Enterococcus Flavescens Sp. Nov., a New Species of Enterococci of Clinical Origin RAFFAELLO POMPEI,L* FRANCESCA BERLUTI’I,’ MARIA C

INTERNATIONALJOURNAL OF SYSTEMATICBACERIOLOGY, July 1992, p. 365-369 Vol. 42, No. 3 0020-7713/92/030365-05$02.00/0 Copyright 0 1992, International Union of Microbiological Societies

Enterococcus flavescens sp. nov., a New Species of Enterococci of Clinical Origin RAFFAELLO POMPEI,l* FRANCESCA BERLUTI’I,’ MARIA C. THALLER,2 ANGELA INGIANNI,’ GIAMPIERO CORTIS,3 AND BENEDETI’O DAINELL14 Istituto di Microbiologia, Universita di Cagliari, and Istituto di Medicina Legale, via Porcell4, Cagliari, Istituto di Microbiologk, Universita di Roma “La Sapienza, ” Rome, ’ and Istituto di Medicina Sperimentale, Universita di Chieti, Chieti, Italy

Four yellow-pigmented group D enterococci of uncertain taxonomic position were isolated from several humans with severe infections. The results of DNA composition, DNA-DNA hybridization, fatty acid content, and biochemical property studies demonstrated that these organisms were slightly related to other previously described yellow-pigmented enterococcal species and constitute a new species, for which we propose the name Enterococcusjlavescens. The type strain of E. jlavescens is strain CCM 439.

Until recently, the taxonomy of the group D streptococci J. Coyette, Lihge, Belgium, and some strains (CA strains) was uncertain and not well defined. In 1984 Schleifer and were derived from the collection of the Cagliari Institute of Kilpper-Balz proposed that the genus Enterococcus should Microbiology. Test strains of four recently described species include the species Enterococcus faecalis and Enterococ- were kindly provided by R. R. Facklam, Centers for Disease cus faecium (17). Since then, other species have been Control, Atlanta, Ga. transferred to this genus, and new species have also been Biochemical tests. Biochemical tests were performed by described. The results of biochemical tests, DNA base using previously described methods (8, 12, 13, 16) and by composition determinations, DNA-DNA hybridization ex- using API 20 Strep and API 50CH systems (Ayerst Italiana) periments, penicillin-binding protein determinations, and following the manufacturer’s instructions. Additional tests long-chain fatty acid content determinations have been used were performed as described by Facklam (5). Bacteriolytic to describe as many as 17 different enterococcal species activity was determined in double-layer Micrococcus luteus (1-3, 9, 11, 16, 17, 19). agar plates as described previously by Pompei et al. (15). Three yellow pigment-producing species belonging to the Numerical taxonomy. The similarity coefficient which we enterococcus group have been described previously; these used was the simple matching coefficient, and cluster anal- are Enterococcus casseliflavus, Enterococcus mundtii, and ysis was performed by using the average linkage method Enterococcus sulfireus (2, 3, 12). These species have been (unweighted pair group using mathematical average) (18). isolated mainly from environmental materials (e.g., grass DNA base composition and DNA-DNA hybridization. DNA silage, plants, and soil, etc.); rarely, they have had a clinical base compositions were estimated by subjecting prepara- origin (4, 16). tions to thermal denaturation in standard saline citrate as During a study on the taxonomy of enterococci, four described by Marmur and Doty (lo), using a Beckman model strains of yellow-pigmented group D enterococci (CA-YPE DU70 spectrophotometer equipped with a jacketed cuvette strains) were isolated from clinical specimens. These organ- chamber. DNA-DNA hybridization experiments were car- isms differed consistently from the previously described ried out by using the filter method under optimal (65°C) and species. Biochemical tests, antibiotic susceptibility tests, suboptimal (75°C) conditions for reassociation, as described and plasmid profiles of these human CA-YPE strains re- previously (7). Thermal binding indices (19) were also deter- vealed several peculiarities (14). In this study, the results of mined, as were the thermal elution midpoints of a homodu- DNA-DNA hybridization tests and fatty acid content deter- plex of E. flavescens CA 2T (T = tqe strain) and heterodu- minations indicated that the four atypical CA-WE strains, plexes of E. flavescens strains CA 2 and CA 3, strains CA which were isolated from several humans with severe infec- 2T and CA 4, and strains CA 2T and CA 8. tions, occupy a unique taxonomic position and represent a Long-chain fatty acid analysis. Fatty acid methyl esters new species, for which we propose the name Enterococcus were prepared from packed freeze-dried cells as described flavescens. by Farrow et al. (6) and were analyzed by using a Perkin- Elmer gas chromatograph.

