Download Characterization of Microaerotolerant Bacteroides Strains Isolated

INTERNATIONAL
JOURNALOF SYSTEMATIC
BACTERIOLOGY,
July 1988, p. 259-264
0020-7713/88/030259-06$02.OO/O
Copyright 0 1988, International Union of Microbiological Societies
Vol. 38, No. 3
Characterization of Microaerotolerant Bacteroides Strains Isolated
from Sewage Sludge and Paramecium caudatum
R. HAMMANNt
Institute for Medical Microbiology and Immunology, University of Bonn, 0-5300Bonn I , Federal Republic of Germany
Microaerotolerant strains of gram-negative, nonsporing rod-shaped bacteria were isolated from two
nonhuman sources (sewage sludge and Paramecium caudatum). These strains were saccharolytic, produced
acetic, propionic, and succinic acids and traces of isobutyric, isovaleric, and lactic acids, contained
sphingolipids, and were metronidazole susceptible. One strain, which was used for menaquinone analysis,
contained MK-9, MK-10, and MK-11 respiratory quinones. The guanine-plus-cytosine contents of the
deoxyribonucleic acids ranged from 38 to 41 mol% I concluded that these strains, although they are able to
grow slowly in an aerobic atmosphere, represent members of the genus Bacteroides.
.
The majority of intestinal Bacteroides species that are
commonly found in humans and warm-blooded animals
belong to the Bacteroides fragilis group, which is characterized by short or pleomorphic, obligately anaerobic, gramnegative rods that produce acetic, propionic, and succinic
acids, together with small amounts of isobutyric and isovaleric acids (15). In the course of a study on various Bacteroides strains, including strains from nonhuman sources, a
strain of gram-negative, fermentative rod-shaped bacteria
not belonging to the Enterobacteriaceae was isolated from a
sewage sludge sample, and similar strains isolated from the
protozoan Paramecium caudatum were also studied. These
strains were originally thought to belong to the genus Bacteroides, but then I realized that they were able to grow
microaerobically, a characteristic that is contradictory to the
present definition of the genus Bacteroides. When the strains
were compared with other nonhuman Bacteroides strains, I
realized that another strain from sewage sludge obtained
from M. P. Bryant through E. M. Barnes labeled Bacteroides ruminicola (2) could also grow microaerobically.
(The results are taken from a thesis written for appointment as a university lecturer at the University of Bonn,
Bonn, Federal Republic of Germany.)
MATERIALS AND METHODS
Origin of strains. Strain M89 was isolated from a sewage
sludge sample (sewage plant of Schladern, near Bonn, Federal Republic of Germany). The sample was collected in a
sterile Erlenmeyer flask plugged with cotton. After arrival in
the laboratory, approximately 1 g of the sample was serially
diluted in sterile Schaedler broth [Trypticase soy broth, 10.0
g/liter; polypeptone peptone, 5.0 g/liter; glucose, 5.0 g/liter;
yeast extract, 5.0 g/liter; (tris)hydroxymethylaminomethane,
3.0 g/liter; hemin, 0.01 g/liter; L-cystine, 0.4 glliter; pH 7.61,
and 0.05-ml volumes were plated onto Schaedler agar
(Becton Dickinson GmbH, Heidelberg, Federal Republic of
Germany) to which 5% sheep blood, 100 mg of kanamycin
per liter, and 10 mg of vancomycin per liter had been added
prior to pouring. The resulting kanamycin-vancomycin agar
is a commonly used selective medium for human Bacteroides species (9). The plates were transferred to GasPak
jars immediately after plating and were incubated for 3 days
at 37°C. After incubation, several single colonies were
t Present address: Becton Dickinson GmbH, Tullastrasse 8-12,
6900 Heidelberg 1, Federal Republic of Germany.
picked from dilution
and were isolated on kanamycinvancomycin agar, on Schaedler agar without antibiotics, and
on Columbia blood agar. The latter plates were incubated in
a CO, incubator (6% CO, in air); all other plates were
incubated under anaerobic conditions for 3 days at 37°C.
