mt ANAEROBIC BACTERIA m m:
LARGE mrssrms or MKCE

Dissertation £0: the Degree of M. S.

MICHGAN STATE UNWERSETY.
MARTHA A. HARRIS
1 9 73

.....

Michigan State
‘[.It1i\r<:1:ii
”L ‘7

~_-._:_
w—w -‘

     

  

‘5' BINBING BY
nuns & SUNS' ;
WHEfi'EQEE‘LJ'iE

2 .‘.- . MIPIIBII

 

ABSTRACT

THE ANAEROBIC BACTERIA IN THE
LARGE INTESTINE OF MICE

BY

Martha A. Harris

The anaerobes in the large intestine of laboratory mice
were cultured by the roll-tube method of Hungate (21). From
a direct microscopic count of a mouse intestinal contents,

10

4.4 x 10 organisms per gram of intestinal contents were

found. An average of the actual cultural counts revealed

3.3 x 1010

organisms per gram of contents. In the present
investigation, specific genera, and where possible individual
species, are characterized.

The genera isolated were Bacteroides, Eubacterium,
Fusobacterium, Lactobacillus, and Peptostreptococcus. The
species isolated included Bacteroides fragilis ss. thetaiotao-
micron, Bacteroides furcosus, Bacteroides pneumosintes,
Bacteroidee rumincola ss. brevis, Eubacterium aerofaciens,

Eubacterium contortum, Eubacterium tenue, Lactobacillus

fermentum, and Peptostreptococcus intermedius.

 

 

A-w

THE ANAEROBIC BACTERIA IN THE
LARGE INTESTINE OF MICE

BY

Martha A. Harris

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Microbiology and Public Health

1973

JQ" ACKNOWLEDGEMENTS

I would like to express sincere gratitude to Dr. Gordon
R. Carter for his guidance in developing this study and for
his advice and encouragement during the study. I am also
grateful to Dr. C. A. Reddy for his willingness to assist in
the development of this investigation and for his suggestions
and advice during the study. Excellent photographs of the
results obtained in this investigation were taken by Mr.
Harold McAllister, to whom I am very thankful. Sincere
gratitude is expressed to the members of the staff of the
Clinical Microbiology Laboratory, Mrs. Dorothy Boettger,
Mr. Harold McAllister, Mr. A. Wayne Roberts, and Mrs. Mary
Grace Shue, for their assistance, suggestions, and help in

technical operations and securing_supplies.

ii

TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . . . .
REVIEW OF THE LITERATURE
Ecology of the Gastrointestinal Tract

General Gastrointestinal Microflora.

The Bacterial Population of the
Small Intestine. .

Predominant Microorganisms of the
Large Intestine.

Induced Changes in the Gastrointestinal
Microflora.

The Effects of Various Diets on
Intestinal Flora . .

The Changes of Microflora by Anti-
bacterial Agents . . . . . .

Possible Functions of Cecal Flora and Products.

The Probable Role of the Fusiform
Bacilli. . . .

The Fatty Acids Produced by
Anaerobic Bacteria . .

Role of Anaerobes in Infections

The Habitats and Infections of
Anaerobes. . .
Pathogenicity of Anaerobes

MATERIAL AND METHODS .

Animals . . . . . . . . .
Cultural Methods. . . . . . . .
Culture Media . .

Isolation Procedures.

Cultural Methodology.
Identification.

iii

Page

O‘Oh-fi-fi

11
12

12
12
13

RESULTS.

Direct Smears of Mouse Intestinal
Cultural Counts

Microscopic Count

Genera of Intestinal Anaerobes.
Bacteroides

Eubacterium .

Fusobacterium

Lactobacillus

Peptostreptococcus.

DISCUSSION .
SUMMARY.
BIBLIOGRAPHY

iv

Flora

Table

LIST OF TABLES
Page

Enumeration of the cultural counts of
anaerobic bacteria in the intestinal
contents of mice. . . . . . . . . . . . . . . . 24

Morphological and staining characteristics
of the anaerobic bacteria isolated from the
large intestine of mice . . . . . . . . . . . . 25

Colonial morphology and acid end products

of peptone-yeast extract-glucose (PYG) broth

of the anaerobic bacteria in the large

intestine of mice . . . . . . . . . . . . . . . 26

Acid end products produced in PYG broth by
certain anaerobes isolated from the mouse
intestine . . . . . . . . . . . . . . . . . . . 27

Other biochemical tests . . . . . . . . . . . . 29

Figure

001430!

10
ll
12
13
14
15
16
17
18
19
20

LIST OF FIGURES

Ether extract of Bacteroides fragilis ss.
thetaiotaomicron. . ... . . . . . . . . .

Methyl extract of Bacteroides fragilis ss.
thetaiotaomicron. . . . . . . . . . . .

Ether extract of Baoteroides furcosus
Methyl extract of Bacteroides furcosus.
Ether extract of Bacteroides pneumosintes
Methyl extract of Bacteroides pneumosintes.

Ether extract of Bacteroides ruminicola ss.
brevis. . . . . . . . . . . . . .

Methyl extract of Bacteroides ruminicola ss.

brevis. . . . . . . . . . . . . .

Ether extract of Bacteroides sp.1

Methyl extract of Bacteroides sp.1.

Ether extract of Bacteroides sp.2

Methyl extract of Bacteroides sp.z.

Ether extract of Eubacterium aerofaciens.
Methyl extract of Eubacterium aerofaciens
Ether extract of Eubacterium contortum.
Methyl extract of Eubacterium contortum
Ether extract of Eubacterium tenue.
Methyl extract of Eubacterium tenue . . .
Ether extract of Fusobacterium 8p.

Methyl extract of Fueobacterium sp.

vi

Page

31

31
32
32
33
33

34

34
35
35
36
36
37
37
38
38
39
39
40
40

Figure Page
21 Ether extract of Lactobacillus fermentum. . . . 41
22 Methyl extract of Lactobacillus fermentum . . . 41

23 Ether extract of Peptostreptococcus
intermedius ‘ O O O ' O O O O ' 0 O O O O 0 0 O O 0 O 42

24 Methyl extract of Peptostreptococcus
intermedius . . . . . . . . . . . . . . . . . . 42

vii

INTRODUCTION

Although the earth is aerobic, anaerobic conditions
prevail in a variety of ecological environments including
the gastrointestinal tract of mammals (7,43). Thus anaerobic
bacteria can proliferate in these ecosystems.

