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INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, July 1987, p. 257-259 0020-7713/87/030257-03$02.00/0 Copyright 0 1987, International Union of Microbiological Societies Vol. 37, No. 3 Characterization and Identification of Enterococcus 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 bacteria whose taxonomy has undergone important changes in the last few years. The enterococci (sensu Sherman [9]), Streptococcus bovis, and Streptococcus equinus have been classified traditionally as serological group D streptococci. However, nucleic acid studies have shown that Streptococcus faecalis and Streptococcus faecium are only distantly related to S. bovis and S. equinus (5,7). These studies resulted in the proposal to transfer S. faecalis and S . faecium to a new genus, Enterococcus, as E. faecalis and E. faecium (8). Other group D streptococci which belong to the enterococcal MATERIALS AND METHODS Strains. A total of 264 strains (Table 1) were selected solely on the basis of their ability to grow in the presence of 40% bile (bile-esculin agar, Difco Laboratories, Detroit, Mich.) and in 6.5% NaCl in brain heart infusion agar (Oxoid Ltd., London, England). The selection was made from a larger collection of catalase-negative, gram-positive cocci and short rods isolated from the intestines of farm animals on mitis salivarius agar (GIBCO, Paisley, Scotland) or brain 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 E. faecalis E. faecium E. hirae E. durans E. gallinarum E. avium E. mundtii E. casselgavus Unknown 25 39 25 13 4 0 0 1 2 21 15 6 0 0 2 1 0 0 22 11 11 17 5 0 0 1 1 0 2 group have since been transferred to this genus (3), and new species have been added (2, 4). In the present study, the biochemical characteristics of Enterococcus strains isolated from the intestines of animals were studied, and an attempt was made to identify them as known Enterococcus species. When necessary, the results were confirmed by deoxyribonucleic acid (DNA) homology studies. * Corresponding author. Horses Sheep Goats Rabbits 4 0 0 0 0 0 2 (Sigma Chemical Co., St. Louis, Mo.) per ml. All strains originated from different animals and from different farms or owners except when more than one species was isolated from a single animal. In such cases, one representative of each species was included. The following Enterococcus strains were obtained from the Czechoslovac Collection of Microorganisms, Brno, the Deutsche Sammlung von Mikroorganismen, Gottingen, Federal Republic of Germany, and the National Collection of Dairy Organisms (now National Collection of Food Bacteria), Reading, United Kingdom: E. faecalis CCM 2541, E. faecium DSM 20477T, CCM 2123, and CCM 2308, E. hirae 257 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 13 May 2017 07:05:48 258 INT.J. SYST.BACTERIOL. DEVRIESE ET AL. TABLE 2. Characteristics of Enterococcus strains from farm animals No. of strains positive" Characteristic E. faecium (76) E . hirae E. durans (13) E . gallinarum (4) E. avium (3) E . mundtii E . casseliJlavus 0 0 94 85 0 0 8 96 0 0 76 62 68 0 69 76 0 0 59 32 54 0 50 59 0 0 13 0 0 0 0 13 4 0 4 3 4 0 4 4 0 0 3 0 0 0 0 0 0 3 3 0 3 0 3 3 1 1 1 0 1 0 1 1 0 96 96 96 96 0 0 96 2 92 25 70 18 72 63 0 34 74 71 0 76 76 0 30 71 0 70 0 7 73 2 20 0 0 0 0 0 59 55 0 15 50 0 57 0 0 59 0 0 0 12 13 0 0 10 0 0 0 0 0 0 0 0 0 4 4 0 4 4 0 4 4 0 3 0 0 4 0 4 0 0 3 3 3 3 3 0 0 0 0 3 3 3 0 2 0 3 3 3 3 3 2 2 2 1 3 0 3 3 2 3 0 0 1 1 0 1 1 1 1 1 0 1 0 1 1 1 1 0 0 E . faecalis (96Y Motility Pigment (yellow) Voges-Proskauer reaction Hippurate hydrolysis a-Galactosidase P-Glucuronidase p-Galactosidase Arginine dihydrolase Acid from: L- Arabinose Mannitol Sorbitol Lactose Trehalose Inulin Raffinose Amidon Glycogen Sucrose Sorbose Rhamnose Melibiose Melezitose D - x ylose Adonitol Production of gelatinase (59) 