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Applied and Environmental Microbiology, July 1999, p. 3158-3163, Vol. 65, No. 7
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Formation of Hyodeoxycholic Acid from Muricholic
Acid and Hyocholic Acid by an Unidentified Gram-Positive Rod Termed
HDCA-1 Isolated from Rat Intestinal Microflora
H. J.
Eyssen,
G.
De
Pauw, and
J.
Van
Eldere*
Rega Institute for Medical Research,
Katholieke Universiteit Leuven, Leuven, Belgium
Received 18 February 1999/Accepted 6 May 1999
 |
ABSTRACT |
From the rat intestinal microflora we isolated a gram-positive rod,
termed HDCA-1, that is a member of a not previously described genomic
species and that is able to transform the 3
,6
,7-trihydroxy bile acid -muricholic acid into hyodeoxycholic acid
(3,6-dihydroxy acid) by dehydroxylation of the 7-hydroxy group
and epimerization of the 6-hydroxy group into a 6-hydroxy group.
Other bile acids that were also transformed into hyodeoxycholic acid
were hyocholic acid (3,6,7-trihydroxy acid), -muricholic
acid (3,6,7-trihydroxy acid), and 
-muricholic acid
(3,6,7-trihydroxy acid). The strain HDCA-1 could not be grown
unless a nonconjugated 7-hydroxylated bile acid and an unidentified
growth factor produced by a Ruminococcus productus strain
that was also isolated from the intestinal microflora were added to the
culture medium. Germfree rats selectively associated with the
strain HDCA-1 plus a bile acid-deconjugating strain and the growth
factor-producing R. productus strain converted
-muricholic acid almost completely into hyodeoxycholic acid.
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INTRODUCTION |
The major biliary bile acids in the
rat are tauro-conjugated cholic acid, chenodeoxycholic acid, and -
and -muricholic acid. In germfree male and female rats fed a
purified casein-starch diet, the major bile acids in the cecum are
cholic acid (16 and 25%, respectively) and -muricholic acid (73 and
46%, respectively) (6). In conventional rats, the bile
acids are extensively modified by the intestinal microflora in the
terminal ileum and the large intestine (15). Bile acids in
feces of female and male conventional rats are deoxycholic acid (9.3 and 8.8%, respectively), 3,6-dihydroxy-5-cholanoic acid (16.8 and 19.1%, respectively), and hyodeoxycholic acid (47.2 and 55.6%,
respectively) (7). Deoxycholic acid is the
7-dehydroxylation product of cholic acid, hyodeoxycholic acid is
thought to derive from -muricholic acid through 7-dehydroxylation
and 6-hydroxy epimerization, and 3,6-dihydroxy-5-cholanoic
acid is the 3-epimer of hyodeoxycholic acid. Feces from conventional
rats also contain variable amounts of -muricholic acid (1, 20,
31), which is thought to derive from -muricholic acid through
epimerization of the 6-hydroxy group (4, 24, 25).
Bacterial 7-dehydroxylation of the biliary bile acids is crucial in the
formation of secondary bile acids. Following 7-dehydroxylation, secondary bile acids may undergo various other oxido-reduction reactions. Bacteria that can 7-dehydroxylate cholic acid and chenodeoxycholic acid are found in many intestinal genera, such as
Clostridium, Eubacterium,
Lactobacillus, and Bacteroides (9, 11,
21). However, 7-dehydroxylating activity is less common. It
was suggested that 7-hydroxy groups had to be epimerized to 7-hydroxy groups prior to dehydroxylation (8). A
7-dehydrogenating Clostridium absonum strain was isolated
by MacDonald and Roach (18). Because many
7-dehydroxylating strains are also capable of reducing 7-keto groups
to 7-hydroxy groups, intestinal bacterial 7-dehydroxylation could
be the result of a combination of the activities of
7-dehydrogenating and 7-dehydroxylating strains. However, White
et al. (30) and Takamine and Imamura (28)
isolated from the rat intestinal tract 7-dehydroxylating
Eubacterium sp. strains that were capable of
7-dehydroxylating the 3,7-dihydroxy bile acid ursodeoxycholic
acid. We also isolated from the rat intestinal microflora a
Clostridium sp. strain with 7- and 7-dehydroxylating activity in the presence of chenodeoxycholic, cholic, ursodeoxycholic, and ursocholic acids (unpublished data). No strain, however, that can
dehydroxylate the 7-hydroxy group of hyocholic acid or the 7-hydroxy group of -muricholic acid has yet been described.