Strains M94 and M96 were isolated from the protozoan P.
caudatum ; they were obtained from C. Martinez-Medina,
Sevilla, Spain. Strain Bryant C12 was an isolate from sewage
sludge, which was originally isolated by M. P. Bryant and
was obtained from E. M. Barnes, Agricultural Research
Centre Food Research Institute, Norwich, England. This
strain, which was originally labeled B. ruminicola, had been
examined by members of the Taxonomic Subcommittee on
Gram-Negative Anaerobic Rods, who concluded that it does
not belong to this species.
Strains of Bacteroides fragilis (NCTC 9343* [T = type
strain] and 1113, a clinical isolate), Bacteroides thetaiotaomicron (ATCC 29741), Bacteroides vulgatus (K-24-2,
normal human intestinal flora), and Bacteroides distasonis
(K-28-8, normal human intestinal flora) were included for
reference purposes.
Maintenance of strains. All strains were maintained in
Rosenow broth containing a piece of calf brain and 0.1 g of
CaCO, per vial under oxygen-free C 0 2 (12, 24). The tubes
were incubated for 3 days at 37°C prior to freezing at -18°C.
Differentiation and identification. The methods used for
physiological tests and gas chromatography of the acid
metabolic products have been described elsewhere (12). In
addition, strains were tested for growth on conventional
Endo and MacConkey agar plates (Becton Dickinson).
Metronidazole susceptibility The agar dilution technique
on Wilkins-Chalgren agar without blood (Becton Dickinson)
was used to determine the metronidazole susceptibility of
the strains. The tests were performed as described by the
National Committee for Clinical Laboratory Standards (22).
Metronidazole was dissolved in saline containing 10% dimethyl sulfoxide (Roth, Karlsruhe, Federal Republic of
Germany). The strains were inoculated by using a multipoint
inoculator. One set of plates was incubated anaerobically,
and the other was incubated in air containing 6% CO,. The
plates were read after 48 h and again after 6 days of
incubation at 37°C.
Differential media and growth conditions. The following
media were used to determine growth of the strains under
various atmospheric conditions: Columbia agar supplemented with 5% sheep blood (Becton Dickinson), Columbia
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260
INT. J .
HAMMANN
agar without blood but with resazurin (1mg/liter) and hemin
(1 mg/liter), and Columbia agar without blood but with
resazurin. Furthermore, rumen fluid-glucose-cellobioseagar
(RGCA) (14), and modified Caldwell-Bryant medium
(MCBM) (3) were used. The MCBM contained (per liter) 0.5
g of glucose, 0.5 g of cellobiose, 0.5 g of soluble starch, 37.5
ml of a mineral salt solution, 0.25 g of cysteine hydrochloride
0.001 g of resazurin, 2.0 g of Trypticase peptone, 0.5 g of
yeast extract, 0.001 g of hemin, 0.001 g of vitamin K,
(menadion), and 16.0 g of agar (pH 6.8). The mineral salt
solution contained (per liter) 0.2 g of CaCl,, 0.2 g of
MgSO, . 7 H,O, 1.0 g of K,HPO,, 1.0 g of KH,PO,, 4.0 g of
KHCO,, and 2.0 g of NaC1. The agar media were sterilized
by autoclaving them for 15 min at 121°C.
For inoculation of the media, one colony from a Schaedler
blood agar plate anaerobic culture was streaked onto the
surface of one plate of each of the media described above for
each strain. One plate of each medium was incubated in a
GasPak jar with a CO, envelope, one plate was incubated
under microaerobic conditions (CampyPak plus Pd catalyst),
and one plate was incubated under anaerobic conditions
(GasPak plus Pd catalyst). In addition, one plate was inoculated and was placed in a GasPak jar supplied with a
CampyPak envelope without catalyst.
Redox measurements. Redox potentials were determined
with an Ingold model Pt-4800 combined electrode and a
model PHM 82 standard pH meter (Radiometer, Copenhagen, Denmark). The electrode was introduced into each
GasPak jar through a hole in the lid. The end of the electrode
was inserted into the surface of the upper agar plate, which
was filled with approximately 40 ml of agar up to the rim of
the plate to give contact between the agar and both electrodes. Then the jar was closed completely, and the space
between the electrode and the lid was sealed with plasticine.
