Synonomy of Peptococcus glycinophilus

INTERNATIONALJOURNAL OF SYSTEMATIC
BACTERIOLOGY,
Apr. 1983, p. 207-210
0020-7713/83/020207-O4$02.OO/O
Copyright 0 1983, International Union of Microbiological Societies
Vol. 33, No. 2
Synonomy of Peptococcus glycinophilus (Cardon and Barker
1946) Douglas 1957 with Peptostreptococcus micros (PrCvot
1933) Smith 1957 and Electrophoretic Differentiation of
Peptostreptococcus micros from Peptococcus magnus (PrCvot
1933) Holdeman and Moore 1972
ELIZABETH P. CATO,* JOHN L. JOHNSON, D. E. HASH, AND LILLIAN V. HOLDEMAN
Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg,
Virginia 24061
The soluble cellular proteins of strains of Peptococcus magnus and Peptostreptococcus micros were examined by polyacrylamide gel electrophoresis. The
protein patterns were distinctive and repeatable, and the two species could be
separated readily. The protein pattern of strain ATCC 23195, the type strain of
Peptococcus glycinophilus (Cardon and Barker 1946) Douglas 1957, was identical
to that of strain ATCC 33270 (= VPI 5464), the type strain of Peptostreptococcus
micros (Prevot 1933) Smith 1957. Because these two strains were 84% homologous as determined by deoxyribonucleic acid-deoxyribonucleic acid homology
experiments, the name Peptococcus glycinophilus is a later subjective synonym of
Peptostreptococcus micros. Preliminary results indicated that “Pep tococcus
variabilis” (Foubert and Douglas 1948) Douglas 1957 may be a valid species, but
reinstatement is not proposed at this time.
Peptococcus magnus and Peptostreptococcus
micros are anaerobic cocci that frequently are
isolated, either in pure culture or together with
other organisms, from a wide range of human
infections. These two species are non-saccharoclastic. The growth of most strains is stimulated
by the addition of Tween 80 to culture media (6).
Most strains of Peptococcus magnus digest gelatin slowly when Tween 80 is present in the
medium, and ammonia is usually produced from
peptone by both Peptococcus magnus and Peptostreptococcus micros. Strains of both species
do not hydrolyze esculin or starch, and they do
not produce indole or reduce nitrate; little or no
gas appears in agar deep cultures in peptoneyeast extract-glucose agar cultures. The principal acid product in broth cultures is acetate,
although trace amounts of lactate and succinate
may be detected. Although glucose may stimulate the growth of many strains, the acids present in peptone-yeast extract-glucose broth cultures appear to be derived from peptone rather
than from glucose, because equal or greater
amounts of products are detected in the same
medium without glucose.
Separation of Peptococcus magnus and Peptostreptococcus micros usually is based on cellular morphology (6); strains with cells more
than 0.6 Fm in diameter that occur singly, in
pairs, or occasionally in short chains of pairs are
207
identified as Peptococcus magnus, whereas
strains with cells less than 0.6 pm in diameter
that occur usually in short chains but occasionally singly and in pairs are identified as Peptostreptococcus micros (6). These parameters
can vary with the age of the culture and the
composition of the medium in which the strain is
grown. Therefore, we have continued to search
for better methods to differentiate these species.
In this paper we present the results of a polyacrylamide gel electrophoretic (PAGE) analysis
of the soluble cellular proteins of the type and
reference strains of Peptococcus magnus and
Peptostreptococcus micros and of strains of
phenotypically similar species.
MATERIALS AND METHODS
Bacterialstrains. Many of the strains used were from
the collection of A.-R. Prdvot (Pasteur Institute, Paris,
France), who first described both Peptococcus magnus and Peptostreptococcus micros (14). All strains
had been characterized and identified previously in
our laboratory by using traditional biochemical tests
(6). The following strains of Peptococcus magnus
were used: ATCC l5794= (type strain) (15) (= VPI
4286 = Prdvot 2974 [bbDiplococcusmagnus”]), from
purulent cystitis; VPI 4288-1 (= Prdvot 1249A [“D.
magnus”]), from umbilicus pus; VPI 4735 and VPI
5026B, products of abortion, Virginia; VPI 5434 (=
Prevot 1269), from a cervical ulcer; ATCC 14956 (=
VPI 5663), from a postpartum uterus, Illinois; VPI
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208
INT. J. SYST.BACTERIOL.
CAT0 ET AL.
