INTERNATIONAL JOURNAL of SYSTEMATIC BACTERIOLOGY Vol. 24, No. 2 April 1974, p. 260-277 Printed in U.S.A. Copyright 0 1974 International Association of Microbiological Societies New Genus, Coprococcus, Twelve New Species, and Emended Descriptions of Four Previously Described Species of Bacteria from Human Feces LILLIAN V. HOLDEMAN and W. E. C. MOORE
Anaerobe Laboratory, Virginia Polytechnic Institute and State University,Blacksburg, Virginia24061
A new genus of anaerobic cocci, Coprococcus, and 12 new species of anaerobes, Coprococcus eutactus, C. catus, C. comes, RuminococCus callidus, R. torques, Streptococcus hansenii, Bacteroides eggerthii, Eubacterium eligens, E. formicigenerans, E. hallii, Lactobacillus rogosae, and Clostridium nexile, are described. Emended descriptions and proposed neotype strains for Strepto- coccus constellatus (Prkvot) comb. nov., S. morbillorum (Prkvot) comb. nov., S. in termedius PrCvot, and Eubacterium biiorme (Eggerth) PrCvot are presented.
During quantitative and qualitative studies of The moles percent guanine plus cytosine (G+C) the human fecal flora, a large number of content of the deoxyribonucleic acid (DNA) prepara- previously undescribed bacterial species was tions was determined by the thermal melting point encountered as predominant members of the (T,) method (7) by using an automatic recording flora. Of these species, those for which we have spectrophotometer (Gilford Instrument Laboratories). DNA from Escherichia coli B was included in each set nine or more isolates from six or more persons of analyses as a standard. are described here. In addition, the taxonomic position of some species of cocci is discussed, RESULTS AND DISCUSSION and for Eubacterium biforme (Eggerth) Prbvot, an emended description is presented and a Anaerobic Cocci neotype strain is designated. Coprococcus gen. nov., and new species of the A discussion of the incidence of these species genus Coprococcus as compared with other kinds of organisms of the intestinal flora is published elsewhere (8). Many strains of the anaerobic, gram-positive cocci isolated in this study did not have characteristics that would permit their inclusion in any of the described genera. These strains do MATERIALS AND METHODS not belong to any presently described genera in the family Peptococcaceae Rogosa (14). They The organisms described in this report were isolated are unlike ruminococci because they produce and characterized according to anaerobic tube culture butyric acid. They do not occur in packets, so methods and procedures previously described (5, 8, they are not members of Sarcina. They are 18). unlike Pep tococcus and Peptostreptococcus Blood agar plates (BAP) were prepared aerobically on the day of use from brain heart infusion agar because carbohydrates appear to be the main (BHIA; Difco) dehydrated base with 0.5% yeast energy source, whereas peptones are used as a extract, vitamin K-hemin solution (5), and 5% sheep nitrogen source. blood added. For this group of organisms we propose the Egg yolk agar plates (EYA) were stored in an name Coprococcus gen. nov. (Co’pro.coc’cus anaerobe jar if not used the day of preparation and Gr. n. copro feces; Gr. n. coccus berry; M.L. were streaked within 5 days of the preparation date. If masc. n. Coprococcus fecal coccus). Copro- growth did not occur, organisms were retested by coccus includes those gram-positive, anaerobic using freshly prepared medium. cocci that actively ferment carbohydrates, Plates were incubated in vented GasPak jars in an atmosphere of 90% hydrogen-10% CO, after three producing butyric and acetic acids with formic series of partial evacuation and filling with the gas or propionic and/or lactic acids. Fermentable mixture. carbohydrates are either required or are highly Production of H,S was determined in prereduced stimulatory for growth and continued subcul- SIM medium (BBL). ture. The G+C content of the DNA of the 260 VOL. 24, 1974 NEW GENUS, NEW SPECIES, AND EMENDED DESCRIPTIONS 261
strains tested is 39 to 42 mol%. The type sediment; there usually was a slight to moderate species is C. eutactus sp. nov. turbidity; after incubation for 5 days the pH Because the coprococci are gram-positive, was 4.7 to 5.0. anaerobic cocci, they are classified in the family Growth occurred equally well at 37 and 45 Peptococcaceae in the order Eubacteriales. C; growth was poor to moderate at 25 and 30 Characteristics useful in differentiating Copro- C. coccus from other genera in the family are Biochemical reactions. The characteristics of given in Table 1. Descriptions of three new 59 isolates of this species are given in Table 2. species of Coprococcus are given in Table 2 and In addition, the type strain and five other in the following discussion. strains tested fermented dextrin, reduced neu- tral red, and produced acetylmethylcarbinol (i) Coprococcus eutactus sp. nov. (Gr. adj. (AMC). The six strains tested did not ferment eu. tac’tus orderly, well disciplined [referring to adonitol, glycerol, inulin, or sorbose and there- the uniform reactions of the different strains] .) fore grow poorly, if at all, in PY media Obligately anaerobic, nonmotile, gram-positive containing these substrates. No reaction ’was cocci which usually occur in pairs. Cells may produced on EYA by five strains; one strain did decolorize easily, particularly in media contain- not grow on the surface of EYA incubated ing a fermentable carbohydrate. Cells were anaerobically. No ammonia was detected from usually round, and 0.7 to 1.3 pm in diameter; cultures in chopped meat, PY, PY-glucose, or they could be slightly elongate in peptone-yeast arginine medium. Hippurate was not hydro- extract (PY)-glucose cultures (Fig. 1). lyzed, and H2 S (SIM) was not produced. Colonies. After incubation for 5 days in Fermentation products. Fermentation pro- rumen fluid-glucose-cellobiose agar (RGCA) roll ducts (average milliequivalents per 100 ml of tubes, colonies were 0.5 to 2 mm in diameter, culture) were as follows. white or tan, lenticular, and translucent; the From PY-glucose: Formic (1.5 to 2.0), centers of the colonies were occasionally butyric (0.9), lactic (0.7), and acetic (0.5) granular or opaque. acids, sometimes with ethanol and trace On anaerobic BAP incubated for 2 days, amounts of pyruvic and succinic acids. Abun- surface colonies of the type strain were dant hydrogen was produced. punctiform, circular, entire, convex, translu- From PY-pyruvate: Formate (1.3) and ace- cent, whitish, smooth, shiny, and without tate (0.8), sometimes with trace amounts of hemolytic activity. There was no growth on lactate, butyrate, and succinate. BAP incubated in a candle jar or in an aerobic From PY: Occasionally trace amounts of atmosphere. acetic, succinic, or butyric acids. Cultural characteristics. There was poor or no Threonine was not converted to propionate. growth in PY broth without fermentable Lactate and gluconate were not used. carbohydrate. PY-glucose cultures had abun- G+C content of the DNA: 41 mol% by T, dant growth and a smooth (occasionally ropy) for the type strain.
TABLE 1. Characteristics of genera of Peptococcaceae Rogosaa
Peptone, a Lactic, the Butyric or other Mol% Cell major energy Carbohydrate sole major 3+ carbon vola- G+C Genus arrangement source fermented !acid product tile acids produced
cbprococcus Ruminococcus Strep tococcus C. eutactus C catus C. comes R. callidus R. torques hansenii Substrate or ATCC 17 ATCC 22 ATCC 18 ATCC 53 reaction 27759 (1 2/10) 27761 (14/9) 27758 (1 2/11) 27760 (27/10)
Am ygdalin a a W- - V Arab in0 se - aw aw Cello biose a W -W - a a Esculin V - - -W - -W Esculin hydrolysis + + V V +- W V Fructose a a a a -W -W a
Glucose a a a a a- a a Lactose a a aw a a- a a Maltose a a a a a a W- Mannitol - V W Mannose a a -W -W -W -W W-
Melezit ose a a - - -a MeIibio se a a V -W a- a Raffinose a a aw a a a Ribose -W -
Salicin aw a -a -W V a- V- Sorbitol - W- W- Starch aw a -W - -W - Starch hydrolysis V W - - - - - Sucrose a a a a a a Trehalose - - - V Xylose - a a -W W-
Milk C- - -C - C- C C- Gas (glucose agar) 3 92 3 432 3 92 - - 3,- Reduction (resazurin) + + + + V + V Products detected Fbla F Lba Lba Lba SA SAf La2
(2,PY 9s) (2) (S,PY) (F~,PY) @YJ) (fs) =All strains were negative for fermentation of erythritol, glycogen, inositol, and rhamnose. No strain completely digested gelatin, digested milk or meat, produced indole or wtalase, reduced nitrate. Symbols: -, Negative reaction; -, no visible growth or only slight growth and negative reaction; +, positive reaction; a (carbohydrate cultures), acid (pH below 5.5); c, curd; v, variable reaction; w, weak reaction or pH between 5.5 and 6; numbers (growth and gas), amount estimated on “- to 4+” scale. Where two reactions are given (e.g., “aw”), the first was the more usual and the second was observed less frequently. Products were analyzed from carbohydrate (usually glucose or fructose) culture. Capital letters indicate 1 meq (or more) per 100 ml of culture; small letters indicate less than 1 meq/lOO ml of culture. Products in parentheses were not uniformly detected. Symbols: a, Acetic acid; b, butyric acid; c, caproic acid; f, formic acid; ib, isobutyric acid; iv, isovderic acid; 1, lactic acid; p, propionic acid; py, pyruvic acid; s, succinic acid; 2, ethanol; 4, butanol. Number of isolates. Nllmhmr nf cnerimenc frnm which isolates camehumber of persons from whom specimens came. VOL. 24, 1974 NEW GENUS, NEW SPECIES, AND EMENDED DESCRIPTIONS 263 Type strain: American Type Culture Collec- tion (ATCC) 27759 (= Virginia Polytechnic Institute [VPI] C33-22); isolated from human feces. This species is easily recognized by its production of formic, lactic, and butyric acids from glucose, by its poor growth in the absence of fermentable carbohydrates, and by its rather FIG. 2. Coprococcus catus. Left, cells from PY- uniform fermentation of the substrates indi- jhctose broth culture. Right, cells from PY-glucose broth culture. See Fig. 16 for scale. cated above.
