INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, Apr. 1986, p. 213-221 Vol. 36, No.

2
0020-7713/86/020213-09$02.00/0
Copyright 0 1986, International Union of Microbiological Societies

Bacteroides forsythus sp. nov. , a Slow-Growing, Fusiform

Bacteroides sp. from the Human Oral Cavity
A. C. R. TANNER,l* M: A. LISTGARTEN,* J. L. EBERSOLE,l AND M. N. STRZEMPKO'
Department of Microbiology, Forsyth Dental Center, Boston, Massachusetts 02115l and University of Pennsylvania,
Philadelphia, Pennsylvania 191M2

The characteristics of a group of slow-growing, fusiform, fastidious anaerobes isolated from advanced
periodontal lesions in human oral cavities were examined. Our results indicated that 12 fusiform Bacteroides
strains belong to a new species in the genus Bacteroides. The name Bacteroides forsythus is proposed for these
isolates. The type strain is strain ATCC 43037.

Anaerobic, gram-negative, fusiform organisms which did G+C contents were determined by the thermal denaturation
not resemble previously recognized speqies were isolated method (1). Levels of DNA-DNA homology were deter-
from deep periodontal pockets of humans that showed mined by using the initial renaturation rates (2).
progressive tissue loss (3, 15, 17). The cells appeared to be API enzyme substrate tests. API ZYM and API An-Ident
long filaments or medium rods with tapered (fusiform) or enzymatic substrate tests were performed in duplicate as
rounded ends. Some cells had central swellings. Large recommended by the manufacturer (Analytab Products)
spheroids were frequently associated with the cells, partic- (18). Cells from agar plates were suspended in sterile water,
ularly the cells with filamentous morphology. These strictly inoculated onto the test strips, and incubated in air for 4 h at
anaerobic fusiform Bacteroides strains grew slowly on blood 35°C.
agar plates and grew poorly in all broth media tested and on PAGE (i) Gel preparation. We prepared 9% sodium dode-
most agar media tested. Biochemical tests gave inconsistent
results and were usually negative. For this reason, the
fusiform Bacteroides strains were characterized without TABLE 1. Strains used and their sources
using most of the conventional biochemical tests, Deoxyri-
Species Strain(s)a Source
bonucleic acid (DNA) guanine-plus-cytosine (G +C) content,
DNA-DNA homology, cell wall ultrastructure, serology, Fusiform Bacteroides ATCC 43037T Periodontal
API enzyme substrate tests (Analytab Products, Plainview, (= FDC 3389, pockets
N.Y.), and protein profiles of whole sonicated cells obtained FDC 42, 148, 293,
by using polyacrylamide gel electrophoresis (PAGE) indi- 334, 436, 2008,
cated that these organisms comprise a distinct species in the and R145
FDC BV16, FDC Periapical tissue
genus Bacteroides. The name Bacteroides forsythus is pro- BV19, FDC
posed for this new species. DL12, and FDC
Strains and inocula. The strains which we used are listed in DL38
Table 1. These organisms were maintained on Trypticase Reference species
soy agar plates supplemented with 5% sheep blood (TSBA; B . fragilis ATCC 25285T Appendix abscess
BBL Microbiology Systems, Cockeysville, Md.) at 35 to ATCC 23745 Pleural fluid
37°C in an atmosphere containing 10% H2, 10% CO2, and B . thetaiotaomicron ATCC 29148T Human feces
80% N2. The fusiform Bacteroides strains were maintained ATCC 12290 Appendix abscess
on TSBA plates; a fusiform Bacteroides strain was streaked B. distasonis ATCC 8503T Human feces
B. ovatus ATCC 8483T Human feces
on one-half of each plate, and a strain of Fusobacterium B. vulgatus ATCC 8482T Human feces
nucleaturn was streaked on the other half of the plate. The B. zoogleoformans ATCC 332MT Gingival sulcus
strains were stored in liquid nitrogen by using previously B. buccae ATCC 33574T and Gingival sulcus
described methods (14). Inocula for tests were obtained by (B. capillus) ATCC 33691
scraping cells from the surfaces of TSBA plates. At least 100 B. oris ATCC 33573T Periodontal pocket
plates of fusiform Bacteroides strains were used to obtain B. oralis ATCC 33269T Periodontal pocket
sufficient cells for DNA extraction. Two to four plates were Bacteroides ATCC 29799T Human feces
used to provide the inoculum for an enzyme-linked immu- capillosus
nosorbent assay and for each of the tests in the API enzyme C. sputigena ATCC 33612T Periodontal pocket
( = FDC 4T)
substrate test series. C. gingivalis ATCC 33624T Periodontal pocket
DNA-DNA hybridization. Cells of reference strains were ( = FDC 27T)
grown in 2- to 3-liter broth cultures (mycoplasma broth C. ochracea FDC 6 Periodontal pocket
[BBL] supplemented with 5 mg of hemin per liter) for F. nucleatum FDC 364 and 40 Periodontal pocket
extraction of DNA. The DNA was extracted by using a F. gonidiaformans ATCC 25563T
method modified (13) from the procedure of Marmur (9). W . recta ATCC 33238T Periodontal pocket
a ATCC, American Type Culture Collection, Rockville, Md.; FDC,Forsyth
* Corresponding author. Dental Center, Boston, Mass.

