Bacterial
ecology
R. E. Hungaze,3
in the small
Ph.D.
ABSTRACT
A modified
intestinal Use
contents
of the
bacteria
in
with
method the
simulating
conditions.
Am.
intubation
intestine I
Journal
of Clinical
Nutrition
method
contamination
has disclosed
small
conditions
no
from
strains of
allowed other
subjects.
the
natural
habitat
suggest
C/in.
Nutr.
31: S 125-S
127,
31: OCTOBER
collection
intestinal
resembling
some
Much current interest centers on microbial activities in the human large intestine and their possible role in the etiology of cancer and other colonic disorders. The microbial ecology of the small intestine may seem unrelated to colonic disease, but digesta from the ileum are a source for part of the colonic microflora, and bacterial growth in the jejunum and ileum may be pertinent to events in the large bowel, particularly if the ileal flora is of considerable magnitude. We studied the human small intestine not only because such studies are needed as the ultimate check on the applicability of information and ideas derived from other experimental animals, but also because this habitat is in itself intriguing and of sufficient intrinsic interest and importance to merit a thorough study of its ecology. The small intestine is the primary site for secretion and action of host enzymes and bile and for absorption of digestion products. Do these processes occur under the stress of competition by bacteria, or is host nutritional success independent of microorganisms? Is the small intestinal flora a factor in the conversions of bile? Can fiber in the diet exert an effect also on events in the small intestine or is its importance limited to the colon? Another question of more general but related nature is, does the small intestine have its own characteristic (autochthonous) flora or are its denizens chance entrants from the stomach or large intestine? An analysis of the microbial ecosystem should provide answers to some of these questions. Conditions in the small intestine differ markedly from those in the colon. Secretion
The American
intestine1’2
of samples
sites
Haemophihus Stimulation that
it
with
as among by
is
and
well
bile
and
adapted
of
human
no exposure the
most
rapid to
small to air.
abundant
growth upper
under intestinal
1978.
and action of host enzymes in the jejunum produce soluble carbohydrates and split protein products readily absorbed by host epitheial cells but not absorbed rapidly enough to prevent accumulation in appreciable concentrations in the lumen of the gut. These substrates are ideal for the growth of many bacteria, and without some mechanism for keeping bacterial numbers down they could seriously compete with the host for the products of digestion. Acidity in the stomach greatly reduces the concentration of ba#{224}teria in digesta. A 10-mm exposure of saliva to an aqueous solution of HC1 at pH 2 kills all the bacteria except a few sporeformers. Thus under some conditions digesta enter the small intestine practically free of accompanying viable bacteria. The problem is to trace their development between stomach and colon. To accomplish this we have attempted to develop a valid sampling procedure for “normal” humans that would require a minimum of transit time between habitat and experimental treatment, that would exclude air during collection and handling, and that would avoid any contamination from other alimentary sites. Intubation was selected as most practical and likely to fulfill these criteria, and the following procedure was developed. The sampling apparatus consisted of a 244and a 366-cm length of polyvinyl chloride
‘From the Department of Bacteriology, California, Davis, California 95616. 2 Dr. Joan Macy, Ida Yu, and Casey contributed port. Professor
1978,
Downloaded from https://academic.oup.com/ajcn/article-abstract/31/10/S125/4656078 by St Bartholomew's & the Royal London School of Medicine and Denistry user on 21 August 2018
the
pp. S 125-S
experimental Emeritus
127.
work
described
University CaIdwell in this
of have re-
of Bacteriology.
Printed
in U.S.A.