MATERIALS AND METHODS RESULTS AND DISCUSSION Strains. Reference strains were obtained from the Ameri- can Type Culture Collection, Rockville, Md., and the Czech- The strains which we used, their sources and characteris- oslovak Collection of Microorganisms, Brno, Czechoslova- tics, and their DNA base compositions are shown in Table 1. kia (Table 1). The CA-YPE strains were obtained from The guanine-plus-cytosine (G+C) contents of the CA-YPE clinical specimens and have been described in detail else- strains ranged from 42 to 43 mol%. Numerical taxonomy, where (14). Some strains of enterococci were obtained from performed by using the simple matching coefficient and the average linkage methods, showed that the 39 strains which we studied could be clustered into several phena and that the * Corresponding author. CA-YPE strains constituted a homogeneous cluster (Fig. 1).

365 366 POMPEI ET AL. INT. J. SYST.BACTERIOL.

TABLE 2. Results of DNA-DNA hybridization studies performed with E. jlavescens and various enterococcal species % Homology with [32P]DNAfrom E. Strain flavescens 2T TABLE 1. Strains and DNA base compositions of Species enterococcal species Optimum Stringent temp conditions G+C Source and/or content Entemoccus flavescens CA 2T 100 100 Species Strain comments (mol%) CA 3 96 72.7 CA 4 71.6 49.8 Entemoccus CA 2T (= CCM 439T) Human (sepsis) 42 CA 8 104 99.7 flavescens CA 3 (= CCM 440) Human (abscess) 42 Entemoccus CasselifIavus ATCC 2578gT 19.8 12.9 CA 4 Human (abscess) 43 ATCC 25789 36.9 19.5 CA 8 (= CCM 4411 Human (osteo- 42 ATCC 14436 10.5 25.7 myelitis) ATCC 12755 8.7 c2 Enterococcus ATCC 2578gT Plant (MUT 20) 43" Enterococcus mundtii CCM 2801 2.4 <2 casseliflavus ATCC 25789 CCM 2479 45" CA 61 4.8 c2 ATCC 14436 43b Enterococcus gallinarum CCM 2518 6.2 c2 ATCC 12755 R. Hugh 491, 42 Enterococcus faecium ATCC 19434= 5.6 c2 motile CA 108 12.6 <2 Enterococcus CCM 2801 39 CA 15 3.3 <2 mundtii CA 61 Human (urine) 39 CA 120 7.2 c2 Entemoccus CCM 2518 40 Enterococcus durans ATCC 19432T 7.7 c2 gallinarum ATCC 6056 9.1 c2 Entemoccus ATCC 19434T 38" ATCC 11576 7.2 c2 faecium S271 5.6 c2 CA 108 Human (hemo- 38 Enterococcus hirae ATCC 9790 7.2 <2 culture) ATCC 8043T 6.4 c2 CA 15 Human (hemo- 37 ENT 86 8.1 <2 culture) SEG 2.9 <2 CA 120 Human (genital 38 A51b <2 c2 swab) Enterococcus faecalis ATCC 27959 2.9 c2 Enterococcus ATCC 19432T 3gb ATCC 19433T 4.2 <2 durans ATCC 6056 38 ATCC 11700 3.8 c2 ATCC 11576 39 CCM 2541 13.4 c2 S271 J. Coyette 39 CCM 2497 5.8 c2 Enterococcus ATCC 9790 37 CA 176 1.1 <2 hirae ATCC 8043= 3gb CA 18b 2.7 c2 ENT 86 38 Enterococcus avium CA 8s 7.5 <2 SEG J. Coyette 38 CA 15s 8.5 c2 A51b J. Coyette 37 CA 20 8.3 <2 Enterococcus ATCC 27959 39 CA 103b 13.6 c2 faecalis ATCC 19433 39 Enterococcus pseudoavium SS 1277 4.4 <2 ATCC 11700 CCM 1875 39 Enterococcus raffinosus SS 1278 3.1 <2 CCM 2541 E. faecalis 38 Entemoccus malodoratus SS 1279 6.7 c2 subsp. zymo- Entemoccus solitarius SS 1266 5.4 <2 genes CCM 2497 38 CA 176 Human (urine) 39 CA 18b Human (urine) 39 Enterococcus CA 8s Human (urine) 40 avium CA 15s Human (urine) 39 This group was linked to the nearest species (E. casselijla- CA 20 Human (urine) 40 vus) at a similarity level of 87%, while the similarity level CA 103b Human (urine) 40 between the CA-YPE strains and most other species of the Enterococcus SS 1277 R. R. Facklam 40" genus Enterococcus was 73% (the exceptions were Entero- pseudoavium coccus avium, Enterococcus pseudoavium, Enterococcus Enterococcus SS 1278 R. R. Facklam 40" malodoratus, and Enterococcus rafinosus). It is worth not- rafinosus ing that Enterococcus hirae and Enterococcus are Enterococcus SS 1279 R. R. Facklam 38" durans solitarius linked at a similarity level of 88% and that these two species Enterococcus SS 1266 R. R. Facklam 40" are linked to E. faecium at a similarity level of 86%. malodoratus The results of the DNA-DNA hybridization studies dem- Enterococcus NCDO 2379 38 onstrated that the four CA-YPE strains were closely related sulfi4mus to each other. The AT, values supported the hypothesis that strains CA 2=, CA 3, CA 4, and CA 8 should be included a Data from reference 4. Data from reference 2. in the same species (AT, is the difference between the Data from reference 1. melting point of a homoduplex and the melting point of a heteroduplex) (data not shown). In contrast, the CA-YPE strains were not closely related to any of the other Entero- coccus strains studied which belonged to different species (Table 2). VOL.42, 1992 EMEROCOCCUS FLAVESCENS SP. NOV. 367