213
214 TANNER ET AL. INT. J. SYST.BACTERIOL.

FIG. 1. PAGE of whole sonicated cells of B. forsythus and oral fusiform species. The upper and lower lines indicate the positions of the
superimposed internal standards. B. forsythus is characterized by two protein bands at molecular weights of more than 205,000. Lane a,
High-molecular-weight standard mixture; lanes 1, 10, and 18, F. nucleaturn FDC 364; lanes 2 through 9 and 11through 14, B. forsythus strains
(lane 2, strain FDC 338T [= ATCC 43037T]; lane 3, strain FDC 42; lane 4, strain FDC 148; lane 5, strain FDC 293; lane 6, strain FDC 334;
lane 7, strain FDC 436; lane 8, strain FDC 2008; lane 9, strain FDC R145; lane 11, strain FDC BV19; lane 12, strain FDC DL12; lane 13, strain
FDC DL38; lane 14,strain FDC BV16); lane 15, C. gingivalis ATCC 33624T;lane 16, C . ochracea FDC 6; lane 17, C.sputigena ATCC 33612=.

cyl sulfate discontinuous polyacrylamide gels (thickness, 1.5
mm) with 20 wells in a vertical slab unit (model SE600; 20 by
20 cm) as recommended by Hoefer Scientific Instruments,
San Francisco, Calif. The stacking gels contained 4% poly-
acrylamide. Polyacrylamide gel solutions were degassed for
15 min in a Brewer Jar at a negative pressure of 0.066 Pa (26
to 28 lb/in2). The gels were left overnight before use.
(ii) Loading and running the gels. Cells were suspended in
0.2 ml of water and disrupted by sonication for 1min with a
microultrasonic cell disrupter (Kontes, Vineland, N.J.) at a
power setting of 2. A treatment buffer containing 0.125 M tris
(hydroxymethy1)aminomethane hydrochloride (pH 6.8), 4%
sodium dodecyl sulfate, 20% glycerol, and 10% 2-
mercaptoethanol was added, the suspensions were boiled for
5 min, and a phenol red tracking dye was added; 2 to 10 ~1 of
each suspension was added to each well. The gels were run
at a constant current of 30 mA per gel until the tracking dye
was within 1 cm of the end of the gel (about 3 to 5 h).
(iii) Staining. The gels were fixed overflight in 50%
methanol-50% distilled water containing 0.1 ml of 37%
(wt/wt) formaldehyde per 100 ml of solution. The fixative
was removed, 500 ml of a 5-pg/ml dithiothreitol solution was
added to each gel, and the gels were agitated for 1.5 h. The
dithiothreitol was poured off, 500 ml of silver stain (a 0.1%
solution of AgN03) was added to each gel, and the prepara-
tions were agitated for 1 h. The gels were washed with
distilled water for 30 s and then washed twice with 100 ml of
developer containing 3% Na2C03and 0.5 ~1 of formaldehyde
per ml. A 200-ml portion of developer was then added to
