 1984 as stan- (deoxyribonucleic acid [DNA]-DNA hybridization, DNA- dards. DNA-DNA hybridizations were performed under ribosomal ribonucleic acid hybridization, and 16s ribosomal optimum conditions (25°C below the melting temperature) by ribonucleic acid oligonucleotide cataloging) studies (6, 13, using the membrane filter method described previously (9). 16), Schleifer and Kilpper-Balz proposed the genus . The peptidoglycan types of two Enterococcus (ex Thiercelin and Jouhaud) to accommodate strains (strains NCDO 2374 and NCDO 2375T [T = type S.faecalis and S. faecium (16). In addition to Enterococcus strain]) were determined. Cells (0.5 g) were disrupted by faecalis and Enterococcus faecium, six other species have using glass beads and a Mickle disintegrator (Mickle Labo- been assigned to this genus (Enterococcus avium, ratory Engineering Co., Gomshall, United Kingdom), and Enterococcus casseliflavus, Enterococcus durans, walls were purified as described by Schleifer and Kandler Enterococcus gallinarum, Enterococcus hirae, and (15). The amino acid compositions of the wall hydrolysates Enterococcus malodoratus) (3, 5). Although the vast major- were determined by using paper chromatography and an ity of enterococci can now be identified with confidence, automatic amino acid analyzer. there are a number of "enterococcus-like" strains whose Long-chain fatty acids. Dry cells (50 mg) were degraded by taxonomic position remains equivocal. These include a acid methanolysis, and fatty acid methyl esters were ana- variety of motile strains isolated from human clinical condi- lyzed by gas-liquid chromatography as described previously tions which have in the past been designated Enterococcus (4). (Streptococcus) faecalis or Enterococcus (Streptococcus) Biochemical tests. Biochemical tests were performed by faecium (2,14) or both, although neither of these species has using the API 20 STREP and API 50CH systems (API been described as motile (16). Similarly, the taxonomic Products, Basingstoke, United Kingdom) according to the position of some yellow-pigmented enterococci isolated instructions of the manufacturer. Test preparations were from soil (19) and plants (20) remains unresolved, although incubated at 37"C, and readings were made after 4, 24, and preliminary DNA-DNA homology studies have indicated 48 h. Motility was tested by using stab inoculation into that they are not related to Enterococcus casselijlavus (20). yeast-glucose-phosphate broth (8) modified to contain 0.1% Other yellow-pigmented enterococci have been isolated glucose, 0.1% lactose, and 0.2% (wthol) agar (Oxoid no. 3; from cows (11) and have been shown by Farrow et al. (6) to Oxoid Ltd., Basingstoke, United Kingdom). Pigment pro- be distinct from previously described Enterococcus species. duction was observed in wet packed cells harvested by In the present investigation, biochemical and chemical centrifugation. studies were performed with representatives of the taxa described above in an attempt to clarify their , Our RESULTS AND DISCUSSION results indicate that the nonpigmented, motile strains cur- The DNA base compositions of the test strains are shown rently listed as variants of Enterococcus faecalis and in Table 1. The nonmotile, nonpigmented strains had gua- Enterococcus faecium are in fact members of Enterococcus nine-plus-cytosine (G + C) contents which ranged from 37 to gallinarum, whereas some yellow-pigmented nonmotile 40 mol%, whereas the motile, nonpigmented strains had a strains from plants and soil represent a new species, for slightly higher G+C content range (40 to 42 mol%). The which the name Enterococcus mundtii is proposed. G+C contents of the motile, yellow-pigmented strains ranged from 42 to 45 mol%, whereas the nonmotile, yellow- MATERIALS AND METHODS pigmented strains had a significantly lower G+C content Strains. Details concerning the test strains and their range (38 to 39 mol%) (Table 1). These values are within the sources are given in Table 1. accepted G+C content range (ca. 37 to 45 mol%) for the DNA base composition and DNA-DNA hybridization. DNA genus Enterococcus (3, 16). Furthermore, the division of the was prepared by using a modification (6) of the method of yellow-pigmented strains into two groups on the basis of DNA base composition is in accordance with the results of Vaughn et al. (20). The results of DNA-DNA hybridization * Corresponding author. experiments are shown in Table 2. The nonmotile,

8 VOL. 36, 1986 ENTEROCOCCUS MUNDTII SP. NOV. 9

TABLE 1. Strain histories, pigment production, motility, and DNA base compositions