E.RAFFINOSUS E.PSEUOOAVIUM E.AVIUM E. SOLlTARlUS

E. FAECALIS t E. HlRAE E. DURANS

E. FAECIUM

E. GALLINARUM E. MALOOORATUS E. MUNOTll

E. FLAVESCENS

E. CASSELIFLAVUS

tI 50 60 70 80 90 100

PERCENT SIMILARITY FIG. 1. Dendrogram of phenotypic relationships among the enterocoocal strains studied, based on simple matching coefficients and unweighted average-linked clustering.

E. casseliflavus ATCC 25789 appeared to be the closest antiserum. AU strains hydroee esculin, are Voges- relative, but the hybridization value which we determined Proskauer, arginine dehydrolase, f3-galactosidase, leucine was far below the level of DNA homology that is consis- arylamidase, and pyrrolidonylarylamidase positive, and pro- tent with a relationship at the species level. Moreover, when duce acid from L-arabinose, ~-xylose,galactose, D-fructose, we determined the hybridization values under stringent D-mannose, mannitol, N-acetylglucosamine, amygdalin, ar- conditions, the distance between the CA-YPE strains and butin, salicin, cellobiose, maltose, lactose, sucrose, tre- the other enterococcal species notably increased. There- halose, D-raffinose, inulin, gluconate, rhamnose, and f3-gen- fore, the CA-YPE strains are not closely related to any of tiobiose. All strains are amylase, protease, pyruvate, the previously described species of enterococci, although p-glucuronidase, and alkaline phosphatase negative and fail they belong to the same genus. We propose that these to produce acid from glycerol, erythritol, ribose, ~-xylose, organisms belong to a new species, Enterncoccus fluvescens adonitol, a-methyl-xyloside, L-sorbose, dulcitol, myo-inosi- sp. nov. tol, sorbitol, a-methyl-mannoside, melezitose, starch, glyco- Description of Entemoccus fkrvescens sp. nov. Enterococ- gen, xylitol, D-turanose, D-~YYCOS~,D-tagatose, D- and ~-f!u- cus flavescens (fla. ve' scens. L. adj. flavescens, showing a cose, D- and L-arabitol, and 2- and 5-ketogluconate. Variable yellow color). Cells are ovoid and occur mostly in pairs or results are obtained for growth at 10°C (one strain is able to short chains. Surface colonies on blood or nutrient agar are grow), growth at 45°C (one strain is not able to grow), and circular and smooth with entire edges. Nonhemolytic. Cat- the litmus milk test. With regard to bacteriolytic activity alase negative. Facultatively anaerobic. Yellow pigmented. patterns, the CA-WE strains fall into lyogroup EIII, just as Motile. Antigenic extracts of all strains react with group D E. casselipavus does (Table 3).

