Streptococcus faecium var. casselifavus, nov. var.

Transcription

1 JOURNAL OF BACTERIOLOGY, June 1968, p American Society for Microbiology Vol. 95, No. 6 Printed in U.S.A. Streptococcus faecium var. casselifavus, nov. var. J. ORVIN MUNDT AND WANDA F. GRAHAM Departments ofmicrobiology and Food Technology, University of Tennessee, Knoxville, Tennessee Received for publication 28 March 1968 Streptococcus faecium var. casseliflavus is a gram-positive, spherical cell. The cells occur chiefly as pairs within chains and elongate to ogive-shaped cells during growth. Growth is good on 5% bile salts-agar and in broth at 10 C, and in broth adjusted to ph 9.6 or containing 6.5% NaCl, but many strains fail to grow at 45 C. Litmus is reduced rapidly prior to formation of an acid curd. Few strains release ammonia from arginine or serine. The organism is not proteolytic and does not produce H2S or acetylmethylcarbinol, reduce nitrate, decarboxylate tyrosine, or produce slime on sucrose-agar. Most strains survive heating to 60 C for 30 min. It produces gray colonies on potassium tellurite agar, reduces 2,3,5-triphenyltetrazolium-HCl to a pink color, and ferments cellobiose, dextrin, maltose, mannose, and sorbitol, thus resembling S. faecalis. Like S. faecium, it produces peroxidase but not catalase on heated blood media, dissimilates malate, and ferments arabinose, melibiose, and salicin, but not melezitose. Like both species, it ferments dextrose, galactose, lactose, mannitol, sucrose, trehalose, and citrate. Properties peculiar to the variant include the high ph limiting initiation and termination of growth; the fermentation of a-methyl-d-glucoside, raffinose, and xylose; motility; and growth without blue button formation in ethyl violet broth. The water-soluble, pale lemon-yellow pigment is released into the aqueous phase only after the cell envelope is altered by fat solvents. The bacterium thrives as an epiphyte on plants. During recent years, yellow-pigmented streptococci have been isolated with frequency and in large numbers from plants (20). They constitute a homogeneous group with properties of both Streptococcus faecalis and S. faecium, but they have attributes of physiology, ecology, and motility which set them apart from these species. It is proposed to recognize them as a taxon of streptococci associated with plants, to which the name, S. faecium var. casseliflavus (casseli-, from Kassel's yellow; flavus, yellow) be given. Graudal (10) encountered yellow-pigmented streptococci among his collection of motile cultures, but he did not specify the sources of his cultures; hence, it is not known how many may have originated from human or animal sources. At the time of his publication, the knowledge leading to differentiation between S. faecalis and S. faecium had not developed to the extent it has today, and the existence of streptococci similar in properties to the enterococci in nature and apart from the animal or human host was unknown. He considered the pigmented strains to be identical with S. faecium. MATERIALS AND METHODS Isolation. Methods used for the isolation of the yellow streptococci have been described (20). The cultures included in this study were obtained from a number of samples of a variety of plants, with not more than 20 of the 361 cultures used in the initial studies taken from any one sample. With very few exceptions, a minimum of 50 cultures gathered from a series of platings were employed for the determination of each characteristic. Media and methods. The base media were phenol red broth, azide-dextrose-0.1% yeast extract (ADYE) broth, and tryptic or Trypticase soy broths and agars. In general, standard media of commercial formulation were used. The ability to decarboxylate arginine, ornithine, and tyrosine was determined by the method of Falkow (8); that of arginine was also determined by the method of Deibel, Lake, and Niven (6). Deamination of arginine and serine was determined by the method of Niven, Smiley, and Sherman (21). Diffused growth throughout the medium of Ball and Sellers (1), in contrast to growth along the line of the stab, was taken as presumptive evidence of motility. The need for folic acid was determined by loop inoculation of folic acid-deficient medium with washed cells. Hydrolysis of sodium hippurate was determined according to the method of Coffey and Foley (4). Extraction and examination of the pigment. The acetone-water extract of the pigment from washed cells was examined for xanthophyll according to the method of Wilson (25) and, after treatment with hexane and elution on a column of Sea Sorb 43, according to the method of Bickhoff et al. (3). The aqueous extract of the pigment obtained upon lysis 2005