Presently, there are four factors that are related
directly and/or indirectly to anaerobiosis. Most obligate
anaerobic bacteria lack the enzyme catalase, which decomposes
hydrogen peroxide into water and oxygen (9,43). Secondly,
certain organic peroxides are reported to be formed, on
exposure to air, in laboratory media that are used for the
cultivation of anaerobes (6,43). These organic peroxides
inhibit the growth of anaerobes. Thirdly, the growth of
oxygen sensitive bacteria is primarily a function of the
oxidation-reduction potential, the measure of the capacity
of a system to accept or donate electrons (31,43). The lower
the oxidation-reduction potential, the greater the ease with
which the anaerobes grow. Finally, recent evidence suggests
that the enzyme, superoxide dismutase, is extremely important
for the survival of microorganisms in the presence of air.
Superoxide dismutase is involved in the dismutation of the

superoxide radical:

+2H+----O +HO

2 2 2 2

2
The superoxide radical, 02-, is a very undesirable physio-
logical one and inhibits a number of biological reactions.
All aerobes and aerotolerant anaerobes are known to possess
this enzyme, while all obligate anaerobes appear to lack
this enzyme.

Spears and Freter (4S) conducted a study of the anaerobic
flora in the ceca of mice to compare the efficiency of the
agar plate-anaerobic jar procedure and the roll-tube method
of Hungate. The roll-tube method of Hungate recovered more
anaerobic bacteria than the agar plate-anaerobic jar procedure.
Gram reaction, morphology, motility, and specific biochemical
tests were used to classify the isolates. The specific genera
recovered by the agar plate-anaerobic jar procedure and the
roll-tube method of Hungate were not identified.

Aranki et al. (2) isolated anaerobes from the ceca of
mice by the anaerobic chamber method. They reported Gram-
positive bacilli, Gram-negative cocco-bacilli and tapered-
end bacilli.

As a result of numerous studies, the oxygen sensitive
bacteria of the gastrointestinal tract of mice have been
classified into genera. Pre-reduced anaerobically sterilized
media were used for specific biochemical tests by some
investigators; however, the specific differential biochemical
tests were not discussed. Using the anaerobic chamber for
primary isolation, investigators identified the genera,
Bacteroides, CZostridium, Eubacterium, Fusobacterium, and

Lactobacillus (18,22,24,36). These genera were not

speciated.

 

~I...

3

The present research is concerned with the enumeration
and identification of the predominant anaerobic bacteria in
the large intestine of the laboratory mouse, an important
experimental animal. The results will be compared with the
results obtained in previous investigations, employing the
anaerobic chamber method.

The current investigation employed a variety of criteria,
the Gram reaction, morphology, motility, aerotolerance,
spore formation, gas production, specific biochemical tests,
and analysis of fermentation acids by gas chromatography,
to identify the isolates from the large intestine of mice.

The significance of this investigation is related to the
fact that anaerobic bacteria are the predominant microbes in
the gastrointestinal tract of mammals and they are frequently
involved in clinical infections. A knowledge of the pre-
dominant genera and species in the large intestine of.healthy
mice is essential to better understand the microbial ecology
of this habitat and specific organisms involved in pathologi-

cal processes.

REVIEW OF THE LITERATURE

Ecology of the Gastrointestinal Tract

 

General Gastrointestinal Microflora

The intestinal tract of mammals is essentially anaerobic
and has an oxidation-reduction potential of 7250 millivolts
or lower (29). Obligately anaerobic bacteria account for
approximately 90% of the gastrointestinal microflora of
mammals.

Bacteria proliferate in the stomach, small intestine,
and large intestine (15,29). It was earlier believed that
when the stomach and small intestine were empty, they were
devoid of microorganisms. This idea prevailed when the
cultural methods were designed only for the enumeration of
aerobic bacteria. Previously, enterococci and other enteric
bacteria were thought to be the predominant gastrointestinal
microflora. Improved cultural methods for the isolation of
anaerobic bacteria have shown that in all areas of the
gastrointestinal tract, anaerobes consistently far outnumber
the aerobic bacteria (15,29,44).

At birth, the gastrointestinal tract of the mammalian
fetus is essentially free of cultivable microorganisms (38).
As soon as suckling begins, various species of bacteria

become established in different parts of the gastrointestinal

5
tract (38). Schaedler, Dubos, and Costello (38) investi-
gated the chronological establishment of the bacterial
flora in the gastrointestinal tract of mice. They found
that lactobacilli, anaerobic streptococci, and flavobacteria
colonized the entire gastrointestinal tract within 24 hours
of birth. Maximum populations were reached by lactobacilli
and the anaerobic streptococci around the twelfth day of
life. The flavobacteria reached maximum populations around
the tenth day, but they completely disappeared twelve days
after the birth of the animal.

The indigenous microflora of the intestinal tract of
mice is made up of organisms exhibiting different types of
relationships in the host (15). Escherichia coli is repre-
sentative of the organisms that possess a degree of infectivity
but their numbers are kept at low levels (15). Some micro-
organisms exemplified by the genus Bacteroides have achieved
a state of symbiosis and play an essential role in the
anatomical development and physiological functions of the
host (15,18). Bacteroides species constitute the autoch-
thonous flora of the mouse and are considered to be relatively
stable for animals of the same species (17,18,36).

Throughout the life span of the animal, the gastroin-
testinal flora remain essentially the same unless the animal
is subjected to stress or antibiotics are administered
(15,23,29,34,35). The organisms of the digestive tract
metabolize complex polysaccharides such as cellulose and

starch, simple sugars, amino acids, and bile acids. Large

6
quantities of fatty acids are produced as end products of
metabolism by these bacteria. Butyric acid, one of the
fatty acids produced from bacterial metabolism, has been
found to have an inhibitory effect on the coliform popula-
tion (23). Some of the volatile and non-volatile fatty
acids are presumably absorbed from the gastrointestinal
tract and utilized by the host (29).

