APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1981, p. 737-745 Vol. 41, No.

3
0099-2240/81/030737-09$02.00/0

Isolation and Characterization of Thirteen Intestinal

Microorganisms Capable of 7a-Dehydroxylating Bile Acids
SEIJU HIRANO,* RYOSEI NAKAMA, MICHIHIRO TAMAKI, NORIYUKI MASUDA, AND
HIROSHI ODA
Department of Bacteriology, Faculty ofMedicine, Kagoshima University, Kagoshima 890, Japan

Thirteen anaerobic bacteria capable of performing the 7a-dehydroxylation of

both cholic acid and chenodeoxycholic acid were isolated from human feces and
also from sewage. Ten organisms from heat-treated samples were species of
Clostridium identical or closely related to the Clostridium bifermentans-C.
sordellii group and consisted of four strains elaborating 7a-dehydroxylase alone
and six strains capable of catalyzing both 7a-dehydrogenation and 7a-dehydrox-
ylation. The remaining three organisms, recovered from fresh human feces, were
gram-positive, nonflagellated, nonsporeforming, anaerobic rods and comprised
two distinct species. Strain HD-17, still unidentified, had both activities, but was
unique in that it exclusively 7a-dehydroxylated cholic acid while biotransforming
chenodeoxycholic acid, preferably through 7a-dehydrogenation. Two unclassified
strains, b-8 and c-25, metabolized both acids through 7a-dehydroxylation and 7a-
dehydrogenation. Except for strains b-8 and c-25, all of the 7a-dehydroxylating
bacteria split the conjugated bile acid series, and hydrolases were detected in cell-
free filtrates of early stationary-phase broth cultures.

