INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1987, p. 257-259 Vol. 37, No. 3 0020-7713/87/030257-03$02.00/0 Copyright 0 1987, International Union of Microbiological Societies

Characterization and Identification of Species Isolated from the Intestines of Animals L. A. DEVRIESE,'* A. VAN DE KERCKHOVE,l R. KILPPER-BALZ,* AND K. H. SCHLEIFER2 Faculty of Veterinary Medicine, University of Ghent, B-9000 Ghent, Belgium,' and Lehrstuhl fiir Mikrobiologie, Technische Universitat, Munich, Federal Republic of Germany2

Tests useful for the identification of Enterococcus strains were applied to a collection of isolates from animal intestines and to reference strains, all of which were capable of growth on 40% bile and in 6.5% NaCl. Most strains could be identified as known species, and their characteristics corresponded, with a few exceptions of minor importance, with those described for Enterococcus hirae, Enterococcus durans, Enterococcus mundtii, Enterococcus gallinarum, Enterococcus avium, and Enterococcus casseliflavus. However, some diagnostically important carbohydrate reactions of Enterococcus faecalis and Enterococcus faecium strains differed from those given in the species descriptions and in recent reports. Production of acid from D-raffinose and D-xylose by E.faecium varied with the host species from which the strains were isolated. E. durans and E. gallinarum were isolated only from poultry, whereas E. avium was found only in mammals.

Enterococci are an important group of intestinal MATERIALS AND METHODS whose has undergone important changes in the last few years. The enterococci (sensu Sherman [9]), Strep- Strains. A total of 264 strains (Table 1) were selected tococcus bovis, and Streptococcus equinus have been clas- solely on the basis of their ability to grow in the presence of sified traditionally as serological group D streptococci. How- 40% bile (bile-esculin agar, Difco Laboratories, Detroit, ever, nucleic acid studies have shown that Streptococcus Mich.) and in 6.5% NaCl in brain heart infusion agar (Oxoid faecalis and Streptococcus faecium are only distantly re- Ltd., London, England). The selection was made from a lated to S. bovis and S. equinus (5,7). These studies resulted larger collection of -negative, gram-positive cocci in the proposal to transfer S. faecalis and S. faecium to a and short rods isolated from the intestines of farm animals on new genus, Enterococcus, as E. faecalis and E. faecium (8). mitis salivarius agar (GIBCO, Paisley, Scotland) or brain Other group D streptococci which belong to the enterococcal heart infusion agar (Oxoid) with 10 pg of oxolinic acid

TABLE 1. Number of strains belonging to different Enterococcus species isolated from animal hosts

~ ~~ No. of strains isolated from: Enterococcus species Poultry Cattle Pigs Dogs Horses Sheep Goats Rabbits E. faecalis 25 21 22 17 E. faecium 39 15 11 5 E. hirae 25 6 11 4 E. durans 13 0 0 0 E. gallinarum 4 0 0 0 E. avium 0 2 1 0 E. mundtii 0 1 1 0 E. casselgavus 1 0 0 0 Unknown 2 0 2 2

group have since been transferred to this genus (3), and new (Sigma Chemical Co., St. Louis, Mo.) per ml. All strains species have been added (2, 4). originated from different animals and from different farms or In the present study, the biochemical characteristics of owners except when more than one species was isolated Enterococcus strains isolated from the intestines of animals from a single animal. In such cases, one representative of were studied, and an attempt was made to identify them as each species was included. known Enterococcus species. When necessary, the results The following Enterococcus strains were obtained from were confirmed by deoxyribonucleic acid (DNA) homology the Czechoslovac Collection of Microorganisms, Brno, the studies. Deutsche Sammlung von Mikroorganismen, Gottingen, Fed- eral Republic of Germany, and the National Collection of Dairy Organisms (now National Collection of Food Bacte- ria), Reading, United Kingdom: E. faecalis CCM 2541, E. * Corresponding author. faecium DSM 20477T, CCM 2123, and CCM 2308, E. hirae

257 258 DEVRIESE ET AL. INT.J. SYST.BACTERIOL.

