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Gallstone formation in the conventional mouse
Gallstone
Formation
Conventional
in the
Mouse
The Role of Bacteria CHARLES F. FREY,
M.D. AND ROLF FRETER,
From the Departments of Surgery and Microbiology, LJniversity of Michigan Medical Center, Ann Arbor, Michigan. This work was supported in full by National Institutes of Health Public Health Service Grant AM 11315.
G and
PH.D., Ann
Arbor, Michigan
by General Biochemicals, Chagrin Falls, Ohio) to which had been added additional cholesterol and cholic acid. The final mixture, containing 1 per cent cholesterol and 0.5 per cent cholic acid by weight, was fed to these mice. They were sacrificed after termination of the high cholesterol diet at two, four, and six weeks. Group II. Twenty mice, ten male and ten female, were fed the L-356 diet without added cholesterol or cholic acid for two, four, and six weeks prior to sacrifice. Group III. Twenty-seven mice, thirteen male and fourteen female, were fed the L-356 diet with added cholesterol and cholic acid for three to four weeks, at which time they were sacrificed. Group IV. Fifteen mice, six male and nine female, were fed the L-356 diet without added cholesterol or cholic acid for three to four weeks, at which time they were sacrificed. The mice were sacrificed by cervical cord transection. The abdomens were prepared with iodine and alcohol, then the skin was incised and peeled off the abdominal musculature. The newly prepared surface was reprepared with iodine and alcohol. The abdomen was opened through a midline incision. The cystic duct was clamped, after which the gallbladder was excised and placed into broth culture
can be induced in the mouse by a diet containing 1 per cent cholesterol O.Ti per cent cholic acid by weight. Frey,
ALLSTONES
Thorpe, and Abrams [I] found gallstones evolved sooner in conventional than in germfree mice fed the same diet. The development of cholecystitis was similar in onset and degree in germ-free and conventional mice and was found attributable to the ingestion of a lithogenie diet rather than to bacterial invasion. Bacteria were postulated to hasten gallstone formation by acting as a nidus for crystal aggregation or by altering cholesterol absorption and metabolism. To test the hypothesis we have attempted to determine whether bacteria exist in the gallbladder wall, bile, or gallstones of conventional mice. MATERIAL AND METHODS
three week old weanling CD-l Swiss River Breeding Laboratory were divided into the following four groups: Group I. Thirty-six mice, half of which were male, were fed the Notre Dame L-356 diet,* (manufactured Ninety-eight
mice from the Charles
124.850, potassium phosphate dibasic 510.75, sodium phosphate dibasic 454.00, sodium chloride (NaCl) 113.500, potassium iodide (Kl) 1.7025, magnesium sulfate 1’70.250, manganese sulfate 28.375, ferric citrate 170.250, copper sulfate 8.7170, cobalt chloride 1.1350,
* Ingredients: lb./100 Ib.-vitamin-free test casein 20.0000, corn oil 5.0000, rice flour 58.0000, nonnutritive fibre 5.0000, desiccated liver 2.0000, ascorbic acid 0.2000, i-inositol 0.1000, yeast extract, Albimi 2.0000, vitamins Ladek 2.0000, salts L-11 5.0000, vitamin B mix 75 0.5000; gm./lOO lb.-vitamin A concentrate 200,000 U/gm. 1.816, vitamin Da Dawe’s sterol 1500 U/gm. 30.260, Mixed tocopherols 68.100, menadione 4.540, corn oil 4,s. to 2 lb. 803.28 gm., calcium carbonate (CaCOa) 681.000, calcium phosphate dibasic
zinc sulfate 2.2700, sodium borate (Borax) 1.1350, aluminum potassium sulfate 1.7025; gm./lOO lb.vitamin B mix: thiamine HCl 2.72, riboflavin 1.36, nicotinamide 2.26, nicotinic acid 2.26, calcium pantothenate 13.60, choline chloride 90.500, pyridoxine HCl 0.9050, pyridoxamine DiHCl 0.1820, biotin 0.0454, folic acid 0.4540, para-aminobenzoic acid 2.260, vitamin B,? in mannitol, 0.1% trituration 11.30, corn starch q.s.. to 0.5 lb. 97.60. 868