To transform -muricholic acid into hyodeoxycholic acid, the
6-hydroxy group of -muricholic acid also has to be epimerized into a 6-hydroxy group. Several intestinal strains capable of performing this reaction have been described (4, 24, 25). A
combined action of intestinal bacteria that perform the
7-dehydroxylation reaction and other strains that epimerize the
6-hydroxy group could therefore also lead to the formation of
hyodeoxycholic acid. Einarsson (2) suggested that
hyodeoxycholic acid was formed from lithocholic acid by hepatic enzymes
that converted lithocholic acid into a 3,6-dihydroxy bile acid
and bacteria that further oxidized it into a 3-hydroxy, 6-keto bile
acid and reduced it to hyodeoxycholic acid. In this paper we report the
isolation of an unidentified strain that can transform hyocholic acid
and -,-, and -muricholic acids directly into hyodeoxycholic acid.
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MATERIALS AND METHODS |
Culture media used and incubation conditions.
Medium A
consisted of 200 mg of freeze-dried feces from male, germfree rats in 5 ml of distilled water, supplemented with 0.5 ml of a 2-mg/ml solution
of -muricholic acid. Medium B contained 5 ml of PN medium
supplemented with 125 mg of freeze-dried, fat-free ground beef and 0.5 ml of the bile salt solution tested (6 mg/ml). The PN medium consisted
of 0.5% (wt/vol) Proteose Peptone 3 (Difco); 1% (wt/vol) Bacto
Tryptone Yeast (Difco); 0.4% (wt/vol) brain heart infusion (BBL);
0.1% (wt/vol) MgSO4 · 7H2O; 1%
(wt/vol) K2HPO4 · 3H2O;
0.1% (wt/vol) Na2CO3 · 10 H2O; 0.15% (wt/vol) Trizma 7.6; 0.02% (wt/vol) Tween 80;
0.0005% (wt/vol) hemin; 0.0001% (wt/vol) vitamin K (Konakion; Roche,
Basel, Switzerland); 0.04% (wt/vol) cysteine HCl; and 4% (vol/vol)
water extract of feces from germfree rats (supernatant of 6 g of
freeze-dried feces from male germfree rats in 100 ml of water
supplemented with 0.2 ml of 30% NaOH, boiled for 10 min and
centrifuged at 3,000 × g for 10 min). Medium C was PN
medium supplemented with 2% agar (Difco) and 0.6 mg of hyocholic acid
per ml. Medium D contained 2% (wt/vol) Special Peptone (Oxoid), 1%
(wt/vol) K2HPO4 · 3H2O,
0.2% (wt/vol) glucose, 0.1% (wt/vol) NaCl, 0.1% (wt/vol)
MgSO4 · 7H2O, 0.002% (wt/vol) hemin,
0.0001% (wt/vol) vitamin K, and 0.04% (wt/vol) cysteine HCl and was
supplemented with 0.5 ml of a 6-mg/ml solution of hyocholic acid. The
dilution solution contained 1% (wt/vol) Bacto Tryptone Yeast, 0.9%
(wt/vol) Na2HPO4 · 2H2O, and
0.05% (wt/vol) dithiothreitol. Autoclaved liquid and solid culture
media were prereduced in the anaerobic chamber (Anaerobic System; Forma Scientific, Marietta, Ohio) for at least 48 h prior to inoculation in the anaerobic chamber. Agar plates were poured inside the anaerobic chamber. The atmosphere in the anaerobic chamber consisted of 90%
N2 and 10% H2. Inoculated liquid and agar
plate cultures were incubated at 37°C for 3 or 4 days.