6217, from a sebaceous cyst, Connecticut; VPI 8532,
from a breast abscess, Georgia; ATCC 29328 (= VPI
9274), from an abdominal wound, California; VPI
11505, from a testicle, Maryland; and ATCC 14955,
the type strain of “Peptococcus variabilis” (= VPI
13647), from a draining sinus, Texas. The strains of
Peptostreptococcus micros used were: VPI 5464, the
parent strain of ATCC 33270T (15) (= Prtvot 3119B
[labeled “Streptococcus micros”]), from purulent
pleuresy; VPI 5462 (= Prtvot 3104A [labeled “ S .
micros”]), from actinomycosis; VPI 5746 (= Prtvot
3330B [labeled “ S . micros”]), from pleural fluid; VPI
5747 (= PrCvot 3383 [labeled “ S . micros”]), from
sputum; VPI 5853 (= Prkvot 3660 [labeled “S. micros”]), from a brain abscess; VPI 10958, from a brain
abscess, Florida; VPI D1A-4, from a gingival sulcus,
moderate periodontitis, Virginia; VPI D7B-28, from a
gingival sulcus, severe periodontitis, Virginia; VPI
E4Y-16, from a gingival sulcus, experimental periodontitis, Virginia; and ATCC 23195T (Peptococcus
glycinophilus; = VPI 67113, from San Francisco Bay
mud. Streptococcus faecalis VPI U4-20 also was included in this study as a control in the PAGE analyses.
Methods. Cells from 24-h cultures were inoculated
into 5 ml of prereduced, anaerobically sterilized supplemented brain heart infusion broth (10) which contained 0.1% (wt/vol) calcium carbonate and 0.025%
(vol/vol) Tween 80. After incubation for 24 h at 37”C,
the cells were harvested by centrifugation at 8,000 x g
for 10 min. The PAGE patterns of soluble cellular
proteins were determined as described by Moore et al.
(10).
Enzyme activities other than those listed in the
Anaerobe Laboratory Manual (6) were determined by
using the API-ZYM system (Analytab Products, Plain-
view, N.Y.) according to the directions of the manufacturer.
Conversion of glycine to acetate and COz was
determined chromatographically (6) after cultures
were incubated in an oxygen-free nitrogen atmosphere
in peptone-yeast extract-1% glycine broth. The results were compared with those obtained with cultures
in peptone-yeast extract broth without glycine. Ammonia production was estimated by using Nessler
reagent (6).
The methods used for isolating deoxyribonucleic
acid (DNA), determining the guanine-plus-cytosine
content of the DNA, and preparing labeled DNA have
been described previously (8). A modification (8) of
the S1 nuclease method was used for the DNA homology experiments.
RESULTS AND DISCUSSION
The PAGE patterns of the soluble cellular
proteins of the type and reference strains of
Peptococcus magnus and Peptostreptococcus
micros are shown in Fig. 1. The patterns of
strains of Peptococcus magnus were more heterogeneous than those of strains of Peprostreptococcus micros. However, the general similarity of the protein patterns of the Peptococcus
magnus strains showed that these strains are
closely related. Similar pattern variability has
been found among genetically homologous
strains within species of BiJidobacterium (1)and
species of Clostridium (3). Markowitz and Lerner have reported immunologically distinct
groups in Peptococcus magnus (9), but we do
FIG. 1. PAGE patterns of strains of P. magnus and P . micros.Lane 1,S.faecalis, control strain (VPI U4-20).
Lanes 2 to 11, P . magnus: 2, VPI 4286 (= ATCC 15794, type strain); 3, VPI 4288-1; 4, VPI 4735; 5 , VPI 5026B; 6,
VPI 5434; 7, VPI 5663;8, VPI 6217; 9, VPI 8532; 10, VPI 9274; 11, VPI 11505. Lanes 12 to 20, P.micros: 12, VPI
5464 (= ATCC 33270, type strain); 13, VPI 5462; 14, VPI 5746; 15, VPI 5747; 16, VPI 5853; 17, VPI DlA4; 18,
VPI D7B28; 19, VPI E4Y16; 20, VPI 10958.
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P. MICROSs AND P. MAGNUS
VOL.33, 1983
not know whether the slight pattern variation
observed correlates with different serotypes.
The bands near the origin (top of gel), the sharp
band for Peptostreptococcus micros near the
center of the gel, and the patterns in the lower
one-quarter of the gel were distinctive for each
species and allowed reliable confirmation of
identification.
All strains of Peptostreptococcus micros tested showed strong acid phosphatase reactions
and moderate alkaline phosphatase reactions
when they were incubated in the semiquantitative API-ZYM system, confirming the report of
Porschen and Spaulding (13a). Only trace to
moderate acid phosphatase activity and no alkaline phosphatase activity were detected in the
strains of Peptococcus magnus. Other enzyme
reactions in this system were similar for both
species.
