Effect of microorganisms upper gut of malnourished intestinal sugar absorption Michael Thomas,
Gracey,4 M.D., F.R.A .C.P., Valerie B.Sc., and Del vs E. Stone, B.Sc.
ABSTRACT
The
malnourished organisms
were
contained
Of
grown
for jejunal
arbutin the
on
of
Burke,
in
were
gram-positive
cocci
affect
lactobacillus,
the
also
studied,
intestinal
only
Pseudomonas
inhibitory
while
considered the
gut
sp.
were
also
C. tropicalis
arbutin
enteropathogenic in
may
excessive
numbers Am.J.
malnutrition.
Clin.
not.
Nutr.
(4-7);
however,
little
attention
has
been paid to the possibility that other microorganisms might also interfere with intestinal function. Microbial contamination of the upper gut by a wide variety of microorganisms was documented recently in a group of 20 Indonesian children with malnutrition (8). The present investigation examines the effect of pure cultures of organisms isolated in excessive numbers in these patients on intestinal sugar The American
Journal
of Clinical
Nutrition
28:
AUGUST
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/8/841/4732930 by University of Wyoming Libraries user on 19 June 2018
Candida results
affect
28:
affect sp.,
to
841-845,
the
was rats.
used The
the
sugar
absorption.
function production
did
when
the species
not
present
diarrhea
a
Klebsiella sp.
C. parapsilosis
and
not
studied, All
microorganisms of
used
studied,
Enterobacteriaciae absorption.
basic
transport.
rod
arbutin
that
as the
saprophyticus,
inhibited
of which
substrate
gram-positive
of
cultures fluid
active
C. albicans
suggest
intestinal
contribute
patients
vlococcus
Of
tract
Pure
supernatant
Wistar
only
variety,
the
These
adversely and
Intestinal malabsorption is characteristic of patients with bacterial overgrowth in the small intestine. Several pathogenetic mechanisms may be involved; these include, for example, reduction in the intraluminal levels of conjugated bile salts causing steatorrhea (1), inhibition of carbohydrate absorption by deconjugated bile salts (2) and competition by bacteria for the vitamin B12-gastric intrinsic factor complex causing vitamin B12 malabsorption (3). Recently, the microorganisms Vibrio cholera and Escherichia co/i have been shown to have deleterious effects on the intestinal handling of fluid and electrolytes
Of
Staph The
sp. did not
Proteus
gastrointestinal
of intestinal
absorption.
a nonpathogenic
effective. was
in the
A.
in vivo.
resultant
marker
arbutin.
Jennifer
upper
adult
saprophyte, of
inhibited
Salmonella paratvphi B, a Shigella and of Escherichia coli studied, including and
the
absorption
significantly
found
the
in rats
the
in anesthetized
a recognized
the
studied
and
to those
done
.C.P.,
from
was broth
numbers
which
(p-hydroxphenyl-fl-glucoside),
adversely
absorption
a nutrient
in similar
F.R.A
isolated
sugar
overnight
perfusions
M.B.,
microorganisms
intestinal
microorganisms
solution was
effect
children
isolated from children on in vivo1’ 2, 3
were generally
in the in
lumen
children
of with
1975
transport in rats in vivo. The results suggest that organisms not usually considered pathogenic may have untoward effects on intestinal absorption when present in the upper gut in pathological numbers and that this may be important in malnourished children. Materials
and methods
There were 20 patients in the study: 10 boys and 10 girls with ages ranging from I month to 4 years: all except two patients were less than 2 years of age. Most of the patients were severely malnourished. Further clinical details are given elsewhere (8). Specimens of intestinal contents were obtained in Jakarta by pernasal intubation of the duodenum after a fast of at least 4 hours. These were each kept in 2 ml of a From
the
Gastroenterological
Research
Unit,
Prin-
cess Margaret Children’s Medical Research Foundation, Perth, Western Australia. 2Supported by a grant from The Wellcome Trust, London. 3Address requests for reprints to Dr. Michael Gracey, Gastroenterological Research U nit, Princess Margaret Children’s Medical Research Foundation, Perth, Western Australia. Adolph Basser Research Fellow of the Royal Australasian College of Physicians.
1975,
pp.
84 1-845.
Printed
in U.S.A.