Bile acids used and analysis of bile acids in culture media and
feces.
Muricholic acids (-, -, and -muricholic acids)
were obtained after hydrolysis of the respective methyl esters in 5%
KOH (23) in methanol at 70°C for 1 h.
3,7-Dihydroxy-6-oxo-5-cholanoic acid was isolated after
enzymatic deconjugation of fecal bile acids from cholesterol-fed
germfree rats (23). Hyocholic acid, chenodeoxycholic acid,
and the tauro- and glycoconjugates of cholic acid and chenodeoxycholic
acid were from Calbiochem-Behring (La Jolla, Calif.). Ursodeoxycholic,
hyodeoxycholic, and 3-hydroxy-6-oxo-5-cholanoic acids were from
Steraloids (Wilton, N.H.); cholic acid was from Aldrich Chemical
Company (Dorset, England); ursocholic and
3,6-dihydroxy-7-oxo-5-cholanoic acids were synthesized as
described (16, 32). The procedures used to quantify bile
acids in fecal samples from rats and mice have already been described
(5, 23). Bile acid transformations in bacterial cultures
were analyzed by mixing 1 ml of culture with 2 ml of distilled water, 1 ml of ethyl alcohol and 1 ml of a 0.1-mg/ml solution of
23-nor-deoxycholic acid in methyl alcohol as an external standard. This
mixture was acidified to a pH of <2 with 2 N HCl and extracted
twice with 6 ml of diethyl ether. Pooled extracts were evaporated to
dryness, and the bile acids were derivatized to methylester
trimethylsilylethers with diazomethane and TriSyl (Pierce, Rockford,
Ill.). The reaction mixtures were washed with 5 ml of 0.1 N HCl and
extracted twice with 4 ml of hexane. Finally, the extracts were
concentrated by partial evaporation.
To analyze conjugated bile acids, samples were subjected to alkaline
deconjugation with 20% (wt/vol) KOH-ethylene glycol as described
(5). Gas-liquid chromatography was used to identify and
quantify bile acids. Methylester trimethylsilylethers were analyzed
isothermally at 260°C on a 3% OV1- or 1% QF1-packed column with
N2 at a flow rate of 30 to 40 ml/min. Identification and quantification of bile acid metabolites was by capillary gas-liquid chromatography with standards as described (5, 23) on a
30 m by 0.32 mm DB-5 ms column (Alltech, Deerfield, Ill.). The
carrier gas was helium and the flow rate was 1.5 ml/min. The
temperature was kept initially at 80°C for 2 min, increased at
30°C/min to 275°C, and after 1 min at 275°C again raised to
280°C at 0.5°C/min. Bile acids were analyzed as acetylated
derivatives or as methylester trimethylsilylether derivatives.
Study of bile acid transformations in rats.
Male inbred
germfree, gnotobiotic, and conventional Fisher rats from our own
germfree animal breeding center (Rega Institute, Leuven, Belgium) were
used. The rats were originally obtained from the Laboratoire des
Animaux sans Germes CNRS, Gif-sur-Yvette, France. The animals were kept
in Trexler flexible-film plastic isolators (Standard Safety Equipment,
Palatine, Ill.). The conventional rats were ex-germfree Fisher rats
that were conventionalized by exposure in the same cage to commercially
acquired Fisher rats (Animalium KULeuven, Leuven, Belgium). Due to
coprophagy, the germfree rats acquired conventional intestinal flora
over the course of 4 to 6 weeks. This was established through analysis of fecal bile acids and comparison of the fecal bile acid patterns to
those already published (6, 7). The gnotobiotic rats were
ex-germfree Fisher rats that received rectal instillation of 1 ml of
2-day-old bacterial culture fluid. Germfree rats were first associated
with Clostridium perfringens ATCC 19574 and subsequently with Ruminococcus productus and strain HDCA-1, and finally,
the rats in one group were also associated with Clostridium
sp. strain S1 (14). After instillation of each strain, feces
were checked for the presence of all introduced strains by microscopic
examination and culture of fecal homogenates. The next strain was
introduced only after confirmation of the presence of the preceding
strain. Fecal bile acid profiles were analyzed every week for several weeks after the introduction of all the species of the defined microflora. A gradual change in the composition of the fecal bile acids
was observed; there was evolution from the pattern found in the
germfree animals towards a climax pattern that remained stable for the
rest of the rats' lives. This climax pattern is reported in Table
1. The gradual change in the fecal bile
acid pattern took from 2 to 3 weeks to develop and spanned the interval from the time of inoculation to the time of the stable climax pattern.