The removal of the 7a-hydroxy substituent on
the steroid nucleus (7a-dehydroxylation) is a
unique reaction confined to microbial action.
The reductive conversion through this reaction
of the primary bile acids, cholic acid (CA) and
chenodeoxycholic acid (CDCA), is the most im-
portant of the bile acid transformations taking
place in the intestinal tract of humans and other
animals. It gives rise to the formation of deoxy-
cholic acid (DCA) and lithocholic acid (LCA),
respectively, both of which play a physiologi-
cally significant role after entering the entero-
hepatic circulation. The reaction is carried out
extensively in the lower part of the bowel and
virtually all of the bile acids excreted in the feces
are products of this reaction (5). Available infor-
mation concerning the microorganisms account-
ing for this phenomenon, however, is incomplete
and conflicting. Although one group of investi-
gators asserted that this property is widely dis-
tributed among the intestinal microorganisms
(1, 2), it is generally accepted that it is difficult
to demonstrate this activity in pure bacterial
cultures (6, 24), and only a few strains of intes-
tinal anaerobes have so far been shown to be
capable of performing this reaction (3, 8, 11, 13-
16, 29).
This study is aimed at evaluating the preva-
lence and variety of 7a-dehydroxylating orga-
nisms among the human intestinal microflora.
When many bacterial strains isolated from hu-
man feces and municipal sewage were screened,
737
the enzymatic activity was found to be not as
rare as had been anticipated. The isolation and
the characterization of 13 dehydroxylating or-
ganisms thus obtained, 10 sporeforming and 3
nonsporing gram-positive anaerobic bacteria,
are reported in this paper.
MATERLALS AND METHODS
Sources and isolation of microorganisms. Bac-
teria were isolated from freshly voided feces from
healthy adults and from influx fluids at a municipal
sewage disposal plant. A 1:10 suspension of feces in
sterile phosphate buffer (0.02 M, pH 7.0, containing
0.1% sodium thioglycolate), or in buffered peptone-
yeast extract broth (see below), or raw sewage was
used as the inoculum after removal of coarse debris by
centrifugation at low speed.
In the first three experiments, the specimens were
heated at 80 to 90°C for 20 min to destroy vegetative
forms of bacteria, and 0.1-ml portions of appropriate
dilutions were spread on GAM agar medium (Nissui
Pharmaceutical Co.). In the fourth experiment, dilu-
tions of a pool of fecal suspensions from three persons
were plated on GAM agar medium supplemented with
150 yg of neomycin sulfate per ml to facilitate the
isolation of Bacteroides spp. In the fifth experiment,
two fecal samples were inoculated individually in bile
acid broth (see below), loopfuls from which were
streaked on plain GAM agar plates after 24 and 48 h
of anaerobic incubation. In the sixth to eighth experi-
ments, serial dilutions from each of three fecal samples
were directly plated on GAM agar for a viable cell
count.
All seeded plates were incubated for 48 h in an
738 HIRANO ET AL.
anaerobic jar under a gas mixture of 90% N2 and 10%
CO2 (purified previously by passing over heated copper
gauze). Plates containing 10 to 100 colonies were ex-
amined, and representative colonies (virtually all col-
onies in experiments VI to VIII) were picked and
stabbed into tubes of semisolid GAM agar medium;
the tubes were then incubated aerobically for 3 days.
After purification by reisolation of colonies, the cul-
tures were tested for aerobic growth on the same GAM
plate, and only obligately anaerobic organisms were
preserved in semisolid GAM tubes for further exami-
nation. In experiments VI to VIII, all of the isolated
cultures, including both facultative and obligate an-
aerobes, were studied. All manipulations except an-
aerobic incubation were carried out under atmospheric
conditions, with precautions taken to minimize the
exposure of samples to air.
Transformation of bile acid by isolated bacte-
rial strains. The basal medium used was a peptone-
yeast extract broth prepared essentially according to
the formula given in the Virginia Polytechnic Institute
manual (19) but modified by using 2% peptone instead
of 1% peptone and by dissolving the ingredients in 0.02
M phosphate buffer at pH 7.5 to maintain the pH
above neutral during the entire period of incubation
(2, 25). No carbohydrate was included, since the pres-