The Bacterial Population of the
small Intestine

 

 

Moore, Cato, and Holdeman (29) reported that there are

6 to 108

approximately 10 organisms per gram of ingesta in
the small intestine. Lactobacilli and anaerobic strepto-
cocci were known to be present in the small intestine but

in smaller concentrations than in the stomach and large
intestine. The flavobacteria are more numerous in the small
intestine of mice than in the other areas of the gastroin-
testinal tract (15). However, they disappear completely
after twelve days of age (15). Bacterial metabolic end
products such as acetic, lactic, and succinic acids are
generally present in the small intestine of mammals.

Predominant Microorganisms of the
Large Intestine

 

In the lower regions of the intestinal tract, investi-

gators observed 1011

viable bacteria per gram of contents
(29,36). After ten days of life, Schaedler et al. (38)
showed that enterococci and coliform bacilli multiplied in
the large intestine of mice and reached levels of 109

organisms per gram. Within a few days, the coliform and

7
enterococci p0pulations decreased from 109 to 104 organisms
per gram and remained at this level throughout the life
span of the mouse (38). The anaerobic, Gram-negative bacilli
colonized the large intestine fifteen or sixteen days after
birth. They reached populations of 109 organisms per gram
and persisted at-this level during the life of the mouse.

Lee et al. (24) used an anaerobic chamber to follow the
bacterial development in the ceca of mice. The results
obtained in this investigation paralleled very closely the
results observed by Schaedler and his co-workers (38) using
selective plating media. However, more anaerobic bacterial
morphotypes were cultured using the anaerobic chamber
method. Bacteroides species and tapered-end bacilli appeared

fourteen days after birth and reached populations of 10.10

and 1011

respectively. Spirochete-like organisms were found
around the twelfth day of the life of the animals.

Using an anaerobic-chamber to examine the ceca of mice,
Lee and Dubos (22) found lactobacilli, coliform, enterococci,
bacteroides and unidentified fusiform rods. Fusiform
organisms were also present in the intestinal contents of
mice.. Since these_organisms are extremely sensitive to
oxygen, they may reside in the mucous layer of the large
bowel (22,36). This was later confirmed by Savage,
McAllister, and Davis (36), who examined frozen sections of
the large bowel under electron microscopy and showed that
anaerobic fusifOrm bacilli probably representing the genera

Fueobacterium, Eubaeterium, and Clostridium are embedded in

the mucosal epithelium of the large bowel of mice. A few

8
spiral-shaped organisms were also seen near the mucin layer
in this study (36).

Gordon and Dubos (18) reported the anaerobic bacteria
isolated from previously cesarean obtained, barrier sustained
mice (COBS). The COBS mice were colonized with bacteria
isolated from normal mice. The anaerobic bacteria isolated
from the COBS mice were cultured using pre-reduced anaerobi-
cally sterilized media. Clostridium species were isolated
from the 10-7 through 10-8 dilutions of cecal contents.
Spiral organisms were present in the 10-9 through 10-10
dilutions of cecal contents. Tapered-end, Gram-positive
bacilli were identified as members of the genus Eubacterium

10 and 1011

and reached levels of 10 organisms per gram of
cecal contents. Doughnut-shaped, Gram-positive organisms
were identified as Catenabacterium contortum, (Eubacterium
contortum). Some unidentified tapered-end rods with visible

-10

flagella were isolated in the 10 to 10-11 dilutions of

cecal contents. Medium to thin rods of the genus Fuso-

-10 to 10"11 dilutions.

bacterium were numerous in the 10
One species was identified as Fusobacterium ruseii.

Barnes et a1. (3,4,5) studied the anaerobic flora from
the ceca of chickens and turkeys using the modified roll-
tube method of Hungate (21). These investigators found that
the Gram-negative, non-sporulating bacilli made up approxi-
mately 40% of the anaerobic population and that this per-

centage was approximately equalled by the Gram-positive,

non-sporing bacilli. Peptostreptococcus species were found

9
to comprise approximately 15% of the cecal population of
chickens and turkeys.

Dubos et al. (15) found differences in the fecal-flora
of various strains of mice. The majority of the mice har-
bored lactobacilli, bacteroides, coliform, and Escherichia
coli as.the predominant types of organisms. Pseudomonas,
Proteus, and Clostridium varied from one mouse colony to

another.
Induced Changes in-the Gastrointestinal Microflora

The Effects of VariOus Diets
on Intestinal Flora"

 

 

The compositions of the intestinal flora of mammals can
be drastically altered by different diets. Dubos and his
colleagues (15) studied the effects of a complete commercially
obtained D G G diet and a casein diet on the bacterial popu-
lation of the gastrointestinal tract of mice. The casein
diet decreased the numbers of lactobacilli in the stomach,
small and large intestine. It is probable that the casein
diet did not providerequired metabolites for the growth of
the lactobacilli (15,24).

Lee and his colleagues (24) studied the effects of milk
as a complete diet on the development of the cecal flora of
mice. The experimental group of mice was given milk as the
only diet throughout the experiment, while the control mice
were maintained on a complete mouse diet throughout the
experiment. Lactobacilli, bacteroides, and numerous fusi-

form bacilli were isolated from the control mice. They

10

9 10

reached.normal population levels of 10 , 10 11

, and 10
organisms per gram respectively. In the mice given only
milk, lactobacilli and bacteroides were isolated and reached

9 10 respectively. In

normal population levels of 10 and 10
contrast, the coliform population persisted at levels of
1010 organisms per gram instead of showing a decline as in
the control group of mice. Fusiform bacilli were not iso-
lated from the mice on the milk diet.

By the use of selective plating media, Dubos et al.

(14) determined the effects of different diets on the fecal
flora of mice. Different groups of mice were given the
complete D G G diet, a Sherman diet containing 33% milk and
66% wheat flour, a diet containing casein, and a diet con-
taining wheat gluten (15). Large numbers of Gram-negative,
cocco-bacilli and fusifOrm bacilli were seen in smears from
the stools of all animals. The D G G and Sherman diets
resulted in larger numbers of lactobacilli in the fecal
flora of mice than the casein and gluten diets (15). It was
concluded from the results that the complete diets produced
more favorable conditions and growth factors than the casein
and gluten diets.

It has been established that the strict anaerobic
bacteria colonize the intestinal tract of mice fourteen to
sixteen days after birth. The period corresponds chrono-
logically to the intake of solid food. The strict anaerobic
bacteria may need reduced conditions or metabolites required

for growth that are present only in solid food before they

will proliferate in their normal habitats (24).