Species Strain Source Other designation(s) and/or comments Yellow Motility G+C content pigment (mol%) Enterococcus NCDO 582 Cow teats and hands Yellow-pigmented Streptococcus; isolated by + 38' mundtii of milkers C. L. Hannaya NCDO 2374 Soil Received from J. 0. Mundt (strain MUTK + 39 564)c NCDO 2375T Soil Received from J. 0. Mundt (strain MUTK + 38 559T)" NCDO 2377 Plant Received from J. 0. Mundt (strain MUTK 6) + - 39 Enterococcus NCDO 2372T Plant ATCC 25788T + + 43 casselifavus NCDO 2725 ATCC 12755 (R. Hugh 491; S. faecium, motile) + + 42 NCDO 4725 Human ATCC 27284 (M. Pownall; Lancefield group n, + + 42 motile) ATCC 12817 Human Motile S. faecalis (isolated from oropharyngeal + + 44 regionld ATCC 13638 Human Motile Streptococcus sp. (P. V. Liu 4179; iso- + + 43 lated from a case of chronic ) 7818 Plant Received from B. M. Lund' + 42 7819 Plant Received from B. M. Lund' + 42 78/10 Plant Received from B. M. Lund' + 45 78111 Plant Received from B. M. Lund' + 44 En terococcus NCDO 942T - 38' faecium NCDO 1625 Human H6; Sharpe serotype 23 39 NCDO 1643 Canned ham CH28; Sharpe serotype 35 37 NCDO 1644 Pig PG9; Sharpe serotype 36 - 38 Enterococcus NCDO 1608 Human N42; Sharpe serotype 10 + 40 gailinarum NCDO 1618 Human B74; Sharpe serotype 15 + 41 NCDO 2153 Received as Streptococcus casselifavus + 40 NCDO 2313T + 40' NCDO 2682 Chicken crop, ce- Isolated by R. Fullelf + 40 cum, or duodenum NCDO 2704 Chicken crop, ce- Isolated by R. Fullerf + 41 cum, or duodenum NCDO 2705 Chicken crop, ce- Isolated by R. Fullelf + 40 cum, or duodenum NCDO 2706 Chicken crop, ce- Isolated by R. Fullelf + 42 cum, or duodenum NCDO 2707 Chicken crop, ce- Isolated by R. Fullelf + 42 cum, or duodenum ATCC 12169 Streptococcus sp. + 40 ATCC 12170 Streptococcus sp. + 40 ATCC 12818 Human Motil S. faecalis (isolated from a patient with + 42 ulcerated epidermoid cancer of the gingival ridge)d Enterococcus NCDO 125€JT NCIB 8191T, ATCC 8043T, DSM 20160T 38' hirae NCDO 2558 Yogurt Received as Streptococcus thermophilus 37 Enterococcus NCDO 2369T ATCC 1402ST 39b avium Enterococcus NCDO 596T ATCC 19432T 38b durans Enterococcus NCDO 581T ATCC 19433T 39' faecalis Enterococcus NCDO 846T Gouda cheese 41 malodoratus Enterococcus sp NCDO 2138 Mastitis Roguinsky strain 47-16 40 NCDO 2379 Plant Received from J. 0. Mundt (strain'MUTK 31) 38 Streptococcus NCDO 21ST Mastitis 38 garvieae

a See reference 11. Data from reference 6. Isolated from soil (19). See reference 12. B. M. Lund, Food Research Institute, Norwich, United Kingdom. R. Fuller, Food Research Institute, Reading, United Kingdom. 10 COLLINS, FARROW, AND JONES INT. J. SYST.BACTERIOL.

TABLE 2. Levels of DNA homology of enterococci under optimum conditions % Homology with t3H1DNA from:

