We describe Peptostreptococcus hydrogenalis sp. nov., which is found in human feces and ... previously classified as Peptostreptococcus group A-1) were.
Vol. 40, No. 3
INTERNATIONAL JOURNALOF SYSTEMATIC BACTERIOLOGY, July 1990, p. 305-306 0020-7713/90/030305-02$02.00/0 Copyright 0 1990, International Union of Microbialogical Societies
Peptostreptococcus hydrogenalis s p . nov. from Human Fecal and Vaginal Flora TAKAYUKI EZAKI," SHU-LIN LIU, YASUHIRO HASHIMOTO, AND EIKO YABUUCHI Department of Microbiology, School of Medicine, Gifu University, 40 Tsukasa-machi, Gifu 500, Japan We describe Peptostreptococcus hydrogenalis sp. nov., which is found in human feces and vaginal discharge. This new species was established on the basis of the results of DNA-DNA hybridization among anaerobic cocci. The results of differential biochemical reactions also are given. The type strain of this species is strain GIFU 7662 (= JCM 7635). cus heliotrinreducens and Peptostreptococcus barnesae were not included) and the three group A-1 isolates confirmed the distinctness of these three strains (Table 1) from the other species studied. The group A-1 strains are unlike P . heliotrinreducens (6) and P . barnesae (9) because group A-1 strains produce indole. In addition, P . heliotrinreducens reduces nitrate and group A-1 strains do not, and P . barnesae does not produce butyrate but group A-1 strains produce
In a previous study (3), we reported that three strains of gram-positive anaerobic cocci (strains GIFU 1244, GIFU 7662= [T = type strain], and GIFU 7872, which were previously classified as Peptostreptococcus group A-1) were genetically distinct from strains of other species studied. These three strains were strictly anaerobic gram-positive cocci that grew in anaerobic peptone-yeast extract medium. Thus, they did not require a fermentable carbohydrate for
TABLE 1. Levels of relatedness among strains of P . hydrogenalis and strains belonging to eight other species of the genus Peptostreptococcusa % Relatedness with labeled DNA from:
rr+r Competitive DNA from:
content (mol%)
P . hydrogenalis GIFU 7662T at 37°C
P . prevotii ATCC 9321T at 37°C
P . hydrogenalis GIFU 7662T at 45°C
P . prevotii ATCC 9321T at 45°C
P . hydrogenalis GIFU 7662T P . hydrogenalis GIFU 1244 P . hydrogenalis GIFU 7872 P . asaccharolyticus ATCC 14936T P . indolicus ATCC 29427T P . prevotii ATCC 9321T P . tetradius GIFU 7672T P . anaerobius ATCC 27337T P . magnus ATCC 15794* P . micros VPI P . productus ATCC 27340T
31b 31 31 32 34 33 32b 33 32 28 45
100 98 85 23 20 24 15 0 10 3 0
10 6 10 0 4 100 39 0 17 0 0
100 90 83 5 0 6 0 0 6 0 0
5 0 10 1 0 100 25 0 4 0 0
a
DNAs were hybridized in 2~ SSC ( I x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) containing 50% formamide for 2 h as described previously (1). The values for strains GIFU 7662T and GIFU 7672T were reported to be 30 mol% in a previous study (3) but have been revised.
butyrate as a major metabolic product from peptone-yeast extract broth supplemented with 1%glucose (PYG). Phenotypically, group A-1 strains most closely resemble Peptostreptococcus asaccharolyticus or Peptostreptococcus indolicus because they produce indole and butyrate. Unlike P . indolicus (4), they do not produce coagulase, and unlike P . asaccharolyticus (3,they produce acid from glucose and abundant hydrogen from PYG. These strains produce more butyrate than strains of other butyrate-producing species. In our studies several times PYG cultures in flasks closed with rubber stoppers produced so much gas that the stoppers burst from the flasks during incubation. From 10 ml of modified Gifu anaerobic medium (GAM; Nissui, Tokyo), the type strain of Peptostreptococcus hydrogenalis sp. nov. produced 5 ml of hydrogen within 24 h, whereas P . asaccharolyticus, P . indolicus, and Peptostreptococcus prevotii strains produced less than 1.5 ml of hydrogen in the same medium. Because group A-1 strains produce copious hydrogen from peptone-yeast extract medium and PYG as determined by gas chromatography, we propose to name them Peptostreptococcus hydrogenalis.
growth. The guanine-plus-cytosine (G+C) content of their DNAs was 31 mol%, which is close to the values reported for other members of the genus Peptostreptococcus (7, 8). On the basis of their G+C contents and their ability to grow in peptone without a fermentable carbohydrate, these strains were considered to be members of the genus Peptostreptococcus (3). Our recent studies of partial 16s rRNA nucleotide sequences of 21 strains of anaerobic cocci representing 20 species (manuscript in preparation) have indicated that the members of this group, which was provisionally classified as Peptostreptococcus group A-1 (3), are members of the genus Peptostreptococcus . Quantitative DNA-DNA hybridization in microdilution plates (1) both at the optimal temperature (37°C in 50% formamide) and under stringent conditions (45°C in 50% formamide) to evaluate the levels of genetic relatedness among the type strains of 8 of the 10 previously described species of the genus Peptostreptococcus (Peptostreptococ-
* Corresponding author. 305
306
NOTES
INT.J. SYST.BACTERIOL. TABLE 2. Differential characteristics of P. hydrogenalis and other Peptostreptococcus species G+C
Species
P. hydrogenalis P. asaccharolyticus P. indolicus P. prevotii P. tetradius P. anaerobius P. magnus P. micros P. productus P. heliotrinreducens P. banesae
content (mo*%)
Production of Butyrate
Gas
Indole
Coagulase
31 30-34 32-34 29-33 30-32 33-34 32-34 28-29 44-45 35-37 34-35
Nitrate
Alkaline
reduction
phosphatase activity
+ ++
Acid produced from: Glucose
Peptidase activitiesa
Pyrrolidone AMD activity
Maltose
b
-b
+ -
ND' ND
a Peptidase activities include arginine AMD, ornithine AMD, lysine AMD, alanine AMD, alanine-arginine AMD, arginine-arginine AMD, and phenylalaninearginine AMD activities. The activities of these peptidases, together with those of alkaline phosphatase and pyrrolidone AMD, were determined as described by Ezaki and Yabuuchi (2). Some strains are positive. Some strains are weakly positive. A trace amount of butyrate is produced. ND, Not determined.