I...

11

The Changes of Microflora by
Antibacterial Agentsr

 

 

Savage and Dubos (34) added the antibiotics, penicillin,
terramycin, and kanamycin, to the drinking water of mice
and studied their effects on the cecal flora. These and
similar studies (34,35) showed that when penicillin was
administered in the drinking water at 0.1, 0.3, and 0.6 g/l
for two days, no cecal flora could be cultured after 12
hours in animals given drinking water containing 0.3 or
0.6 g/l. After 24 hours, the dry weights of all ceca of
mice on the three concentrations of penicillin increased.
Savage.and Dubos (34) found that after approximately.thirty
days of penicillin administration, the major flora in these
mice consisted of KZebsieZZa, Aerobacter, and enterococci.

Administration of terramycin and kanamycin, which are
broad spectrum antibacterial drugs, also resulted in an
increase of the dry weights of the ceca of mice (34). In
these mice, enterococci, bacteroides, and fusiform bacilli
were mainly observed.

Lee and Gemmell (23) found that administration of
penicillin eliminated the fusiform bacilli from the intestinal
tracts of mice. Butyric acid associated with the presence
of these bacteria was not present in the ceca of these mice.
Acetic acid and small traces of propionic acid, isobutyric
acid, and isovaleric acid were present. The coliform popu-

4 10

lation increased from 10 to 10 organisms per gram. In

the control mice, the coliform population remained at 104

organisms per gram. Although acetic and butyric acids were

12
in highest concentrations, propionic, isobutyric, isovaleric,

and valeric acids also were present in the ceca.

Possible Functions of Cecal Flora and Products

 

The Probable Role of the Fusiform Bacilli

The fusiform bacilli, tapered-end rods, apparently
represent four genera of anaerobic bacteria in the large
intestine of normal adult mice, viz., Bacteroides, Clostridium,
Eubacterium, and Fusobacterium (36). Many of the species of
the genus Clostridium did not readily form spores. Many
species of the genera Clostridium and Eubacterium were easily
decolorized and appeared Gram-negative in the Gram-stain.

Profound changes were seen in the cecal flora of the
mice that were treated with specific antibacterial agents
as compared to those of theuntreated group (34,35). The
enlargement of the ceca occurred at the end of two weeks in
mice given antibacterial agents from birth (35). It is
during this period that the anaerobic, Gram-negative bacilli
are beginning to reach maximum populations (18,36). Savage
and McAllister (35) reported that the enlargement of ceca
after administration of penicillin may be related directly
to the function of the numerous Gram-negative, fusiform
bacilli that are embedded in the mucosal epithelium of the
large bowel of mice (36).

The Fatty Acids Produced by
Anaerobic Bacteria

 

Bacteroides species are generally non-motile, non-

sporulating, Gram-negative bacilli that produce acetic acid

 

l3
and varying quantities of propionic, isobutyric, butyric,
isovaleric, and succinic acids from the fermentation of
carbohydrates and amino acids. The Gram-positive, non-
spore forming rods, Eubacterium, produce butyric acid and
small amounts of other acids. Fusobacterium, the Gram-
negative, non-sporulating bacilli, produce butyric acid as
a major product from the fermentation of carbohydrates and
amino acids (20).

Lee and Gemmell (23) reported that acetic acid is
detected in the intestine of mice usually seven days after
birth. Butyric acid was present twelve to fourteen days
after birth of the animal. Approximately sixteen days after
birth, propionic, isobutyric, isovaleric, and valeric acids
were present. Further investigations of Lee and Gemmell
(23) revealed that as the fusiform bacilli decreased in
numbers, the coliform bacteria increased in numbers. This
suggested that butyric acid produced by the fusiform bacilli
usually maintained the coliform population at minimal levels

in the intestinal tract of adult mice.
Role of Anaerobes in Infections

The Habitats and Infections of Anaerobes

Oxygen sensitive bacteria comprise the predominant flora
in certain cavities and mucous membranes of man and animals
(16,43). They outnumber the aerobic bacteria roughly one
thousand to one in habitats such as the intestinal tract of
humans (43). Anaerobes are potential pathogens and, given

certain opportunities, they can cause infections. Many

14
species of anaerobes that are present in the intestinal
tract are encountered in clinical infections (29). Although
actinomycotic and clostridial infections are well known, the
anaerobic bacteria are, in general, neglected in human and
veterinary microbiology (7,43).

Anaerobic cocci are found in the gastrointestinal and
genital tracts of man and animals (16,43). The genera of
Gram-positive cocci are Peptococcus and Peptostreptococcus
and the Gram-negative cocci are classified as Acidaminc-
coccus and VeiZZoneZZa. The anerobic cocci can cause abscess
formation, subacute bacterial endocarditis, and suppurative
myositis (16).

The anaerobic, Gram-positive, non-sporulating bacilli
are isolated from clinical infections. Among these are
Actinomyces, Bifidobacterium, Eubacterium and Propioni-
bacterium. Some species of these genera are found in the
gastrointestinal tract and mouth of man and animals and
they may cause abscess formation and septicemias in associa-
tion with other organisms (43).

Endospores are produced by anaerobic, Gram-positive
bacilli of the genus Clostridium. Some species are found
in soil, food, and the intestinal tract of mammals. Patho-
genic species can cause food poisoning, necrotic enteritis,
wound infections, and a variety of other infections (43).

The Gram-negative, non-sporing bacilli are found in
the mouth, upper respiratory tract and intestinal tract of
man and animals (43) and most often represent the genera

Bacteroides and Fusobacterium. These organisms cause

15
abscess formation, septic thrombophlebitis, and many other

infections (16).

Pathogenicity of Anaerobes

 

It has been difficult to establish the pathogenicity of
a single anaerobe in experimental animals. Frequently,
anaerobes are secondary invaders in infections and often
they are associated with other organisms (7,29,43).

Macdonald, Gibbons, and Socransky (7,42) showed that a
fatal infection could be established in guinea pigs by four
organisms collectively, Bacteroides melaninogenicus, another
bacteroides, a motile, Gram-negative bacillus, and a facul-
tative diphtheroid. The infection was not fatal if the
organisms were given individually or in combinations of three
to the guinea pigs.