each gel for 2 min or until the bands were sufficiently stained.
Gel staining was stopped by adding 2.3 M citric acid (9.66 g
of citric acid in 20 ml of water for each gel) and shaking the
preparations for 10 min. The gels were stored wet in plastic
wrap in plastic bags.
(iv) Gel analysis. Only gels prepared from the same batch
of acrylamide and electrophoresed at the same time in the
same tank were compared. The protein bands on the wet gels
were scanned by using a model 2202 Ultroscan laser spec-
trophotometer (LKB, Stockholm, Sweden). Bacterial pro-
tein profiles were compared by performing cluster analysis
of correlation coefficients as described by Kersters and De
Ley (7). The low- and high-molecular-weightstandards used
were protein bands of F. nucleaturn FDC 364, which was put
on three places on the gel. The selected bands were con-
nected with black lines (Fig. 1 and 2). Preliminary experi-
ments revealed that the fusiform Bacteroides strains were
characterized by proteins having mdlecular weights of more
than 205,000, the molecular weight of a myosin standard. As
previously noted by Weber and Osborn (19), myosin is
unsuitable as an internal standard because it yields several
electrophoretic bands. The molecular weights of protein
bands were estimated by reference to a high-molecular-
weight standard mixture (Sigma Chemical Co., St. Louis,
Mo.).
Electron microscopy. Cell wall ultrastructure was deter-
mined as described previously (16). Cells of fusiform Bacte-
roides strains from 6-day-old colonies on TSBA plates were
fixed for 2 h in a mixture containing 5% (vol/vol) glutaralde-
hyde and 4% (vol/vol) formaldehyde buffered to pH 7.3 with
sodium cacodylate (6). Reference strains were harvested
VOL. 36, 1986 BACTEROIDES FORS YTHUS SP. NOV. 215

FIG. 2. PAGE of whole sonicated cells of reference Bacteroides and other oral species. The upper and lower lines indicate the position
of the superimposed F . nucleatum internal standards. Lane a, High-molecular-weightstandard mixture; Lanes 1, 10, and 18, F . nucleaturn
FDC 364; lanes 2 and 3, B . fragilis ATCC 252tUTand ATCC 23745, respectively; lanes 4 and 5, B . thetaiotaomicron ATCC 2914gTand ATCC
12290, respectively; lane 6, B . distasonis ATCC 8503T;lane 7, B . vulgatus ATCC 8482T;lane 8, B . zoogfeoforrnans ATCC 33285T;lane 9,
Bacteroides capillosus ATCC 29799T;lanes 11 and 12, B . buccae ATCC 33574Tand ATCC 33691, respectively; lane 13, B . oris ATCC 33573T;
lane 14, B . orafis ATCC 33269T;lane 15, F . gonidiafurrnans ATCC 25563; lane 16, W . recta ATCC 3323gT;lane 17, F . nucleaturn FDC 40.