841
842 transport glycerol)
GRACEY medium ( I .8 ml of glucose at - 20 C until transported
broth on dry
and 0.2 ml of ice personally
by air to Perth where the laboratory studies were performed. Specimens were cultured on the following selective media: MacConkey’ agar, Hektoen enteric agar, strontium selenite and strontium chloride enrichment broths, horse blood agar, De Man Rogosa and Sharpe medium, Sabouraud’s dextrose agar and Mitis Salivarius agar. Bacteria and yeasts occurring in numbers greater than lO”/ml in duodenal aspirates, were isolated and grown in pure culture. Stock cultures of these organisms grown overnight in a transport medium containing 10% glycerol were stored at -70 C. Selected bacteria and yeasts were grown in pure culture in ‘aerobic conditions in Krebs-Henseleit (9) bicarbonate buffer with 1% peptone in Erlenmeyer flasks to give a large surface area to volume ratio for optimal growth. For growing Candida sp. the pH of the buffer was lowered to 5.6 then raised to 7.4 immediately before the perfusions. The organisms were incubated at 37 C for 18 hours in a Dubnoff-type shaking water bath at 50 oscillations/mm. At the end of the incubation period specimens of broth were plated onto blood agar for bacteria and Sabouraud’s dextrose agar for yeasts, and subsequently’ examined microscopically to check for contamination. The remainder of the broth was centrifuged at 3,000 rpm for 30 mm and the supernatant used as the basic solution for the perfusion experiments mentioned below. This fluid was diluted in 0.2 ml aliquots in 1.8 ml oftransport medium and serial tenfold dilutions from 10’ to l0 made in nutrient glucose broth. Known volumes of these dilutions were cultured on blood agar and Sabouraud’s dextrose agar for 24 hours and subsequent growth quantitated (see Table I). In the control experiments, all the procedures mentioned above were done except the addition of microorganisms to the fluid prior to overnight incubation: the control perfusate was, therefore, simply a broth culture in which microorganisms had not been grown. The perfusate was based on the broth supernatant adjusted to pH 7.4 to which was added 2 mg/mI of polyethylene glycol 4,000 (British Drug Houses) as a nonabsorbable marker of changes in fluid volume. The substrate studied was arbutin (p-hydroxyphenyl--gIucoside), an analogue of D-glucose which shares with it the intestinal active sugar transport pathway (10) and has the advantage over glucose of not being metabolized by the small-intestinal epithelium. This substrate was added to the perfusate at a concentration of 10 m. Incubation experiments were done which indicated that the microorganisms studied did not significantly metabolize this substrate over the time involved in these experiments. The experiments were done in adult Wistar rats weighing 150 300 g and obtained from a long-established colony in the University of Western Australia. The animals were anesthetized with open ether and the abdomen opened by a midline incision. A 20-cm segment of jejunum was selected and entry and exit cannulas (external diameter 3.0 cm, internal diameter 2.0 cm) introduced into the lumen of the gut through transverse incisions in its wall and fixed by means of black silk ligatures. The perfusate was delivered by a constant-rate perfusion pump (Paton Industries, Adelaide, South Australia) at 2 mI/hour in a peristaltic direction. The
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/8/841/4732930 by University of Wyoming Libraries user on 19 June 2018
ET
AL.
perfused segment was returned to the abdominal cavity and the perfusate collected continuously from the draining exit cannula. The first 30 mm of the experiment were used for equilibration conditions to be achieved and the draining fluid discarded: perfusate collected over the subsequent 60 mm was used for the assays. Arbutin was assayed as free phenol (II, 12) and polyethylene glycol by a micromodification of Hyd#{233}n’s turbidometric method (13). The rate of absorption of the substrate in vivo is expressed as imoles per centimeter per hour as measured by its disappearance from the lumen after appropriate adjustments for changes in volume indicated by concentrations of polyethylene glycol
before and after the experiments. Standard mathematical methods were used to calculate means and standard deviations (SD). Levels of statistical significance were obtained by Student’s t-test: P values of < 0.05 are taken to be significant.