Moore et al. (28,29) established the mechanisms by
which the liver of turkeys is infected by intestinal anaerobes.
Anaerobes were consistently isolated from liver granulomas.

A virulent strain of Streptococcus faecalis was found.to pro-
duce lesions of the intestinal wall. Then the intestinal
anaerobes gained entrance to the circulatory system through
these lesions and then established themselves in the liver.

If the gastrointestinal anaerobic bacteria, or anaerobes
from any cavities or membranes, gain entrance to the circula-
tory system,they can infect other tissues and organs. They
are capable of causing damage to the host once they are

displaced from their normal habitats.

MATERIAL AND METHODS

Animals

Male albino mice were obtained from the Spartan Research
Animal laboratories (Haslett, Michigan). The mice were
housed in individual steel cages with wood shaving for bedding
in an animal room associated with the Clinical Microbiology
Laboratory (Michigan State University). The mice were fed
Wayne Mouse Breeder Blox, a complete diet which was obtained
commercially from the Allied Mills, Incorporated (Chicago,
Illinois). The principal ingredients in the mouse pellets
were wheat, skimmed milk, whey, casein, fish meal, vitamins,
and a variety of salts. The mice were provided with drinking

water from individual glass bottles.

Cultural Methods

 

The anaerobic roll-tube technique as described by
Hungate (21) and later modified by Moore (27) was used
except where indicated otherwise. This technique was devel-
oped to completely exclude oxygen in preparing, dispensing,
and inoculating tubed media.

A gas mixture containing 85% nitrogen, 12% carbon
dioxide, and 3% hydrogen was used to exclude air in prepar-
ing media and during various cultural manipulations. Traces
of oxygen in the gas mixture were removed by passing it
over a cold catalyst, Deoxo. The Anaerobic Culture

16

17

Apparatus used in this investigation is the same as that
described by Moore (20) and it essentially consisted of 3
stationary 6 inch, 16 gauge hypodermic needles bent at
right angles. The gas mixture which provided a continuous
stream of oxygen free gas for maintenance of anaerobic
conditions while preparing media and inoculating tubes was
connected to the hypodermic needles by y-shaped connectors

and rubber tubing.

Culture Media

 

Pre-reduced anaerobically sterilzed media (PRAS) were
autoclaved, then dispensed, and inoculated in the complete
absence of oxygen. All PRAS media contained the oxidation-
reduction indicator, resazurin. The ingredients were boiled
under the gas mixture in a round bottomed flask to remove
the dissolved oxygen. The flask was stoppered, wired, and
autoclaved. After autoclaving and cooling, cysteine hydro-
chloride, a reducing agent, and sodium carbonate were added
to the medium.

Dilution blanks were made with the anaerobic salts
solution described in the Anaerobe Laboratory Manual (20).
The anaerobic salts solution was used in blending a weighed
amount.of intestinal contents in a Waring blendor. Ten-
fold dilutions of the blended intestinal contents were made
using 9 ml blanks of the anaerobic salts solution.

The medium used in this study for agar roll-tubes and
maintenance slants was a modification of Medium 10 of

Caldwell and Bryant (8,20). Glucose, cellobiose, soluble

18
starch, trypticase, yeast extract, and a variety of mineral
salts were present in the medium to provide growth factors.
Hemin solution was incorporated into the medium because it
is required by some anaerobes in cytochrome formation.
Sodium carbonate was a part of the buffer system in the
medium, and cysteine hydrochloride was used as a reducing
agent. The dye, resazurin indicated the oxidation-reduction
potential of the medium.

Commercial pre-reduced anaerobically sterilized media
purchased from the Robbin Laboratories, Incorporated (Chapel
Hill, N.C.), were used for biochemical tests. Carbohydrates,
supplemented brain heart infusion broth, milk, nitrate broth,
bile broth, and gelatin were some of the media used to clas-

sify organisms into genera and species.

Isolation Procedures

Adult male mice were sacrificed by the use of anhydrous
ether and pinned on a dissecting board. The large intestine
was removed aseptically. The intestinal contents were
squeezed out of the intestine of the mice and immediately
weighed. Complete anaerobiosis was maintained after this
step. The weighed intestinal contents were blended anaerobi-
cally under the gas mixture in a Waring blendor containing
a known volume of the anaerobic salts solution for approxi-
mately 1 minute.

One milliliter of the blended intestinal contents was
transferred aseptically and anaerobically to a tube con-

taining 9 ml of sterile anaerobic salts solution. Ten-fold

19

dilutions were made to 1011.

Separate pipettes were.used
for mixing and transferring 1 ml of the diluted intestinal
contents from one dilution tube to another.

Nine milliliters of the pre-reduced modified Medium 10,
which had been made prior to opening the mouse, were-kept in
a water bath at 48 C. One milliliter of a specific labeled
dilution was added to a labeled tube of modified Medium 10.

Four agar roll-tubes were made of each dilution from 10.7

to 10'11.

The tubes were stoppered, and then rolled under
a continuous stream of tap water until the agar was solidi-
fied around the tubes.

The tubes were incubated at 37 C for seven days...Counts
were made in the tube dilution containing 30 to 300 colonies.
The procedures for analysis of the cultural counts were the
same as that given in the Anaerobe Laboratory Manual (20).

A direct microscopic.count was made from the 10-3
dilution of the blended intestinal contents. The procedures
were those given in the Anaerobe Laboratory Manual (20).

A Gram-stained smear wasxnade from the 10-1 dilution

of the intestinal contents of mice. All morphotypes were

1

recorded. A wet mount was made from the 10- dilution and

examined for motility with a phase-contrast microscope.

Cultural Methodology

 

A platinum or stainless steel transfer needle was used

to pick representative colonies from the 10-9, 10'10

-11

, and
10 dilution tubes. Isolated colonies were stabbed into

slants of pre-reduced modified Medium 10. After 24 hours,

20
the water of syneresis was examined for morphology, culture
purity, and Gram reaction. Mixed cultures were streaked
on a rolletube to obtain discrete colonies.

Each organism was examined for aerotolerance by streak-
ing 1/4 of a blood agar plate and incubating it at 37 C
aerobically for 24 hours.