2 2006 MUNDT AND GRAHAM J. BACTERIOL. of washed cells with ethylenediaminetetraacetic acid (EDTA) and lysozyme was scanned with a Beckman DBG recording spectrophotometer over a range of 320 to 750 m,u. RESULTS Morphology. The bacteria are gram-positive, occurring as single pairs and as pairs within chains. The cells are usually not truly spherical (Fig. la) but rather are bluntly pointed, 0.8,i in diameter and elongating to a length of 1.2,u during growth. Graudal (10) refers to a "constriction in the middle" which is a characteristic feature of the elongated cells. This may be interpreted as occurring in cells undergoing the process of division; however, the feature is observed also in resting cells, and the constriction may be due to the formation of two cells within a common cell wall (Fig. lb). Infrequently during the initial steps of isolation, quite elongated cells, fluctuating in diameter throughout the length of the cell, are observed. Such cells appear, apparently spontaneously, on rare occasions during continued cultivation. Characteristics shared with the enterococci. With few exceptions, cultures of S. faecium var. casseliflavus grow readily on 5% bile salts-agar, in broth at 10 C, and in broth adjusted to ph 9.6 or containing 6.5% NaCl (Table 1). These cultures reduce litmus rapidly prior to formation of an acid curd. Nearly all cultures survive heating in broth adjusted to ph 7.0 for 30 min at 60 C; however, 31.3% of the cultures fail to grow in broth at 45 C, and only 53% of the cultures grow at 48 C. All cultures ferment dextrose, fructose, galactose, lactose, mannitol, sucrose, and trehalose, and utilize citrate, without production of gas. None produces H2S or acetylmethylcarbinol, digests casein or gelatin, hydrolyzes urea, reduces nitrate, or decarboxylates arginine, ornithine, or tyrosine. None utilizes lactic, propionic, tartaric, or acetic acids, or produces slime on sucrose agar. All of Graudal's cultures released ammonia b} All FIG. 1. Electron micrographs ofstreptococcusfaecium var. casseliflavus stained with uranyl nitrate. (a) X 52,500 (upper) and X 60,000 (lower); negative stain (PTA). (b) X 52,500.

3 VOL. 95, 1968 TABLE 1. S. FAECIUM VAR. CASSELIFLAVUS NOV. VAR. Properties of Streptococcus faecium var. casseliflavus Property No. of ~~cultures Percentage positive Growth in broth At 10 C At 45 C At ph With 6.5% NaCl Growth on agar With 5% bile salt With potassium tellurite Reduction of Litmus Methylene blue Triphenyltetrazolium- HCl Deamination of arginine Dissimilation of malate Production of peroxidase Hydrolysis of hippurate Motility Minimal ph for growth Terminal ph 4.5 to Fermentation of a-methyl-d-glucoside Raffinose Xylose from arginine; however, only 14% of the cultures in this study did so. It is a matter of speculation as to whether the earlier bacteriologists rejected cultures as being S. faecium which were negative to this criterion or whether the ability to deaminate arginine may be a property associated with animal residence. Nearly one-fourth of the cultures released ammonia from serine. Characteristics shared with S. faecalis. The variant grows to produce gray, rather than black, punctate colonies on ADYE agar containing potassium tellurite at a concentration of 1:2,500. This feature is also observed in the motile enterococci studied by Lund (16). The variant tolerates % crystal violet. It reduces 2,3, 5-triphenyltetrazolium-HCl to an intermediate pink, rather than to the magenta color, both on the solid medium of Barnes (2) and in the liquid medium of Whittenbury (24). Folic acid is not required for growth. It ferments cellobiose, dextrin, maltose, mannose, and sorbitol. Approximately half the cultures ferment glycerol anaerobically. Characteristics shared with S. faecium. Peroxidase is produced on heated blood-dianisidineagar. Catalase is not produced, either in broth or on heated blood-agar. Malate is dissimilated 200J7 vigorously with production of gas. Arabinose, melibiose, and salicin are fermented by all cultures, and some cultures ferment starch slowly. Melezitose is not fermented, and gluconate is not oxidized. a-hemolysis is apparent on 5% human blood-agar. Characteristics peculiar to the variant. All cultures of the variant ferment raffinose, a sugar fermented also by S. bovis. Enterococci rarely ferment this sugar (18, 22). The variant also ferments a-methyl-d-glucoside and xylose; most cultures ferment inulin, and a small number ferment dulcitol. None of the cultures ferments L-xylose or L-sorbose. Growth is good in the ethyl violet medium of Litsky, Mallman, and Fifield (15), but without production of the blue button characteristic of the enterococci. The terminal ph in 2% dextrose broth after 7 days of incubation is ph 4.5 to 4.7. Growth is rarely initiated below ph 5.0. The majority of the cultures produce an arborescent or diffuse growth when cultured in the medium of Ball and Sellers (1). Pigment. The color of the colony on agar media is a very pale lemon yellow, intensified if the growth of several colonies is raked together. Within the cell and upon extraction, the pigment is stable to oxygen and to treatment with 3 and 30% H202. It differs, therefore, from the pigment noted by Hannay (13) in S. faecalis, which developed a primrose color and then turned brown upon exposure to oxygen. The pigment is watersoluble but not diffusible. It is liberated into the aqueous phase upon treatment of the cells with fat solvents and upon rupture of the cells through treatment with EDTA, lysozyme, and centrifugation. The extracted pigment is not precipitated with 10% trichloroacetic acid or upon heating the solution. The absorption of light begins at 365 m,u and is complete at 377 min. Molisch tests performed on the supernatant fluids ofthe lysozymetreated cells are positive. The properties and the spectra eliminate identity of the pigment as a xanthophyll or a carotinoid, and they do suggest possible identity as a glycosidic flavanol or flavanone. Production of the pigment is a constant property, persisting unchanged in intensity through numerous transfers of cultures and surviving in cultures maintained for several years in frozen storage. Serology. The properties of the bacterium place it with the group D streptococci; however, a poor reaction is obtained with type D antisera. Antigens extracted from the cells by either Lancefield's (14) or Fuller's (9) procedures failed to react with several preparations of commercially prepared sera. M. D. Moody (personal communication)