TABLE 2. Characteristics of Enterococcus strains from farm animals No. of strains positive" Characteristic E. faecalis E. faecium E. hirae E. durans E. gallinarum E. avium E. mundtii E. casseliJlavus (96Y (76) (59) (13) (4) (3) (3) (1) Motility 0 0 0 0 4 0 0 1 Pigment (yellow) 0 0 0 0 0 0 3 1 Voges-Proskauer reaction 94 76 59 13 4 3 3 1 Hippurate hydrolysis 85 62 32 0 3 0 0 0 a-Galactosidase 0 68 54 0 4 0 3 1 P-Glucuronidase 0 0 0 0 0 0 0 0 p-Galactosidase 8 69 50 0 4 0 3 1 Arginine dihydrolase 96 76 59 13 4 0 3 1 Acid from: L- Arabinose 0 74 0 0 4 3 3 1 Mannitol 96 71 0 0 4 3 3 1 Sorbitol 96 0 0 0 0 3 3 0 Lactose 96 76 59 12 4 3 3 1 Trehalose 96 76 55 13 4 3 3 1 Inulin 0 0 0 0 0 0 2 1 Raffinose 0 30 15 0 4 0 2 1 Amidon 96 71 50 10 4 0 2 1 Glycogen 2 0 0 0 0 0 1 0 Sucrose 92 70 57 0 3 3 3 1 Sorbose 25 0 0 0 0 3 0 0 Rhamnose 70 7 0 0 0 3 3 1 Melibiose 18 73 59 0 4 0 3 1 Melezitose 72 2 0 0 0 2 2 1 D-xylose 63 20 1 0 4 0 3 1 Adonitol 0 0 0 0 0 2 0 0 Production of gelatinase 34 0 3 0 0 0 0 0

a All strains grew in 6.5% NaCl and 40% bile; all produced pyrrolidonylarylamidase and leucine arylamidase. None was alkaline phosphatase positive; all hydrolyzed esculin and produced acid from ribose. The numbers in parentheses are the numbers of strains tested.

CCM 2423, CCM 2424, DSM 2O16OT, and NCDO 2708, E. occurring species, were isolated from all, or nearly all, of the durans CCM 5612T, E. gallinarum NCDO 2313T, NCDO animal hosts investigated. 2311, and CCM 2518, E. casselijlavus CCM 247gT, E. avium The biochemical reactions of the animal bacterial strains DSM 20063, and E. malodoratus NCDO 847. All of these are shown in Table 2. Some of these reactions differed with strains, except CCM 2541, CCM 2518, and DSM 20063, had the hosts from which the strains were isolated. This was been studied previously by DNA-DNA hybridization analy- particularly noticeable for acid production from D-raifinose ses (3, 4, 5, 8). by strains identified as E. faecium. Of the 39 E. faecium Biochemical tests. Acid production from 0.5% sucrose, strains isolated from poultry, 30 produced acid from D- sorbose, rhamnose, melibiose, D-xylose, adonitol, and raffinose (D-raffinose positive), whereas all 37 of the other E. melezitose was tested in phenol red broth base (GIBCO), faecium strains isolated from different mammalian host and the results were read after 1,2, and 3 days at 37°C. Other species and the 3 collection strains studied were D-raffinose carbohydrates were tested in API Strep galleries (API, Montaillieu-Vercier, France). Pigment production was ex- amined on tryptic soy agar (Oxoid), and motility was tested TABLE 3. DNA homology values for various enterococcia by stab inoculation into medium containing 1% tryptose, % Homology with 35S-labeled 0.5% NaC1, and 0.25% Noble agar (Difco). Dextran and DNA from: levan formation were tested on agar medium containing 5% Source of sucrose (SAC agar; API), and gelatinase production was filter-bound DNA Enterococcus E. gallinarum tested on a medium containing 1% tryptone, 0.1% yeast sp. strain 804acb CCM 2518 extract, 0.3% meat extract, 0.3% gelatin, and 1.5% agar, to Enterococcus sp. strain 804acb 100 16 which Frazier reagent was added after 2 days of incubation. Enterococcus sp. strain AlOa 78 14 Other tests were performed by using API 20 Strep galleries E. faecium DSM 20477T 82 13 in accordance with the instructions of the manufacturer. E. gallinarum CCM 2518 13 100 DNA-DNA hybridization studies were performed as previ- Enterococcus sp. strain A20 13 89 ously described (7). Enterococcus sp. strain A13 12 96 E. gallinarum NCDO 2313T 13 84 E. casselifavus CCM 2479 12 21 RESULTS AND DISCUSSION E. faecalis DSM 20478T 10 10 E. avium DSM 20063 10 11 Of the 264 isolates from animals, 255 could be identified as Lactococcus lactis DSM 20481T 4 6 known Enterococcus species. E. durans and E. gallinarum Streptococcus pyogenes NCTC 8198T 5 6 were found only in poultry, whereas E. avium was isolated ~~ Hybridization was performed under optimal conditions (25OC below the from two cows and one pig but not from poultry (Table 1). E. melting point of DNA). faecalis, E. hirae, and E. faecium, the most frequently Identified as E. faecium by biochemical tests. VOL.37, 1987 CHARACTERIZATION AND IDENTIFICATION OF ENTEROCOCCI 259