All rats used in our experiments received a steam-sterilized commercial
diet (SRMA1210; Hope Farms, Woerden, The Netherlands)
and water ad
libitum. For the analysis of bile acids, feces were
collected every 2 or 3 days from 30 rats in each group, homogenized
with equal volumes of
water, and freeze-dried.
16S rDNA sequence analysis of HDCA-1.
Genomic DNA was
prepared according to the protocol of Niemann et al. (22).
16S rRNA genes were amplified by PCR by using the following primers:
5'-AGTTTGATCCTGGCTCAG-3' and
5'-TACCTTGTTACGACTTCACCCCA-3'. PCR-amplified 16S rDNAs were
purified by using a QIAquick PCR Purification kit (Qiagen GmbH, Hilden,
Germany). Complete sequencing was performed by using an Applied
Biosystems Inc. 377 DNA Sequencer and the protocols of the manufacturer
(Perkin-Elmer, Foster City, Calif.) by using an ABI Prism Dye
Terminator Cycle Sequencing Ready Reaction kit. The following five
forward primers and three reverse primers were used to get an optimal
overlap of sequences, enhancing the reliability of the assembled data:
5'-CTCCTACGGGAGGCAGT-3', 5'-CAGCAGCCGCGGTAATAC-3',
5'-AACTCAAAGGAATTGACGG-3',
5'-AGTCCCGCAACGAGCGCAAC-3', 5'-GCTACACACGTGCTACAATG-3',
5'-ACTGCTGCCTCCCGTAGGAG-3',
5'-GTATTACCGCGGCTGCTG-3', and
5'-GTTGCGCTCGTTGCGGGACT-3'. Sequence assembly was performed by using the program AutoAssembler (Perkin-Elmer). The resulting consensus sequence was 1,443 bp long. Phylogenetic analysis was performed by using the software package GeneCompar (Applied Maths, Kortrijk, Belgium) after including the 1,443-bp-long consensus sequence
in an alignment of small ribosomal subunit sequences collected from the
international nucleotide sequence library of the European Molecular
Biology Laboratory (EMBL) via the FASTA search program. This alignment
was pairwise calculated with an open gap penalty of 100% and a unit
gap penalty of 0%. A similarity matrix was created by homology
calculation with a gap penalty of 0% and after discarding unknown
bases. The resulting tree was constructed by using the neighbor-joining method.
| |
RESULTS |
Isolation of a minimal hyodeoxycholic acid-forming culture.
Fresh feces from 2- to 4-month-old ex-germfree conventionalized Fisher
rats were transferred to the anaerobic chamber, and approximately
0.5 g was homogenized in 10 ml of the dilution fluid. Anaerobic
subcultures of 0.5 ml of this fecal suspension in medium A converted
-muricholic acid and hyocholic acid into hyodeoxycholic acid,
whereas aerobic subcultures did not. Heating of the fecal suspension in
the dilution fluid for 20 min at 80°C followed by anaerobic
subculturing did not impede transformation of -muricholic acid and
hyocholic acid into hyodeoxycholic acid. However, the hyodeoxycholic
acid-producing microflora could not be maintained on any of the
commonly used culture media, such as brain heart infusion broth,
Columbia broth, or thioglycolate broth. Two essential conditions for
maintaining the hyodeoxycholic acid-producing microflora were a pH
of > 6.5 and the presence in the culture medium of 7-hydroxy bile
acids. Although bile acids were found to be essential to sustain the
hyodeoxycholic acid-producing microflora, bile acid concentrations of
0.1 mg/ml or more led to the disappearance of the hyodeoxycholic
acid-forming activity. Addition of 125 mg of freeze-dried ground beef
per 5 ml of the strongly buffered PN culture medium (medium B)
permitted addition of up to 1 mg of bile acids per ml and led to stable
hyodeoxycholic acid formation.