Obligate anaerobic and microaerophilic bacteria were
grown in peptone-yeast-extract-glucose (PYG) broth for 48
hours. The volatile and non-volatile fatty acids produced
from the PYG broth were analyzed by a Dohrmann Gas Chromato-
graph employing a resoflex column. Helium was used as the
carrier gas for the system with a flow rate of 120 cc/min.
The column and thermal conductivity detector were maintained
at approximately 118-120 C, while the injection port was
maintained at 145 C. The procedures for the ether.and
methyl extracts of the acidified cultures were those given
in the Anaerobe Laboratory Manual (20).

Supplemented brain heart infusion broths were inocu-
lated from pure slant cultures. After growth in the supple-
mented brain heart infusion broth, differential biochemical
media were inoculated using Pasteur pipettes. Approximately
four to five drops of the broth culture were inoculated into
each tube for the requisite biochemical tests.

The carbohydrate media used in the present study were
arabinose, cellobiose, fructose, galactose, glucose, lactose,
maltose, mannitol, mannose, melezitose, raffinose, rhamnose,
starch, sucrose, and xylose. The final pH in these media

was determined by a Beckman pH meter. A pH of 6.0-5.5 was

21
read as weak acid for most cultures, while a pH of below 5.5
was read as strong acid. A pH of below 5.7 was read as
acid for arabinose and xylose, since the pH of these media
is 5.9 after inoculation under C02.

Bile, esculin, gelatin, heme requirement, hydrogen pro-
duction, indol, milk, and nitrate media were used for other
differential tests. The selective bile medium was used to
determine if growth of the organism was stimulated or inhibited
by bile. Esculin hydrolysis was determined by adding a few
drops of ferric ammonium citrate solution to the culture.

The development of a black color was read as a positive test.
Gelatin cultures were refrigerated with control uninoculated
gelatin tubes. The uninoculated gelatin controls solidified.
Failure of the gelatin cultures to solidify was read as
positive. The gelatin cultures were read as weak if they
liquefied in 1/2 the time required for the gelatin controls
to liquefy. An uninoculated gelatin tube usually liquefied
in 15 to 20 minutes. Heme requirement was tested by incor-
porating heme solution in PYG broth. Growth in the PYG +
heme broth was compared to growth in the PYG broth. Hydrogen
production was determined by breaks produced in the agar of
the maintenance slants. The presence of indol was detected
by mixing 2 ml of a nitrate culture with xylene and then
adding Ehrlich's reagent. A pink ring was read as positive.
Inoculated milk cultures were observed for the development

of acid, curd, and/or digestion. Nitrate reduction was
tested by adding the conventional nitrite reagents (20).

Development of a red color was considered a positive test.

22
If no color developed, after the addition of the reagents,
then zinc dust was added. The development of a red color
after zinc addition was read as negative. No color after
the addition of zinc dust was read as positive in that
complete nitrate reduction had occurred.

All Gram-positive bacilli were subjected to a heat
test. A tube of starch broth was inoculated and heated at
80 C for 10 minutes and then incubated at 37 C for 24 hours.
Growth in the starch broth after the heat test confirmed
the presence of a sporulating Clostridium.

Young cultures, 12 hours old, grown in peptone-yeast
broth, were Gram-stained to determine the Gram reaction.

A phase-contrast microscope was used to determine motility

from wet mounts prepared from peptone-yeast broth cultures.

Identification

 

Anaerobic bacteria isolated from the large intestine of
mice were identified as to the species when possible, by
the system of classification proposed in the Anaerobe
Laboratory Manual (20). The basic criteria used for the
identification of the anaerobic bacteria were the Gram
reaction, morphology of the organism, motility, acid end
products in PYG broth, aerotolerance, spore formation,
pigment production, and specific differential biochemical

reactions.

RESULTS

Direct Smears of Mouse Intestinal Flora
1

 

Smears of the 10- dilution of the mouse intestinal
contents in the anaerobic salts solution were Gram-stained.
Elongated to ovoid, Gram-positive and Gram-negative cocci
were observed singly, in chains, and in small clusters.
Numerous tapered-end, closed-end, and irregular-shaped,
Gram-positive bacilli were arranged singly, in clusters, and
in palisades. Tiny, Gram-negative, cocco-bacilli, rounded-
end bacilli, and.tapered-end bacilli were found in the mouse
smears.

1 dilution of the mouse intestinal

Wet mounts of the 10-
contents, mentioned above, were examined under the phase-
contrast microscope for observing bacterial motility. Many
motile bacteria were present including motile bacilli with

rounded-ends and tapered-ends, and spiral~shaped organisms.

Cultural Counts

 

In the intestinal tract of the mice examined in this

study, 1.3-1.6 x 1010

viable bacteria per gram of contents
were observed and presented in Table l. Colony counts were
made in roll-tubes of the mouse intestinal contents contain—

ing 30 to 300 colonies.

23

24

Table 1. Enumeration3 of the cultural counts of anaerobic
bacteria in the intestinal contents of mice

 

 

Animal Weight of Average counts in Bacteria per
Number contents 108 dilution gram

10
Mouse I 0.22 g 32 1.3 x 10
Mouse II 0.31 g 53 1.6 x 1010
Mouse III 0.52 g 78 6.7 x 1010

 

aProcedures in the Anaerobe Laboratory Manual (20) were
used in enumerating the bacteria. Other experimental details
are given in the text.

Microscopic Count

 

A direct microscopic clump count made from the 10-3

dilution of the intestinal contents revealed 4.4 x 1010

bacteria per gram of intestinal contents.

Genera of Intestinal Anaerobes

 

Representative colonial morphotypes were isolated pri~

9 -10 '11

marily from the 10- , 10 , and 10 dilutions of intestinal

11 dilution tubes

contents of three mice. Several of the 10—

for two mice did not contain any colonies.
The genera isolated from the 10.9 dilution were Lacto-

bacillus and Peptostreptococcus. Bacteroides, Eubacterium,

-10 and 10-11

and Fusobacterium were present in the 10 dilu-
tions of the intestinal contents of the mouse.