ence of a fermentable carbohydrate seemed to sup-
press the dehydroxylation (presumably due to acidifi-
cation of the medium), although it stimulated bacterial
growth. Bile acid, free or conjugated, was added to
this medium to a final concentration of 200 ,uM from
a stock solution of 10 mg of bile acid per ml of ethanol.
The medium was then dispensed in 4-ml quantities in
12- by 105-mm test tubes (for incubation for a definite
period of time) or in 30-ml amounts in 100-ml flasks
(for successive sampling). The medium was prepared
on the day of use and inoculated immediately after
autoclaving and cooling.
The inoculum was a 0.1-ml portion from an over-
night culture of the test strain in peptone-yeast ex-
tract-glucose broth. The inoculated tubes were as-
sayed for bile acids after 4 days of incubation in an
anaerobic jar (under an atmosphere of pure N2 to
avoid acidification of the culture medium by C02).
The medium in the flasks was inoculated under flush-
ing with N2 through the liquid phase, and 4-ml samples
were removed for assay at various time intervals dur-
ing incubation under N2 for up to 7 days. After incu-
bation, bacterial growth was assessed by measuring
the transmittance of light at 660 nm on a photoelectric
colorimeter, and the final pH of the culture medium
was checked by a pH meter.
Preparation for GLC. The spent culture medium
was extracted three times with an equal volume of
ethyl acetate after acidification to pH 2.0 with 6 N
HCI. The combined solvent fractions were washed
repeatedly with distilled water until the washing fluid
became neutral in reaction. After evaporation to dry-
ness under a stream of air at 45°C, the bile acid residue
was reconstituted with 2 ml of methanol and 1 drop of
concentrated H2SO4, and the solution was allowed to
stand at room temperature overnight (methylation).
After addition of 4 ml of distilled water and adjustment
to pH 10 with saturated NaHCO3, the methylated bile
acids were extracted thoroughly with ethyl acetate,
APPL. ENVIRON. MICROBIOL.
and the extract was washed and evaporated in the
manner described above. The final residue was dis-
solved in 0.5 ml of chloroform, and 3-,ul portions were
injected into a gas-liquid chromatography (GLC) col-
umn (3% QF-1). After analysis, the chloroform was
evaporated and the methylated bile acid residue was
exposed to silylating reagents to convert all free hy-
droxy groups on the steroid nucleus to trimethylsilyl
ethers, as recommended by Makita and Wells (22).
Three-microliter portions of the reaction mixture were
directly injected into a 3% Hi Eff-8B column.
Analysis of bile acids by GLC. A Hitachi 163
gas-liquid chromatograph equipped with a flame ion-
ization detector was used isothermally. A U-shaped
glass tube (3-mm inside diameter by 2 mj packed with
3% QF-1 coated on Chromosorb WAW DMCS, 80 to
100 mesh, was used for separation of methyl cholan-
oates, and a similar tube packed with 3% Hi Eff-8B
coated on 80- to 100-mesh Gas-Chrom Q was used for
analysis of the silyl ethers of methylated bile acids.
The column oven was held at 260°C for QF-1 and at
230°C for Hi Eff-8B. The injector and detector were
held at 50°C higher than the respective column tem-
peratures. N2 was used as the carrier gas at a flow rate
of 30 to 40 ml/min. H2 and air were at 1.1 and 1.3 kg/
cm 2 , respectively.
Individual GLC peaks were identified by retention
time relative to that of methyl deoxycholate or its silyl
ether depending upon the derivation of the test sample
(= relative retention time). A standard pool of authen-
tic bile acids was analyzed in parallel, and the relative
retention times of the sample and the standard acids
were compared. For bile acids such as 3-keto- and 12-
ketocholanoates, for which we have no authentic com-
paratives, the published relative retention times (9,
10) were consulted for comparison.
Quantitative estimation was performed by measur-
ing the peak area with an electronic integrator (Shi-
madzu Chromatopac EIA) and relating it to the peak
area of a known amount of the corresponding deriva-
tive of deoxycholate. Quantities of individual bile acids
were expressed as percent composition, after it had
been confirmed that the recovery of the total bile acids
in each sample amounted to 80 to 90% of the substrate
bile acid added. Blank tubes containing bile acid in
sterile broth showed the same rate of recovery, and
tubes inoculated without substrate showed no bile acid