4 2008 MUNDT AND GRAHAM J. BACTERIOL. detected the antigen in several cultures sent to him. Graudal also obtained a reaction for group D antigen with his cultures (10). Ecology. S. faecium var. casseliflavus has been isolated from edible greens, legumes, corn plants, grasses, cucumbers, several varieties of squash, maturing grains, broccoli, and a variety of leaves and vegetables over a period of several years. The level of population ranges from near 0 to 108/g of plant material. It is frequently the most numerous, and sometimes the only, member of the lactic acid-producing bacteria isolated from plants. DIscussIoN The pigmented organism studied clearly is a member of the group known as the enterococci. Because of essential properties such as the dissimilation of malate, production of peroxidase but not of catalase, and the fermentation of arabinose, melibiose, and salicin, but not of melezitose, they are closely related to S. faecium. Although the factors of pigmentation and ecology per se do not justify status as a variant, other properties do justify the proposal. These include the high ph at which growth is initiated and terminated, as compared with the limits for the enterococci. The majority of the cultures are motile, whereas true enterococci are not (5). Among the sugars, the pigmented bacteria ferment seven sugars also fermented by S. faecalis and S. faecium, five which are fermented by S. faecalis, three which are fermented by S. faecium, and three sugars which neither species ferments. Nuances of traits are observed which further suggest differentiation. S. faecium tolerates a high temperature of incubation very well; the variant does not. Strains of the variant able to grow at 45 C in broth will grow on agar surfaces at this temperature only if the media are incubated in a water bath and not in an air incubator. Therefore, the need for a high environmental relative humidity is indicated. Seasonally, the pignented bacteria thrive during the spring and early summer months, and again in midfall, whereas the enterococci are relatively few in the natural environment until midsummer, after which both incidence and numbers increase (19). The variant grows rapidly at 10 C; many cultures are well developed in broth at 48 hr, whereas 4 or more days are required by S. faecalis and S. faecium. The organism studied appears to be a normal resident of plants and is apparently rarely associated with the human or with animals. None of these organisms exists among our stock cultures of S. faecium isolated from animals; however, 82% of all S. faecium cultures isolated from plants are the pigmented variant. Very astute and observant workers of recent years with modern knowledge and culture media at their command, among them Eichel (7), Guthof (11), Haenel and Mueller-Buethow (12), Medrek and Barnes (17), Mieth (18), Deibel (5), and Rogers and Sarles (23), make no mention of pigmented streptococci in their communications discussing intestinal microflora. One may conclude that these pigmented organisms are not found within the body or in its wastes. Being widespread in nature, these organisms obviously must be ingested, but apparently they have little or no potential to become established in the animal or human intestinal tract. The proposed variant does not account for all strains related to, but not conforming with the properties of, S. faecium. Nonmotile, nonpigmented, unconstricted cultures exist in our stocks which are atypical. Also, Lund (16) studied a group of motile enterococci, one of which was pignented, and concluded that a distinct group of enterococci, differing from S. faecalis and S. faecium, is indicated by the electrophoretic patterns of protein and lipase. There is a suggestion that between the present classical species there exist recognizable islands of concentration to which names may be assigned for taxonomic and other purposes or alternatively that the spectra of properties will lead to a continuum of strains with ill-defined taxonomic boundaries. ACKNOWLEDGMENTS This investigation was supported by Public Health Service research grant UI from the Division of Environmental Engineering and Food Protection. We express our appreciation to Max D. Moody, National Communicable Disease Center, Atlanta, Ga., for detection of the group D antigen; to Brenda L. Brandt, now with Walter Reed Army Medical Center, for the initial studies of the pigmented streptococci; to J. K. Blettner, The University of Tennessee, for screening the extracts of the pigment for xanthophyll; and to Frances L. Ball, MAN Program, Oak Ridge, Tenn., for the electron micrographs. LrrERATURE CITED 1. Ball, R. J., and W. Sellers Improved motility medium. Appl. Microbiol. 14: Barnes, E. M Tetrazolium reduction as a means of differentiating Streptococcus faecalis from Streptococcus faecium. J. Gen. Microbiol. 14: Bickhoff, E. M., L. I. Livingston, G. F. Bailey, and C. R. Thompson Xanthophyll determination in dehydrated alfalfa. J. Assoc. Offic. Agr. Chemists 37: Coffey, J. M., and G. F. Foley An im-

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