after 3 to 4 days. After a 20-min wash in s-collidine buffer, and embedded in Epon (8). Sections (0.1 pm) were cut,
the cells were postfixed in 2% s-collidine-buffered osmic acid mounted on bare 300-mesh grids, stained with uranyl acetate
for 90 min. The fixed cells were stored overnight in cold and lead citrate ( l l ) , and examined and photomicrographed
s-collidine buffer, dehydrated in graded ethanol solutions, with a Philips model EM-300 transmission electron micro-
scope. Negatively stained preparations of whole cells were
obtained by applying single drops of cellular material sus-
TABLE 2. G + C contents and levels of DNA-DNA homology pended in 2% phosphotungstic acid to carbon-reinforced
among B . forsythus strains Formvar-coated grids. The excess liquid was removed by
Level of DNA homology (%) with blotting the edge of the grid and lifting the grid to air dry.
G+C B . forsythus strain: Serologic methods. Fusiform Bacteroides strain FDC 33gT
Isolate content (T = type strain) harvested from 100 blood agar plates was
(mol%)" ATCC FDC FDC FDC FDC FDC
43037= 42 293 334 BV19 DL12 used to prepare antisera. The cells were suspended in
phosphate-buffered saline (0.02 M phosphate, pH 7.5) con-
ATCC 43037T 46 loob 100 100 94 taining 1 mM ethylenediaminetetraacetate, washed with the
FDC 42 46 100 100 95 same solution and killed with 0.5% neutral Formol-saline
FDC 148 46 100 (0.5% formaldehyde-0.15 M NaCI [pH 7.01 supersaturated
FDC 293 100 100 75
FDC 334 44 95 95 75 100
with MgC03) (4). Rabbit antisera were produced by two
FDC 436 47 89 98 75 subcutaneous injections (14 days apart) of lo9 Formalin-
FDC 2008 47 86 100 fixed cells. The globulin fraction was separated from the
FDC R145 48 92 97 88 serum by precipitation with 40% (NH4)2S04, conjugated
FDC BV16 46 100 100 with horseradish peroxidase (lo), and stored at -20°C until
FDC BV19 45 96 100 100 99 it was used. The conjugate was tested for antibody activity
FDC DL12 47 99 100 by using an enzyme-linked immunosorbent assay (5). After
FDC DL38 47 94 75 100 preliminary tests with strains belonging to 16 oral species,
Determined from the midpoint of thermal denaturation. G + C contents dilutions that did not exhibit cross-reactions were chosen for
were calculated from the values for reference strains of other Bacteroides species comparisons.
species that were 2 mol% higher than previously published values. Therefore, The enzyme-linked immunosorbent assay was performed
a 2 mol% correction was used for the B . forsythus isolates to determine their by using cultures obtained from blood agar plates and
G + C contents.
Mean of at least two determinations obtained by using the initial renatura- suspended in phosphate-buffered saline. Cell suspensions
tion rate (2). were standardized to an optical density at 580 nm of approx-
216 TANNER ET AL. INT. J. SYST.BACTERIOL.

Correlation Coefficient
0.4 0.5 0.6 0.7 0.8 0.9 1 .o
I I I I I 1 1
ATCC 33574' - B. buccae
ATCC 3326gT - 8. oralis
ATCC 33691 - B. buccae
ATCC 33573' - B. oris
- B. capillosis

n
ATCC 29799
40
364
3F. nucleatum
ATCC 25285'3B. fras,is
ATCC 23745
12290 B. thetaiotamicron
ATCC 29148'3
ATCC 33285 - B. ~ 0 0 8 l m m
ATCC 8503' - B. distasonis
ATCC 8482f - B. vulgatus
ATCC 33238' - W. recta
ATCC 25563 - F. gonidi4fonnanr
ATCC 33624' - C. gbgiwlis
6 - C. ochracea
ATCC 33612' - C. spurigena
DL38

42
436 B. jbrsythus
334
293
148
R145
BV16
FIG. 3. Cluster analysis of the protein profiles of B . forsythus and selected oral and reference Bacteroides species.