TABLE 3. Bacteriolytic activities of E. flavescens and some other species of enteromxi

Species THO + TA + TA + TA SSA + LYOPUP THD 0.02% SA 0.04% BS 0.05% SUR (pH 9.6) Ml-ca Enterococcus fzavescens 2+ - 1+ 2+ - 2+ EIII Enterococcus casseLi'vus 3+ - 1+ 2+ - 2+ EIII Enterococcus gallinanrm 2+ 2+ - 1+ 1+ 2+ EW Enterococcus mundtii 2+ 1+ 1+ - 2+ - EM1 Enterococcus faecium 3+ 1+ 1+ 2+ - 3+ EII Enterococcus faecalis 3+ 3+ 2+ - 4+ 2+ EI Abbreviations: THD, Todd-Hewitt medium (Difw);THO, Todd-Hewitt medium (Oxoid); TA, tryptose agar; SSA, streptoaxcus selective agar (Biolire); SA, sodium wide; BS, bile salts; SUR, suramin; MI-Ca, Micrococcus luteus Ml-Ca used as a substrate for lysis. 368 POMPEI ET AL. hm. J. SYST.BA~RIOL.

TABLE 4. Long-chain fatty acid compositions of E. jhvescens ACKNOWLEDGMENTS strains and E. casselijhvus ATCC 25789 This work was supported by grant 91.00221.PF41 from CNR Fatty acid content (%'.)" target projects FATMA to R.P. and by Biotechnology and Bioin- Strain strumentation Grant 90.00098.70 to M.C.T. c141 c140 C160 C180 We thank Eli0 Dazzi for performing the gas chromatographic 2;; analyses. Enterococcus casseli@vus 6.5 41.0 47.9 4.4 ATCC 25789 REFERENCES Enterococcus frczvescens 1. Collins, M. D., R R Facklam, J. A. E. Farrow, and R. CA 2= 0.6 11.3 30.1 39.6 14.5 Williamson. 1989. Enterococcus mmussp. nov., Enterococ- CA 3 12.6 24.0 33.2 14.4 cus solitanus sp. nov., and Entemoccus psebavium sp. nov. CA 4 0.4 9.7 25.6 40.5 13.7 FEMS Microbiol. Lett. 52283-288. CA 8 4.5 35.7 48.1 8.5 2. Collins, M. D., J. A. E. Farrow, and D. Jones. 1986. Enterococ- cus mundtii sp. nov. Int. J. Syst. Bacteriol. 36:&12. a Examples of abbreviations for fatty acids: Clk0, saturated hexadecanoic Collins, U. M. Rodriguez, N. E. Pigott, and R. R acid; Clel, monounsaturated hexadecanoic acid. None of the strains con- 3. M. D., tained Cis:o acid, C16:1acid, or cyclo-C19 acid (cis-ll,12-methylenoctade- Facklam. 1991. Enternomus &par sp. nov., a new Enterococ- canoic acid). cus species from human sources. Lett. Appl. Microbiol. 12:95- 98. 4. De Vriese, L A., K. Ceyssens, U. M. Rodriguez, and M. D. Collins. 1990. Enterococcus columbae, a species from pigeon The major fatty acids are hexadecanoic acid and monoun- intestine. FEMS Microbiol. Lett. 71:247-252. saturated octadecenoic acid (All); no unsaturated hexade- 5. Facklam, R. R. 1972. Recognition of group D streptococcal canoic fatty acids are detected (Table 4). A minor, but not species of human origin by biochemical and physiological tests. significant, difference in fatty acid composition occurs in Appl. Microbiol. 23: 113 1-1 139. strain CA 8. E. fEavescens is distinguished from E. cusselifla- 6. Farrow, J. A. E., D. Jones, B. A. Philip, and M. D. Collins. v#r by being unable to ferment ribose and by lacking 1983. Taxonomic studies on some group D streptococci. J. Gen. hemolysis on sheep blood. Other tests for separating E. Microbiol. 1291423-1432. 7. Garvie, E. I. 1976. Hybridization between the deoxyribonucleic jhvescens from E. faecium, E. mundtii, and E. sulfureus are acids of some strains of heterofennentative lactic acid bacteria. shown in Table 5. The type strain is strain CA 2 (= CCM Int. J. Syst. Bacteriol. 26:11&122. 439). 8. HenMckson, D. A. 1985. Reagents and stains, p. 1093-1107. In Description of the type strain. The description of the type E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. strain is the same as the description of the species. Shadomy (ed.), Manual of clinical microbiology, 4th ed. Amer-