S125
SI 26
HUNGATE
(Tygon) tubing, each attached proximally to a 16-gauge needle fitted to a BD syringe stopcock, in turn fitted to a l-mh or other appropriately sized syringe. By closing the stopcock, syringes (with dead space occupied by anaerobic sterile water) could be exchanged without extrance of air or contamination. Each tube was filled with anaerobic sterile water except the distal end into which 0.2 ml of melted sterile 4% agar was drawn and allowed to solidify. At the distal end of the longer tube a plastic cylindrical plug (8 x 1 .6 mm diameter) was inserted, pushing the agar up the tube a distance of 4 mm. A lead bag was tied with nylon dental floss to the exposed end of the plug. The bag consisted of the little finger from a size 6 surgical glove, filled with 7 g of iron powder plus 2 ml of glycerol, and tied off with the floss, leaving a 6- to 8-mm length of floss between plug and bag. The shorter tube was placed alongside the proximal portion of the long tube and tied to it with nylon floss at 30-cm intervals. Its distal end contained the solidified agar but no plastic plug or other attachment except the floss binding it near its tip to the other tube. The double tube permitted simultaneous sampling from two sites 122 cm apart in the alimentary tract. The lead bag was swallowed and the subject lay on the right side for an hour or two to assist entrance of the bag into the duodenum, then resumed activities as normal as possible. Food was taken at regular meal times, and the distance of penetration into the gut estimated from the length of tubing drawn in by peristaltic action on the lead bag. After the desired sampling site was reached the stopcocks were opened, and the 1 ml of water in the syringe was forcibly injected to detach the plastic and agar closures from the distal ends of the tubes. A 10-mm period was allowed to permit the ejected agar and water to be displaced from the open end of the tubes before samples were taken. To collect the sample, a 5-mi syringe was substituted and its plunger slowly withdrawn to suck in the intestinal contents. Very gentle force was used to prevent solid digesta from blocking the small opening into the tube. After the samples had been collected, the stopcocks were closed and the tubes withdrawn from the subject. The distal ends of the sampling
Downloaded from https://academic.oup.com/ajcn/article-abstract/31/10/S125/4656078 by St Bartholomew's & the Royal London School of Medicine and Denistry user on 21 August 2018
tubes were wiped dry, the exteriors were sterilized with 70% ethanol, and the distal 10 cm was cut off aseptically to eliminate any contamination acquired as the tube was withdrawn. The yellowish color of the intestinal contents disclosed the juncture in the tube between water and digesta. The collected sample often included some gas bubbles. In most cases the sample volume was less than that of the tube lumen (3.2 ml for the shorter tube). Most of it was ejected into a butyl-stoppered tube (75 x 10 mm) gassed with 80% N2-20% CO2, but the proximal 10cm length of the sample was placed in a separate tube for immediate pH determination. The main sample was split into two portions. One was frozen for later analysis of sugars or for use as a medium supplement; the other was incubated anaerobically at 38 C and sampled for culture counts and sugar determinations after 0, 2, and 4 hr of incubation to estimate the growth rate under conditions simulating those in the small intestine. The counts were made at 38 C on Casman medium both aerobically and anaerobically under N2-CO2. The sample was also plated on EMB agar to detect cohiforms. In initial experiments involving single samphing sites, digesta were taken from the stomach, duodenum, jejunum, and esophagus. No colonies developed from 0. 1 ml of inoculum from duodenal digesta from two subjects or from a single stomach sample. Ajejunal sampie for subject 1 showed 106 aerobes and
ecology
R. E. Hungaze,3
in the small
Ph.D.
ABSTRACT
A modified
intestinal Use
contents
of the
bacteria
in
with
method the
simulating
conditions.
Am.
intubation
intestine I
Journal
of Clinical
Nutrition
method
contamination
has disclosed
small
conditions
no
from
strains of
allowed other
subjects.
the
natural
habitat
suggest
C/in.
Nutr.
31: S 125-S
127,
31: OCTOBER
collection
intestinal
resembling
some
Much current interest centers on microbial activities in the human large intestine and their possible role in the etiology of cancer and other colonic disorders. The microbial ecology of the small intestine may seem unrelated to colonic disease, but digesta from the ileum are a source for part of the colonic microflora, and bacterial growth in the jejunum and ileum may be pertinent to events in the large bowel, particularly if the ileal flora is of considerable magnitude. We studied the human small intestine not only because such studies are needed as the ultimate check on the applicability of information and ideas derived from other experimental animals, but also because this habitat is in itself intriguing and of sufficient intrinsic interest and importance to merit a thorough study of its ecology. The small intestine is the primary site for secretion and action of host enzymes and bile and for absorption of digestion products. Do these processes occur under the stress of competition by bacteria, or is host nutritional success independent of microorganisms? Is the small intestinal flora a factor in the conversions of bile? Can fiber in the diet exert an effect also on events in the small intestine or is its importance limited to the colon? Another question of more general but related nature is, does the small intestine have its own characteristic (autochthonous) flora or are its denizens chance entrants from the stomach or large intestine? An analysis of the microbial ecosystem should provide answers to some of these questions. Conditions in the small intestine differ markedly from those in the colon. Secretion
The American
intestine1’2
of samples
sites
Haemophihus Stimulation that
it
with
as among by
is
and
well
bile
and
adapted
of
human
no exposure the
most
rapid to
small to air.
abundant
growth upper
under intestinal
1978.