imately 0.3, and 50-pl samples were dispensed into wells of
microtiter plates. The plates were centrifuged, and the cells
were fixed to the plates with 0.25% glutaraldehyde. The
exposed binding sites on the plates were blocked with 1%
bovine serum albumin in 100 mM glycine buffer, and the
plates were washed three times with phosphate-buffered
saline supplemented with 0.05% Tween 20 (Fisher Scientific
Co., Fairhaven, N. J.). Specific horseradish peroxidase-
globulin was then incubated for 30 min with the bacterial
strains, and the plates were washed three times with phos-
phate-buffered saline supplemented with 0.05% Tween 20.
The presence of peroxidase bound to the test strain was
determined by adding 0.03% H202 and N,N,N',N'-tetra-
methylbenzidine. (The N,N,N' ,N' ,-tetramethylbenzidine
was dissolved in a solution containing 15 mg of glacial acetic
acid per ml and diluted 1 5 0 in 0.05 M sodium citrate buffer
[pH 4.21; 0.1 ml of this solution was added to each well.)
After 15 to 20 min at 25"C, NaSCN was added to a final
concentration of 0.075 M. Formation of a blue precipitate
indicated a positive reaction.
RESULTS AND DISCUSSION
DNAs from 12 fusiform Bacteroides strains showed more
than 75% homology with each other, indicating that they
belong to a single species (Table 2).These isolates were
gram-negative, strictly anaerobic, and rod shaped and had a
median G+C content of 46 mol% (Table 2), which is within
2 mol% of the G+C content of the type strain of the genus
Bacteroides. The strains were nonmotile. The metabolic
products included acetate and succinate (when the orga-
nisms were grown in basal medium containing 17 g of
Trypticase [BBL] per liter, 3 g of yeast extract [BBL] per
liter, 3 g of NaCl per liter, 3 g of KN03 per liter, and 3 mg of
hemin per liter [15]) or acetate and propionate (when the
organisms were grown in mycoplasma broth containing 0.5%
glucose, 4 mg of sodium acetate per ml, 10 pg of sodium
sulfite per ml, 100 pg of sodium succinate per ml, 0.5 kg of
L-cysteine per ml, and 3 mg of hemin per liter). There was
little or no DNA-DNA homology between the fusiform
Bacteroides strains and the other Bacteroides species tested.
The B. forsythus strain showed 0 to 31% homology with
Bacteroides fragilis ATCC 25285T, 0 to 28% homology with
Bacteroides thetaiotaomicron ATCC 29148=, 0 to 48% ho-
mology with Bacteroides distasonis ATCC 8503T,0 to 20%
homology with Bacteroides vulgatus ATCC 8482T, 0 to 30%
homology with Bacteroides oris ATCC 33573T, 5 to 14%
homology with Bacteroides oralis ATCC 3326gT, and 11 to
17% homology with Bacteroides buccae ATCC 33574T.The
protein profiles were similar within the new species but
different from the protein profiles of reference strains be-
longing to other species (Fig. 1 through 3). Thus, the
fusiform Bacteroides strains appear to belong in the genus
Bacteroides, and we propose the new species described
below for these strains.
Bacteroides forsythus sp. nov. Bacteroides forsythus
(for.sy'thus. N. L. n. forsythus referring to the Forsyth
Dental Center, where the species was first isolated) cells are
gram negative, fusiform with pointed ends (Fig. 4 and 5 ) , and
0.30 to 0.50 km in diameter (mean 5 standard deviation, 0.40
2 0.05 km). The cell length varies between 1 and 30 pm
(average, 5 Fm). Filaments (Fig. 6), cells with central
swellings that are up to 3 pm in diameter (Fig. 7), and
spheres are frequently observed.
The cells are nonmotile and have no flagella.
Cell wall structure. The outer membrane consists of an
VOL. 36, 1986 BACTEROIDES FORSYTHUS SP. NOV. 217

FIG. 4. B . forsythus strain FDC 293. ~ 3 2 , 8 0 0Bar

. = 1 pm.
FIG. 5. B . forsythus strain FDC 334. Ultrastructure of peripheral layers and invagination of cell periphery in the region of septum
formation (arrow). ~ 6 8 , 0 0 0Bar
. = 0.1 pm.
218 TANNER ET AL. INT. J. SYST.BACTERIOL.

FIG. 6. B . forsythus strain FDC 334 filamentous form x20,400. Bar = 1 pm.
FIG. 7. Negatively stained preparation of a B . forsythus strain FDC 334 cell1 with central swelling. x6,400. Bar = 1 pm.
FIG. 8. B . forsythus strain ATCC 43037=. Cell cross section with well-defined inner membrane (I), outer membrane (2), and distinctive
external layer (3) with closely packed dome-shaped structural subunits. There is no distinct peptidoglycan layer between the inner and outer
membranes. X136,OOO. Bar = 0.1 pm.

outer layer which is approximately 4 nm thick and is
separated by a 2-nm electron-lucent space from an inner
layer that is 1 nm thick (Fig. 8 and 9). The inner membrane
consists of two electron-dense layers, each approximately 2
nm wide, with a 2-nm intervening electron-lucent layer. The
inner and outer membranes are separated by a moderately
electron-dense zone that is about 16 nm wide, which appears
to lack a distinct peptidoglycan layer. A regularly structured
external layer which surrounds the outer membrane consists
of subunits?which in some cross-sections resemble adjacent
arches that are 10 nm wide and 10 nm high. These subunits
are separated from the outer membrane by a 12-nm electron-
lucent zone. Electron-dense radial connections appear to
extend from the subunits to the outer surface of the outer
membrane. Negative staining of tangential sections through
the cell periphery indicates that the subunits in the outer-
VOL. 36, 1986 BACTEROIDES FORSYTHUS SP. NOV. 219