Descriptionof Peptostreptococcus hydrogenalis sp. nov. Peptostreptococcus hydrogenalis (hy.dro.ge.na'lis . chemical term hydrogen; L. suff. -alis, pertaining to; N . L. masc. adj. hydrogenalis, pertaining to hydrogen, because this organism produces hydrogen). Cells are strictly anaerobic, grampositive cocci (diameter, 0.7 to 1.8 pm) and occur in short chains or in masses. Colonies on sheep blood agar range from 0.5 to 2 mm in diameter and are not hemolytic. The three strains which we studied do not produce coagulase or reduce nitrate. Abundant hydrogen gas and weak acid (pH range, 5.6 to 6.0) are produced from PYG. No acid is produced from L-( +)-rhamnose, D-( +)-xylose, maltose, D(+)-mannose, D-( -)-mannitol, sucrose, D-( -)-fructose, D(+)-cellobiose, or D-( +)-trehalose. Indole and alkaline phosphatase are produced. Catalase activity is weak or negative. Pyrrolidone arylamidase (AMD) activity is positive. The major metabolic products from prereduced PYG are butyrate and acetate. P. hydrogenalis has been isolated from human feces and vaginal discharge. The pathogenicity of this organism is unknown. The G+C content of the DNAs of the type strain and two other strains is 31 mol%. The type strain is strain GIFU 7662 (= JCM 7635), which was isolated from human feces. Characteristics useful for differentiating P. hydrogenalis from other species of peptostreptococci are shown in Table 2. Positive alkaline phosphatase and pyrrolidone AMD activities and acid production from glucose are useful characteristics for differentiating this species from P . asaccharolyticus. P . hydrogenalis has week a-glucosidase, p-glucronidase, p-glucosidase, and P-galactosidase activities; the other two indole-positive species do not have these enzyme activities. The oligopeptidase activities of P. hydrogenalis as determined by using a commercial API-ZYM kit (kindly supplied by Aska Pure Chemical Co., Tokyo, Japan) were distinct from those of P. asaccharolyticus. P. hydrogenalis and P . asaccharolyticus have the same aspartate-alanine AMD, serine-tryptophan AMD, lysine-lysine AMD, benzyl-cysteine AMD, and lysine-alanine AMD activities, but the aspartate-arginine AMD activity of P. hydrogenalis is positive and that of P. asaccharolyticus is negative or very weak (2). Of the 20 amino acid AMD activities tested, P. hydro-
genalis showed only pyrrolidone AMD activity, whereas P. asaccharolyticus was active on most of the substrates tested (2) but did not show pyrrolidone AMD activity. We thank T. 0. MacAdoo (Virginia Polytechnic Institute and State University, Blacksburg) for suggesting the specific epithet and W. E. C. Moore for his kind advice. LITERATURE CITED
1. Ezaki, T., H. Hashimoto, and E. Yabuuchi. 1989. Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used for labeling to quantitate genetic relatedness among bacterial strains. Int. J. Syst. Bacterial. 39:224-229. 2. Ezaki, T., and E. Yabuuchi. 1985. Oligopeptidase activity of Gram-positive anaerobic cocci used for rapid identification. J. Gen. Appl. Microbiol. 31:255-265. 3. Ezaki, T., N. Yamamoto, K. Ninomiya, S. Suzuki, and E. Yabuuchi. 1983. Transfer of Peptococcus indolicus, Peptococcus asaccharolyticus, Peptococcus prevotii, and Peptococcus magnus to the genus Peptostreptococcus and proposal of Peptostreptococcus tetradius sp. nov. Int. J. Syst. Bacteriol. 33:683498. 4. Hoi-Sbrenson, G. 1973. Micrococcus indolicus. Some biochemical properties, and the demonstration of six antigenically different types. Acta Vet. Scand. 14:301-326. 5. Holdeman, L. V., E. P. Cato, and W. E. C. Moore (ed.). 1977. Anaerobe laboratory manual, 4th ed. Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg. 6. Lanigan, G. W. 1976. Peptococcus heliotrinreducens sp. nov., a cytochrome-reducing anaerobe which metabolizes pyrrolidizine alkaloids. J. Gen. Microbiol. 94:l-10. 7. Moore, L. V. H., J. L. Johnson, and W. E. C. Moore. 1986. Genus Peptostreptococcus Kluyver and van Niell936, p. 1083-1092. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 2. The Williams & Wilkins Co., Baltimore. 8. 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. 21:234-237. 9. Schiefer-Ullrich,H., and J. R. Andreesen. 1985. Peptostreptococcus barnesae sp. nov., a Gram-positive, anaerobic, obligately purine utilizing coccus from chicken feces. Arch. Microbiol. 143:26-3 1.