peaks.
Combined gas chromatography-mass spec-
trometry. The silylated compounds separated on Hi
Eff-8B were analyzed by mass spectrometry with a
Hitachi M-60 gas chromatograph-mass spectrometer.
The column was kept at 240°C, the injector was held
at 280°C, and the separator was kept at 320°C. The
helium inlet pressure was 1.2 kg/cm2. The ionizing
voltage was kept at 20 eV, and the accelerating voltage
was held at 3.2 kV. Recording was done from mle 0 to
700 in 4 s. Background spectra, mainly from the sta-
tionary phase, were recorded and subtracted from the
sample spectra.
Examination of bacteriological properties. Co-
lonial morphology and the lecithinase reaction were
observed on GAM agar plates supplemented with 10%
egg yolk after anaerobic incubation for 2 days. Urease
and catalase tests were applied to the bacterial growth
VOL. 41, 1981 INTESTINAL 7a-DEHYDROXYLATING BACTERIA 739
on the same plate according to the techniques of from Gaschro-Kogyo Co.
Sutter et al. (31). Other biochemical and fermentative
reactions were examined as described by Holdeman RESULTS
and Moore (19), using ordinary peptone-yeast extract
as the basal medium. Final acidity of the carbohydrate
Screening of intestinal isolates for bile
broth was determined with a pH meter on day 3 of acid transformation. A total of 347 strains
anaerobic incubation. Volatile and non-volatile fatty isolated by three procedures were examined for
acids produced in 2-day cultures in peptone-yeast ex- their ability to transform CDCA in broth cul-
tract or peptone-yeast extract-glucose were analyzed tures. The overall results are summarized in
by GLC in an ethylene glycol adipate column under Table 1. The 7a-dehydroxylating conversion of
temperature programming, as recommended by Hir- CDCA into LCA was shown by 10 of the 77
akawa (17). For electron microscopy, cells grown on presumed clostridial strains isolated from heat-
GAM agar plates were suspended in a small amount treated samples (experiments I to III) and 3 of
of sterile distilled water. A drop of the suspension was the 240 isolates obtained by plating without
mounted on a Formvar-carbon-coated grid. After neg-
ative staining with 2% potassium phosphotungstate, selective treatment (experiments V to VIII).
the specimen was examined in a Hitachi H-300 elec- None of the 30 presumed bacteroides strains
tron microscope. recovered from plates containing neomycin (ex-
Bile acids. Bile acids are referred to in the text by periment IV) showed the activity. The 7a-de-
the following abbreviations for their trivial names: hydrogenation giving rise to the formation of
CDCA, chenodeoxycholic acid (3a,7a-dihydroxy-5,f- 7KL was found in 19 (25%) of the clostridial, 13
cholanoic acid); CA, cholic acid (3a,7a,12a-trihydroxy- (43%) of the bacteroides, and 100 (42%) of the
5,B-cholanoic acid); DCA, deoxycholic acid (3a,12a-di- unselected isolates. Furthernore, 31 (40%) of the
hydroxy-5,8-cholanoic acid); 7KD, 7-ketodeoxycho- clostridial strains and 8 (3%) of the unselected
lic acid (3a,12a-dihydroxy-7-keto-5fl-cholanoic acid);
7KL, 7-ketolithocholic acid (3a-hydroxy-7-keto-5,8- isolates were found to epimerize the 3a-hydroxy
cholanoic acid); and LCA, lithocholic acid (3a-hy- group of CDCA into a fl-configuration (3a-epi-
droxy-5fl-cholanoic acid). merization).
The free bile acids used as substrates were: CDCA Transformation of CDCA and CA by 7a-
(99.9% pure), a gift from Tokyo Tanabe Pharmaceu- dehydroxylating bacteria. The 13 strains
tical Co.; CA (99.1% pure), purchased from Sigma found to dehydroxylate CDCA in the above test
Chemical Co.; and 7KL (lot no. 322), obtained from were compared for their activities against CDCA
Gaschro-Kogyo Co., all of which proved to be pure and CA, the two major components of human
when tested by GLC. Glycine and taurine conjugates bile (Table 2). Clostridium sordellii 4709, a
of bile acids (sodium salt, A grade) were obtained from
Calbiochem. The conjugates were contaminated with stock culture originally obtained from Kanazawa
small amounts of free bile acids; therefore, parallel University (N. Nishida), was also capable of 7a-
cultures containing each conjugate (but uninoculated) dehydroxylating CDCA and CA and was in-
were set up and served as controls. The methyl esters cluded in the study for comparison. Table 2
of bile acids used as GLC references were all obtained shows the percentages of substrate converted