The American
Journal
of Surgery
Gallstone
RESULTS
tubes. Subcultures were grown in the same medium if the tubes showed evidence of growth. At four to five months all the remaining tubes in group I and II which had not become turbid were subcultured. The medium used for groups I and II consisted of trypticase soy broth (Baltimore Biological Laboratory) contaiiing, in addition, 0.5 per cent yeast extract. (I.0 I per cent menadione, 0.01 per cent Hemin, 0.0% per cent NaCO,? and 0.05 per cent cysteineHCI. Since in their earlier work Spears and Freter [2] had shown that the predominant intestinal flora of the mouse is anaerobic, precautions were taken to avoid contact with air of the specimen to be cultured. Tubes were filled with the broth in a chamber containing an atmosphere of 10 per cent Hn and 5 per cent COe in Nz containing less than 10 parts per million 02. The test tubes containing the medium were stoppered with sterile rubber. The specimen was introduced into the tube by lifting the stopper momentarily. During the time the stopper was lifted a stream of sterile gas of the aforementioned composition was admitted into the tube via a hypodermic needle. The inoculated tubes were incubated at ::Pc. in an atmosphere of the above composition. The medium used for groups III and IV consisted of chopped meat medium (Difco) containing I.7 per cent normal rabbit serum. The medium was dispersed in screw cap tubes and incubated at 37~. with the caps tightly closed but without a special gas l)hase overlaying the medium. This type of medium supported the growth of strict aerobes (for examl,le. Pseudomonas) and of many anaerobes.
The broth which contained the gallbladder and its contents from the fifty-six mice in groups I and II remained clear except for five tubes which became turbid after thirteen to fourteen days. These were from three mice (group I) which had been on the high cholesterol diet, and two mice (group II) which had been on the L-356 diet for four weeks. (Tables I and II.) Gram-positive rods with granules morphologically resembling “diptheroids” were isolated from four of the turbid tubes. Short gram-positive rods having a different appearance from the diptheroids on colony formation were isolated from the other turbid tubes. The latter organism was considered a contaminant. Four to five months after the gallbladders were first excised all media, which had remained clear, were subculturerl. The subcultures remained sterile. Gallstones were not noted in any gallbladders of the mice with positive cultures. At six weeks four of eight mice on the lithogenic diet had grossly visible gallstones. (Table I.) Cultures of the gallbladders of mice in groups III and IV, which had been maintained for three to four weeks on the high cholesterol or L-356 diet, remained sterile on incubation in all but one instance. (Tables III and IV.) From one
TABLE RESULTS
Mouse
Number _-____. __~ .i 3
2 :! :i
3
Sex Male
Female Male Female Male
Female
OF GALLBLADDER
CULTURES
FROM
Time on Diet (wk.)
Bacterial Growth
2 2 4
Xone Kane 1
4
1
6 6
None Sone
Y(i!b
Formation
II
MICE
Oh‘
L-356 STANDARDRATION(GROUPII J
Subcultured .___~
Gross Gallstones
_..
Gram-positive rods (diptheroids) Gram-positive rods; short
Nnnc None None Kane sme NOilt!
Frey and Freter
8’70
TABLE III RESULTSOF GALLBLADDERCULTURESFROM MICE ON HIGH CHOLESTEROL DIET (GROUP III) Time on Diet (wk.)
Mouse
Number
Sex
Bacterial Growth None None 1 None None None
Male Female Male Female Male Female
male
mouse
on the lithogenic
gram-positive further male the
diet
cocci were grown
identified.
(Table
III.)
Nine
mice and six of fourteen lithogenic
diet
had
(group
III)
which were not of thirteen
female
gross
mice on
gallstones.