TABLE 5. Differential characteristics of Enterococcus jhvescens, Enterococcus casselipavus, Enterococcus mundtii, Enterococcus gallinanun, Enterococcus sulfureus, and Enterococcus faeciwn Characteristic E. j%avescens E. casselij%avus E. mundtii E. gaUinanun E. faecium E. sdfureur

Group D antigen +a + + + + Acid produced from: Adonitol - - Amidon -(+I +(-I L-Arabinose + + Glycerol V V Glycogen -(+I Gluconate + Inulin + Mannitol + a-Methyl-D-mannoside -(+I a-Methyl-D-glucoside + D-Raffinose + Rhamnose -(+I Ribose + Sorbitol - Sucrose + + D-Tagatose + - D-Tur anose + ND D-Xylose + - Hydrolysis of hippurate + Production of: Arginine dehydrolase + + - a-Galactosidase V + + fl-Galactosidase + + + fl-Glucuronidase V - Yellow pigment - + Motility + - Hemolysisb B" ND

a +, positive; -, negative; V, variable; +(-), most strains are positive; -(+), most strains are negative; ND, not determined. On Columbia CNA blood agar (sheep blood). On horse blood. VOL. 42,1992 ENTEROCOCCUS FLAVESCENS SP. NOV. 369

ican Society for Microbiology, Washington, D.C. human infections. J. Clin. Microbiol. 29:288&2886. 9. Kilpper-Biilz, R, U. Bnhl, and K. H. Schleifer. 1980. Nucleic 15. Pompei, R, M. C. Thaller, F. Pittaluga, 0. More, and G. Satta. acid homology studies between Peptococcus saccharnlyticus 1992. Analysis of bacterioiytic activity patterns, a new method and various anaerobic and facultatively anaerobic Gram-psi- for the taxonomy of enterococci. Int. J. Syst. Bacteriol. 4237- tive cocci. FEMS Microbiol. Lett. 8205-210. 43. 10. Marmur, J., and P. bow. 1%2. Determination of the base 16. Ruoff, K. I,., L. de la Maza, M. J. Murtagh, J. D. Spargo, and composition of deoxyribonucleic acid from its thermal denatur- M. J. Ferraro. 1990. Species identification of enterococci iso- ation temperature. J. Mol. Biol. 3:208-218. lated from clinical specimens. J. Clin. Microbiol. 2tk435-437. 11. Martinez-Murcia, A. J., and M. D. Collins. 1991. Entemoccus 17. Schleifer, K. H., and R Kilpper-Biilz. 1984. Transfer of Strep- sulfureus, a new yellow pigmented Enterncoccus species. tococcus faecalis and Streptococcus faecium to the genus FEMS Microbiol. Lett. 80:69-74. Entemoccus nom. rev. as Enterncoccus faecalb comb. nov. 12. Mundt, J. O., and W. F. Graham. 1968. Strepttococusfaecium and Entemcocus faecium comb. nov. Int. J. Syst. Bacteriol. var. ciuselifla us, nov. var. J. Bacteriol. 95:2005-2009. 34:31-34. 13. Phillips, E., a& P. Nash. 1985. Culture media, p. 1051-1092. In 18. Sneath, P. H. A., and R R Sokal. 1973. Numerical taxonomy. B. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. W. H. Freeman and Co., San Francisco. Shadomy (ed,), Manual of clinical microbiology, 4th ed. Amer- 19. Vaughan, D. H., W. S. Riggsby, and J. 0. Mundt. 1979. ican Society for Microbiology, Washington, D.C. Deoxyribonucleic acid relatedness of strains of yellow-pig- 14. Pompei, R, G. Lampis, F. Berlutti, and M. C. Thdler. 1991. mented, group D streptococci. Int. J. Syst. Bacteriol. 29:204 Characterization of yellow-pigmented enterococci from severe 212.