The jar was incubated at 37°C for 5 days. Millivolt readings
were recorded after 30 min and again after 5 days. The
following formula, given by the manufacturer of the electrode, was used to convert the potentiometer readings (E)
into the standard hydrogen electrode redox potential (Eh):
Determination of oxygen concentrations. Oxygen concentrations were determined by gas chromatography, using a
Shimadzu model GC 8A chromatograph equipped with a
thermal conductivity detector on Molecular Sieve 5A columns. The injector temperature was 8O"C, and the column
temperature was 50°C. The detector current was 140 mA.
Helium was used as a carrier gas. Air and different gas
mixtures were used as references. Gas samples from GasPak
jars were aspirated with gas-tight syringes (Hamilton, Darmstadt, Federal Republic of Germany) through elastic rubber
stoppers that had been inserted in holes in the lids of the jars.
Determination of G+C content of DNA. The deoxyribonucleic acid (DNA) guanine-plus-cytosine (G +C) content was
determined spectrophotometrically by the thermal melting
point method. DNA was isolated as described by Marmur
(19). Purification and spectrophotometric determinations
were done as described elsewhere (13).
DNA-DNA homology. Levels of DNA homology were
determined by spectrophotometric measurement of the reassociation rates of individual DNA types and their mixtures
(7, 8). The measurements were done with a model 250
spectrophotometer (Gilford Instrument Laboratories, Inc.,
Oberlin, Ohio) equipped with a model 2527 thermoprogrammer (Gilford). DNA of B . fragilis NCTC 9343T and
SYST.
BACTERIOL.
Escherichiu coli DSM 30083 were included as standards. All
measurements were done five times.
Sphingolipid content. Strains were grown in prereduced
brucella broth (Becton Dickinson) that was inoculated from
anaerobically incubated Columbia blood agar plates. The
broth was incubated under anaerobic conditions at 37°C until
heavy growth appeared (usually 3 days). Media were centrifuged, and the pellets were washed three times with distilled
water. The pellets were treated as described by Dees et al.
(6). Briefly, the cells were hydrolyzed with 25% (vol/vol)
HC1 in methanol. The mixture was incubated for 18 h in a
water bath at 60°C in screw-cap bottles. After cooling, the
hydrolyzed cells were extracted twice with diethyl etherhexane (1:l). Both diethyl ether-hexane layers were isolated
and discarded. A 2-ml volume of distilled water was added,
as well as enough NaOH pellets to adjust the pH to 11to 12
(read electrometrically). After 5 min of shaking, the longchain bases were extracted twice with diethyl ether-hexane
(1:l). The two extracts were combined and were concentrated to 0.1- to 0.2-ml volumes under a stream of nitrogen.
Portions (20 to 40 pl) of the concentrated extracts were
applied to silica gel thin-layer plates (catalog no. 5721; E.
Merck AG, Darmstadt, Federal Republic of Germany) that
had been activated for 45 min at 105°C prior to use. The
plates were developed with chloroform-methanol-water (65:
25:4) and were sprayed with ninhydrin (0.2% in acetone)
after air drying. Spots were developed by heating for about
3 min at 105°C. DL-Sphingosine sulfate, erythro-DL-dihydrosphingosine, and cell material from E. coli DSM 30083
and B . fragilis NCTC 9343T (both grown in brucella broth)
were used as references.
Menaquinofies of strain M89. Determination of menaquinones was done by R. M. Kroppenstedt, Deutsche Sammlung von Mikroorganismen (DSM), Braunschweig, Federal
Republic of Germany, as described previously (18).
RESULTS
Growth and morphology. The microaerotolerant isolates
(strains M89, M94, and M96) and strain Bryant C12 grew on
anaerobically incubated media (Columbia, Schaedlet-, and
kanamycin-vancomycin agar plates, all supplemented with
5% sheep blood) as grey, slightly convex colonies of different sizes. The growth was similar to that of B . frugilis
group organisms. On three different modifications of Columbia agar (one with sheep blood, one with hemin, and one
without any supplement) growth was best for all of the
microaerotolerant strains in the anaerobic atmosphere; however, weak to normal growth also occurred in the microaerobic atmosphere (CampyPak envelope with the catalyst
included in the jar). Growth in air enriched with 5 to 8% CO,
was weak, but very small or pinpoint colonies were observed
after 5 days of incubation.