TABLE 4. Differences between species descriptions and recent should be identified as E. gallinarum. With few exceptions, reports and results obtained in this work with strains the characteristics of the collection strains studied corre- from animals and international collections sponded to the descriptions given in the literature (3,4,5,8). In our hands, the type strain of E.fuecium, DSM 20477, was Species Characteristic Species Other (reference[s]) description" reports" Of this rhamnose positive and weakly amidon positive and E. study fuecium CCM 2123 was hippurate negative and a-galactosi- E. faecalis Acid from: dase positive. The motility, arginine dehydrolase, and sor- (3, 8) L-Sorbose 25/97b bitol reactions of the E. gallinarum strains did not corre- D-Xylose 63/97 spond with those given in the original description (1) but did Melibiose - - 19/97 agree with those given by Collins et al. (3), who transferred Amidon V V 96/97 this species to the genus Enterococcus. Only the negative Sorbitol V V 97/97 inulin reactions of the four animal strains investigated and of strain CCM 2518 differed from the descriptions given by the Hydrolysis of + + 62/79 hippurate latter investigators. Production of: Most of the differences between our results and those a-Galactosidase NI - 68/79 published in the species descriptions and in other recent P-Galactosidase NI + 69/79 reports can probably be explained by the fact that a much Acid from: larger number of strains from different origins were included D-Rafbose 24/79' in our study. Only acid production from amidon appears Amidon 71/79 significantly more common among the enterococci we exam- Rhamnose 8/79 ined, irrespective of species and origin. Differences between D-Xylose 20179' results obtained in this work and species descriptions are given in Table 4. Only the few E. avium and E. casselijavus E. hirae (4) Acid from melezitose V 0163 strains examined here did not differ from the published E. durans Acid from amidon 10114 descriptions. (3, 8) LITERATURE CITED E. gallinarum Acid from inulin +d 217 1. Bridge, P. D., and P. H. A. Sneath. 1982. Streptococcus gal- (1, 3) linarurn sp. nov. and Streptococcus oralis sp. nov. Int. J. Syst. Bactenol. 32:410-415. E. rnundtii (2) Acid from glycogen 113 2. Collins, M. D., J. A. E. Farrow, andD. Jones. 1986. Enterococcus mundtii sp. nov. Int. J. Syst. Bacteriol. 36:&12. + , Positive reaction; -, negative reaction; NI, not indicated; V, variable. Collins, D., D. Jones, J. A. E. Farrow, R. Kilpper-Balz, and Number of strains positive/number of strains tested. 3. M. Dependent on origin of the strains (see the text). K. H. Schleifer. 1984. Enterococcus avium nom. rev., comb. From Collins et al. (3). nov.; E. cusselifluvus nom. rev., comb. nov.; E. durans nom. rev., comb. nov.; E. gallinarum comb. nov.; and E. malodoratus sp. nov. Int. J. Syst. Bacteriol. 34:220-223. 4. Farrow, J. A. E., and M. D. Collins. 1985. Enterococcus hirae, a negative. The identification of one of the raffinose-positive new species that includes amino acid assay strain NCDO 1258 and strains causing growth depression in young chickens. Int. J. poultry strains, AlOa, was confirmed as E. fuecium by Syst. Bacteriol. 3573-75. DNA-DNA hybridization (Table 3). There was also a corre- 5. Farrow, J. A., D. Jones, B. A. Phillips, and M. D. Collins. 1983. lation between acid production from D-xylose by E.faecium Taxonomical studies on some group D streptococci. J. Gen. and the origin of the strains: most strains from cattle (12 of Microbiol. 129:1423-1432. 15) and dogs (4 of 5) were xylose positive, whereas few 6. Graudal, H. 1952. Motile streptococci. Acta Pathol. Microbiol. strains from poultry (2 of 39) and pigs (2 of ll), none of the Scand. 31:46-50. strains from other species, and none of the collection strains 7. Kilpper-Balz, R., B. L. Williams, R. Lutticken, and K. H. produced acid from D-xylose. The relationship of D-xylose- Schleifer. 1984. Relatedness of "Streptococcus milleri" with negative (DSM 20477T and Wac) and -positive (AlOa) Streptococcus anginosus and Streptococcus constellatus. Syst. Appl. Microbiol. 5596500. strains was confirmed by DNA-DNA hybridization (Table 8. Schleifer, K. H., and R. Kilpper-Balz. 1984. Transfer of Strepto- 3). coccus faecalis and Streptococcus faecium to the genus The results of a DNA homology study of two nonpig- Enterococcus nom. rev. as Enterococcus faecalis comb. nov. mented motile strains from poultry (A20 and A13) and a and Enterococcus fuecium comb. nov. Int. J. Syst. Bacteriol. collection strain, CCM 2518, which was originally classified 34: 31-34. as motile E. fueculis (6) showed that all of these strains 9. Sherman, J. M. 1937. The streptococci. Bacteriol. Rev. 1:3-97.