Results The
details
of the
results
are given
in Table
2. Of the gram-positive cocci examined, only the known saprophyte Staphylococcus saprophyticus did not adversely affect the intestinal absorption of arbutin. The only gram-positive rod studied, a lactobacillus, significantly inhibited absorption of the substrate. Seven species of Enterobacteriaciae were studied. Salmonella paratyphi B, a Shigella and Proteus did not affect arbutin absorption. All the species of E. co/i studied, including a species classified by traditional means as “nonpathogenic,” impaired absorption, as did Kiebsiella. The gram-negative rod PseuTABLE Microbial
I populations Oganism
Staph. saprophvticus Staph. pyogenes Strep. viridans Strep.faecalis Lactobacillus Nonpathogenic E. coli E.coli 055 E.coliOlll Salm.paratvphiB Shigella Proteus Klebsiella Pseudomonas C.albicans
C. tropicalis C.parapsilosis
of broth
supernatants Range
Mean 7.8 4.8 6.1 6.8 4.3 6.6 7.1 6.7 8.3 6.9 7.8 9.2 8.3 4.6 3.3 5.7
1.0 6.0 3.0 1.8 1.3 1.9 I.l 6.4x 7.5 5.2 lOx 8.7 4.4 7.9x 7.8 4.8
x I0”-2.5 x
x x x x x
x x x
x x
l0-2.5 l0”-2.5 l0’8.6 I0-3.3 1066.0 l0-8.3 103-2.6x I0-4.8 l0’-I.2 I0-2.5x l0-6.6 10-3.2 l03-2.9x 102_5.3
x I0”-3.3
Results expressed as the log10 of colony counts and range of four cultures specimens in each instance.
x 10’ x
106
x l0 x l0 x 10’ x 106 x l0 108
x
108
x 108 10’ x l010
x
108
10’ x 10’ x 106
the mean viable per milliliter of
EFFECT TABLE 2 Effect of enteric microorganisms of arbutin in vivo Organism Controls
Gram-positive cocci Staph. saprophvticus Siaph.pvogenes(12) Strep. viridans(12) Strep.faecalis(7) rod (11)
Enterobacteriaciae Nonpathogenic E. coli (II) E. coli 055 (9) E.coliOlll(9) E.coliOl42(7) SaIm. paratv phi Shigella(7) Proteus (8) Klebsiella(8)
on intestinal
Absorption”
(8)
Gram-positive Lactobacillus
OF
B (20)
Gram-negative rod Pseudomonas(I0) Candida sp. C. albicans(7) C.tropicalis(20) C.parapsilosis(8) “Absorption expressed meter intestine per hour. standard deviations. The the numbers of experiments.
(8)
MICROORGANISMS
absorption
P
0.46
±
0.13
0.46 0.36 0.30 0.32
±
±
0.11 0.13 0.14 0.06
ns
Gracey,4 M.D., F.R.A .C.P., Valerie B.Sc., and Del vs E. Stone, B.Sc.
ABSTRACT
The
malnourished organisms
were
contained
Of
grown
for jejunal
arbutin the
on
of
Burke,
in
were
gram-positive
cocci
affect
lactobacillus,
the
also
studied,
intestinal
only
Pseudomonas
inhibitory
while
considered the
gut
sp.
were
also
C. tropicalis
arbutin
enteropathogenic in
may
excessive
numbers Am.J.
malnutrition.
Clin.
not.
Nutr.
(4-7);
however,
little
attention
has
been paid to the possibility that other microorganisms might also interfere with intestinal function. Microbial contamination of the upper gut by a wide variety of microorganisms was documented recently in a group of 20 Indonesian children with malnutrition (8). The present investigation examines the effect of pure cultures of organisms isolated in excessive numbers in these patients on intestinal sugar The American
Journal
of Clinical
Nutrition
28:
AUGUST
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/8/841/4732930 by University of Wyoming Libraries user on 19 June 2018
Candida results
affect
28:
affect sp.,
to
841-845,
the
was rats.
used The
the
sugar
absorption.
function production
did
when
the species
not
present
diarrhea
a
Klebsiella sp.
C. parapsilosis
and
not
studied, All
microorganisms of
used
studied,
Enterobacteriaciae absorption.
basic
transport.
rod
arbutin
that
as the
saprophyticus,
inhibited
of which
substrate
gram-positive
of
cultures fluid
active
C. albicans
suggest
intestinal
contribute
patients
vlococcus
Of
tract
Pure
supernatant
Wistar
only
variety,
the
These
adversely and
Intestinal malabsorption is characteristic of patients with bacterial overgrowth in the small intestine. Several pathogenetic mechanisms may be involved; these include, for example, reduction in the intraluminal levels of conjugated bile salts causing steatorrhea (1), inhibition of carbohydrate absorption by deconjugated bile salts (2) and competition by bacteria for the vitamin B12-gastric intrinsic factor complex causing vitamin B12 malabsorption (3). Recently, the microorganisms Vibrio cholera and Escherichia co/i have been shown to have deleterious effects on the intestinal handling of fluid and electrolytes
Of
Staph The
sp. did not
Proteus
gastrointestinal
of intestinal
absorption.
a nonpathogenic
effective. was
in the
A.
in vivo.
resultant
marker
arbutin.