(ii) Coprococcus catus sp. nov. (L. adj. ca’tus clever [referring to the unusual property of growth occurred at 37 and 45 C. producing large quantities of both propionic Biochemical reactions. The characteristics of and butyric acids] .) Obligately anaerobic, non- 17 isolates of this species are given in Table 2. motile , gram-posit ive, slightly elongat e cocci In addition, the type strain and four other which usually occur in pairs that form long strains tested did not ferment adonitol, dextrin, chains (Fig. 2). Cells of unequal size could dulcitol, glycerol, inulin, or sorbose; did not occur in the pairs. The average cell size in produce H2 S (SIM), lecithinase, lipase (EYA), PY-glucose cultures was 1.1 by 1.7 pm; the AMC, or urease; and did not hydrolyze hip- range of cell size was 0.8 to 1.4 by 1.6 to 1.9 purate. Neutral red was reduced. Galactose was very weakly fermented by two strains but not pm. Colonies. After incubation for 5 days in by the type strain and two other strains tested. RGCA roll tubes, colonies were 0.5 to 1 mm in Ammonia was not produced from cultures in diameter, lenticular (occasionally trifoleate), chopped meat, PY, or arginine medium, but and white to tan. small amounts were sometimes detected in On anaerobic BAP incubated for 2 days, PY-glucose and PY-gluconate cultures. surface colonies of the type strain were 0.5 to 1 Fermentation products. Fermentation prod- mm in diameter, circular, erose, umbonate, ucts (average milliequivalents per 100 ml of opaque, smooth, shiny, and white, and pro- culture) were as follows. duced slight alpha-hemolysis. There was no From PY-fructose: Butyric (5 to 7) and growth on BAP incubated in a candle jar or in propionic (1 to 2) acids with trace amounts of an aerobic at-mosphere. acetic acid and sometimes lactic and succinic Cultural characteristics. Slight to moderate acids. Abundant hydrogen was produced. growth occurred in PY broth. PY-fructose From PY-lactate: Propionate (greater than cultures had abundant growth with some 3.5), acetate (2), and butyrate (1 S),sometimes turbidity and a smooth, crumb-like, or slightly with very slight amounts of succinate, pyruvate, ropy sediment; after incubation for 5 days, the isobutyra te, or isovalerate. pH was 4.8 to 5.5. Although the pH in glucose From PY-pyruvate: Acetate (3.5), butyrate broth did not go below 6.0, growth usually was (2), and propionate (l), sometimes with trace stimulated by glucose, and propionate and amounts of lactate, succinate, isobutyrate, or butyrate production exceeded that of PY valerate. cultures. From PY: Butyrate (0.3), with trace amounts There was little growth in PY-6.5% NaC1- of propionate, succinate, and acetate. glucose broth. Growth in PY-glucose was not Threonine was not converted to propionate; affected by 20% bile, 0.1% Tween 80, or 10% gluconate was not used. rumen fluid. G+C content of the DNA: 41 mol% by T, There was usually no growth at 30 C; good for the type strain and 39 mol% for VPI C1-32. Type strain: ATCC 27761 (= VPI C6-61); isolated from human feces. This species is easily recognized by its production of large amounts of butyric and propionic acids from fructose.
(iii) Coprococcus comes sp. nov. (L. no. co’mes companion, fellow traveler [referring to the presence of this species in human feces].) FIG. 1. Coprococcus eutactus. Left, cells $+omPY Obligately anaerobic, nonmotile, gram-positive, broth culture. Right, cells from PY-glucose broth elongate cocci with tapered ends. Cells occurred culture. See Fig. 16 for scale. singly and in pairs and chains of 4 to 20 264 HOLDEMAN AND MOORE INT. J. SYST. BACTERIOL. elements. The average cell size was 1.6 by 2.3 pm (Fig. 3); the range of cell size was 0.8 to 2.0 by 0.8 to 5 pm. Colonies. After incubation for 5 days in RGCA roll tubes, the colonies were 0.5 to 1.0 mm in diameter, lenticular or bifoleate, and translucent. FIG. 4. Ruminococcus callidus. Left, cells from PY On anaerobic BAP incubated for 2 to 3 days, broth culture. Right, cells from PY-glucose broth, surface colonies of the type strain were 0.5 to culture. See Fig. 16 for scale. 1.0 mm in diameter, circular, entire, convex, opaque, smooth, shiny, and white with slight From PY-glucose: D(-) lactic (3.0), butyric alpha-hemolysis. Other strains of the species (0.7), and acetic (0.5) acids, sometimes with were nonhemolytic on sheep blood agar. There small amounts of succinic and pyruvic acids. No was no growth on BAP incubated in a candle jar hydrogen was detected. or in an aerobic atmosphere. From PY-pyruvate: Acetate (5) and butyrate Cultural characteristics. There was only mod- (1 to 2), sometimes with formate. erate growth in PY broth without fermentable From PY: Acetic (1.5) and butyric (0.2) carbohydrate. PY-glucose cultures had a acids, sometimes with trace amounts of pyru- smooth (sometimes stringy) sediment with no vic, lactic, and succinic acids. or only moderate turbidity; after incubation for Threonine was not converted to propionate. Lactate and gluconate were not utilized. 1 day, the pH was 4.8 to 5.2. No growth occurred in PY-6.5% NaC1-glucose G+C content of the DNA: 40 mol% by T, broth. Growth was usually slightly inhibited by for the type strain; 42 mol% for VPI B2-5 1. 20% bile. Growth in PY-glucose was unaffected Type strain: ATCC 27758 (= VPI C1-38); by 0.1% Tween 80 or 10% rumen fluid. isolated from human feces. The temperature for optimal growth was 37 This species is easily recognized by its C; most strains grew well at 45 but not 30 C. production of lactic and butyric acids from Biochemical reactions. The characteristics of glucose. Its fermentation of arabinose and 22 isolates of this species are given in Table 2. xylose help differentiate it from the other two In addition, the type strain and six other strains species in the genus. tested did not ferment adonitol, dextrin, glycerol, or sorbose and did not hydrolyze New species of the genus Ruminococcus hippurate or produce AMC. Neutral red was Descriptions of new species of Ruminococcus reduced. Small to moderate amounts of am- are given in Table 2 and in the following monia were produced in chopped meat and PY discussion. cultures; no ammonia was detected from arginine or PY-glucose cultures. The type strain (i) Rurninococcus callidus sp. nov. (G. Adj. and five of six other strains tested fermented caZ'2i.dus clever, expert [referring to the procliv- galactose. H2S (SIM) was not produced by the ity of this species to attack di- and trisaccha- type strain or by four of six other strains rides] .) Obligately anaerobic, nonmotile, gram- tested. The fermentation of inulin was variable. positive cocci, 1.0 to 1.6 by 1.8 to 2.0 pm, Growth on EYA was variable; no lecithinase or usually occurring in pairs (sometimes short lipase was produced by strains that grew on this chains) with the adjacent ends of the pair medium. rounded and the other ends slightly tapered Fermentation products. Fermentation prod- (Fig. 4). ucts (average milliequivalents per 100 ml of Colonies. After incubation for 5 days in culture) were as follows. RGCA roll tubes, colonies were 0.2 to 1.0 mm in diameter, white to tan, translucent to transparent, and lenticular. Surface colonies were 2 to 3 mm in diameter, flat, and translucent with scalloped edges. On anaerobic BAP incubated for 5 days, surface colonies are punctiform to 1.5 mm in diameter, circular to irregular, umbonate, white with yellowish center, clear, with an entire or FIG. 3. Coprococcus comes. Left, cells from PY undulate edge, and beta-hemolytic. No growth broth culture. Right, cells from PY-glucose broth occurred on BAP incubated in a candle jar or in culture. See Fig. 16 for scale. an aerobic atmosphere. VOL. 24, 1974 NEW GENUS, NEW SPECIES, AND EMENDED DESCRIPTIONS 265