In
none of the six male or nine female
mice on the
L-356
(Table
diet did gallstones
develop.
III.)
Actinomycetes center and
Burnett
formation defined.
[4].
The
Rains
Gallstones occurs
the
acid in
mice
Cholecystitis
diet.
on
the
was
of the
from
tional
and
L-356
ration.
In
this
bladder
study
wall
been in
in mice fed a high
same a
the
we
formation
mice
lithogenic
similar
found
and
conven-
diet. Inflammation on
of conventhe
no
1 2
a lithogenic under
diet.
aerobic
of
in the gall-
of conventional
mice fed
Rich
and
The few organisms
media
anaerobic
were
employed
conditions
[Z].
which grew were considered
to be contaminants
commonly
encountered
in
our laboratory. Our
inability
gallbladder
to culture
wall
or bile
tion
diet makes
in the
mouse.
The
and cultures
obtained
initiation
the
period
of
crystal
in the mouse a role
mice
were
diet.
gallbladder. cultured
them
support
rate
of gallstone
a
bacterial
for crystal
to
aggregation.
and
acid absorption,
have
at this time.
the
hypothesis
formation
cholecystitis
flora of the intestine
this
did play
germ-free
bacterial bile
a
apparent
we would
do not compared
During
If bacteria
aggregation,
results
nidus
that forma-
sacrificed
is often
the higher to
the mice
three to four weeks after
lithogenic
to have
conventional due
from
it unlikely
aggregation
in crystal
Our that
bacteria
of conventional
nidus has any role in gallstone
or
More
in
mice
is
bacterial likely
the
alter cholesterol
thus hastening
gall-
stone formation.
standard
evidence
or any other bacteria or bile
in
diet.
degree and
gallbladders mice
than
1
.
expected
germ-free
germ-free
actinomycetes
in the
not
actinomycetes
conventional
in both
absent
has
Gallstone
tional mice on the lithogenic was
[3]
could develop.
can be induced
sooner
incidence
the
role of bacteria gallstones
believes
bile
germ-free
from et al.
could act as a nidus about which
aggregation
cholesterol
by Rains
8
Gram-positive cocci
bacterial
been cultured
gallstones
of human
the gallbladder crystal
have
of human
None Sane
.
fed a lithogenic
COMMENTS
Gross Gallstones
Subcultured
SUMK4RY Aerobic bladder
and anaerobic
cultures
of the
wall and bile of conventional
lithogenic
diet did not show
gall-
mice on a
growth.
It is un-
TABLE IV RESULTS OF GALLBLADDER CULTURES FROM MICE ON THE STA~VD.~RDL-356 DIET (GROUP IV) Mouse Number
Sex
Time on Diet (wk.)
Bacterial Growth
4 6
Female Male Female
None None None None
1 2
Male Female
None None
1 1
Male
Subcultured
. . . . ..
The American
Gross Gallstones None None None None None None
Journal
of Surgery
Gallstone likely that bacterial cholecystitis nidus ha5 any role in gallstone the mouse.
or a bacterial formation in
REFERENCES 1. FRET, C. F., THORPE, C., and ABRAMS, G. Gallstone
formation in the germ-free mouse. Am. J. Surg., IIf,: 75, 1968. 2. SPEARS, R. W. and FRETER, R. Improved isolation of
Vol. 116, December 1968
Formation
371
anerobic bacteria from the mouse cecum by mail1 taining continuous strict anarrobiosis Pro, .%. Exper. Riol. & &‘ed.. 124: 903, 196i. 3. RAEIS, A. J. H., BARSON, G. J., CRAWFORI). S . and SHREWSBURG, J. F. D. Achievement and bacterologic study of gallstones. The presence of actinomycete. Lancet, 2: 614, 1960. 4. BURNET.~, W. The pathogenesis of gal1stonc.s. In: The Biliary System: A Symposium of the NATO Advance Study Institute, p. 610. I’hilad~elphia, 1963. F. .A. Davis Co.