TABLE 1. Distribution of bile acid-transforrning activities among bacterial strains isolated from human
feces and municipal sewage
Isolation' No. of No. of isolates producing:c
Viable isolates LCA 7a-dehy-
Designation ofstrains
Expt
Source Treatment
countb exam- a
and
333-
7KL
7KL anld 3/s- CC CDCA
droxylating
ined C
ind
7KL CDCA
I Feces Heat 12 2 2 6 I-55, I-102
Sewage Heat 15 2 4 4 I-B6, II-27
II Feces Heat 18 3 3 4 N-9-2, N-9-4, N-9-10
III Feces Heat 8 x 104 15 2 1 9 HU-2, HU-6
Sewage Heat 5 X 104 17 1 3 8 S-il
IV Feces Neomycin 2 x 109 30 13
V Feces None 59 1 24 8 HD-17
VI Feces None 5 x 109 57d 20
VII Feces None 2 x 1010 64d 1 23 b-8
VIII Feces None 2 x 10'0 6od 1 22 c-25
a For bacterial isolation, see text.
b
Units per gram (wet weight) of feces (or 1 ml of sewage).
c Four-day cultures originally containing CDCA were assayed by GLC for metabolic bile acids.
d Preliminary screening of mixed cultures (five strains each); only strains from positive cultures were tested
individually.
740 HIRANO ET AL. APPL. ENVIRON. MICROBIOL.
TABLE 2. Comparative activity of 7a-dehydroxylating bacteria against CDCA and CA
% of CDCA converted to:b % of CA converted to:b
Bacterial strain'
LCA 7KL DCA 7KD
I-55 42,56, 15,32 0,0,0,0 29, 10, 7 0,0,0
I-102 36,53,21, 18 0,0,0,0 8,6,6 0,0,0
I-B6 22,56,20,16 0,0,0,0 13,7,5 0,0,0
II-27 42,42,29,26 0,0,0,0 9,12,4 0,0,0
N-9-2 1, 2, 1, 1 27, 30, 24, 38 12, 4 18, 30
N-9-4 2,3, 1, 1 24,30,27,27 14, 3 20, 29
N-9-10 1, 2 26, 27 4 24
HU-2 3,1 32, 32 5, 5 25, 32
HU-6 2,1 32, 30 8, 6 28, 31
S-11 2,8 15,39 6,9 14, 19
4709 2, 9, 3, 3 44, 60, 29,41 13,17,10,17 20, 18, 30, 24
HD-17 2, 2, 3, 1 25, 44, 36, 52 100, 65, 76 0, 11, 6
b-8 6,8,4 5,6,21 13,11,20 7,8,20
c-25 2,10,4 17, 6, 20 11, 7,18 26, 21, 14
For sources of strains, see Table 1. Strain 4709 is a reference culture of C. sordellii.
b Four-day cultures containing CDCA or CA were assayed for bile acids, and percentages of each substrate
converted into LCA or DCA through 7a-dehydroxylation and into 7KL or 7KD by 7a-dehydrogenation were
calculated. Values from repeated tests are presented.