Jennifer
upper
adult
saprophyte, of
inhibited
Salmonella paratvphi B, a Shigella and of Escherichia coli studied, including and
the
absorption
significantly
found
the
in rats
the
in anesthetized
a recognized
the
studied
and
to those
done
.C.P.,
from
was broth
numbers
which
(p-hydroxphenyl-fl-glucoside),
adversely
absorption
a nutrient
in similar
F.R.A
isolated
sugar
overnight
perfusions
M.B.,
microorganisms
intestinal
microorganisms
solution was
effect
children
isolated from children on in vivo1’ 2, 3
were generally
in the in
lumen
children
of with
1975
transport in rats in vivo. The results suggest that organisms not usually considered pathogenic may have untoward effects on intestinal absorption when present in the upper gut in pathological numbers and that this may be important in malnourished children. Materials
and methods
There were 20 patients in the study: 10 boys and 10 girls with ages ranging from I month to 4 years: all except two patients were less than 2 years of age. Most of the patients were severely malnourished. Further clinical details are given elsewhere (8). Specimens of intestinal contents were obtained in Jakarta by pernasal intubation of the duodenum after a fast of at least 4 hours. These were each kept in 2 ml of a From
the
Gastroenterological
Research
Unit,
Prin-
cess Margaret Children’s Medical Research Foundation, Perth, Western Australia. 2Supported by a grant from The Wellcome Trust, London. 3Address requests for reprints to Dr. Michael Gracey, Gastroenterological Research U nit, Princess Margaret Children’s Medical Research Foundation, Perth, Western Australia. Adolph Basser Research Fellow of the Royal Australasian College of Physicians.
1975,
pp.
84 1-845.
Printed
in U.S.A.
841
842 transport glycerol)
GRACEY medium ( I .8 ml of glucose at - 20 C until transported
broth on dry
and 0.2 ml of ice personally
by air to Perth where the laboratory studies were performed. Specimens were cultured on the following selective media: MacConkey’ agar, Hektoen enteric agar, strontium selenite and strontium chloride enrichment broths, horse blood agar, De Man Rogosa and Sharpe medium, Sabouraud’s dextrose agar and Mitis Salivarius agar. Bacteria and yeasts occurring in numbers greater than lO”/ml in duodenal aspirates, were isolated and grown in pure culture. Stock cultures of these organisms grown overnight in a transport medium containing 10% glycerol were stored at -70 C. Selected bacteria and yeasts were grown in pure culture in ‘aerobic conditions in Krebs-Henseleit (9) bicarbonate buffer with 1% peptone in Erlenmeyer flasks to give a large surface area to volume ratio for optimal growth. For growing Candida sp. the pH of the buffer was lowered to 5.6 then raised to 7.4 immediately before the perfusions. The organisms were incubated at 37 C for 18 hours in a Dubnoff-type shaking water bath at 50 oscillations/mm. At the end of the incubation period specimens of broth were plated onto blood agar for bacteria and Sabouraud’s dextrose agar for yeasts, and subsequently’ examined microscopically to check for contamination. The remainder of the broth was centrifuged at 3,000 rpm for 30 mm and the supernatant used as the basic solution for the perfusion experiments mentioned below. This fluid was diluted in 0.2 ml aliquots in 1.8 ml oftransport medium and serial tenfold dilutions from 10’ to l0 made in nutrient glucose broth. Known volumes of these dilutions were cultured on blood agar and Sabouraud’s dextrose agar for 24 hours and subsequent growth quantitated (see Table I). In the control experiments, all the procedures mentioned above were done except the addition of microorganisms to the fluid prior to overnight incubation: the control perfusate was, therefore, simply a broth culture in which microorganisms had not been grown. The perfusate was based on the broth supernatant adjusted to pH 7.4 to which was added 2 mg/mI of polyethylene glycol 4,000 (British Drug Houses) as a nonabsorbable marker of changes in fluid volume. The substrate studied was arbutin (p-hydroxyphenyl--gIucoside), an analogue of D-glucose which shares with it the intestinal active sugar transport pathway (10) and has the advantage over glucose of not being metabolized by the small-intestinal epithelium. This substrate was added to the perfusate at a concentration of 10 m. Incubation experiments were done which indicated that the microorganisms studied did not significantly metabolize this substrate over the time involved in these experiments. The experiments were done in adult Wistar rats weighing 150 300 g and obtained from a long-established colony in the University of Western Australia. The animals were anesthetized with open ether and the abdomen opened by a midline incision. A 20-cm segment of jejunum was selected and entry and exit cannulas (external diameter 3.0 cm, internal diameter 2.0 cm) introduced into the lumen of the gut through transverse incisions in its wall and fixed by means of black silk ligatures. The perfusate was delivered by a constant-rate perfusion pump (Paton Industries, Adelaide, South Australia) at 2 mI/hour in a peristaltic direction. The
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/8/841/4732930 by University of Wyoming Libraries user on 19 June 2018
ET
AL.