Formation
Conventional
in the
Mouse
The Role of Bacteria CHARLES F. FREY,
M.D. AND ROLF FRETER,
From the Departments of Surgery and Microbiology, LJniversity of Michigan Medical Center, Ann Arbor, Michigan. This work was supported in full by National Institutes of Health Public Health Service Grant AM 11315.
G and
PH.D., Ann
Arbor, Michigan
by General Biochemicals, Chagrin Falls, Ohio) to which had been added additional cholesterol and cholic acid. The final mixture, containing 1 per cent cholesterol and 0.5 per cent cholic acid by weight, was fed to these mice. They were sacrificed after termination of the high cholesterol diet at two, four, and six weeks. Group II. Twenty mice, ten male and ten female, were fed the L-356 diet without added cholesterol or cholic acid for two, four, and six weeks prior to sacrifice. Group III. Twenty-seven mice, thirteen male and fourteen female, were fed the L-356 diet with added cholesterol and cholic acid for three to four weeks, at which time they were sacrificed. Group IV. Fifteen mice, six male and nine female, were fed the L-356 diet without added cholesterol or cholic acid for three to four weeks, at which time they were sacrificed. The mice were sacrificed by cervical cord transection. The abdomens were prepared with iodine and alcohol, then the skin was incised and peeled off the abdominal musculature. The newly prepared surface was reprepared with iodine and alcohol. The abdomen was opened through a midline incision. The cystic duct was clamped, after which the gallbladder was excised and placed into broth culture
can be induced in the mouse by a diet containing 1 per cent cholesterol O.Ti per cent cholic acid by weight. Frey,
ALLSTONES
Thorpe, and Abrams [I] found gallstones evolved sooner in conventional than in germfree mice fed the same diet. The development of cholecystitis was similar in onset and degree in germ-free and conventional mice and was found attributable to the ingestion of a lithogenie diet rather than to bacterial invasion. Bacteria were postulated to hasten gallstone formation by acting as a nidus for crystal aggregation or by altering cholesterol absorption and metabolism. To test the hypothesis we have attempted to determine whether bacteria exist in the gallbladder wall, bile, or gallstones of conventional mice. MATERIAL AND METHODS
three week old weanling CD-l Swiss River Breeding Laboratory were divided into the following four groups: Group I. Thirty-six mice, half of which were male, were fed the Notre Dame L-356 diet,* (manufactured Ninety-eight
mice from the Charles
124.850, potassium phosphate dibasic 510.75, sodium phosphate dibasic 454.00, sodium chloride (NaCl) 113.500, potassium iodide (Kl) 1.7025, magnesium sulfate 1’70.250, manganese sulfate 28.375, ferric citrate 170.250, copper sulfate 8.7170, cobalt chloride 1.1350,
* Ingredients: lb./100 Ib.-vitamin-free test casein 20.0000, corn oil 5.0000, rice flour 58.0000, nonnutritive fibre 5.0000, desiccated liver 2.0000, ascorbic acid 0.2000, i-inositol 0.1000, yeast extract, Albimi 2.0000, vitamins Ladek 2.0000, salts L-11 5.0000, vitamin B mix 75 0.5000; gm./lOO lb.-vitamin A concentrate 200,000 U/gm. 1.816, vitamin Da Dawe’s sterol 1500 U/gm. 30.260, Mixed tocopherols 68.100, menadione 4.540, corn oil 4,s. to 2 lb. 803.28 gm., calcium carbonate (CaCOa) 681.000, calcium phosphate dibasic
zinc sulfate 2.2700, sodium borate (Borax) 1.1350, aluminum potassium sulfate 1.7025; gm./lOO lb.vitamin B mix: thiamine HCl 2.72, riboflavin 1.36, nicotinamide 2.26, nicotinic acid 2.26, calcium pantothenate 13.60, choline chloride 90.500, pyridoxine HCl 0.9050, pyridoxamine DiHCl 0.1820, biotin 0.0454, folic acid 0.4540, para-aminobenzoic acid 2.260, vitamin B,? in mannitol, 0.1% trituration 11.30, corn starch q.s.. to 0.5 lb. 97.60. 868