Cultural characteristics. There was no or poor Colonies. After incubation for 5 days in growth in PY broth without fermentable RGCA roll tubes, colonies were 0.5 to 1.0 mm carbohydrate. PY-maltose cultures had a in diameter, white to tan, translucent (some- smooth to grainy sediment with little or no times transparent) and lenticular to multifole- turbidity; after incubation for 5 days, the pH ate. was 4.8 to 5.5. On anaerobic BAP incubated for 2 days, Growth in PY-glucose was somewhat in- surface colonies of the type strain were hibited by 20% bile, but it was not affected by punctiform to 1.5 mm in diameter, circular, 0.1% Tween 80 or 10% rumen fluid. entire, low-convex, white, translucent, shiny, The temperature for optimal growth was 37 smooth, and nonhemolytic. There was no C; growth was irregular at 30 and 45 C. growth on BAP incubated in a candle jar or in Biochemical reactions. The characteristics of an aerobic atmosphere. 18 isolates of this species are given in Table 2. Cultura! characteristics. Slight to moderate In addition, the type strain and five other growth occurred in PY broth. PY-glucose strains tested did not produce ammonia in PY, cultures had a smooth or ropy sediment and PY-glucose, or arginine cultures, did not pro- often there was no turbidity; after incubation duce H2S (SIM), did not hydrolyze hippurate, for 1 to 3 days, the pH was 4.6 to 5.0. and did not produce lecithinase or lipase Growth in PY-glucose usually was unaffected (EYA). The type and one of five other strains by addition of rumen fluid, 20% bile, or 0.1% tested did not produce AMC, but four strains Tween 80. However, the pH was slightly lower did. The type and five of five other strains in the presence of Tween 80. tested did not produce acid from adonitol, The temperature for optimal growth was 37 dulcitol, galactose, or sorbitol. Two strains C; strains usually grew well at 45 C, but growth weakly fermented dextrin. Of six strains tested, was variable at 30 C. the type and one other strain fermented inulin. Biochemical reactions. The characteristics of Fermentation products. Fermentation prod- 53 isolates of this species are given in Table 2. ucts (average milliequivalents per 100 ml of In addition, the type strain and three other culture) were as follows. strains tested fermented galactose and reduced From PY-fermentable carbohydrate (maltose, neutral red. They did not ferment adonitol, glucose, fructose, or cellobiose): Succinic (3 .O) dextrin, dulcitol, glycerol, inulin, or sorbose and acetic (1.4) acids, usually with formic (0.6 and did not produce AMC, lecithinase or lipase to 1.7) and trace to moderate amounts of lactic (EYA), hydrolyze hippurate, or grow in PY- and pyruvic acids. Abundant hydrogen was 6.5% NaC1-glucose broth. The type strain and produced from maltose. one of three other strains tested produced a Essentially no products were detected from small amount of ammonia from chopped meat. PY cultures (poor growth). Lactate, pyruvate, Ferment a ti on products . Ferment ation prod- and gluconate were not used. ucts (average milliequivalents per 100 ml of G+C content of the DNA: 43 mol% by T, culture) were as follows. for the type strain. From PY-glucose: DL-LaCtiC (2.4) and acetic Type strain: ATCC 27760 (= VPI S7-31); (0.7) acids and ethanol, usually with formic isolated from feces. (0.7) and sometimes with trace amounts of Because this species requires a fermentable succinic acids. Abundant hydrogen was pro- carbohydrate for good growth or continued duced. subculture and produces succinic and acetic From PY-pyruvate: Acetate (2.7) and for- acids, it is placed in the genus Rurninococcus. It mate (1.7), sometimes with lactate (0.9). differs from other described species in the From PY: Little or no acetic, lactic, and/or genus by fermenting raffinose and from R. succinic acids. flavefaciens, which it most closely resembles, by fermenting maltose.
(ii) Ruminococcus torques sp. nov. (L. n. torlques twisted necklace [referring to appear- ance of the chains from broth cultures].) Obligately anaerobic, nonmotile, gram-positive, spherical to oval cells occurring in pairs and short chains. Cells of the type strain were 0.3 to FIG. 5. Ruminococcus torques. Left, cells from PY 0.6 pm in diameter. Cell size ranged from 0.3 to broth culture. Right, cells from PY-glucose broth 0.9 pm (Fig. 5). culture. See Fig. Id for scale. - 266 HOLDEMAN AND MOORE INT. J. SYST. BACTERIOL.
Lactate and gluconate were not utilized. No surface colonies of the type strain were 2 mm propionate was produced from threonine. in diameter, circular, entire, convex, opaque, GtC content of the DNA: 42 mol% by T, shiny, smooth, and nonhemolytic. There was for the type strain; 40 mol% for VPI C14-52. no growth on BAP incubated in a candle jar or Type strain: ATCC 27756 (= VPI B2-51); in an aerobic atmosphere. isolated from human feces. Cultural characteristics. PY-glucose broth The production of abundant hydrogen and cultures were usually turbid with a smooth or often relatively large amounts of acetic acid stringy sediment; after incubation for 5 days, exclude this organism from the genus Strepto- the pH was 4.9 to 5.2. coccus. Growth was moderate to poor in PY broth. Growth in PY-glucose is unaffected by 0.1% Tween 80, 10% rumen fluid, or 20% bile. New species, new combinations, and emended There was good growth in PY-glucose at 37 descriptions of anaerobic members of the genus and 45 C, poor to moderate growth at 30 C, Streptococcus and usually no growth at 25 C. Although the species of the genus Strepto- Biochemical reactions. The characteristics of coccus as listed in the 7th edition of Bergey’s 17 isolates of this species are given in Table 2. Manual of Determinative Bacteriology are all In addition, the type strain and four other facultatively anaerobic, we concur with the strains tested fermented galactose and inulin recommendation of Rogosa (14) that obligately and reduced neutral red. They did not produce anaerobic species of cocci which occur in chains acid in adonitol, dextrin, dulcitol, glycerol, or and that are fermentative and produce lactic sorbose and did not hydrolyze hippurate. They acid as the major product (with or without did not grow in 6.5% NaC1-glucose broth and small amounts of acetic or formic acids, did not produce lecithinase or lipase (EYA). No ethanol, COz) should properly be included in ammonia, or only a slight amount, was pro- the genus Streptococcus. Following this recom- duced in chopped meat cultures. The type and mendation, the species presently known as one of four other strains tested produced H2S Peptostreptococcus in termedius is returned to (SIM). AMC was produced by one of four the genus Streptococcus, where it was originally strains tested. placed by PrCvot (10). The species Peptostrep- Fermentation products. Fermentation prod- tococcus rnorbillorum (16), placed in Diplo- ucts (average milliequivalents per 100 ml of coccus (11) and Peptococcus (4), and the culture) were as follows. species Peptococcus constellatus ( 1), originally From PY-glucose: D (-)-Lactic (4.0), acetic placed in Diplococcus (9), also produce lactic (OS), and succinic (0.2) acids. No hydrogen acid as the sole major fermentation product and was detected. therefore are placed in the genus Streptococcus, From PY-pyruvate: Lactate (1.3) and acetate along with Streptococcus hansenii sp. nov., a (1.8). species isolated from the intestinal tract. From PY: Acetic (0.3) acid, with trace A description of Streptococcus hansenii sp. amounts of succinic and lactic acids. nov. is given in Table 2 and in the following Lactate and gluconate were not utilized. No discussion. Emended descriptions of Strepto- propionate was produced from threonine. coccus constellatus (Prkvot) comb. nov., Strep- G+C content of the DNA: 38 mol% by Tm tococcus intermedius PrCvot, and Streptococcus for the type strain and 37 mol% for VPI morbillorum (PrCvot) comb. nov. are given in C9-30. Table 3 and in the following discussion. Type strain: ATCC 27752 (= VPI C7-24); isolated from human feces. (i) Streptococcus hansenii sp. nov. (M.L.gen. n. han.sen’i.i of Hansen (named for P. Arne This species ferments lactose, unlike S. constellatus and S. morbillorum, and does not Hansen, a Danish-American bacteriologist ] .) Dbligately anaerobic, nonmotile, gram-positive ferment cellobiose, unlike S. intermedius. cocci, with rounded or slightly tapered ends, (ii) Streptococcus constellatus (PrCvot) comb. which occurred in pairs or chains of pairs. Cells nov. [ Syns.: Peptococcus constellatus (PrCvot) of the type strain in glucose broth cultures were Douglas 1957; Diplococcus constellatus Prkvot 1.6 to 2,3 pm in diameter (Fig. 6). 19241 . Anaerobic to aerotolerant, nonmotile, Colonies. After incubation for 5 days in nonsporesorming, gram-positive cocci which are RGCA roll tubes, colonies were 0.5 to 2 mm in round to slightly oval and occur in pairs and diameter, lenticular, and translucent or white short chains. Cells in the pairs could be of very with translucent edges. unequal size; some appeared to be “budding.” On anaerobic BAP incubated for 2 days, The average size of cells was 0.8 to 1.3 pm; the VOL. 24, 1974 NEW GENUS, NEW SPECIES, AND EMENDED DESCRIPTIONS 267
TAB LE 3. Reactions of species of anaerobic-to-a ero to1era n t str eptoco ccia S. constellatus S. intermedius S. morbillorum
~ Substrate Original Original Original or descrip- Type 7 descrip- Type 63 9 reaction tionb strain strains tionC strain strains descrT-tion strains
Am ygdalin V -a W- Arabinose - - - Cellobiose a aw -W Dextrin W- V -W Esculin V -a - Esculin hydrolysis + + -
Fructose a a Vf V Galactose a a J V Glucose a a a aw Glycerol W- -W Vf - Glycogen - - -W indin - -a - Lactose a a - Maltose a a a aw Mannitol - -a - Man nose a a V
Melezitose W- -W -W Melibio se - V - Raffinose - V -W Ribose -W -a V Salicin a a- -
Starch V V Vf W- Starch hydrolysis - V - Sucrose a a a aw Trehalo se a a- - Xylose - -W -W
Gelatin digestion -W -W -W Milk C C -c - - - Gas - 92 Acet ylmethylcarbinol +- -+ -W Neutral red reduction - V V
a None of these strains tested in our laboratory produced acid from adonitol, dulcitol, erythritol, inositol, rhamnose, sorbitol, or sorbose; none produced H, S, reduced nitrate, or digested meat. See Table 2 for symbols. Privot (9). PrCvot (10). ti PrCvot (1 1). Privot, 1933 (1 1); arabinose attacked by strains from appendicitis. From PrCvot et al. (13).