Morphological, cultural, and biochemical characteristics
of the bacterial isolates are presented in Tables 2, 3, 4,

and 5 respectively. The gas chromatograms of the ether and

25

Table 2. Morphological and staining characteristics of the
anaerobic bacteria isolated from the large intestine

 

 

of mice
A Gram

Organism Reaction Microscopic Morphology

Bacteroides fragilis - large blunt rods

55. thetaiotaomicron

Bacteroides furcosus - short to long
pleomorphic rods

Bacteroides pneumosintes - bacilli with

. ' . tapered-ends

Bacteroides ruminicola - small cocco-bacilli

ss. brevis

Bacteroides sp.1 - medium-sized
pleomorphic rods

Bacteroides sp.2 - medium to long
pleomorphic rods

Eubacterium aerofaciens + bacilli in clumps

Eubacterium contortum + medium-sized rods
with curved ends

Eubacterium tenue + short rods in
clumps and chains

Fusobacterium sp. - large blunt rods

Lactobacillus fermentum + bacilli in chains

Peptostreptococcus + cocci in chains

intermedius

 

26

 

 

Table 3. Colonial morphology and acid end products of
peptone-yeast extract-glucose (PYG) broth of the
anaerobic bacteria in the large intestine of mice

Colonial Acid end

Organism morphology products of PYG

Bacteroides fragilis tiny colorless acid, propionic,

ss. thetaiotaomicron round colonies lactic, succinic

Bacteroides furcosus circular colonies acetic, lactic,

with dark colored succinic
edges

Bacteroides white circular alcohol, acetic,

pneumosintes colonies with lactic, succinic

filamentous edges

Bacteroides ruminicoZa smooth edged acetic, lactic,

ss. brevis elongated colonies succinic

Bacteroides sp.1 elongated white acetic, pro-

colonies pionic, lactic,
succinic

Bacteroides sp.2 white mucoid acetic, lactic,

irregular-shaped succinic
colonies

Eubacterium aerofaciens opaque circular acetic, lactic,

yellow colonies succinic

Eubacterium contortum white colonies acetic, lactic,

with indented succinic
edges

Eubacterium tenue small circular acetic, pro-

Fusobacterium sp.

Lactobacillus

fermentum

Peptostreptococcus

intermedius

opaque colonies
circular white
colonies

round mucoid
white colonies

yellowish-beige
colonies

pionic, lactic,

succinic

acetic, butyric,
lactic, succinic

acetic, lactic,
succinic

acetic, pro—
pionic, lactic,
succinic

 

27

mavfimEsmcre

I + + + + + + I exoooooummsnmocmom
I I I + 3 + 3 I Esnrmssmk meNwooQonooq
I I I + + I 3 I .mm Estmuoecomsm
I + + + 3 + I I mason Sawsouoecsm
I + + + + + + I Sansoneoo Eswsmuoecsm
I + + + 3 + I I memwookosmo Erasmuoocsm
I I I + + I 3 I N.mm nonwosmeoem
I I I + 3 + 3 I H.mm nonwosmpoem

mwnmsc .mm
I + + + + + 3 + eNoowrwsss monsosmuoom
I I I I I I I I mmntwmossmem nonwosmcoom
I I I + 3 3 3 I mzmoossk monsosmnoom

§O&O®50690%U%N£fi .mm

I + + + 3 3 I + mwNwmohk

nonwosmnoom

 

HOH QmOH GmOH mmOU OmO“ OmD“ OmOflQ 0m0fl
-Aeesz -Hsz -osq -sfio -uaHsu -ossm -oHHou -Anss<

EmfiemMMO

 

onflumopaw omzoe
ecu Eoum woumfiomfi monOHome nwmuhou 3n guano own :a couscoum muosw0hm.©:o.wwo< .v canoe

28

xmo33 .o>flpewoz .o>wpfimom+

 

msswmssmnew
msooooonmmsumocmmm

EscemEsm% mmewoecouoeq
.mm Eswsmcoocomsm
mason Seamuoocsm

Esusouroo Essmwoecsm
meewoekosmo Exmsmcoecsm
N.mm wmnwosmnoem

H.mm nonwosmwoem

ownmsc .mm
oNoowrwsss mmwwosmuoom

mmnrwmoszmrm nonwosmcoem
mxmooszk mmwwosmnoem

eosowsoeuowonoxc .mm
ewNmmesk mmwmosmuoem

 

omofixx

omOHusm cohmum

:fio
Imawm

QmOfl
-Esem

OmOfl

Itwmmmm

Qmou
-ANoHsz

omoe
Inez

Emwemmuo

 

A.e.ueoov a oases

29

:ofiumomfim .wuzuo .xmo33 .o>flpmmoz .o>flufimom
I +

 

 

c
exhumesmcrw
+ o + I + + I msooooocmmscwonmmm
+ I I I + I + Espressmk msNMwoocouoeq
I c I + 3 + I .mm Eswsmcoecomsm
I cu I + + I + were» Esasmwcecsm
+ o I I I + + Exesswrno Esssmcmecsm
+ O I I I + I mtmwoekosmd sewsmeoecsm
I U0 I + I I + ~.mm mannonemoom
I we I + 3 I 4 H.Qm mmhwcxmwoem
mmsmsc .mm
I co I + I + I omoowewess mmcwosmwoem
I I I + I I I emcewmoszmrm nonwosmuoem
I I I + I I I mxmoosxk nonwosmuodm
rosomsoeuoweumnu .mm
I do I + I + + mwNwmesk nonwosmuoem
oumuufiz Ma“: HoveH eowuosw peosohflzv :wpmaoo mfimxaouwx: :p3OHm Emwemmso

Iona N: Ion mam: eflflsomm oawm

 

mummy Hmowaonuown Honuo .m canoe

30
methylated extracts of the acid end products of the PYG broth

cultures of the intestinal isolates are presented in Figures

1-24.

Bacteroides

 

Morphology of the anaerobic, Gram-negative, non-
sporulating bacilli of the genus Bacteroides ranged from
tiny cocco-bacilli with rounded ends to medium-sized.p1eo-
morphic bacilli with rounded and tapered-ends. All isolates
of this genus are obligate anaerobes. The major acid end
products of these isolates are acetic and succinic acids
with varying amounts of lactic and propionic acids being
produced by some species. The addition of the heme to the
peptone-yeast extract-glucose broth (PYG), greatly enhanced
the growth of all isolates in comparison to the same medium
without heme.