The American
Journal
of Surgery
Gallstone
RESULTS
tubes. Subcultures were grown in the same medium if the tubes showed evidence of growth. At four to five months all the remaining tubes in group I and II which had not become turbid were subcultured. The medium used for groups I and II consisted of trypticase soy broth (Baltimore Biological Laboratory) contaiiing, in addition, 0.5 per cent yeast extract. (I.0 I per cent menadione, 0.01 per cent Hemin, 0.0% per cent NaCO,? and 0.05 per cent cysteineHCI. Since in their earlier work Spears and Freter [2] had shown that the predominant intestinal flora of the mouse is anaerobic, precautions were taken to avoid contact with air of the specimen to be cultured. Tubes were filled with the broth in a chamber containing an atmosphere of 10 per cent Hn and 5 per cent COe in Nz containing less than 10 parts per million 02. The test tubes containing the medium were stoppered with sterile rubber. The specimen was introduced into the tube by lifting the stopper momentarily. During the time the stopper was lifted a stream of sterile gas of the aforementioned composition was admitted into the tube via a hypodermic needle. The inoculated tubes were incubated at ::Pc. in an atmosphere of the above composition. The medium used for groups III and IV consisted of chopped meat medium (Difco) containing I.7 per cent normal rabbit serum. The medium was dispersed in screw cap tubes and incubated at 37~. with the caps tightly closed but without a special gas l)hase overlaying the medium. This type of medium supported the growth of strict aerobes (for examl,le. Pseudomonas) and of many anaerobes.
The broth which contained the gallbladder and its contents from the fifty-six mice in groups I and II remained clear except for five tubes which became turbid after thirteen to fourteen days. These were from three mice (group I) which had been on the high cholesterol diet, and two mice (group II) which had been on the L-356 diet for four weeks. (Tables I and II.) Gram-positive rods with granules morphologically resembling “diptheroids” were isolated from four of the turbid tubes. Short gram-positive rods having a different appearance from the diptheroids on colony formation were isolated from the other turbid tubes. The latter organism was considered a contaminant. Four to five months after the gallbladders were first excised all media, which had remained clear, were subculturerl. The subcultures remained sterile. Gallstones were not noted in any gallbladders of the mice with positive cultures. At six weeks four of eight mice on the lithogenic diet had grossly visible gallstones. (Table I.) Cultures of the gallbladders of mice in groups III and IV, which had been maintained for three to four weeks on the high cholesterol or L-356 diet, remained sterile on incubation in all but one instance. (Tables III and IV.) From one
TABLE RESULTS
Mouse
Number _-____. __~ .i 3
2 :! :i
3
Sex Male
Female Male Female Male
Female
OF GALLBLADDER
CULTURES
FROM
Time on Diet (wk.)
Bacterial Growth
2 2 4
Xone Kane 1
4
1
6 6
None Sone
Y(i!b
Formation
II
MICE
Oh‘
L-356 STANDARDRATION(GROUPII J
Subcultured .___~
Gross Gallstones
_..
Gram-positive rods (diptheroids) Gram-positive rods; short
Nnnc None None Kane sme NOilt!
Frey and Freter
8’70
TABLE III RESULTSOF GALLBLADDERCULTURESFROM MICE ON HIGH CHOLESTEROL DIET (GROUP III) Time on Diet (wk.)
Mouse
Number
Sex
Bacterial Growth None None 1 None None None
Male Female Male Female Male Female
male
mouse
on the lithogenic
gram-positive further male the
diet
cocci were grown
identified.
(Table
III.)
Nine
mice and six of fourteen lithogenic
diet
had
(group
III)
which were not of thirteen
female
gross
mice on
gallstones.