perfused segment was returned to the abdominal cavity and the perfusate collected continuously from the draining exit cannula. The first 30 mm of the experiment were used for equilibration conditions to be achieved and the draining fluid discarded: perfusate collected over the subsequent 60 mm was used for the assays. Arbutin was assayed as free phenol (II, 12) and polyethylene glycol by a micromodification of Hyd#{233}n’s turbidometric method (13). The rate of absorption of the substrate in vivo is expressed as imoles per centimeter per hour as measured by its disappearance from the lumen after appropriate adjustments for changes in volume indicated by concentrations of polyethylene glycol
before and after the experiments. Standard mathematical methods were used to calculate means and standard deviations (SD). Levels of statistical significance were obtained by Student’s t-test: P values of < 0.05 are taken to be significant.
Results The
details
of the
results
are given
in Table
2. Of the gram-positive cocci examined, only the known saprophyte Staphylococcus saprophyticus did not adversely affect the intestinal absorption of arbutin. The only gram-positive rod studied, a lactobacillus, significantly inhibited absorption of the substrate. Seven species of Enterobacteriaciae were studied. Salmonella paratyphi B, a Shigella and Proteus did not affect arbutin absorption. All the species of E. co/i studied, including a species classified by traditional means as “nonpathogenic,” impaired absorption, as did Kiebsiella. The gram-negative rod PseuTABLE Microbial
I populations Oganism
Staph. saprophvticus Staph. pyogenes Strep. viridans Strep.faecalis Lactobacillus Nonpathogenic E. coli E.coli 055 E.coliOlll Salm.paratvphiB Shigella Proteus Klebsiella Pseudomonas C.albicans
C. tropicalis C.parapsilosis
of broth
supernatants Range
Mean 7.8 4.8 6.1 6.8 4.3 6.6 7.1 6.7 8.3 6.9 7.8 9.2 8.3 4.6 3.3 5.7
1.0 6.0 3.0 1.8 1.3 1.9 I.l 6.4x 7.5 5.2 lOx 8.7 4.4 7.9x 7.8 4.8
x I0”-2.5 x
x x x x x
x x x
x x
l0-2.5 l0”-2.5 l0’8.6 I0-3.3 1066.0 l0-8.3 103-2.6x I0-4.8 l0’-I.2 I0-2.5x l0-6.6 10-3.2 l03-2.9x 102_5.3
x I0”-3.3
Results expressed as the log10 of colony counts and range of four cultures specimens in each instance.
x 10’ x
106
x l0 x l0 x 10’ x 106 x l0 108
x
108
x 108 10’ x l010
x
108
10’ x 10’ x 106
the mean viable per milliliter of
EFFECT TABLE 2 Effect of enteric microorganisms of arbutin in vivo Organism Controls
Gram-positive cocci Staph. saprophvticus Siaph.pvogenes(12) Strep. viridans(12) Strep.faecalis(7) rod (11)
Enterobacteriaciae Nonpathogenic E. coli (II) E. coli 055 (9) E.coliOlll(9) E.coliOl42(7) SaIm. paratv phi Shigella(7) Proteus (8) Klebsiella(8)
on intestinal
Absorption”
(8)
Gram-positive Lactobacillus
OF
B (20)
Gram-negative rod Pseudomonas(I0) Candida sp. C. albicans(7) C.tropicalis(20) C.parapsilosis(8) “Absorption expressed meter intestine per hour. standard deviations. The the numbers of experiments.
(8)
MICROORGANISMS
absorption
P
0.46
±
0.13
0.46 0.36 0.30 0.32
±
±
0.11 0.13 0.14 0.06
ns