FIG. 6. Streptococcus hansenii. Left, cells from PY' FIG. 7. Streptococcus constellatus. Left, cells from broth culture. Right, cells from PY-glucose broth PY broth culture. Right, cells fiom PY-glucose broth culture. See Fig. 16 for scale. culture. See Fig. I6 for scale. 268 HOLDEMAN AND MOORE INT. J. SYST. BACTERIOL. range of cell size was 0.5 to 1.3 by 0.8 to 2.4 Isolated from clinical specimens and vaginal pm in PY-glucose broth cultures (Fig. 7). swabs. Colonies. On anaerobic BAP incubated for 2 This species is most easily recognized by its to 3 days, surface colonies of the type strain production of lactic acid as a major product, its were 0.5 to 2 mm in diameter, circular, entire fermentation of glucose, maltose, and sucrose to slightly umbonate, convex, translucent, without fermentation of lactose, and its hy- shiny, smooth, and nonhernolytic. Slight hemol- drolysis of esculin. Salicin fermentation has also ysis was observed with other strains (some been uniform in the strains tested. tested on horse blood agar). Pr6vot first recognized this species by its Cultural characteristics. PY broth cultures formation of satellite colonies around a large had light to moderate turbidity. PY-glucose central colony in deep agar cultures. The cultures had abundant growth with granular or satellite phenomenon has not been noted when smooth sediment; after incubation for 5 to 7 these strains were grown as surface colonies nor days, the pH was 4.4 to 5.0. There was poor with the neotype strain in agar deep cultures in growth in PY-6.5% NaCl-glucose broth and BHIA supplemented with yeast extract, vitamin PY-glucose-bile broth. Growth usually was not K, and hemin. Hare (3) also noted no satellite enhanced by Tween 80. colonies. After several laboratory transfers, three of In other characteristics, the reactions of the seven strains tested grew on the surface of BAP neotype strain conform to those given by incubated aerobically, and four of five strains PrCvot et al. (1 3) except that we did not detect tested grew on the surface of BAP incubated in fermentation of arabinose or production of a candle jar. The neotype strain did not grow AMC. We found no fermentation of either on the aerobic plates but sometimes produced L-arabinose or D-arabinose. Rogosa (Bergey ’s slight growth on the surface of plates incu- Manual, 8th ed., in press) stated that “a positive bated in the candle jar. The ability to grow in arabinose and a negative trehalose fermentation an aerobic atmosphere may vary with the age [as originally reported (9)l are in error; modern and amount of inoculum. techniques show arabinose is not fermented and The temperature for optimal growth was 35 trehalose is fermented.” to 37 C; good growth occurred at 30 C, but only slight growth occurred at 25 and 45 C. (iii) Streptococcus intermedius PrCvot 1925, Biochemical reactions. In addition to the emended description. [ Syn. : Peptostrepto- reactions listed in Table 3, the neotype strain coccus interrnedius (PrCvot) Smith 19571. and six other strains of this species did not Anaerobic to aerotolerant, nonmotile, non- produce Hz S, catalase, lecithinase or lipase sporeforming, gram-positive cocci occurring in (EYA), or hydrolyze hippurate. Ammonia was chains. Cells are often somewhat elongate; cells usually detected from cultures in chopped in pairs may be of unequal size. The average meat, arginine, and PY. The neotype strain did size is 0.5 by 0.9 pm; the range of cell size is not produce urease. No diaminopimelic acid 0.3 to 0.8 by 0.5 to 1.5 ,um (Fig. 8). was detected in whole-cell hydrolysates of the Colonies. On anaerobic BAP incubated for 2 neotype strain, indicating that none is present days, surface colonies of the type strain are 0.5 in the cell wall (C. S. Cummins, personal mm in diameter, circular, entire, convex, communication). opaque, white, shiny, smooth, and nonhemo- Fermentation products. Fermentation prod- lytic. ucts (average milliequivalents per 100 ml of Cultural characteristics. There was moderate culture) were as follows. growth in PY broth, sometimes with a sedi- From PY-glucose: Lactic (5.5) and acetic ment. PY-glucose cultures had abundant growth (0.2) acids, sometimes with trace amounts of and usually were turbid and had a sediment formic and succinic acids. A trace of hydrogen (smooth, grainy, flocculent, or stringy). After was produced. incubation for 3 to 5 days, the pH was 4.5 to From PY-pyruvate: Formate (6.5) and ace- tate (5.0), usually with trace amounts of succinate and lactate. From PY: Trace amounts of lactic and acetic acids, sometimes with small amounts of formic, succinic, and pyruvic acids. Lactate was not utilized. Threonine was not converted to propionate. Neotype strain: ATCC 27823 (= VPI 3810 = FIG. 8. Streptococcus intermedius. Left, cells from PrCvot 4055); a PrCvot strain probably isolated PY broth culture. Right, cells from PY-glucose broth from a case of purulent pleurisy. culture. See Fig. I6 for scale. VOL. 24, 1974 NEW GENUS, NEW SPECIES, AND EMENDED DESCRIPTIONS 269
5.0 in PY-glucose cultures. Tween 80 enhanced growth of some strains, particularly fresh isolates. There was no or only slight growth in PY-6.5% NaC1-glucose and PY-glucose-bile broths. Of 50 strains of this species tested, four grew slightly on the surface of BAP incubated FIG. 9. Streptococcus morbillorum. Left, cells from aerobically and two grew moderately well. Of PY broth culture. Right, cells from PY-glucose broth 18 strains tested for surface growth on BAP culture. See Fig. 16 for scale. incubated in a candle jar, one grew slightly, three grew moderately well, and one grew very PrCvot 19331 . Anaerobic to aerotolerant, non- well. The neotype strain produced moderate motile, nonsporeforming, gram-positive cocci growth on BAP incubated in a candle jar, but which were often elongate and occurred in pairs there was no growth on BAP incubated in an and short chains. The cells in the pairs often aerobic atmosphere. The amount of growth in were of unequal size. The average cell size in the candle jar varied with the age and amount PY-glucose cultures was 0.5 by 1.2 pm; the of inoculum. Several strains were obligately range of cell size was 0.3 to 0.8 by 0.5 to 1.4 anaerobic on initial isolation but grew rather pm (Fig. 9). well in the candle jar after several transfers. Colonies. On anaerobic BAP incubated for 2 The temperature for optimal growth was 35 days, surface colonies of the neotype strain to 37 C. Most strains grew well at 30 and 45 C. were pinpoint to 0.5 mm in diameter, circular, Only moderate or poor growth occurred at 25 entire, convex, translucent, shiny, and smooth. C. The neotype strain was not hemolytic, but Biochemical reactions. In addition to the some of the other strains produced slight characteristics given for this species in Table 3, alpha-hem olysis. the neotype strain and 63 other strains tested Cultural characteristics. Only one of nine did not produce H2S (SIM). AMC production strains grew on the surface of BAP incubated was variable by the neotype strain as well as by aerobically or in a candle jar. The neotype and other strains. The neotype strain did not reduce seven other strains tested did not grow on BAP neutral red; some of the other strains did. The incubated aerobically or in a candle jar. neotype strain did not produce urease. However, aerotolerant strains may occur more Fermentation products. Fermentation prod- frequently than these results indicate because ucts (average milliequivalents per 100 ml of strains of facultative cocci usually are not culture) were as follows. referred to our laboratory for identification. From PY-glucose: DL-Lactic acid [the Strains may be obligately anaerobic on first amount of L(+)-lactic exceeds the amount of isolation. Not all strains, however, have become D(-)-lactic acid] (6.0) often with acetic acid aerotolerant; the neotype strain still does not (0.25) and sometimes with a small amount of grow aerobically, even after numerous serial succinic acid. No hydrogen was produced. transfers in the laboratory and after freeze From PY-pyruvate: Formate (4.0) and ace- drying at least two times. tate (3.5), sometimes with moderate amounts Fermentable carbohydrate and Tween 80 of lactate, and sometimes with small amounts enhanced growth. There was only poor to of succinate. moderate growth in PY-glucose cultures, but From PY: Moderate amounts of lactic and abundant growth with granular or smooth acetic acids usually were produced, often with sediment was produced in PY-glucose-Tween 80 traces of succinic or formic acids. broth. Some strains had only sediment with no G+C content of the DNA: 38.7 mol% by turbidity in PY-glucose-Tween 80 cultures. chemical analysis (1 5) for the neotype strain. After incubation for 5 to 7 days, the pH was Neotype strain: ATCC 27335 (= VPI 3372A 4.8 to 5.4 in PY-glucose-Tween 80 cultures. = PrCvot 1877); the original source is unkown. Only moderate growth was* produced in Isolated from numerous kinds of human PY-Tween 80 broth medium. There was usually clinical specimens and from feces. no growth in PY-6.5% NaCl-glucose broth; The hydrolysis of esculin and the fermenta- many strains grew poorly in PY-glucose-bile tion of cellobiose and lactose help differentiate broth. S. intermedius from closely related species. The temperature for optimal growth was 35 to 37 c. (iv) Streptococcus morbillorum (Prbvot) Biochemical reactions. In addition to the comb. nov. [ Syns.: Peptostreptococcus morbil- characteristics for this species given in Table 3, lorum (PrCvot) Smith 1957; Diplococcus rube- the neotype strain and eight other strains tested olae Tunicliff 1933; Diplococcus morbillorum did not produce H2S (SIM), AMC, catalase, or 270 HOLDEMAN AND MOORE INT. J. SYST. BACTERIOL.