A slight stimulation by hemin was observed with strains
M89 and M96 in the CampyPak atmosphere and with B .
thetaiotaomicron in the anaerobic atmosphere.
Table 1shows the results of the redox determinations with
Columbia blood agar, RCGA, and MCBM media in four
different atmospheres. The CampyPak atmosphere with and
without the Pd catalyst, the GasPak anaerobic atmosphere
(including the catalyst), and the aerobic, C0,-enriched atmosphere were evaluated. In addition, the oxygen concentrations were determined in the jars containing strains M89,
M94, M96, and Bryant C12, as well as B . fragilis and B .
thetaiotaomicron. The use of the CampyPak atmosphere
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VOL. 38, 1988
MICROAEROTOLERANT BACTEROIDES STRAINS
261
TABLE 1. Aerotolerance of microaerobic isolates and reference strains of Bucteroides on three media under different conditions
Oxygen
Medium
Eh
Atmosphere
(mV)"
(%,concn
v o l , o l ~ ~ M89
M94
M96
Growth of strain:
Bryant B. fragilis
c12
Columbia agar +
5% sheep blood
RGCA
MCBM
a
Air + 5 to 8% CO,
CampyPak without catalyst
CampyPak with catalyst
GasPak with catalyst
Air + 5 to 8% CO,
CampyPak without catalyst
CampyPak with catalyst
GasPak with catalyst
Air + 5 to 8% CO,
CampyPak without catalyst
CampyPak with catalyst
GasPak with catalyst
+317
+43
-96
-250
+233
+ 104
-45
- 195
+200
-31
- 100
-315
Values after 5 days of incubation.
-, No growth; (+), very weak growth, often pinpoint colonies;
19
16
9.5
1.2
17
15
8
0.8
18
16
8.5
1.0
+
+
++ ++
+
++ ++
+++ +++ +++
(+I
(+>
(+I
+
+
(+I
+
+
++ +++ +++
(+)
+
+
+
++ ++ ++
++ ++ ++
(+I6
+
B. thetaiotamicron
1113
(+)
(+)
ATCC 29741
-
-
+
++
-
+++
+++
-
(+)
(+>
-
-
++
+++
++
-
-
-
-
++
++
+
-
(+)
+
++
-
-
+, weak growth; + +, normal growth; + + +, luxurious growth.
without catalyst in this experiment showed that the microaerotolerant strains can grow under these atmospheric conditions better than in air enriched with C02. The redox
potentials in the aerobic, C0,-enriched atmosphere were the
highest, but they still were not sufficiently high to inhibit the
growth of the microaerotolerant strains on Columbia agar
supplemented with sheep blood or on RCGA. A pink coloration of the resazurin redox indicator present in the
semisynthetic media (MCBM, RCGA) was observed during
incubation in all atmospheres containing more than 1.2%
oxygen.
The oxygen determinations showed that strains M94 and
M96 were generally more oxygen tolerant than strains Bryant C12 and M89 and were able to grow slowly in the
presence of 15 to 19% oxygen, whereas the latter two strains
grew only slightly or not at all with such oxygen concentrations. B . fragilis and B . thetaiotaomicron did not grow at all
in an atmosphere containing more than 1.2% oxygen.
None of the strains grew on MacConkey or Endo agar,
either anaerobically, microaerobically, or aerobically.
Metronidazole susceptibility. Under strictly anaerobic conditions (GasPak anaerobic atmosphere), the minimal inhibitory concentrations for strains M89, M94, M96, and Bryant
C12 were 0.5, 2.0, 1.0, and 1.0 pg/ml, respectively (Table 2);
in the C0,-enriched atmosphere the minimal inhibitory
concentrations were 16.0, 128, 8.0, and 1.0 pg/ml, respectively. Growth with the highest subinhibitory concentration
was very weak.