into the respective major metabolites on day 4 and 7a-hydroxy and 7-keto acids were used in-
of anaerobic incubation, when the metabolites terchangeably, producing the same metabolites
had reached practically their maximum concen- (Fig. 2).
trations, as shown in Fig. 1, in which the changes Besides the principal metabolites produced
in the composition of bile acids during the course through 7a-dehydroxylation and 7a-dehydro-
of incubation for up to 7 to 10 days are illus- genation, small amounts of a 3-keto derivative
trated. The values in Table 2 represent the of CDCA were frequently produced by certain
results of tests successively performed during strains (Fig. 1). It can be seen in Fig. 1 that
the in vitro transfers of the organisms covering strain HD-17, which extensively dehydroxylates
a period of >1 year. CA, is also capable of metabolizing the resulting
All of the strains consistently dehydroxylated DCA into 12-keto and 3-keto derivatives.
both CDCA and CA, yielding LCA and DCA, These bile acid-transforming activities have
respectively (Table 2). The 7a-dehydroxylation been maintained almost unchanged from the
took place early in the incubation and ceased time of isolation onwards.
after 24 h, when bacterial growth had reached a Identification of 7a-dehydroxylation
maximum level. The concentration of LCA or products. In the preceding experiments, bile
DCA remained undiminished upon prolonged acid metabolites were separated as methyl esters
incubation, suggesting an end product nature on QF-1 and tentatively identified on the basis
(Fig. 1). The relative activity against both 7a- of relative retention times. To confirm their
hydroxy bile acids varied considerably with dif- identity, the methylated samples were rechro-
ferent bacterial strains: the 1-55 group (I-55, matographed after trimethylsilylation, and the
I-102, I-B6, and II-27) constantly dehydroxy- separated peaks were analyzed by mass spec-
lated CDCA to a greater extent than CA, trometry. The suspected 7a-dehydroxylation
whereas HD-17 was unique for an extensive products, LCA and DCA, showed the same GLC
dehydroxylation of CA and restricted activity mobilities and mass spectra as those of the same
against CDCA (Table 2; Fig. 1). derivatives of the corresponding authentic acids
Most of the dehydroxylating organisms cata- simultaneously processed for comparison. The
lyzed 7a-dehydrogenation also, giving rise to the mass spectra were also compatible with the spec-
formation of a 7-keto acid, 7KL or 7KD, with tra published by Sjovall et al. (27) for known
the exception of the I-55 group, which showed references.
no indication of the presence of an oxidative Deconjugation by 7a-dehydroxylating
process at C-7 (Table 2). In contrast to dehy- bacteria. Except for b-8 and c-25, the 7a-dehy-
droxylation, dehydrogenation was reversible, droxylating strains were capable of deconjugat-
 CDCA CA
1001 100'
I -102 ................1-102 C