Subculturing of pure cultures of bacterial strains picked after plating
out of the mixed hyodeoxycholic acid-forming cultures
on medium C never
led to the isolation of pure cultures of hyodeoxycholic
acid-forming
bacteria. The minimal mixed culture, in which stable
hyodeoxycholic
acid-forming activity was found, consisted of two
strains. The first of
these two strains was a gram-positive coccus
identified as
R. productus, and the other strain was an unidentified
gram-variable
rod named HDCA-1. Identification of
R. productus was based
on the
VPI Anaerobe Laboratory Manual (
12) and
Bergey's Manual of Systematic Bacteriology (
26).
These bacteria presented
as gram-positive coccobacilli, lying in pairs
or short chains.
They fermented arabinose, cellobiose, fructose,
glucose, lactose,
maltose, mannose, melibiose, raffinose, rhamnose,
ribose, sorbitol,
sucrose, trehalose, and xylose. Gas-liquid
chromatography of fermentation
products in peptone-yeast
extract-glucose broth showed that these
bacteria produced acetate,
succinate, and lactic acid (
13).
Gas-liquid chromatography
of whole-cell fatty acids and comparison
to the database via the
Microbial Identification System (MIDI,
Inc., Newark, Del.) confirmed
the identification. The
R. productus strain was grown as a
pure culture but did not transform hyocholic
acid or
-muricholic
acid.
Characteristics and phylogeny of strain HDCA-1.
Cells of
strain HDCA-1 presented as small, gram-variable, nonmotile, tapered
rods lying in pairs or short chains. HDCA-1 in fecal suspensions
obtained from gnotobiotic rats selectively associated with HDCA-1,
R. productus, and C. perfringens ATCC 19574 survived heat shock for 20 min at 80°C. Spore formation could not be
induced in vitro in any of the culture media that we tested. Colonies on medium C were less than 0.1 mm in diameter, circular, entire, and
glistening, and consisted of a monolayer of bacteria (Fig. 1).
Growth of strain HDCA-1 in liquid culture medium was obtained only if
the
R. productus strain was cocultured or if 10 drops
of
culture supernatant of a 48-h-old
R. productus culture were
added to the culture medium. Addition of 5.5% (vol/vol) of the
R. productus culture supernatant led to an HDCA-1 cell
density
of around 10
6 per ml after 3 days of incubation on
medium B. Reduction of the
volume of supernatant added to below 2.7%
(vol/vol) led to loss
of the HDCA-1 culture if subcultures were made
every 3 days. On
solid media, growth of strain HDCA-1 could be obtained
only after
streak inoculation of
R. productus, presumably
because of rapid
inactivation of the culture supernatant by trace
amounts of oxygen.
The growth factor was prepared by boiling of the
entire culture
or filtration-centrifugation of a 48-h-old
R. productus culture.
It was resistant to boiling for 5 min under
anaerobic conditions
but was rapidly inactivated by contact with
oxygen. After inactivation
by oxygen it could be reactivated by
addition of 6 mM dithiothreitol
in the anaerobic isolator. At
20°C
it could be kept for at least
6 months. It was, however, inactivated at
a pH of <6.5. The growth
factor was also produced by the
R. productus type strain (ATCC
27340). It was not present in horse
blood or feces from germfree
rats. Vitamins, pantethine, glutathione,
lipoic acid, or coenzyme
A could not replace the activity of the growth
factor. Other growth
requirements of strain HDCA-1 were strict
anaerobiosis (E
h below
250 mV) and a pH between 7.0 and
7.75 at the time of inoculation.