Physiology and biochemical characteristics. The results of
the carbon source fermentation tests are shown in Table 3.
All of the strains were strongly saccharolytic. All of the
strains hydrolyzed esculin, but only strains M94 and ATCC
29741 (B. thetaiotaomicron) produced indole. Nitrates were
not reduced to nitrites, and H2S was formed only by strain
K-24-2 (B. vulgatus). The fermentation end products of the
microaerotolerant strains were similar to those of B . fragilis
group organisms; acetic, propionic, lactic, and succinic acids
and minor amounts or traces of isobutyric and isovaleric
acids were produced.
Strains M89 and Bryant C12 had carbon source fermentation patterns similar but not totally identical to those of B .
vulgatus K-24-2. Strain M94 behaved like B . thetaiotaomicron ATCC 29741, while strain M96 behaved like B . distasonis K-28-8. Strains M89, M94, and M96 were sent to the
Anaerobe Laboratory, Virginia Polytechnic Institute and
State University, Blacksburg, for external identification, and
nearly identical results were obtained. These organisms
were characterized as gram-negative, facultatively anaerobic
rods that could not be identified further (L. V. Holdeman,
personal communication). Catalase reaction was positive for
all strains when they were tested in broth cultures (peptoneyeast extract-glucose) supplemented with 3 and 10% H,O,.
Oxidase was found to be negative in our laboratory, but was
found to be weakly positive by L. V. Holdeman for the
three strains tested in her laboratory. We were not able to
reproduce these results with different methods (trimethylpara-phenylenediamine, dimethyl-para-phenylendiamine,or
Taxo-N-Discs [Becton Dickinson]).
Base composition and DNA homology. As shown in Table
3, the G + C contents of the DNAs of the strains ranged from
38 to 44 mol%. Despite the phenotypic similarity to various
Bacteroides strains in fermentation patterns and G + C contents, DNA homology experiments revealed only negligible
homology between strains M89 and K-24-2 (37% homology),
between strains M94 and ATCC 29741 (23% homology), or
TABLE 2. Metronidazole susceptibility of aerotolerant strains
M89, M94, M96, and Bryant C12 (agar dilution) under
reducing conditions (GasPak anaerobic system)
and in air containing 6% CO,
Metronidazole
128
64
32
16
8
4
2
1
0.5
Growth of strain:
Atmosphere
concn (pglrnl)
GasPak
Air + 6%CO,
GasPak
Air + 6% CO,
GasPak
Air + 6% CO,
GasPak
Air + 6% CO,
GasPak
Air + 6% CO,
GasPak
A i r + 6%CO,
GasPak
Air + 6 % C 0 2
GasPak
Air + 6%CO,
GasPak
Air+6%C02
M89
M94
M%
Bryant C12
-a
-
-
-
-
-
-
-
-
(+)
-
(+)
-
-
-
(+)
+
++
-
-
-
-
-
-
-
-
++
(+)
(+)
++
(+)
(+)
-
++
-
++
+
++
++
++
-
++
(+)
++
('Growth compared with antibiotic-free control: -, no growth;
weak growth; +, visible growth; + +, good growth.
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-
(+)
(+I
(+I, very
262
INT. J. SYST. BACTERIOL.
HAMMANN
coli DSM 30083 extracts did not produce any ninhydrinpositive spots when they were treated in the same way.
Menaquinone content. Strain M89 contained MK-9 (5%),
MK-10 (35%), and MK-11 (60%).