Species Strain Enterococcus Enterococcus Enterococrus casseliJIavus Enterococcus Enterococcus sp. mundtii faecium gallinarum strain NCDO NCDO 582 NCDO 1643 ATCC 12817 NCDO 23727’ NCDO 2704 2379 Enterococcus mundtii NCDO 582 100 47 23 24 52 27 NCDO 2374 83 NCDO 2375T 93 25 40 NCDO 2377 79 30 Enterococcus casselifavus NCDO 2372T 40 97 100 59 41 NCDO 2725 91 80 NCTC 4725 79 73 ATCC 12817 100 58 44 ATCC 13638 88 77 7818 95 67 7819 84 66 78/10 80 58 7811 1 80 68 Enterococcus faecium NCDO 942T 44 100 17 25 30 NCDO 1625 37 88 NCDO 1643 31 100 15 17 NCDO 1644 45 118 22 Enterococcus gallinarum NCDO 1608 31 43 107 NCDO 1618 31 43 98 NCDO 2153 29 37 28 77 NCDO 2313T 35 39 103 37 NCDO 2682 26 34 78 NCDO 2704 27 42 29 34 100 NCDO 2705 27 40 82 NCDO 2706 25 31 90 NCDO 2707 29 29 90 ATCC 12169 39 77 ATCC 12170 29 88 ATCC 12818 24 28 86 Enterococcus durans NCDO 596T 46 18 24 37 Enterococcus faecalis NCDO 581T 27 28 21 42 Enterococcus hirae NCDO 125gT 37 14 20 29 Enterococcus malodoratus NCDO 846T 24 13 21 31 Enterococcus sp. NCDO 2138 13 15 31 NCDO 2379 27 100 Streptococcus garvieae NCDO 2MT 10 2 8 18

nonpigmented strains of Sharpe and associates (serotypes from soil (19), one (strain NCDO 2377) was isolated from 23, 35, and 36) (17, 18) were found to be highly related to plants (20), while strain NCDO 582 was isolated from cow Enterococcus faecium. In contrast, the motile, non- teats (11). Strain NCDO 582 was ca. 25 to 50% related to pigmented strains formed a single homology group that was other enterococcal species and 10% related to Streptococcus 77 to 107% related to chicken crop isolate NCDO 2704; in garvieae NCDO 2WT; S. garvieae is also nonmotile, and addition to other chicken isolates, this homology group one strain of this species is known to be pigmented (9). included strains from human and clinical sources and the Although strain NCDO 2379 was nonmotile and yellow type strain of Enterococcus gallinarum (strain NCDO 2313). pigmented, it was not related to the homology group de- Enterococcus gallinarum was described as nonmotile by scribed above or any of the other Enterococcus species Bridge and Sneath (2), but all members of this homology examined. Similarly, strain NCDO 2138, which was isolated group (including the type strain of Enterococcus gallinarum) from mastitis, was found to be distinct from the other were found to be motile in this study. The description of enterococci examined and thus also remains unclassified Enterococcus gallinarum (2) should be amended accord- (Table 2). ingly. Our investigation clarified the taxonomic relationships of a The yellow-pigmented, motile strains with DNA base number of “problematic” enterococci. In particular, the compositions ranging from 42 to 45 mol% G+C formed a identification of Enterococcus casseliflavus and Enterococ- single homology group that was 58 to 80% related to cus gallinarum strains from human sources resolved the Enterococcus casseliflavus NCDO 2372T and 79 to 97% taxonomy of strains previously thought to be motile “vari- related to Enterococcus casseliflavus ATCC 12817. This ants” of Enterococcus faecalis and Enterococcus faecium homology group included three strains from human sources (1, 12, 14). The results of this study also demonstrated that (strains ATCC 12817, ATCC 13638, and NCTC 4725) and nonmotile yellow-pigmented strains NCDO 582, NCDO four strains from vegetable material (10). Four nonmotile, 2374, NCDO 2375T, and NCDO 2377 represent a distinct yellow-pigmented strains formed a distinct homology group species, for which the name Enterococcus mundtii is pro- that was 79 to 93% related to strain NCDO 582. Two of these posed. strains (strains NCDO 2374 and NCDO 2375T) were derived Description of Enterococcus mundtii sp. nov. Enterococcus VOL.36, 1986 ENTEROCOCCUS MUNDTll SP. NOV. 11

TABLE 3. Long-chain fatty acid compositions of Enterococcus mundtii strains Fatty acid composition (%)" Strain

NCDO 582 5.0 1.0 11.0 28.5 1.8 1.o 33.5 2.0 16.2 NCDO 2374 5.0 0.8 10.8 27.9 1.7 0.8 31.5 1.7 19.8 NCDO 2375= 4.1 0.6 9.3 28.0 0.2 0.8 35.0 1.4 20.6 NCDO 2377 4.5 0.5 10.0 27.0 0.8 1.2 34.0 2.0 20.0