Addition of dithiothreitol at
concentrations up to 12 mM reduced
the E
h to below
300
mV. Although this was not toxic to the HDCA-1,
it did not improve
growth, nor did it lead to increased bile acid
transformation. Growth
was impaired at pHs below 7.0, and no growth
was observed at pHs below
6.5. In addition, supplementation of
the culture medium with a
carbohydrate (glucose, fructose, ribose,
maltose, lactose, melibiose,
and to a lesser extent sucrose, trehalose,
and starch) and the presence
of a nonconjugated 7-hydroxy bile
acid were also necessary for growth.
The freeze-dried ground beef
(125 mg/5 ml) that was added to allow
addition of bile acids in
concentrations greater than 0.1 mg/ml could
be replaced by 0.002%
(wt/vol) hemin (medium D). However, even under
optimal conditions,
the number of CFU of strain HDCA-1 never exceeded
10
6/ml. Replacing the standard 90% N
2-10%
H
2 atmosphere in the anaerobic
chamber with an 80%
N
2-10% H
2-10% CO
2 atmosphere
did not stimulate
growth.
Identification of HDCA-1 through conventional methods was not possible
because of our inability to grow the strain on the
necessary culture
media. Gas-liquid chromatography of whole-cell
fatty acids and
comparison to the database via the Microbial Identification
System did
not lead to detection of similarities with known patterns
sufficient to
allow identification. Sequence analysis of the 16S
rRNA genes (EMBL
accession no.
AJ238611) and alignment with
the other sequences from the
EMBL sequence library by using the
software package GeneCompar (Applied
Maths) made it possible to
determine the phylogenetic position of
HDCA-1 within the spectrum
of low G+C content gram-positive bacteria.
HDCA-1 showed the greatest
sequence similarities (91 to 92%) with
unidentified rumen isolates
(
27). Other close relatives were
Termitobacter aceticus (89.6%),
Eubacterium
desmolans ATCC 43058 (89.2%),
Clostridium viride DSM
6836 (89.1%),
Ruminococcus flavefaciens NCFB 2213 (87.6%),
and
Ruminococcus albus OR108 (87.5%) (Fig.
2). The low sequence similarities
(less
than 97%) clearly indicate that HDCA-1 is a member of a
new, not
previously described genomic species (
29).
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FIG. 2.
Phylogenetic tree of HDCA-1 (ID3154) based on the
alignment of the most similar 16S rDNA sequences, using the GeneCompar
software package (Applied Maths). Type species are shown in bold,
designations of type strains are followed by a T, and invalid species
names are given in quotes.
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Bile acid transformations by strain HDCA-1.
Between 70 and
80% of 0.6-mg/ml solutions of the 7- and 7-trihydroxy bile acids
-muricholic acid, -muricholic acid, -muricholic acid,
ursocholic acid, and cholic acid were 7-dehydroxylated by strain
HDCA-1. Identical concentrations of the 7-dihydroxy bile acid
chenodeoxycholic and the 7-dihydroxy bile acid ursodeoxycholic acid
were less than 50% 7-dehydroxylated.
In addition to the dehydroxylation of the 7-hydroxy group, strain
HDCA-1 also epimerized the 6
-hydroxy groups of
-muricholic
acid
and
-muricholic acid into 6
-hydroxy groups. As a result
of this
combined 7-dehydroxylating and 6
-epimerizing activity,
strain HDCA-1
transformed
-muricholic,
-muricholic,
-muricholic,
and
hyocholic acid into hyodeoxycholic acid. We did not find
dehydroxylation
of 3-, 6-, or 12-hydroxy groups by strain HDCA-1.
Study of the time course of
-muricholic acid and hyocholic acid
transformation by strain HDCA-1 showed a rather slow formation
of
hyodeoxycholic acid which went on well into the stationary
growth phase
(Fig.
3). We also observed a weak
transient formation
of 3
,6
-dihydroxy-7-oxo-5
-cholanoic acid
both with
-muricholic
and with hyocholic acid and the slow
accumulation of 3
-hydroxy-6-oxo-5
-cholanoic
acid starting after 2 days of incubation.