lecithinase or lipase reactions (EYA); they did demonstrated that, at least among these orga- not hydrolyze hippurate. Ammonia usually was nisms, different phenotypic patterns of carbo- detected in chopped meat and in arginine broth hydrates fermented do indicate different ge- cultures, occasionally from PY cultures. The netic groups. This is fortunate, because much neotype strain did not produce urease. previous taxonomy has been based primarily Fermentat ion products. Fermentation prod- upon carbohydrate utilization. We now have ucts (average milliequivalents per 100 ml of evidence and more confidence that such pheno- culture) were as follows. typic observations do, in fact, correlate with From PY-glucose-Tween 80: Lactic (4.0) and genetically distinct kinds of bacteria. Unfortu- acetic (0.6) acids, sometimes with formic and nately, within present phenotypic groups, there trace amounts of succinic and pyruvic acids, or are sometimes more than one distinct genetic ethanol. No hydrogen was produced. group. This indicates that there are even more From PY-pyruvate-Tween 80: Acetate (4.0), distinctly different organisms than now can be usually with formate (2.0), sometimes with differentiated phenotypically. lactate (0.7) and trace amounts of succinate. Among the gram-positive species, there are a From PY-Tween 80: Small amounts of acetic number with strains that easily decolorize in acid, often with a trace of lactic acid, some- older cultures and therefore may be among times with a trace of formic, succinic, or those that appear to be gram-negative in the pyruvic acids. original smears. Often there is considerable Lactate and gluconate were not utilized; pleomorphism in cultures, but this varies with threonine was not converted to propionate. age of culture and from substrate to substrate Neotype strain: ATCC 27824 (= VPI 5424 = and may, in part, reflect the higher oxidation- Prdvot 2917B); a Prevot strain isolated from reduction potential of the media as compared lung abscess. with that in the colon or rectum. The earlier Isolated from clinical specimens. descriptions by Eggerth (2) were exceedingly This species is most easily recognized by its helpful and comprehensive enough to allow production of lactic acid as a major product, differentiation of morphologically similar yet and fermentation of glucose, maltose, and physiologically distinct species described here. sucrose without fermentation of lactose or hydrolysis of esculin. New species of Bacteroides The characteristics of the neotype strain, Characteristics o f Bac tero ides egger thii sp . which was isolated and identified by Prhot as nov. are given in Table 4 and in the following D. morbillorum, conform to those given by discussion. Prhot et al. (13) except that we did not detect Hz S or propionate production. H2 S production (i) Bacteroides eggerthii sp. nov. (L. gen. n. varies considerably with different tests. We do eg.gerth'i.i of Eggerth [named for Arnold H. not know which test was originally used. The Eggerth, an American bacteriologist] .) Obli- gas chromatographic procedures currently used gat ely anaerobic, nonmotile, gram-n ega t ive, ple- for analyses of fermentation acids give more omorphic rods, ranging from coccoid to large precise identification of the compounds than rods with vacuoles or swellings, occurring singly did the earlier methods (presumably Duclaux or in pairs. Cells of the type strain were 1.1 to distillation). From experience with other cul- 1.6 by 1.6 to 9.4 pm; the sizes of six strains tures, we know that identification by Duclaux measured ranged from 0.4 to 1.1 by 0.8 to 14.0 analyses can be in error by one to two carbons. pm in PY-glucose broth cultures (Fig. 10). The propionate reported originally may have Colonies. After incubation for 5 days in been formate. We therefore feel that the RGCA roll tubes, colonies were 0.5 to 1.0 mm neotype strain here designated is representative in diameter, white to tan, translucent, lenticu- of the species as originally described. lar, occasionally multifoleate, and could have Anaerobic Rods dark centers or spots. On anaerobic BAP incubated for 2 days, A number of kinds of anaerobic rods that surface colonies were punctiform, circular, have not previously been descriked were en- entire, convex, translucent, grey-white, shiny, countered in the predominant flora. Among the and smooth, and were nonhemolytic. No black gram-negative organisms, the presence of a pigment was observed on laked blood roll fermentative Bacteroides species that failed to streaks after incubation for 8 days. No growth attack sucrose is an example. The application of occurred on BAP incubated in a candle jar or in DNA-DNA homology by competition experi- an aerobic atmosphere. ments by Johnson (6 ;personal communication) Cultural characteristics. Moderate to good to many strains of Bacteroides species has growth occurred in PY-broth. PY-glucose broth TABLE 4. Reactions of some new species of anaerobic rodP
Eubacterium Bacteroides Lactobacillus Clostridium eggerth ii E. biforme E. eligens 5. formicigenerans E. halii rogosae nexile
Substrate 3riginal or ATCC 1e scr iz- ATCC ATCC 22 4TCC ATCC ATCC 11 4TCC reaction 27 754 tion 27806 27750 (1 9/16) 17755 27751 27753 :7/71 27757
Am ygdalin -W -W V - - W Arabinose a- a - V - Cellobio se -a aw V aw a - Esculin V wa V -W -W W &ulin hydrolysis + + -+ + -+ +
Fructose V W a a a a W Glucose aw a a a- a a a Glycogen a- a V - Lactose aw a V a- wa a- a Maltose a a V -W - aw - Mannitol - - a -
Mannose aw a a -a -W -W - Melibiose -W - W Ra ffinose - - - a Rhamnose V -a - -a - Ribose - - -a - Salicin - L V -a a W
Sorbitol - - - - Starch a a V -W -W W Stach hydrolysis + + - - Sucrose - - V -a a Trehalose - - a - Xylose a a - -a a -a a
Gelatin digestion +------W - Milk C C C- C- C -C - Indole + + - - - - -
Gas (glucose agar) - - - 92 - 4 92 4 Reduction (resaz.) + + - - + +
Growth: PY 293 3 -91 1 2,- 1 Growth: bileglucom 4Y3 3 -,3 - 491 2 Motility - - +- + - - Products detected Sa Sa FA2 Fa21 AF12 Af 2 (pibivl) (pivibl) (WPY) (s) (SYPY)
a NO strain fermented erythritol, inositol, or melibiose. No strain digested meat, produced catalase, or reduced nitrate. See Table 2 for symbols used. Number of isolates. Number of specimens from which isolates came/number of Dersons from whnm rnerimPnc mm- Em-~r+h(3' 272 HOLDEMAN AND MOORE INT. J. SYST. BACTERIOL.