0 1 0 3 2 0 (3) (1) 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. durans CCM 5612T, E. gallinarum NCDO 2313T, NCDO 2311, and CCM 2518, E. casselijlavus CCM 247gT,E. avium DSM 20063, and E. malodoratus NCDO 847. All of these strains, except CCM 2541, CCM 2518, and DSM 20063, had been studied previously by DNA-DNA hybridization analyses (3, 4, 5, 8). Biochemical tests. Acid production from 0.5% sucrose, sorbose, rhamnose, melibiose, D-xylose, adonitol, and melezitose was tested in phenol red broth base (GIBCO), and the results were read after 1,2, and 3 days at 37°C. Other carbohydrates were tested in API Strep galleries (API, Montaillieu-Vercier, France). Pigment production was examined on tryptic soy agar (Oxoid), and motility was tested by stab inoculation into medium containing 1% tryptose, 0.5% NaC1, and 0.25% Noble agar (Difco). Dextran and levan formation were tested on agar medium containing 5% sucrose (SAC agar; API), and gelatinase production was tested on a medium containing 1% tryptone, 0.1% yeast extract, 0.3% meat extract, 0.3% gelatin, and 1.5% agar, to which Frazier reagent was added after 2 days of incubation. Other tests were performed by using API 20 Strep galleries in accordance with the instructions of the manufacturer. DNA-DNA hybridization studies were performed as previously described (7). RESULTS AND DISCUSSION Of the 264 isolates from animals, 255 could be identified as known Enterococcus species. E. durans and E . gallinarum were found only in poultry, whereas E. avium was isolated from two cows and one pig but not from poultry (Table 1). E. faecalis, E . hirae, and E. faecium, the most frequently occurring species, were isolated from all, or nearly all, of the animal hosts investigated. The biochemical reactions of the animal bacterial strains are shown in Table 2. Some of these reactions differed with the hosts from which the strains were isolated. This was particularly noticeable for acid production from D-raifinose by strains identified as E . faecium. Of the 39 E. faecium strains isolated from poultry, 30 produced acid from Draffinose (D-raffinose positive), whereas all 37 of the other E. faecium strains isolated from different mammalian host species and the 3 collection strains studied were D-raffinose TABLE 3. DNA homology values for various enterococcia % Homology with 35S-labeled Source of filter-bound DNA Enterococcus sp. strain 804acb Enterococcus sp. strain AlOa E. faecium DSM 20477T E. gallinarum CCM 2518 Enterococcus sp. strain A20 Enterococcus sp. strain A13 E. gallinarum NCDO 2313T E. casselifavus CCM 2479 E. faecalis DSM 20478T E. avium DSM 20063 Lactococcus lactis DSM 20481T Streptococcus pyogenes NCTC 8198T DNA from: Enterococcus E . gallinarum sp. strain 804acb CCM 2518 16 14 13 100 89 96 84 21 10 11 6 6 100 78 82 13 13 12 13 12 10 10 4 5 ~~ Hybridization was performed under optimal conditions (25OC below the melting point of DNA). Identified as E. faecium by biochemical tests. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 13 May 2017 07:05:48 CHARACTERIZATION AND IDENTIFICATION OF ENTEROCOCCI VOL.37, 1987 TABLE 4. Differences between species descriptions and recent reports and results obtained in this work with strains from animals and international collections Species (reference[s]) E. faecalis (3, 8) Characteristic Acid from: L-Sorbose D-Xylose Melibiose Amidon Sorbitol Hydrolysis of hippurate Production of: a-Galactosidase P-Galactosidase Acid from: D-Rafbose Amidon Rhamnose D-Xylose E. hirae (4) Acid from melezitose E. durans (3, 8) Acid from amidon E. gallinarum Acid from inulin (1, 3) E. rnundtii (2) Acid from glycogen Species Other description" reports" this study Of V V V V 25/97b 63/97 19/97 96/97 97/97 + + 62/79 NI NI + - 68/79 69/79 - 24/79' 71/79 8/79 20179' V 0163 10114 +d 217 113 + ,Positive reaction; -, negative reaction; NI, not