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FIG. 3.
Transformation of -muricholic acid (a) and hyocholic
acid (b) by strain HDCA-1 as a function of time.  , -muricholic
acid (panel a) and hyocholic acid (panel b);  , hyodeoxycholic acid;
 , 3,6-dihydroxy-7-oxo-5-cholanoic acid; and  ,
3-hydroxy-6-oxo-5-cholanoic acid.
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Association of germfree rats with strain HDCA-1.
Strain HDCA-1
alone could not be associated with germfree rats, most likely because
of its absolute requirements for 7-hydroxylated nonconjugated bile
acids, for the growth factor produced by R. productus, and
for a low Eh. Prior association of germfree rats with the
growth factor-producing R. productus strain and the bile acid-deconjugating C. perfringens strain ATCC 19574 allowed
the association of these gnotobiotic rats with strain HDCA-1. Analysis of fecal bile acids from these gnotobiotic rats and comparison with
fecal bile acids from germfree rats showed a 50% reduction in
-muricholic acid, a 16% reduction in cholic acid, and a 53% reduction in chenodeoxycholic acid (Table 1). Concomitantly, we found
in the feces of these gnotobiotic rats 19.9% hyodeoxycholic acid,
7.6% deoxycholic acid and 2% lithocholic acid in contrast to a total
absence of these bile acids for the germfree rats. -Muricholic acid,
because it is almost completely sulfated, appeared not to be
transformed in the gnotobiotic rats, judging from its relative
concentrations in gnotobiotic and germfree rats. Plating of serial
dilutions of fresh fecal homogenates from three rats showed that
R. productus and C. perfringens were present at
densities of 109 per g. The numbers of cells of HDCA-1 in
the feces were estimated at 103 per g as judged from the
formation of hyodeoxycholic acid in serial dilutions of the same fresh
fecal homogenates.
Additional association of the
R. productus plus
C. perfringens ATCC 19574 plus HDCA-1-associated gnotobiotic rats
with the
bile acid desulfating and deconjugating
Clostridium
sp. S1 strain
(
14) increased deconjugation of
-muricholic
acid and cholic
acid. Consequently, the transformation of
-muricholic acid and
cholic acid into hyodeoxycholic and deoxycholic
acid was also
increased to levels that were even higher than those
found in
conventional rats (Table
1). In addition, 0.5% of total fecal
bile acids was identified as
-muricholic acid, and 11.3% was
identified as a
-muricholic acid metabolite with a double bond
in
the side chain, between C-22 and C-23, as already described
by Robben
et al. (
24) and Kayahara et al. (
17).
Germfree CH3 mice (Rega Institute) were kept for up to 3 months in the
same cage as the gnotobiotic rats associated with the
complex
microflora (
R. productus,
C. perfringens ATCC
19574, HDCA-1,
and
Clostridium sp. S1). Interestingly, we
found that all the
strains except for HDCA-1 became established in the
ex-germfree
mice. Rectal instillation of bacteria in germfree CH3 mice
in
the same way as used for the germfree Fisher rats also did not
lead
to establishment of HDCA-1 in the
mice.
| |
DISCUSSION |
The transformation of - and -muricholic acids into
hyodeoxycholic acid includes a 7- or 7-dehydroxylation reaction
and a 6-epimerization reaction. Hypothetical reaction mechanisms for
these transformations are diaxial transelimination of the 7-hydroxyl
group and the 6-hydrogen for -muricholic acid and diequatorial
transelimination of the 7-hydroxyl group and the 6-hydrogen for
-muricholic acid. The resulting 3-hydroxy-
6-enol intermediate
could subsequently be reduced to a 6-hydroxyl moiety by
transhydrogenation. Time course experiments on -muricholic acid
metabolism by strain HDCA-1 showed transient formation of a
3,6-dihydroxy-7-oxo-5-cholanoic acid during the exponential growth phase and accumulation of 3-hydroxy-6-oxo-5-cholanoic acid
in the stationary growth phase of the culture.