indole and by having a pH of 6.0 or above in sucrose and raffinose cultures. Emended description and new species of Eubac- terium Characteristics of Eubacterium biforme and FIG. 10. Bacteroides eggerthii. Left, cells porn PY four new species of Eubacterium are given in broth culture. Right, cells from PY-glucose broth Table 4 and in the following discussion. culture. See Fig. I6 for scale. (i) Eubacterium biforme (Eggerth) Prkvot, cultures were turbid with smooth (occasionally 1938. Emended description. Obligately anaero- stringy) sediment, and the pH was 4.9 to 5.6 bic, gram-positive, nonsporeforming, nonmotile after incubation for 1 to 2 days. Growth in coccobacilli occurring in pairs and chains. PY-glucose broth was generally unaffected by Cells of the type strain were 0.8 to 1.1’by 1.1 the addition of 10% rumen fluid or 0.1 % Tween 30 3.1 pm in PY-glucose broth cultures (Fig. 80. 11). Sizes of three other strains measured range The temperature for optimal growth was 37 from 0.6 to 2.7 by 1.6 to 15 pm. Rods and C. Good growth occurred at 30 and 45 C; coccoid forms often occurred in the same moderate growth occurred at 25 C. chain. Central swellings (“pelton de jardinier”) Biochemical reactions. The characteristics for were seen in some cultures, particularly among 38 isolates of this species are given in Table 4. cells from growth on solid media. In addition, the type strain and seven other Colonies. After incubation for 5 days in strains tested fermented dextrin and galactose RGCA roll tubes, colonies were 0.5 to 1.0 mm in diameter, white to tan, and usually lenticular and reduced neutral red. They did not ferment and translucent, opaque, or translucent with adonitol, dulcitol, glycerol, inulin, or sorbose. opaque centers, sometimes with diffuse edges. The eight strains tested did not produce AMC or urease, hydrolyze hippurate, grow in 6.5% Occasional colonies looked like white “woolly NaC1-glucose broth, or produce any reaction on balls .” On anaerobic BAP after incubation for 2 EYA. Moderate ammonia was produced by the days, surface colonies were 0.5 to 2.0 mrn in type strain and three of seven other strains tested. Moderate H2S was detected in SIM diameter, circular, entire to slightly erose, flat to low convex, white, shiny, smooth, translu- medium with the type strain and four of seven cent with dense centers, and nonhemolytic. other strains tested. Fermentation products. Fermentation prod- There was no growth on BAP incubated in a ucts (average milliequivalents per 100 ml of candle jar or in an aerobic atmosphere. culture) were as follows. Cultural characteristics. No growth, or very From PY-glucose: Succinic (3.8) and acetic poor growth, occurred in media without a (0.9) acids, often with a small amount of fermentable carbohydrate. PY-glucose-Tween- propionic and traces of lactic, isovaleric, and 80 broth cultures had abundant growth with isobutyric acids. A trace amount of hydrogen smooth sediment, usually without turbidity, was detected from eight of eight cultures and a pH of 4.5 to 5.0 in 1 to 2 days. Growth tested. was enhanced by 0.1% Tween-80, but was not From PY-pyruvate: Acetate (2.0) and suc- affected by addition of rumen fluid. cinate (0.8) with trace to moderate amounts of The temperature for optimal growth was 37 propionate, isobutyrate, and isovalerate were C; some strains grew at 45 C, but most did not usually produced. grow at 30 C. From PY: Propionic (0.8), acetic (0.4), Biochemical reactions. The characteristics for isovaleric (0.3), succinic (0.1 ), and isobutyric 88 isolates of this species are given in Table 4. (0.1) acids. In addition, the type strain and eight of nine Lactate and gluconate were not utilized. Small amounts of propionate were irregularly produced from threonine. G+C content of the DNA: 46 mol% by T, for the type strain and 44 mol% for VPI B8-30. Type strain: ATCC 27754 (= VPI T5-42B); isolated from human feces. Although this organism morphologically re- FIG. 11. Eubacterium biforme. Left, cells from PY sembles subspecies of Bacteroides frugilis, it is broth culture. Right, cells from PY-glucose broth most easily recognized by its production of culture. See Fig. I6 for scale. VOL. 24, 1974 NEW GENUS, NEW SPECIES, AND EMENDED DESCRIPTIONS 273
other strains tested produced acid from galac- tose; all 10 reduced neutral red. The type and nine other strains tested did not ferment adonitol, dextrin, dulcitol, glycerol, inulin, or sorbose, and grew poorly in PY media to which these substrates had been added. The 10 strains tested did not produce AMC, hydrolyze hip- purate, or produce H2S in SIM medium. The FIG. 12. Eubacterium eligens. Left, cells from PY type strain and four of nine other strains tested broth culture. Right, cells from PY-glucose broth culture. See Fig. 16 for scale. produced a small amount of ammonia in chopped meat cultures; none produced am- diameter, white to tan, and lenticular. Surface monia in PY-glucose cultures. Only 8 of 10 colonies on BHIA streak tubes were puncti- strains tested grew on EYA; they gave no form, circular, entire, transparent to translu- reaction. cent, white to tan, and smooth. Surface Fermentation products. Fermentation prod- colonies on 2-day-old anaerobic BAP were 0.5 ucts (average milliequivalents per 100 ml of to 1 .O mm in diameter, circular, entire, convex, culture) were as follows. smooth, translucent to semi-opaque, shiny, From PY-glucose-Tween 80: D L-Lactic (4.4) white, and nonhemolytic. No growth occurred and butyric (0.7) acids, moderate ethanol, on inoculated BAP incubated in a candle jar or usually with caproic acid (0. l), occasionally in an aerobic atmosphere. with trace amounts of succinic, pyruvic, and Cultural characteristics. No growth, or only acetic acids. Abundant hydrogen was produced. slight growth, was produced in PY broth (Caproic acid was produced irregularly; produc- without fermentable carbohydrate. PY-fructose tion probably was dependent on the amount of broth cultures were turbid with smooth sedi- growth and culture age. Most chromatographic ment and a pH of 4.6 to 5.8 in 5 days. Tween analyses were made on 5-day-old cultures.) 80 (0.1%) sometimes enhanced growth. Growth From PY-pyruvate-Tween 80: Butyrate (1.5), was not usually affected by addition of 10 to often with lactate (0.14), and acetate (0.7), 30% rumen fluid. sometimes with formate and a small amount of The temperature for optimal growth was 37 caproate. C. Most strains grew well at 45 C; little or no Lactate and gluconate were not utilized; growth was produced at 30 C. threonine was not converted to propionate. Biochemical reactions. The characteristics for G+C content of the DNA: 32 mol% by T, 69 isolates of this species are given in Table 4. for the neotype strain. In addition, the type strain and five other Neotype strain: ATCC 27806 (= VPI C 17-5); strains tested reduced neutral red. One pro- isolated from human feces. duced weak acid from dextrin. They did not This species is most easily recognized by its ferment adonitol, dulcitol, galactose, glycerol, production of butyric and caproic (when inulin, or sorbose, and did not grow well in PY detected) acids, along with a major amount of media to which these substrates had been lactic acid. Lack of fermentation of arabinose added. The six strains tested did not produce and maltose help differentiate it from species HZS (SIM) or ammonia. The type and three which it most closely resembles. other strains tested did not produce AMC or hydrolyze hippurate. Only three of six strains (ii) Eubacteriurn eligens sp. nov. (L. adj. tested grew on EYA; they gave no reaction. eZ'i.gens choosy [referring to its generally poor Fermentation products. Fermentation prod- growth without fermentable carbohydrate] .) ucts (average milliequivalents per 100 ml of Obligately anaerobic, gram-posit ive, nonspore- culture) were as follows. forming, straight to slightly curved rods, occur- From PY-glucose: Formic (1.8) and acetic ring singly or in pairs, occasionally in short (1.0) acids, and ethanol, often with lactic acid chains of three to six cells. Central or eccentric (0.35) and a trace amount of succinic acid, swellings predominated in some strains. sometimes with traces of butyric or pyruvic Twenty-four of 33 strains tested were motile acids. No hydrogen was detected. and peritrichous. Cells decolorized easily in From PY-pyruvate: Acetate (greater than older cultures, but some gram-positive cells 5.0), formate (greater than 3.0), lactate (1.2), were usually seen. Cells of the type strain were and a moderate amount of ethanol. 0.3 to 0.8 by 1.9 to 4.9 pm in PY-glucose From PY-gluconate: Formate (4.8), acetate cultures (Fig. 12). (3.5), and ethanol, often with lactate (0.3) and Colonies. After incubation for 5 days in a trace of succinate, sometimes with traces of RGCA, colonies were 0.5 to 1.0 mm in pyruvate or butyrate. 274 HOLDEMAN AND MOORE INT. J. SYST. BACTERIOL.