' Abbreviations for fatty acids are illustrated by the following examples: Clh:O,straight-chain saturated hexadecanoic acid; ClnI, monounsaturated octadecenoic acid; cyclo-Cly, cis-11,12-methylenoctadecanoicacid. mundtii (mund'ti.i. L. gen. n. mundtii of Mundt; named after melezitose, a-methyl-D-glucoside, a-methyl-xyloside, L- the late J. 0. Mundt, an American microbiologist) cells are sorbose, D-tagatose, D-turanose, xylitol, or L-xylose. All ovoid and occur mostly in pairs or short chains. The cells are strains are Voges-Proskauer positive and hydrolyze esculin; gram positive and nonmotile. Surface colonies on blood agar hippurate is not hydrolyzed. All strains are arginine or nutrient agar are circular, smooth, and entire. A yellow dehydrolase, P-galactosidase, leucine arylamidase, and pigment is produced. Nonhemolytic. Catalase negative. pyrolidonylarylamidase positive; most strains are a- Growth occurs at 10 and 45T, in 6.5% NaCl, and at pH 9.6. galactosidase positive. Alkaline phosphatase negative. Facultatively anaerobic. L-Lactic acid is the major product Reacts with Lancefield group D antisera. Group A type of of glucose fermentation. Acid is produced from N-acetyl- peptidoglycan based upon lysine (LYs-D-As~).The long- glucosamine, amygdalin, L-arabinose, arbutin, cellobiose, chain fatty acids are of the straight-chain saturated, mono- D-frUCtOSe, galactose, P-gentiobiose, lactose, maltose, D- unsaturated, and cyclopropane ring types (Table 3). The mannose, mannitol, melibiose, ribose, salicin, sucrose, major fatty acids are hexadecanoic, octadecenoic (All), and trehalose, and D-xylose. Most strains produce acid from cis-ll,12-rnethylenoctadecanoicacids. The G +C content of a-methyl-D-mannoside, D-raffinose, and rhamnose; variable the DNA ranges from 38 to 39 mol%, as determined by the results are obtained for glycerol and sorbitol. Acid is not melting temperature method. The strains form a distinct produced from adonitol, D-arabinose, D-arabitol, L-arabitol, homology group, as determined by DNA-DNA hybridiza- dulcital, erythritol, b-fucose, L-fucose, glycogen, gluconate, tion. Isolated from cows, the hands of milkers, plants, and inositol, 2-keto-gluconate, 5-keto-gluconate, D-lyxose, soil. The type strain is strain NCDO 2375.

TABLE 4. Differential characteristics of Enterococcus mundtii, Enterococcus cusseliJluvus, Enterococcus duruns, Enterococcus fueciurn, and Enterococcus gallinarum

Characteristic Enterococcus Enterococcus Enterococc~~s Enterococciw Enterococcus mcrndtii ccisseli'jhvi~s durcrns fcrecium gallinurum Acid produced from:" Amidon -(+Ih V +(-I L-Arabinose + + + Glycerol V V V Glycogen - -(+I Gluconate + + Inulin -(+I + + Mannitol + + + Melibiose + + + a-Methyl-D-mannoside +i-1 +(-I -(+I a-Met b yl-D-glucoside - + + D-Raffinose +(-) +(-) + Rhamnose +(-) +(-I --(+I - Sorbitol V - Sucrose + + + D-Tagatose -(+I +(-I D-Turanose +(-I + D-xylose + + + Hydrolysis of hippurate" + Production of" Arginine dehydrolase + +(-) a-Galactosidase +i-) + P-Galact osidase + + P-Glucuronidase Yellow pigment produced t + Motility + P-Hem01 ytic - '' Determined by using the API 50CH system with phenol red as the indicator. +, Positive; -, negative; v, variable; +(-), most strains positive but occasional strains negative; -(+), most strains negative but a few strains positive. ' Hippurate and enzymatic tests were performed with the API 20s system. 12 COLLINS, FARROW, AND JONES INT. J. SYST. BACTERIOL.