TABLE 3. Biochemical characteristics of the aerotolerant
isolates and some Bacteroides reference strains
Strain
F: ; ;
Test
M89
Acid from:
Glucose
Fructose
Galactose
Lactose
Maltose
Mannose
Sucrose
Tre halose
Cellobiose
Melezitose
Raffinose
Inulin
Dextrin
Starch
Glycogen
Arabinose
Xylose
Rhamnose
Xylan
Glycerol
Adonitol
Mannitol
Sorbito1
Inosi to1
Salicin
Amygdalin
Esculin
Hydrolysis of
esculin
Production of:
Indole
H,S
NO,- from NO,Catalase
Products from PYG'
Acetic acid
Propionic acid
Isobutyric acid
Isovaleric acid
Lactic acid
Succinic acid
G+C content of
DNA (moI%)
C12 K-24-2 M94
+
+ " +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ v (
+ +
+ +
+
v
+ +
+
+
+
+
-
+
-
-
+
+
+
+
+ +
( + ) +
+ +
(+) (+)
+ +
38
40
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
NDb
-
ND
-
+
+
+
+
+ )
+
+
(
DISCUSSION
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
v
+
M96 K-28-8
+
(+)
+
-
+
+
+
+
+
+
+
40
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
(+)
39
+
+
+
+
+
+
+
+
+
+
-
+
+ ( + ) +
+
)
(+)
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
41
+
ND
ND
+
+
+
+
+
-
+
+
+
+
+
+
(+)
41
+
+
+
-
(+)
+
+
+
+
+
+
44
a For carbohydrates and similar compounds: +, pH 5 5.4; (+), pH 5.5 to
5.8; -, pH 2 5.9; v, variable. For other tests: +, positive reaction, (+), weak
reaction; -, negative reaction.
ND, Not determined.
PYG, Peptone-yeast extract-glucose medium.
between strains M96 and K-28-8 (10% homology). However,
considerable DNA homology was detected between strains
M89 and Bryant C12 (94% homology).
Sphingolipid content. The alkaline diethyl ether-hexane
extracts of strains M89, Bryant C12, M94, and M96 yielded
the same ninhydrin-positive spots as B. fragilis NCTC 9343T
and 1113 (a clinical isolate), with Rf values of 0.25 and 0.19.
In addition, a spot with an Rfvalue of 0.1 was detected for
strains M96, Bryant C12, and M89. Threo-DL-dihydrosphingosine and erythro-DL-dihydrosphingosineeach produced two spots with &values of 0.19 and 0.1 that cochromatographed with the respective probes. DL-Sphingosine
produced a spot with an Rfvalue of approximately 0.2. E.
By definition, the genus Bacteroides comprises only
strictly anaerobic, gram-negative, nonsporing rods (15). The
isolates described here (strains M89, M94, M96) all possess
more or less microaerotolerant growth characteristics, especially when they are grown on complex, blood-containing
media. Strain Bryant C12, which was originally isolated by
Bryant as a strain of B. ruminicola subsp. ruminicola, also
grew microaerobically. Aerotolerant strains labeled Bacteroides have also been isolated by Iannotti and co-workers
(16) from swine manure digestors and by Mead and Jones
(20) from ovine rumina, The pattern of acid end products of
the strains described here, their ability to grow on anaerobically incubated kanamycin-vancomycin agar, which is a
selective medium for Bacteroides (9), their susceptibility to
metronidazole, their sphingolipid content, and the menaquinone pattern of strain M89, which is similar to that of B.
fragilis group organisms (4, 23), suggest a relationship to the
genus Bacteroides. As Johnson (17) demonstrated, there are
several DNA homology groups within B . fragilis, B. thetaiotaomicron, B. vulgatus, and B . ovatus. Therefore, it is
possible that the strains examined here are members of
as-yet-unknown homology groups of Bacteroides or that the
reference strains used here for DNA hybridization were
members of another homology group, although they were
phenotypically similar.
All true facultatively anaerobic gram-negative rods, including members of the Enterobacteriaceae, are resistant to
nitroimidazoles, even under anaerobic conditions (5). However, the microaerotolerant isolates were susceptible to
metronidazole under anaerobic conditions. In air enriched
with CO, there was an increase in the minimal inhibitory
concentration, but only strain M94 reached a value of 128
Pdml*
Furthermore, fatty acids with more than two carbon atoms
are not produced by members of the Enterobacteriaceae or
other facultative anaerobes. The most striking fact for the
assignment of the isolates to the genus Bacteroides is the
presence of sphingolipids in the cells. The presence of these
membrane constituents is quite uncommon in bacteria and
seems to be restricted to the aerobic genus Sphingobacterium (6, 27) and the anaerobic genera Bacteroides (10, 25)
and Fusobacterium (11).Although there are species without
sphingolipids currently assigned to the genus Bacteroides,
their taxonomic position is quite unclear (23) and awaits
further taxonomic studies.