Usually no growth was produced in PY-lac- tate or in PY-threonine. G+C content of the DNA: 36 mol% by Tm for the type strain (motile) and for VPI T1-52 (nonmotile). Type strain: ATCC 27750 (= VPI C1548); isolated from human feces. FIG. 13. Eubacterium formicigenerans. Left, cells E. eligens differs from E. aerofaciens by (i) from PY broth culture. Right, cells from PY-glucose the relatively small amount of lactate produced, broth culture. See Fig. I6 for scale. (ii) extremely poor growth without fermentable carbohydrate, (iii) cellular morphology, (iv) dextrin, glycerol, inulin, or sorbose. They did motility (of some strains), and (v) lack of not produce H2S (SIM), AMC, ammonia, or hydrogen production. urease. They did not grow in PY-6.5% NaC1- glucose broth. The type strain and five of six (iii) Eubacterium formicigenerans sp. nov. other strains tested grew on EYA; they gave no (N. L. adj. for.mi. ci.gen’er.ans fonnic-acid- react ion. producing [referring to its production of large Fermentation products. Ferment ation prod- amounts of formic acid from fermentation of ucts (average milliequivalents per 100 ml of carbohydrates] .) Obligately anaerobic, gram- culture) were as follows. positive, nonsporeforming, nonmotile rods oc- From PY-glucose: Acetic (1.9), formic (1.6), curring in chains or pairs. Cells of the type and lactic (0.9) acids and ethanol, sometimes strain were 1.1 to 1.4 by 1.6 to 3.5 pm in with small amounts of succinic and pyruvic PY-glucose cultures (Fig. 13). The sizes of the acids. Moderate to abundant hydrogen was five other strains measured ranged from 0.6 to produced. 1.4 by 0.8 to 4.7 pm. From PY-pyruvate: Acetate (4.7), formate Colonies. After incubation for 5 days in (1.7), and ethanol, usually with lactate (0.3) RGCA, colonies were 0.5 to 1.0 mm in and sometimes with a trace of succinate. diameter, white to tan, circular to lenticular, From PY: Acetic acid (0.5), sometimes with and often had fuzzy edges or a “woolly ball” small amounts of lactic, succinic, or pyruvic appearance. Surface colonies on BHIA streak acids. tubes or anaerobic BAP were 0.5 to 3.0 mm in Lactate and gluconate were not utilized; diameter, circular to slightly irregular, entire to threonine was not converted to propionate. slightly erose, convex to umbonate, opaque, G+C content of the DNA: 40 mol% by Tm white to tan, shiny, and smooth. Slight greening for the type strain and 44 mol% for VPI was produced on sheep BAP by the type strain, C34-11. but the other six strains tested were nonhemo- Type strain: ATCC 27755 (= VPI C8-13); lytic. No growth occurred on BAP incubated in isolated from human feces. a candle jar or in an aerobic atmosphere. The fermentation acids and ethanol produced Cultural characteristics. Poor to moderate and lack of fermentation of cellobiose help growth was produced in PY broth. PY-glucose differentiate E. formicigenerans from other broth cultures had stringy or flocculent (occa- species with which it might be confused. sionally smooth) sediment with little or no turbidity and a pH of 4.7 to 5.0 in 5 days. (iv) Eubacterium hal2ii sp. nov. (M.L. gen. n. Growth in PY-glucose was generally not af- haZ1’i.i of Hall [named for Ivan C. Hall, an fected by 0.1% Tween 80 or 10% rumen fluid. American bacteriologist] .) Obligately anaero- The temperature for optimal growth was 37 bic, gram-positivey nonmotile rods occurring C. Most strains grew moderately well at 30 C singly and in pairs, occasionally in chains. Cells and 45 C, but usually not at 25 C. of the type strain were 0.8 to 1.7 by 4.7 to Biochemical reactions. In addition to the more than 25.0 pm (filaments) in PY-glucose characteristics given for 22 isolates of this cultures (Fig. 14). The sizes of six strains species in Table 4, the type strain and six other measured ranged from 0.8 to 2.4 by 4.7 to strains tested reduced neutral red and produced more than 25.0 pm. Subterminal and terminal acid from galactose. The type strain and two of swellings were seen, but the cultures did not six other strains tested hydrolyzed hippurate. survive heating at 70 C for 10 min. The type strain and five of six others strains Colonies. After incubation for 5 days in tested did not ferment dulcitol; one strain RGCA roll tubes, colonies were 0.5 to 1.0 mm fermented dulcitol slightly. The type and six in diameter and lenticular or “woolly balls.” other strains tested did not ferment adonitol, Surface colonies (BHIA streak tubes or anaero- VOL. 24, 1974 NEW GENUS, NEW SPECIES, AND EMENDED DESCRIPTIONS 275
\ New anaerobic species of Lactobacillus Characteristics of Lactobacillus rogosae sp. nov. are given in Table 4 and in the following discussion.
(i) Lactobacillus rogosae sp. nov. (M.L. gen. FIG. 14. Eubacterium hallii. Left, cells from PY broth culture. Right, cells from PY-glucose broth n. ro.go'sae of Rogosa [named for Morrison culture. See Fig. 16 for scale. Rogosa, an American bacteriologist I .) Obli- gately anaerobic, gram-p ositive, nonsporef orm- bic BAP) were 1 to 2 mm in diameter, circular ing rods, occurring singly and in pairs and short to slightly irregular, entire to slightly erose, low chains. Cells of the type strain and of four convex, semi-opaque, white to yellowish, other strains measured were 0.4 to 0.65 by 1.1 smooth, shiny, and nonhemolytic. No growth to 7.9 ym in PY-fructose cultures (Fig. 15). The occurred on BAP incubated in a candle jar or in type strain and most other strains were motile an aerobic atmosphere. arid peritrichous; nonmotile strains without Cultural characteristics. There was moderate flagella also have been isolated. growth in PY broth. PY-glucose cultures had Colonies. After incubation for 5 days in abundant growth, smooth or ropy sediment RGCA, colonies were 0.5 to 1.0 mm in with little or no turbidity, and a pH of 4.7 to diameter, translucent to transparent, lenticular 5.5 in 5 days. The addition of 0.1% Tween 80 to diffuse, and tan to colorless. Surface colonies or 10% rumen fluid did not affect growth in on Sweet E agar (5) streak tubes were 1 mm in PY -glu cose. diameter, circular, raised to convex, entire, The temperature for optimal growth was 37 translucent to opaque, dull, and smooth. Only C. Most strains grew at 30 C, but less well than one of six strains tested grew on BAP incubated at 37 C. The strains usually did not grow at 25 anaerobically; none grew on BAP incubated in C and often did not grow at 45 C. a candle jar or in an aerobic atmosphere. Biochemical react ions. The characteristics for Cultural characteristics. There was poor 13 isolates of this species are given in Table 4. growth in PY broth. PY-fructose cultures had In addition, the type strain and five of five moderate to abundant growth with smooth other strains tested produced acid from galac- sediment, usually with turbidity, and a pH of tose and reduced neutral red. They did not 4.9 to 5.5 in 5 days. Addition of 0.1% Tween ferment adonitol, dextrin, glycerol, inulin, or 80 or 10% rumen fluid generally did not affect sorbose; did not produce AMC, ammonia, growth in PY-glucose broth. urease, H2S (SIM), lecithinase or lipase (EYA); The temperature for optimal growth was 37 and did not hydrolyze hippurate or grow in C. Generally there was poorer growth, or no PY-6.5 % NaC1-glucose broth. growth, at 30 and 45 C. Fermentation products. Fermentation prod- Biochemical reactions. The characteristics of ucts (average milliequivalents per 100 ml of nine isolates of this species are given in Table 4. culture) were as follows. In addition, the type and five other strains From PY-glucose: Butyric (3.4) and acetic tested produced a small amount of ammonia in (0.4) acids and butanol, sometimes with small chopped meat cultures and reduced neutral red. amounts of formic, succinic, or lactic acids. They did not produce H2S (SIM) or AMC or Abundant hydrogen was produced. hydrolyze hippurate. They did not ferment From PY: Acetic acid (0.4). adonitol, dextrin, glycerol, inulin, or sorbose Some strains converted pyruvate to moderate and often did not grow in PY broth to which amounts of butyrate and acetate. Lactate and these substrates had been added. gluconate were not utilized; threonine was not converted to propionate. G + C content of the DNA:38 mol% by T, for the type strain. Type strain: ATCC 27751 (= VPI B4-27); isolated from human feces. Production of large amounts of butyric acid and butanol, morphological characteristics of the cells, and carbohydrates fermented help FIG. 15. Lactobacillus rogosae. Left, cells fiom differentiate E. hallii from other species with PY-fructose broth culture. Right, flagella stain, cells which it might be confused. from PY-fructose broth xulture. See Fig. I6 for scale. 276 HOLDEMAN AND MOORE INT. J. SYST. BACTERIOL.