indicated; V, variable. Number of strains positive/number of strains tested. Dependent on origin of the strains (see the text). From Collins et al. (3). negative. The identification of one of the raffinose-positive poultry strains, AlOa, was confirmed as E . fuecium by DNA-DNA hybridization (Table 3). There was also a correlation between acid production from D-xylose by E. faecium and the origin of the strains: most strains from cattle (12 of 15) and dogs (4 of 5) were xylose positive, whereas few strains from poultry (2 of 39) and pigs (2 of l l ) , none of the strains from other species, and none of the collection strains produced acid from D-xylose. The relationship of D-xylosenegative (DSM 20477T and W a c ) and -positive (AlOa) strains was confirmed by DNA-DNA hybridization (Table 3). The results of a DNA homology study of two nonpigmented motile strains from poultry (A20 and A13) and a collection strain, CCM 2518, which was originally classified as motile E. fueculis (6) showed that all of these strains 259 should be identified as E. gallinarum. With few exceptions, the characteristics of the collection strains studied corresponded to the descriptions given in the literature (3,4,5,8). In our hands, the type strain of E . fuecium, DSM 20477, was rhamnose positive and weakly amidon positive and E. fuecium CCM 2123 was hippurate negative and a-galactosidase positive. The motility, arginine dehydrolase, and sorbitol reactions of the E. gallinarum strains did not correspond with those given in the original description (1)but did agree with those given by Collins et al. (3), who transferred this species to the genus Enterococcus. Only the negative inulin reactions of the four animal strains investigated and of strain CCM 2518 differed from the descriptions given by the latter investigators. Most of the differences between our results and those published in the species descriptions and in other recent reports can probably be explained by the fact that a much larger number of strains from different origins were included in our study. Only acid production from amidon appears significantly more common among the enterococci we examined, irrespective of species and origin. Differences between results obtained in this work and species descriptions are given in Table 4. Only the few E. avium and E . casselijavus strains examined here did not differ from the published descriptions. LITERATURE CITED 1. Bridge, P. D., and P. H. A. Sneath. 1982. Streptococcus gallinarurn sp. nov. and Streptococcus oralis sp. nov. Int. J. Syst. Bactenol. 32:410-415. 2. Collins, M. D., J. A. E. Farrow, andD. Jones. 1986. Enterococcus mundtii sp. nov. Int. J. Syst. Bacteriol. 36:&12. 3. Collins, M. D., D. Jones, J. A. E. Farrow, R. Kilpper-Balz, and K. H. Schleifer. 1984. Enterococcus avium nom. rev., comb. 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 new species that includes amino acid assay strain NCDO 1258 and strains causing growth depression in young chickens. Int. J. Syst. Bacteriol. 3573-75. 5. Farrow, J. A., D. Jones, B. A. Phillips, and M. D. Collins. 1983. Taxonomical studies on some group D streptococci. J. Gen. Microbiol. 129:1423-1432. 6. Graudal, H. 1952. Motile streptococci. Acta Pathol. Microbiol. Scand. 31:46-50. 7. Kilpper-Balz, R., B. L. Williams, R. Lutticken, and K. H. Schleifer. 1984. Relatedness of "Streptococcus milleri" with Streptococcus anginosus and Streptococcus constellatus. Syst. Appl. Microbiol. 5596500. 8. Schleifer, K. H., and R. Kilpper-Balz. 1984. Transfer of Streptococcus faecalis and Streptococcus faecium to the genus Enterococcus nom. rev. as Enterococcus faecalis comb. nov. and Enterococcus fuecium comb. nov. Int. J. Syst. Bacteriol. 34: 31-34. 9. Sherman, J. M. 1937. The streptococci. Bacteriol. Rev. 1:3-97. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 13 May 2017 07:05:48