Because the 3-hydroxy-6-oxo-5-cholanoic acid could not be
transformed by strain HDCA-1 into hyodeoxycholic acid, we suggest that
this is not an intermediate in the formation of hyodeoxycholic acid but
is a metabolite of hyodeoxycholic acid. -Muricholic acid was not
found as an intermediate of the transformation of -muricholic acid
into hyodeoxycholic acid. The efficiency with which strain HDCA-1
transforms muricholic acids into hyodeoxycholic acid both in vitro and
in vivo seems to exclude the combined bacterial-hepatic transformation
pathway proposed by Einarsson (2). Because hyodeoxycholic acid is not very efficiently absorbed from the intestinal tract in
rats, Madsen et al. (19) suggested that in the rat
hyodeoxycholic acid formation might be an important mechanism for
controlling the body cholesterol pools. Hence, transformation of
muricholic acid by strain HDCA-1 in the rat might also be important in
controlling cholesterol levels. The traces of -muricholic acid found
in the feces of the gnotobiotic rats associated with strain HDCA-1
suggest that this bile acid could indeed be the result of a further
hepatic modification of reabsorbed hyodeoxycholic acid, as suggested by Madsen et al. (19).
The special growth factors required by HDCA-1 complicated its isolation
from feces and may explain why previous efforts to isolate
hyodeoxycholic acid-forming bacteria from feces did not succeed. The
fact that bile acids with a 7-hydroxy group are a necessary growth
factor suggests that this 7-hydroxyl functions as an electron acceptor
for these bacteria. The requirement for 7-dehydroxylation of a special
factor produced by another bacterial strain has been reported before
(10, 28).
The sensitivity of strain HDCA-1 to changes in pH and particularly its
inability to grow at pHs below 7.0 might explain the fall in
hyodeoxycholic acid production in rats fed a lactose-containing diet
(3). Bacterial fermentation of lactose leads to a reduced colonic pH, as reported by Eyssen et al. (3), and
consequently to a reduced formation of hyodeoxycholic acid.
In conclusion, the isolation of strain HDCA-1 is an illustration of the
complex interactions and metabolic interdependence that exist among the
members of the intestinal microflora and again demonstrates the
enormous metabolic diversity and versatility of the bacteria that
constitute the intestinal microflora.
| |
APPENDIX |
Trivial names for bile acids are as follows:
3-hydroxy-5-cholanoic acid, lithocholic acid;
3-hydroxy-5-cholanoic acid, allolithocholic acid;
3,6-dihydroxy-5-cholanoic acid, hyodeoxycholic acid;
3,6-dihydroxy-5-cholanoic acid, muricholic acid;
3,7-dihydroxy-5-cholanoic acid, chenodeoxycholic acid;
3,7-dihydroxy-5-cholanoic acid, ursodeoxycholic acid;
3,12-dihydroxy-5-cholanoic acid, deoxycholic acid;
3,7,12-trihydroxy-5-cholanoic acid, cholic acid;
3,6,7-trihydroxy-5-cholanoic acid, hyocholic acid;
3,6,7-trihydroxy-5-cholanoic acid, -muricholic acid;
3,6,7-trihydroxy-5-cholanoic acid, -muricholic acid; 3,6,7-trihydroxy-5-cholanoic acid, -muricholic acid;
3,7,12-trihydroxy-5-cholanoic acid, ursocholic acid;
3,12-dihydroxy-23-nor-5-cholanoic acid, 23-nor-deoxycholic acid.
| |
ACKNOWLEDGMENTS |
We thank M. Vancanneyt and D. Janssens of the BCCM/LMG Culture
Collection, Laboratory of Microbiology, University of Ghent, for
performing the 16S rDNA sequence analysis of HDCA-1 and the phylogenetic analysis.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Rega Institute,
Minderbroederstraat 10, B-3000 Leuven, Belgium. Phone: 16 33 73 72. Fax: 16 33 73 40. E-mail:
johan.vaneldere{at}rega.kuleuven.ac.be.
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Abstract
Introduction