and action of host enzymes in the jejunum produce soluble carbohydrates and split protein products readily absorbed by host epitheial cells but not absorbed rapidly enough to prevent accumulation in appreciable concentrations in the lumen of the gut. These substrates are ideal for the growth of many bacteria, and without some mechanism for keeping bacterial numbers down they could seriously compete with the host for the products of digestion. Acidity in the stomach greatly reduces the concentration of ba#{224}teria in digesta. A 10-mm exposure of saliva to an aqueous solution of HC1 at pH 2 kills all the bacteria except a few sporeformers. Thus under some conditions digesta enter the small intestine practically free of accompanying viable bacteria. The problem is to trace their development between stomach and colon. To accomplish this we have attempted to develop a valid sampling procedure for “normal” humans that would require a minimum of transit time between habitat and experimental treatment, that would exclude air during collection and handling, and that would avoid any contamination from other alimentary sites. Intubation was selected as most practical and likely to fulfill these criteria, and the following procedure was developed. The sampling apparatus consisted of a 244and a 366-cm length of polyvinyl chloride
‘From the Department of Bacteriology, California, Davis, California 95616. 2 Dr. Joan Macy, Ida Yu, and Casey contributed port. Professor
1978,
Downloaded from https://academic.oup.com/ajcn/article-abstract/31/10/S125/4656078 by St Bartholomew's & the Royal London School of Medicine and Denistry user on 21 August 2018
the
pp. S 125-S
experimental Emeritus
127.
work
described
University CaIdwell in this
of have re-
of Bacteriology.
Printed
in U.S.A.
S125
SI 26
HUNGATE
(Tygon) tubing, each attached proximally to a 16-gauge needle fitted to a BD syringe stopcock, in turn fitted to a l-mh or other appropriately sized syringe. By closing the stopcock, syringes (with dead space occupied by anaerobic sterile water) could be exchanged without extrance of air or contamination. Each tube was filled with anaerobic sterile water except the distal end into which 0.2 ml of melted sterile 4% agar was drawn and allowed to solidify. At the distal end of the longer tube a plastic cylindrical plug (8 x 1 .6 mm diameter) was inserted, pushing the agar up the tube a distance of 4 mm. A lead bag was tied with nylon dental floss to the exposed end of the plug. The bag consisted of the little finger from a size 6 surgical glove, filled with 7 g of iron powder plus 2 ml of glycerol, and tied off with the floss, leaving a 6- to 8-mm length of floss between plug and bag. The shorter tube was placed alongside the proximal portion of the long tube and tied to it with nylon floss at 30-cm intervals. Its distal end contained the solidified agar but no plastic plug or other attachment except the floss binding it near its tip to the other tube. The double tube permitted simultaneous sampling from two sites 122 cm apart in the alimentary tract. The lead bag was swallowed and the subject lay on the right side for an hour or two to assist entrance of the bag into the duodenum, then resumed activities as normal as possible. Food was taken at regular meal times, and the distance of penetration into the gut estimated from the length of tubing drawn in by peristaltic action on the lead bag. After the desired sampling site was reached the stopcocks were opened, and the 1 ml of water in the syringe was forcibly injected to detach the plastic and agar closures from the distal ends of the tubes. A 10-mm period was allowed to permit the ejected agar and water to be displaced from the open end of the tubes before samples were taken. To collect the sample, a 5-mi syringe was substituted and its plunger slowly withdrawn to suck in the intestinal contents. Very gentle force was used to prevent solid digesta from blocking the small opening into the tube. After the samples had been collected, the stopcocks were closed and the tubes withdrawn from the subject. The distal ends of the sampling
Downloaded from https://academic.oup.com/ajcn/article-abstract/31/10/S125/4656078 by St Bartholomew's & the Royal London School of Medicine and Denistry user on 21 August 2018
tubes were wiped dry, the exteriors were sterilized with 70% ethanol, and the distal 10 cm was cut off aseptically to eliminate any contamination acquired as the tube was withdrawn. The yellowish color of the intestinal contents disclosed the juncture in the tube between water and digesta. The collected sample often included some gas bubbles. In most cases the sample volume was less than that of the tube lumen (3.2 ml for the shorter tube). Most of it was ejected into a butyl-stoppered tube (75 x 10 mm) gassed with 80% N2-20% CO2, but the proximal 10cm length of the sample was placed in a separate tube for immediate pH determination. The main sample was split into two portions. One was frozen for later analysis of sugars or for use as a medium supplement; the other was incubated anaerobically at 38 C and sampled for culture counts and sugar determinations after 0, 2, and 4 hr of incubation to estimate the growth rate under conditions simulating those in the small intestine. The counts were made at 38 C on Casman medium both aerobically and anaerobically under N2-CO2. The sample was also plated on EMB agar to detect cohiforms. In initial experiments involving single samphing sites, digesta were taken from the stomach, duodenum, jejunum, and esophagus. No colonies developed from 0. 1 ml of inoculum from duodenal digesta from two subjects or from a single stomach sample. Ajejunal sampie for subject 1 showed 106 aerobes and