Ferment at ion products. Ferment ation prod- ucts (average milliequivalents per 100 ml of culture) were as follows. From PY-fructose: DL-Lactic (2.3) and acetic (0.9) acids, usually with ethanol and small amounts of succinic or formic acids. No hydrogen was detected . FIG. 16. Clostridium nexile. Left, cells from PY From py: amounts Of acetic acid, broth culture. Right, cells from PY-glucose broth usually with traces of lactic or succinic acids, or culture. Bar represents I 0 Fm. both. From PY-pyruvate: Acetate (4.3), formate diameter, circular, entire, convex to raised, (2.3), and lactate (0.9), often with a trace of semi-opaque or with opaque centers and trans- succinate. Lactate and gluconate were not utilized; lucent edges, smooth, shiny, white or yellowish, threonine was not converted to propionate. and nonhemolytic. G+C content of the DNA. This was not Cultural characteristics. There was slight to determined for the type strain because of poor moderate growth in PY broth. PY-glucose cultures had abundant growth with smooth or growth in large culture and insufficient harvest of DNA; a value of 59 mol% by T, for VPI ropy sediment, sometimes without turbidity, C36-52 (nonmotile strain) was obtained. and a pH of 4.9 to 5.3 in 5 days. Growth was not affected by the addition of 0.1% Tween 80 Type strain: ATCC 27753 (= VPT C37-38); isolated from human feces. or 10%rumen fluid to PY-glucose broth. Fermentation of fructose with little or no The temperature for optimal growth was 37 fermentation of other carbohydrates help dif- C. Strains usually grew well at 30 and 45 C but ferentiate L. rogosae from other species of poorly at 25 C. Biochemical reactions. The characteristics of lactobacilli. 11 isolates of this species are given in Table 4. In addition, the type and five other strains New species of Clostridium tested fermented galactose and reduced neutral red. They did not ferment adonitol, dulcitol, Characteristics of Clostridium nexile sp. nov. glycerol, or sorbose. They did not produce are given in Table 4 and in the following AMC, urease, ammonia, lecithinase or lipase discussion. (EYA), or hydrolyze orotic acid. The type strain produced weak acid from dextrin; the other five strains did not. Inulin was not (i) Clostridium nexile sp. nov. (L. adj. fermented by the type or by two of five other nex’.i.le tied together [referring to its chain strains tested. Hippurate was not hydrolyzed by formation] .) Obligately anaerobic, gram-posi- the type and four of five other strains tested. tive, nonmotile coccobacilli to rods, occurring The type strain and two of five other strains in pairs and chains. Cells of the type strain were tested produced a small amount of H,S (SIM). 0.8 to 1.6 by 1.3 to 6.3 pm in PY-glucose Fermentation products. Fermentation prod- cultures. The sizes of four other strains mea- ucts (average milliequivalents per 100 ml of sured range from 0.8 to 1.6 by 0.8 to 4.7 pm. culture) were as follows. The type strain resisted heating at 80 C for 10 From PY-glucose: Acetic (2.4) and formic min, but no spores were ever seen. Round (1.4) acids and ethanol, often with trace to subterminal (nearly terminal) spores were seen moderate amounts of succinic or lactic acids, or in E broth (5) cultures of strain VPI C35-9A both. Abundant hydrogen was produced. (Fig. 16), but this culture did not survive From PY-pyruvate: Acetate (2.6) and etha- heating at 70 C for 10 min. Of five strains nol, usually with formate (0.5), occasionally carefully checked, three survived heating at 70 with small amounts of lactate. C for 10 min, but no spores were seen; spores From PY: Acetic acid (0.3), sometimes with were seen in a strain that did not survive, and traces of lactic and succinic acids. one similar strain did not survive and no spores G+C content of the DNA: 40 mol% by T, were seen. for the type strain and 41 mol% for VPI Colonies. After incubation for 5 days in C35-9A. RGCA, colonies were 0.5 to 1.0 mm in Type strain: ATCC 27757 (= VPI C48-37); diameter, tan, lenticular, and translucent to isolated from human feces. semi-opaque. Surface colonies (anaerobic BAP C. nexile differs from Clostridium oroticum, or BHIA streak tube) were 0.5 to 1.0 mm in which in some cultures it resembles morpho- VOL. 24, 1974 NEW GENUS, NEW SPECIES, AND EMENDED DESCRIPTIONS 277 logically, by not hydrolyzing orotic acid and by Moore (ed.), Anaerobe laboratory manual. An- not producing acid from arabinose, dulcitol, aerobe Laboratory, Virginia Polytechnic Institute maltose, and rhamnose. and State University, Blacksburg, Va. 5. Holdeman, L. V., and W. E. C. Moore (ed.) 1973. Anaerobe laboratory manual, 2nd ed. Anaerobe ACKNOWLEDGMENTS Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Va. This work was supported primarily by Public Health 6. Johnson, J. L. 1973. Use of nucleic-acid homol- Service grant 14604 from the National Institute of ogies in the taxonomy of anaerobic bacteria. Int. General Medical Sciences and by Public Health Service J. Syst. Bacteriol. 23:308-315. contract 71-2427 from the National Cancer Institute. 7. Marmur, J., and P. Doty. 1962. Determination of We also wish to thank Barbara Francis and John L. the base composition of deoxyribonucleic acid Johnson for determinations of the DNA G+C ratios; from its thermal denaturation temperature. J. Thomas 0. MacAdoo of the Foreign Language Mol. Biol. 5: 109-1 18. Department of this institution for assistance with 8. Moore, W. E. C., and ‘L. V. Holdeman. 1974. nomenclatural terms and derivations; Loretta Albert, Human fecal flora: the normal flora of 20 Maeve Crowgey, Ann Donnelly, Luba Fabrycky, Ed Japanese-Hawaiians. Appl. Microbiol. 27: 96 1-979. Hanna, Linda Knight, Carolyn Hubbard, Susan Sieg, 9. Privot, A. R. 1924. Diplococcus constellatus (n. Gail Selph, and others of our laboratory who sp.). C. R. SOC.Biol. (Paris) 91:426428. performed work on or for this project; and Elizabeth 10. Prthot, A. R. 1925. Les streptocoques anairobies. Cat0 and Ruth Beyer for help in compilation of the Ann. Inst. Pasteur (Paris),39:415447. data. 11. Privot, A. R. 1933. Etudes de systimatique bact6rienne. 1. Lois generals. 11. Cocci anairobies. Ann. SOC.Nat. 15:23-260. REPRINT REQUESTS 12. Privot, A. R. 1938. Etudes de systkmatique bactirienne. Ann. Inst. Pasteur (Paris) Address reprint requests to: Lillian V. Holdeman, 60: 285-307. Anaerobe Laboratory, Virginia Polytechnic Institute 13. Privot, A. R., A. Turpin, and P. Kaiser. 1967. Les and State University, P. 0. Box 49, Blacksburg, Va. bactiries anairobies. Dunod, Paris. 24060. 14. Rogosa, M. 1971. Peptococcaceae, a new family to include the gram-positive, anaerobic cocci of the genera Peptococcus, Peptostreptococcus, and Ruminococcus. Int. J. Syst. Bacteriol. LITERATURE CITED 21~234-237. 15. Romond, C., R. Sartory, J. Malgras. 1966. Le 1. Douglas, H. C. 1957. Peptococcus Kluyver and coefficient de ShapiroChargaff des streptocoques Van Neil, 1936, p. 477. In R. S. Breed, E. D. G. anairobies. Ann. Inst. Pasteur (Paris) Murray, and N. R. Smith (ed.), Bergey’s manual 11 1:I 10-1 18. of determinative bacteriology, 7 th ed. Williams 16. Smith, L. DS. 1957. Peptostreptococcus Kluyver and Wilkins Co., Baltimore, Md. and Van Neil, 1936, p. 540 In R. S. Breed, E. D. 2. Eggerth, A. H. 1935. The gram-positive non- G. Murray, and N. R. Smith (ed.), Bergey’s sporebearing anaerobic bacilli of human feces. J. manual of determinative bacteriology, 7th ed. Bacteriol. 30:277-299. Williams and Wilkins Co., Baltimore, Md. 3. Hare, R. 1967. The anaerobic cocci, p. 204-317. 17. Tunnicliff, R. 1933. Colony formation of Diplo- In H. P. Waterson (ed.), Recent advances in coccus rubeolae. J. Infect. Dis. 52-53:39-53. medical microbiology. Little, Brown & Co., 18. Wachsman, J. T., and H. A. Barker. 1954. Boston. Characterization of an orotic acid fermenting 4. Holdeman, L. V., and W. E. C. Moore. 1972. bacterium, Zymobacterium oroticum, nov. gen., Anaerobic cocci. In L. V. Holdeman and W. E. C. nov. spec. J. Bacteriol. 68:400404.