In
none of the six male or nine female
mice on the
L-356
(Table
diet did gallstones
develop.
III.)
Actinomycetes center and
Burnett
formation defined.
[4].
The
Rains
Gallstones occurs
the
acid in
mice
Cholecystitis
diet.
on
the
was
of the
from
tional
and
L-356
ration.
In
this
bladder
study
wall
been in
in mice fed a high
same a
the
we
formation
mice
lithogenic
similar
found
and
conven-
diet. Inflammation on
of conventhe
no
1 2
a lithogenic under
diet.
aerobic
of
in the gall-
of conventional
mice fed
Rich
and
The few organisms
media
anaerobic
were
employed
conditions
[Z].
which grew were considered
to be contaminants
commonly
encountered
in
our laboratory. Our
inability
gallbladder
to culture
wall
or bile
tion
diet makes
in the
mouse.
The
and cultures
obtained
initiation
the
period
of
crystal
in the mouse a role
mice
were
diet.
gallbladder. cultured
them
support
rate
of gallstone
a
bacterial
for crystal
to
aggregation.
and
acid absorption,
have
at this time.
the
hypothesis
formation
cholecystitis
flora of the intestine
this
did play
germ-free
bacterial bile
a
apparent
we would
do not compared
During
If bacteria
aggregation,
results
nidus
that forma-
sacrificed
is often
the higher to
the mice
three to four weeks after
lithogenic
to have
conventional due
from
it unlikely
aggregation
in crystal
Our that
bacteria
of conventional
nidus has any role in gallstone
or
More
in
mice
is
bacterial likely
the
alter cholesterol
thus hastening
gall-
stone formation.
standard
evidence
or any other bacteria or bile
in
diet.
degree and
gallbladders mice
than
1
.
expected
germ-free
germ-free
actinomycetes
in the
not
actinomycetes
conventional
in both
absent
has
Gallstone
tional mice on the lithogenic was
[3]
could develop.
can be induced
sooner
incidence
the
role of bacteria gallstones
believes
bile
germ-free
from et al.
could act as a nidus about which
aggregation
cholesterol
by Rains
8
Gram-positive cocci
bacterial
been cultured
gallstones
of human
the gallbladder crystal
have
of human
None Sane
.
fed a lithogenic
COMMENTS
Gross Gallstones
Subcultured
SUMK4RY Aerobic bladder
and anaerobic
cultures
of the
wall and bile of conventional
lithogenic
diet did not show
gall-
mice on a
growth.
It is un-
TABLE IV RESULTS OF GALLBLADDER CULTURES FROM MICE ON THE STA~VD.~RDL-356 DIET (GROUP IV) Mouse Number
Sex
Time on Diet (wk.)
Bacterial Growth
4 6
Female Male Female
None None None None
1 2
Male Female
None None
1 1
Male
Subcultured
. . . . ..
The American
Gross Gallstones None None None None None None
Journal
of Surgery
Gallstone likely that bacterial cholecystitis nidus ha5 any role in gallstone the mouse.
or a bacterial formation in
REFERENCES 1. FRET, C. F., THORPE, C., and ABRAMS, G. Gallstone
formation in the germ-free mouse. Am. J. Surg., IIf,: 75, 1968. 2. SPEARS, R. W. and FRETER, R. Improved isolation of
Vol. 116, December 1968
Formation
371
anerobic bacteria from the mouse cecum by mail1 taining continuous strict anarrobiosis Pro, .%. Exper. Riol. & &‘ed.. 124: 903, 196i. 3. RAEIS, A. J. H., BARSON, G. J., CRAWFORI). S . and SHREWSBURG, J. F. D. Achievement and bacterologic study of gallstones. The presence of actinomycete. Lancet, 2: 614, 1960. 4. BURNET.~, W. The pathogenesis of gal1stonc.s. In: The Biliary System: A Symposium of the NATO Advance Study Institute, p. 610. I’hilad~elphia, 1963. F. .A. Davis Co.