FIG. 9. Fragment of outer membrane (2) and external layer (3), showing the similarity of the unusual external layer and the inner surface
of the outer membrane (4). ~272,000.Bar = 0.1 pm.
FIG. 10. Tangential section through the external layer of B. forsyrhus ATCC 43037=, showing the orthogonal packing of the structural
subunits. ~132,000.Bar = 0.1 km.
FIG. 11. Negatively stained preparation of B. forsythus, showing the orthogonal arrangement of structural subunits in the external layer.
~132,000.Bar = 0.1 pm.

most layer are packed in an orthogonal array (Fig. 10 and Biochemical characteristics: API ZYM and API An-Ident
11). reactions. Strains are positive in both API ZYM and API
Colonies. Colonies on Trypticase soy blood agar plates An-Ident tests for a-fucosidase, P-glucosidase, a-glucuron-
after 7 to 10 days are pale pink, speckled, and 1 to 2 mm idase, N-acetyl-P-glucosaminidase, and a-galactosidase ac-
diameter on primary isolation and when they are growing tivities. In addition, the strains are positive in API ZYM
well (Fig. 12). Colonies of poorly growing cultures are C0.5 tests for trypsin and p-galactosidase activities and in API
mm in diameter. An-Ident tests for a-arabinosidase, phosphatase, indoxyl-
Cultural characteristics. These organisms are strict acetate, and arginine, alanine, histidine, and phenylalanine
anaerobes and require C02 and H2 in the atmosphere for
growth on agar surfaces (preliminary experiments have
revealed no growth on agar under 100% N2; 90% N2-10%
C 0 2 ;or 90% N2-10% H2). To produce visible growth on agar
plates, cultures require 4 to 6 days. Complex media are
required for growth. Whole blood (e.g., 5% sheep blood) is
required for optimal growth on agar. Strains grow on
Levinthal agar. Growth on agar plates is stimulated by the
presence of F. nucleaturn (and strains of Streptococcus
sanguis, B. fragilis, Wolinella r e c t a , Bacteroides
intermedius, and Veillonella parvula [unpublished data] in a
satellite pattern.
Visible growth is produced in mycoplasma broth contain-
ing glucose (0.5%), sodium acetate (4mg/ml), sodium sulfite
(10 pg/ml), sodium succinate (100 pg/ml), L-cysteine (0.5
pg/ml), and hemin (3 mg/liter) (unpublished data; S. S.
Socransky , personal communication).
Cultures also grow in Trypticase soy or mycoplasma broth
supplemented with 8 to 12% gelatin and in biphasic cultures
(10 ml of Trypticase soy agar in the solid phase and 1 5 ml of FIG. 12. Colonies of B. forsythus strain ATCC 43037=. The cells
Trypticase soy or mycoplasma broth in the liquid phase), were grown on TSBA that was incubated anaerobically for 10 days
suggesting that agar or gelatin may be a detoxifying agent. without a F. nucleaturn strain. Bar = 1 mm.
220 TANNER ET AL. INT. J. SYST. BACTERIOL.

FIG. 13. B. oris ATCC 33573T. Typical gram-negative cell wall structure with a distinct peptidoglycan layer. x54,OOO. Bar = 0.1 pm.
FIG. 14. B. zoogleoforrnans ATCC 33285*. The cell periphery lacks a distinct peptidoglycan layer. An external layer is present, which
probably corresponds to a portion of the mucoid capsule. ~54,000.Bar = 0.1 pm.