502

DCA
1 4 C 7

100'.
4709
CA

500

,^______ 7KD_
z P.- ---
-
DCA
2
rA 12 3 i 7
100- 100,-
z c-25 C-25
0
0

CD0CA ^

50 50-

le
I
/-

[
-

/3k7a
--
5S. CP-

-^~~~ r ~S~~~ -
- :*
1 4 7 1 4 7

INCUBATION (DAYS)
FIG. 1. Changes in percent composition of metabolites during the course of incubation. The test strains
(I-102, 4709, c-25, and HD-1 7) were grown in broth containing CDCA or CA for 7 to 10 days, and samples were
removed for assay at the indicated times. The percent composition of individual bile acids was calculated by
GLC of the methylated samples on QF-1. 3k7a, 3-Keto derivative of CDCA; 3k12a and 3al2k, 3-keto and 12-
keto derivatives of DCA.
741
742 HIRANO ET AL.
100 4709
z CDCA
0
Z I
o 50
7KL
CA
1 2 3 5
INCUBATION (DAYS)
FIG. 2. Changes in percent composition of metabolites formed from 7KL by strains 4709 and HU-2 during
7 days of anaerobic incubation. Cultural and analytical procedures are the same as for Fig. 1.
ing the series of glycine and taurine conjugates
of bile acids in growing cultures. Strong hydro-
lytic activities were also detected in cell-free
supernatant fractions from early stationary-
phase cultures in GAM broth containing no bile
salt, indicating that the hydrolases were consti-
tutively formed in cultures and excreted extra-
cellularly in an early phase of bacterial growth.
Bacteriological characterization of 7a-
dehydroxylating bacteria. The main charac-
teristics of 7a-dehydroxylating bacteria are sum-
marized in Table 3. All of the organisms are
gram-positive, obligately anaerobic rods; the 10
organisms isolated from heated specimens
formed abundant spores in in vitro cultures and
were therefore assigned to the Clostridium ge-
nus. These clostridial strains resemble each
other in the bacteriological features tested so far
and are very similar, if not identical, to the C.
bifermentans-C. sordellii group, with the possi-
ble exception of strain S-11, which should be
placed in a separate species because of its failure
to liquefy gelatin. The remaining three orga-
nisms, isolated from fresh feces, are nonflagel-
lated and nonsporing and comprise two distinct
species. Strain HD-17 assumed large curved
forms, 0.8 by 1.5 to 4.0 pm (Fig. 3), and fermented
glucose and other carbohydrates. Abundant
growth was attained in GAM broth, and colonies
on GAM plates were 5 mm or more in diameter,
irregular, umbilicate, glistening, and gray.
On the other hand, strains b-8 and c-25, which
are identical in all criteria examined, are much
more slender than HD-17, measuring 0.5 by 1.0
to 1.5 ,um, and occur in chains of two or three
elements with a conspicuous cellular appearance
similar to that presented for Eubacterium len-
APPL. ENVIRON. MICROBIOL.
100 [ HU-2
CDCA
50
- LCA
7 1 2 3 5 7
tum by Bokkenheuser et al. (4) (Fig. 3). The
organisms lacked any activity against carbohy-
drates and proteins, and growth in broth was
poor and was not improved by the addition of
2% (wt/vol) arginine. Colonies on GAM agar
plates were less than 1 mm in diameter, circular,
entire, convex, and translucent.
DISCUSSION
Distribution of 7a-dehydroxylase activ-
ity among intestinal bacteria. In this study,
three categories of human intestinal bacteria
were screened for bile acid-transforming activity:
77 heat-resistant presumed clostridial strains,
30 neomycin-tolerant presumed bacteroides
strains, and 240 unselected strains. 7a-Dehy-
droxylase activity was demonstrated most fre-
quently in the first group. Of the 77 clostridial
strains, 10 were found to be capable of catalyzing
this reaction, restricted to certain species re-
lated, if not identical, to C. bifermentans-C. sor-
dellii. Some of these active strains were also
recovered from municipal sewage. The results
agree with those of Hayakawa and Hattori (16),
Hayakawa (15), and Ferrari and colleagues (11,
12), who reported that certain strains of C. sor-
dellii and C. bifermentans possess this activity.
It is certain that 7a-dehydroxylation is not a
rare property among specified species of Clos-
tridium, but this cannot explain the extensive
occurrence of this reaction in vivo since it does
not seem likely that clostridia of this kind would
be indigenous to the human gut in any apprecia-
ble amount. It may be only an indication of the
diversity of metabolic activity in this genus. Not
a single strain of C. perfringens, which is indig-
enous to the normal intestine in significant num-
VOL. 41, 1981 INTESTINAL 7a-DEHYDROXYLATING BACTERIA 743
TABLE 3. Bacteriological characteristics of 7a-dehydroxylating strains
Strains
Characteristic I-55, I-B6, I-102, N-9-2 N-9-4 b-85
11-27 N-9-10 'HU-2, HU-6 4709 5-11 HD-17 b-85
Gram stain and form +, rod +, rod +, rod +, rod +, rod +, rod +, rod
Flagella + + + + + - -
Spores + + + + +
Indole production + + + +
Gelatin liquefaction + + + +
Glucose, acid + + + + + +
Lactose, acid
Sucrose, acid - - - - - +
Fructose, acid - + + + +
Maltose, acid + + + + +
Mannose, acid + - + _ + +
Esculin hydrolyzed + + + - + +
Starch hydrolyzed
Lecithinase + + + + + - -
Catalase
Urease - - + + +
Fatty acids' in:
PY a,p,b,ib,iv,ic a,p,ib,iv,ic a,p,ib,iv,ic a,p,ib,iv,ic a,p,ib,iv a,p,b,iv a
PYG a, ic a, ic a, ic a, ic a, iv a, ib, iv a
a Fatty acids produced by 2-day cultures: a, acetic; p, propionic; b, butyric; ib,
isobutyric; iv, isovaleric; ic, isocaproic. PY,
Peptone-yeast extract; PYG, peptone-yeast extract-glucose.