Another striking fact is the presence of menaquinones
typical of strictly anaerobic bacteria. The pattern found in
strain M89 is typical for B. fragilis group organisms and for
some B. ruminicola group strains (4, 23). Members of the
Enterobacteriaceae contain only menaquinones with six to
nine isoprene units, if they contain menaquinones at all (4).
The reason for the microaerotolerance of the strains is at
this time quite unclear. Although the strains were catalase
positive, this is no reason for their aerobic growth, and
catalase is also produced by many of the B. fragilis group
species when they are grown in the presence of heme or
hemin (26).
The failure or ability of bacteria to grow in the presence of
oxygen could, up to now, never be explained by the absence
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MICROAEROTOLERANT BACTEROIDES STRAINS
or presence of oxidase, catalase, or superoxide dismutase
(21). Furthermore, the ability to grow microaerobically or
aerobically should not exclude the assignment of a certain
strain to a certain genus. Clostridium perfringens strains
quite often can grow in the presence of oxygen, and, on the
other hand, Enterobacteriaceae strains unable to grow in a
normal atmosphere have been isolated even from clinical
specimens (1).
All of these facts seem to be sufficient to assign the isolates
described in this paper to the genus Bacteroides. The strains
described here will not be assigned to any existing species or
to new species until their taxonomic position is further
clarified. However, strains M89, M94, and M96 have been
deposited as Bacteroides strains in the DSM. Descriptions
are given below.
Bacteroides species strain MS9. Gram-negative, pleomorphic rods, occurring singly or in short chains. Spores not
demonstrated. Anaerobic to microaerobic growth on complex and semisynthetic media. Colonies on anaerobically
incubated Schaedler agar after 3 days of incubation are grey
to white and 2 to 3.5 mm in diameter. Colonies on different
complex or semisynthetic agar media under various microaerobic atmospheres are smaller (Table 1).Very small colonies develop after 5 days of incubation in air enriched with 5
to 8% CO, on Columbia agar without sheep blood. The
redox potential that still supports weak growth is +317 mV
(determined after the incubation period). Good growth still
occurs at a redox potential of +43 mV. Physiological characteristics are given in Table 3. Cells contain sphingophospholipids. The G+C content of the DNA is 40 mol%.
Isolated from sewage sludge. Deposited in the DSM as strain
DSM 4557.
Bacteroides species strain M94. Gram-negative, pleomorphic rods, occurring singly or in short chains. Spores not
demonstrated. Anaerobic to microaerobic growth on complex or semisynthetic media. Good growth still occurs at a
redox potential of +317 mV. At +43 mV, colony sizes
similar to those occurring under strictly anaerobic conditions
occur. Colonies under anaerobic or microaerobic conditions
after 3 days of incubation on Columbia agar and Schaedler
agar are grey to white, mucous, shiny, and convex. Colony
diameter under these conditions is 2 to 3.5 mm. Physiological activities are shown in Table 3. Cells contain sphingophospholipids. The G+C content of the DNA is 41 mol%.
Isolated from P. caudatum. Deposited in the DSM as strain
DSM 4558.
Bacteroides species strain M96. The morphological and
growth characteristics are the same as those described above
for strain M89, but good growth still occurs at a redox
potential of + 317 mV. Physiological characteristics are
shown in Table 3. Cells contain sphingophospholipids. The
G+C content of the DNA is 40 mol%. Isolated from P .
caudatum. Deposited in the DSM as strain DSM 4559.
ACKNOWLEDGMENTS
I acknowledge the technical assistance of B. Amendt and thank
J. R. Andreesen, Gottingen, Federal Republic of Germany, for his
help with the DNA homology experiments, R. M. Kroppenstedt,
DSM, Braunschwcig Federal Republic of Germany, for determining
the menaquinone content, L. V. Holdeman, Virginia Polytechnic
Institute and State University, Blacksburg, for testing some of the
isolates, and H. Werner, Tubingen, Federal Republic of Germany,
for constant advice and critical discussions.
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