Description of the type strain. In most respects the descrip- Syst. Bacteriol. 28:190-193. tion of the type strain resembles the description of the 9. Garvie, E. I., J. A. E. Farrow, and B. A. Phillips. 1981. A species. The type strain produces acid from glycerol, a- taxonomic study of some strains of streptococci which grow at 10°C but not at 45"C, including Streptococcus lactis and Strep- methyl-D-mannoside, sorbitol, rhamnose, and b-raffinose; tococcus cremoris Zentralbl. Bakteriol. Parasitenkd. acid is not produced from amidon or inulin. The type strain Infektionskr. Hyg. Abt. 1 Orig. Reihe C 2:151-165. is a-galactosidase positive. 10. Graham, A. F., and B. M. Lund. 1983. The effect of alkaline pH Enterococcus mundtii is distinguished from Enterococcus on growth and metabolic products of a motile, yellow- casselijlavus, Enterococcus durans, Enterococcus faecium, pigmented Streptococcus sp. J. Gen. Microbiol. 129:2429-2435. and Enterococcus gallinarum by the tests shown in Table 4. 11. Hannay, C. L. 1950. The seralogical identity of a yellow- pigmented Streptococcus. J. Gen. Microbiol. 4:294-297. 12. Hugh, R. 1959. Motile streptococci isolated from the LITERATURE CITED oropharyngeal region. Can. J. Microbiol. 5:351-354. 1. American Type Culture Collection. 1982. Catalogue of strains, 13. Kilpper,Balz, R., G. Fischer, and K. 8. Schleifer. 1982. Nucleic 15th ed. American Type Culture Collection, Rockville, Md. acid hybridication of group N and group D streptococci. Curr. 2. Bridge, P. D., and P. H. A. Sneath. 1982. Streptococcus gal- Microbiol. 7:245-250. Iinarum sp. nov. and Streptococcus oralis sp. nov. Int. J. Syst. 14. Pownall, M. 1935. A motile streptococcus. Br. J. Exp. Pathol. Bacteriol. 32:410415. 16:155-158. 3. Collins, M. D., D. Jones, J. A. E. Farrow, R. Kilpper-Balz, and 15. Schleifer, K. H., and 0. Kandler. 1972. Peptidoglycan types of K. H. Schleifer. 1984. Enterococcus avium nom. rev., comb. bacterial cell walls and their taxonomic implications. Bacteriol. nov.; E. casselijlavus nom. rev., comb. nov.; E. durans nom. Rev. 36:407-477. rev., comb. nov.; E. gallinarum comb. nov.; and E. 16. Schleifer, K. H., and R. Kilpper-Bllz. 1984. Transfer of Strep- malodoratus sp. nov. Int. J. Syst. Bacteriol. 34:220-223. tococcus faeculis and Streptococcus faecium to the genus 4. Farrow, J. A. E., and M. b. Collins. 1984. DNA base composi- Enterococcus nov. rev. as Enterococcus faecalis comb. nov. tion, DNA-DNA homology and long-chain fatty acid studies on and Enterococcus faecium comb. nov. Int. J. Syst. Bacteriol. Streptococcus thermophilus and Streptococcus salivarius. J. 34:3 1-34. Gen. Microbiol. 130:357-362. 17. Sharpe, M. E., and B. G. Fewins. 1960. Serological typing of 5. Farrow, J. A. E., and M. D. Collins. 1985. Enterococcus hirae, Streptococcus faecium and unclassified group D streptococci a new species that includes amino acid assay strain NCDO 1258 from canned hams and pig intestines. J. Gen. Microbiol. and strains causing growth depression in young chickens. Int. J. 23:621-630. Syst. Bacteriol. 3573-75. 18. Sharpe, M. E., and P. M. F. Shattock. 1952. The serological 6. Farrow, J. A. E., D. Jones, B. A. Phillips, and M. D. Collins. typing of group D streptococci associated with outbreaks of 1983. Taxonomic studies on some group D streptococci. J. Gen. neonatal diarrhoea. J. Gen. Microbiol. 6:150-165. Microbiol. 129:1423-1432. 19. Taylor, R. F., M. Ikawa, ahd W. Chesbro. 1971. Carotenoids in 7. Garvie, E. I. 1976. Hybridization between the deoxyribonucleic yellow-pigmented enterococci. J. Bacteriol. 105:676-678. acids of some strains of heterofermentative lactic acid . 20. Vaughan, D. H., W. S. Riggsby, and J. 0. Mundt. 1979. Int. J. Syst. Bacteriol. 26:116-122. Deoxyribonucleic acid relatedness of strains of yellow- 8. Garvie, E. I. 1978. Streptococcus rafinolactis (Orla-Jensen and pigmented, group D streptococci. Int. J. Syst. Bacteriol. Hansen), a group N streptococcus found in raw milk. Int. J. 29:204-212.