C.-

FIG 3 Electron micrographs of representative 7a-dehydroxylating bacteri. (A) 155 (B) HD 17 (C) HU-
6; (D) c 25. Bar 1 ,km.

bers and accounted for the majority of the clos- The second group, which consisted of pre-
tridial isolates studied, showed this dehydroxy- sumed bacteroides, included no dehydroxylatingr
lation activity. organisms. Similar negative results were ob-
744 HIRANO ET AL.
tained in our previous experiment with 64 taxo-
nomically defined Bacteroides fragilis strains
from human intestinal contents (18). Stellwag
and Hylemon (29) were also unable to find any
active strains among 10 reference and 70 labo-
ratory strains of intestinal Bacteroides. Al-
though several organisms ascribed to the Bac-
teroides genus have been reported to be capable
of 7a-dehydroxylating cholic acid (3, 8, 14),
whether this predominant intestinal species
plays any primary role in in vivo dehydroxyla-
tion seems open to question. An isolate from
human feces, which was previously designated
Bacteroides 28S (14), has been reclassified as C.
leptum (29, 30).
From the third group, consisting of nonselec-
tively isolated strains, three dehydroxylating or-
ganisms were obtained: strain HD-17 from the
59 anaerobes isolated indirectly from broth cul-
tures of fresh feces and strains b-8 and c-25 from
the 181 colonies isolated on anaerobic plates for
viable counts. All three of these organisms are
gram-positive, nonsporeforming, anaerobic rods.
Of the organisms about which data have been
published, the lactobacilli isolated by Gustafsson
et al. (13) and described in detail by Midtvedt
and Norman (23, 25) are the sole bacteria be-
longing to this category. As for bile acid-trans-
forming activity, however, the lactobacilli failed
to split conjugated bile acids, in contrast to the
strong hydrolysis by HD-17, and the former
bacilli dehydroxylated CA and CDCA to a sim-
ilar extent whereas the 7a-dehydroxylation by
HD-17 showed an obvious preference for CA
over CDCA. Both equally elaborated 3a-, 7a-,
and 12a-dehydrogenases in addition to 7a-de-
hydroxylase.
Strains b-8 and c-25 share some negative char-
acteristics with E. lentum bacteria (26), but the
cultures failed to respond to arginine (28), and
their accurate taxonomy, particularly the rela-
tion to E. lentum, remains to be determined.
Concerning the metabolism of bile acids by Eu-
bacterium, Midtvedt and Norman (24) reported
3a- and 7a-dehydrogenation by three strains of
Eubacterium species, including one E. lentum
strain, whereas Macdonald et al. (20, 21) dem-
onstrated 3a- and 12a-dehydrogenases but no
7a-dehydrogenation in many strains of E. len-
tum. Bokkenheuser et al. (4) revealed the pres-
ence of a corticoid 21-dehydroxylase in E. len-
tum, but no 7a-dehydroxylation of bile acids has
ever been reported with bacteria belonging to
this genus. Strains b-8 and c-25 in this study
apparently possess not only 3a- and 7a-dehydro-
genases, but also 7a-dehydroxylase activity.
Comparative activity against CA and
CDCA. The 7a-dehydroxylating organisms in
this study consistently dehydroxylated both CA
APPL. ENVIRON. MICROBIOL.
and CDCA, and most of these strains metabo-
lized the two acids to similar extents, as reported
by Midtvedt and Norman (25) for culture of
Lactobacillus strain II and by Aries and Hill (2)
with cell-free preparations of various bacterial
species. In this regard, strain HD-17 was excep-
tional in that it dehydroxylated CA almost ex-
clusively, with little action on CDCA. A similar
result was also reported by Ferrari and Beretta
(12) with a cell-free extract from C. bifermen-
tans.
Dehydroxylation and dehydrogenation
of the 7a-hydroxy group. Except for four
strains of the 1-55 group, the 7a-dehydroxylating
organisms, including nonsporeforming ones,
were also capable of oxidizing the same hydroxy
group in CA and CDCA, like all the previously
reported bacteria (2, 3, 12, 16, 25). C. leptum,
although reported by Stellwag and Hylemon
(29) to lack the oxidative process, was capable
of converting CA into 7KD at the time of isola-
tion (14). It should be noted in this connection
that 7a-dehydrogenation, in contrast to reduc-
tive dehydroxylation, is an oxidative process.
Consequently, the relative rates of these two
reactions may vary according to the degree of
anaerobiosis of the environment. The anaerobic
conditions in this study, in which oxidative keto
formation was invariably demonstrated, occa-
sionally even to a greater extent than dehydrox-
ylation, do not seem to have reproduced the
strictly anaerobic circumstances in the colon,
where virtually all the bile acid metabolites are
in the reduced form. Drasar and Hill (7) have
emphasized that there should be little keto for-
mation under ideal conditions for 7a-dehydrox-
ylation. In this regard, the I-55 group is unique
and unprecedented. The strains have never dem-
onstrated any detectable 7-keto formation since
the time of isolation.
ACKNOWLEDGMENT
We thank N. Akimori, Naka Works, Hitachi Ltd., Katsuta,
Japan, for excellent assistance with the mass spectrometry.
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