, Peptococcus glycinophilus (Cardon and Barker) Douglas 1957, which was isolated from San
Francisco Bay mud, is phenotypically similar to
Peptostreptococcus micros and Peptococcus
magnus. Strains of all three species are nonreactive in conventional biochemical tests, and similar acid products are detected chromatographically from peptone-yeast extract-glucose
medium (2, 6). Peptococcus glycinophilus was
proposed originally as a separate species because of its ability to convert glycine to acetate,
ammonia, and COz (2). The PAGE protein pattern of the type strain of Peptococcus glycinophilus, strain ATCC 23195, wai identical to that of
the type strain of Peptostreptococcus micros,
strain ATCC 33270 (Fig. 2). This strain of Peptococcus glycinophilus produced amounts of acid
and alkaline phosphatases that were equal to the
amounts produced by the strains of Peptostreptococcus micros tested. In addition, the type
strains of Peptostreptococcus micros and Peptococcus magnus converted glycine to acetate,
ammonia, and COz when they were incubated in
peptone-yeast extract-1% glycine broth in an
oxygen-free nitrogen atmosphere. The guanineplus-cytosine content of the DNA of the type
strain of Peptostreptococcus micros was 27
mol% (as determined by thermal denaturation),
whereas that of the type strain of Peptococcus
glycinophilus was 28 mol%. DNA preparations
from the type strain of Peptococcus glycinophilus were 84% homologous with DNA from the
type strain of Peptostreptococcus micros.
Therefore, we propose that Peptococcus glycinophilus be considered a later synonym of Peptostreptococcus micros (Prkvot 1933) Smith
1957.
In 1948, Foubert and Douglas proposed a new
species of anaerobic cocci, “Micrococcus variabilis” (5). Douglas later transferred this species
to the genus Peptococcus (4). Two of the strains
209
included in the original study, strain BU from a
draining sinus and strain U3 from a postpartum
uterus, were deposited in the American Type
Culture Collection by Foubert as strains ATCC
14955 and ATCC 14956, respectively; strain
ATCC 14955 was designated the type strain of
the species. When these strains were tested in
our laboratory, their phenotypic reactions were
indistinguishable from the reactions of Peptococcus magnus, and in 1973 West and Holdeman suggested that “Peptococcus variabilis’ ’ be
considered a later synonym of Peptococcus
magnus (16). However, when cultures of these
two strains were subjected to PAGE analysis,
the protein pattern of strain ATCC 14956 was
like that of the type strain of Peptococcus magnus, but the electrophoretic protein pattern of
the designated type strain of “Peptococcus variabilis,” strain ATCC 14955, was distinct (Fig.
2). One band, which was 50 mm from the origin,
did not appear in any other strain of Peptococcus magnus that we tested. This difference could
indicate that strains ATCC 15794T and ATCC
149ST represent distinct species. However, we
do not wish to propose that the name “Peptococcus variabilis” (Foubert and Douglas) Douglas 1957 be revived until DNA-DNA homology
experiments provide a definitive answer.
Although strains of Peptostreptococcus micros (Peptococcus glycinophilus) were isolated
FIG. 2. PAGE patterns of type and reference
strains of some anaerobic cocci. Lane 1, S. faecafis,
control strain (VPI U4-20). Lanes 2 and 3, P . micros
ATCC 33270 (type strain); lanes 4 and 5, P. gfycinophifus ATCC 23195 (type strain); lanes 6 and 7, P .
magnus ATCC 15794 (type strain); lanes 8 and 9, P .
magnus ATCC 14956 (reference strain); lanes 10 and
11, “P.variabilis” ATCC 14955.
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210
INT. J. SYST. BACTERIOL.
CAT0 ET AL.
from enrichment cultures of San Francisco Bay Dental Research and the National Institute of Allergy and
Diseases, respectively, and by Commonwealth of
mud (2), the principal habitat of this species is Infectious
Virginia project 2022820.
the human oral cavity. In an investigation of the
floras of the gingival crevices of healthy and
LITERATURE CITED
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more prevalent in samples from diseased peri2. Cardon, B. P., and H. A. Barker. 1946. Two new amino
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acid-fermenting bacteria, Clostridium propionicum and
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previously identified as Peptococcus magnus
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technic Institute and State University Anaerobe Laborastrains were from oral or pleural regions, and 1
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recovered no strains of Peptostreptococcus miHuman fecal flora: variation in bacterial composition and
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ACKNOWLEDGMENTS
We are grateful to W. E. C.Moore for critically reviewing
the manuscript and to Leesa Miller, Ann Ridpath, and Carol
Phelps for excellent technical assistance.
This work was supported by Public Health Service grants
DE 05218-01 and A1 15244-01 from the National Institute of
15. Skerman, V. B. D., V. McGowan, and P. H. A. Sneath
(ed.). 1980. Approved lists of bacterial names. Int. J. Syst.
Bacteriol. 30:225-420.
16. West, S. E. H., and L. V. Holdeman. 1973. Placement of
the name Peptococcus anaerobius (Hamm) Douglas on
the list of nomina rejicienda. Request for an opinion. Int.
J. Syst. Bacteriol. 23:283-289.
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