aminopeptidase activities (18). The API ZYM and API
An-Ident enzymatic substrate tests are the only biochemical
tests in which B . forsythus consistently demonstrates clear
positive reactions. Strains do not react in the API 20A or
API 20E test series. There is no detectable pH decrease in
media supplemented with carbohydrates.
Esculin is hydrolyzed.
Serologic reactions. The strains of B . forsythus isolated
from seven different humans are members of a single
serogroup and have no antigenic relationship to F .
nucleatum FDC 364 or Capnocytophaga sputigena ATCC
33612T, which are morphologically similar.
PAGE of soluble cellular proteins. B . forsythus is charac-
terized by two major protein bands at molecular weights of
more than 205,000 (Fig. 1 and 2).
Isolated from human periodontal pockets.
G + C content, 46 mol%.
The type strain is strain FDC 338 (= ATCC 43037).
Differential characteristics. A peptidoglycan layer between
the outer and inner membranes was readily identified in B .
oris (Fig. 13), Bacteroides ovatus, and B . thetaiotaomicron,
but was not detected (or was absent) in Bacteroides
zoogleoformans (Fig. 14), B . distasonis, B . buccae, and B .
forsythus. The outer membrane was smooth and tightly
adapted to the cell periphery in all species which we exam-
ined except B . buccae. In addition, B . zoogleoformans, B .
distasonis, B . buccae, and B . forsythus had an external layer
that was peripheral to the outer membrane (Fig. 8 and 14).
Of the other Species tested, only B . distasonis resembles
B . forsythus in cell ultrastructure. Both of these species have
cell walls with similar inner and outer membranes. However,
the highly structured outermost layer of B . forsythus is
replaced in B . distasonis by a uniform moderately electron-
dense layer that is approximately 17 nm thick and is in direct
contact with the thicker outer layer of the outer membrane.
The mean diameter of B . distasonis cells is distinctly greater
than that of B . forsythus cells.
The API ZYM and API An-Ident reactions of species of
three genera of oral fusiform organisms differ. By using these
tests, Capnocytophaga species could be differentiated by
their consistent aminopeptidase activity, and F. nucleaturn
strains could be differentiated by their lack of reactivity in
VOL.36, 1986
any enzyme substrate test (18). B . forsythus was the only
species tested which showed trypsin activity. Trypsin activ-
ity has also been detected in Bacteroides gingivalis (12).
B . forsythus ATCC 43047T showed no enzyme-linked
immunosorbent assay cross-reactivity with B . gingivalis (5
strains), B . intermedius (strain FDC 581 and 6 other strains),
B . interrnedius VPI 8944, Bacteroides rnelaninogenicus (2
strains), Bacteroides denticola ATCC 10043, Bacteroides
loesheii ATCC 15930, C . sputigena (strain ATCC 33612Tand
6 other strains), Capnocytophaga ochracea (9 strains),
Capnocytophaga gingivalis (strain ATCC 33624T and 1other
strain), W. recta (strain ATCC 33238T and 2 other strains),
Bacteroides gracilis (strain ATCC 33236T and 2 other
strains), B. oralis ATCC 33321, Eikenella corrodens (8
strains), Haemophilus actinomycetemcornitans (10 strains),
or Haernophilus aphrophilus (1 strain).
All of the species examined produced different protein
profiles (Fig. 1 and 2). As determined by cluster analysis, B.
forsythus isolates show 88% similarity to each other and less
than 55% similarity to other species (Fig. 3). Only B .
forsythus produced two major protein bands at molecular
weights of more than 205,000.
B . forsythus is easily differentiated from the fusiform oral
gram-negative organisms F . nucleatum and Fusobacterium
gonidiaforrnans (which produce butyric acid) and
Capnocytophaga species (which ferment carbohydrates) by
the presence of swollen cells, the appearance of the colonies,
the absence of growth in glucose broth, the presence of API
ZYM trypsin activity, the electrophoretic pattern of cellular
proteins, and serologic reactions.
ACKNOWLEDGMENTS
We thank G. McKinley for providing the API ZYM and API
An-Ident plates, S. S. Socransky for help with analysis of
polyacrylamide gels, and Carol Des Roches for technical assistance.
This study was supported by Public Health Service grants
DE-03488, DE-06085, DE-02623, and DE-04881 from the National
Institute of Dental Research.
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