One isolate, strain 5, was designated as Bacteroides
fragilis ss. thetaiotaomicron. This organism, unlike the
previously tested strains, did not produce indol.

Two intestinal anaerobes were identified as strains of
Bacteroides furcosus. One isolate, strain L, appeared
similar to the previously described strains of this species
but the second isolate, strain C, which was lost during
subculturing, differed from the present and previously
tested strains in not fermenting fructose and glucose, and
by producing indol.

Strains 9B35, S, and 4A were similar to Bacteroides

pneumosintes in all of the biochemical reactions described

31

.wflom oficwuosmum .wfiom ufipomHnAI.ueommosum .vflom oficofimopmum .wflum ofluoomu< .hocwonm

.zosomsoecowoeoxu .mm mwNwmosK mmpwosmc .rosowsoeeoweponu .mm mwNwmosk convenes
Ioem mo uomuuxo axnpoz .N ousmfim . Ieem mo pomppxo Honum .H ousmfim

mouseae :fi mafia peomowmoe moumeflwso Hmueonpoc
one ofiflnz moflpwpemsc ueomopmoh moumeflwuo Hmowpuo> HH< ”opoz

ooHE mo oewumouefi owhma one Eosm woumaomfi monopomcm
so sweep omOUSHquompuxo ummooneoumoa ea woosnoem meoswopm was ufiom
on» mo uumuuxo wopmenuoE wee genes we mEmumoumEopco mew

32

.vfiom ofiefioosmum .wfiom
oflpumfiuq .peommohum .mswooss% monsoson
Ioem mo pomuuxo axnuoz .v shaman

ofluoomn< .hocuoum

.eaos
.msmoos3% mmpwosmu

Ioem mo pumsuxo ponum .m oeamwm

33

.wfioe owqfiuusmnm .Ufium owuoma
"A .ucowwohum .menewmoszeem mowmosou
Ioem mo pomsuxo asses: .o ousmflm

 

 

 

 

.honpoum

.kum ofiuoowu<
.mmutvmosxmrm mmwmosmu

Ioem mo womhuxo Rheum .m ousmwm

 

 

34

.uwom owewoozmum .wfiom uwuumaua .wwum ofiuoomu<
.ueommopnm .mwamsc .mm eNoomeesss monsosos .quuonm .mmamsa .mm eNoom2w53s emuwosos
Ioem mo pomppxo axnpoz .m shaman Ioom we pumnuxo Honum .n ohswwm

 

 

 

 

. i I... .I.III4I..I.II11,-J.:II-,_1.Ifl ., 1T. I.II
ELI ., _ |..Il-.II.II1_.lIT III .I _
1 c
1

 

 

 

 

 

 

 

 

35

.kum afinfioo=mum

.cfiom uauumauq .pcommouum H.mw nonwosms

Ioem mo uomsuxo assuoz

 

 

.OH unease

 

 

 

 

 

 

 

.wfium ofieowmosmum
.wfiom ofluoomu< .uonuoum H.mm mmnmosms
Ioem mo pumHuXo Hocum .m ousmfim

 

 

 

 

 

 

 

 

 

 

 

36

.wmum oaefioUSmum
N.mm monsosoc
.NH museum

.eflom Ufiuomauq .uqommoeum
Ioem mo uomuuxo axnpoz

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.wwom uHuoumu< .Honuoum
Ioem mo uomuuxo Honpm

{a

N.mm mmwmosmu
.HH unease

37

ufiuumHuq
Ioocxm mo

.wwom ufiew663mum .vMoe
.pcowmohum .msmwookosmo Samson

pomsuxo Hznuoz .vH ossmfim

 

.esus
.mtmwoekosme Samson

oauoomu< .Honpoum
.mH menusa

IOUQRM MO HUMHHKQ Hmfium

\

 

 

 

 

 

38

.wwom owefluoamum .vfium
vauowanq .ucmwmouwm wsznsounoo Eaves»
Inseam mo uomuuxo HxApoz .oH oasmflm

 

 

C’"

ufluoomu< .nonpoum
Ioeazm mo uumeuXo Assam

.esus
.Exfifiofifiou Sawflwu
.mH unease

 

 

 

 

 

39

.wfiow uficw663mnm

.cwum ofiuomauq .ucowmosum .ssemn smegma
Inseam mo pompuxo Hague: .wH mnemHm

 

,. ,e
1.11 - -WI
w III.;ILJI
-.. I.- 1- .

 

 

 

.Ufiue owe0w90hmum

.wmom uwuoowu< .Honuoum .osrmu Basses

Inseam mo pomnuxo nonum

 

.AH seamen

 

 

 

 

 

 

 

 

 

 

 

 

40

.wfium oaewoo=m .wfium uwuxusn

um .kum ofiuomauq .ueommohnm .mm Samson um .wfium owpouwu< .uonuoum .mm finesse

Ioocomzm mo poohuxo axnuoz

 

.om mnemam Ioecoesm mo pumhuxo Hospm .mH opswfim

 

 

 

 

 

 

 

41

.wwom uwcfioosmnm .wwum
ufipumauq .ueommonum .Esseoesok mnmeooc

.wflom
Iosoeu mo pomHuXo Henna: .NN opswflm

owuoomu< .Hocuoum .Esuemssmk mzNNmooc
Iosoeu mo pumpaxo ponum .HN ohsmfim

 

 

 

 

 

 

1 I 1 1-7 1.
.II 1 1
. 221.
.1 II
1
u . Ifir
-1 1
1 1 I II
1 11 1
1 1 . I T
1 1 .
:1..-
1 . 1.. ,
.II'LI._1 I .1
T.-1% 1
1 11 I .
+. . 1
..1.-M.I “I1... . .1
11. ...,Iu1
1111......" 1.1
-..1fl.11_flmw 1

 

 

 

42

 

.wwom Owcfloosmum .wwom ufiuumauq
.ucmmmohnm .msmwmssmcem msooooocmmsum

.wmom Umcow90hmum .Uwom ofiuoom
Iocemm mo pumepxm axnuoz .eN opdmwm

u< .Hmauoum .mswwmssmusw wsooooonmmsnm
Iosmmm we pumpuxo spasm .mN ohsmflm

 

 

 

 

 

 

EH51.

43
previously. These isolates, however, were medium-sized,
tapered-end rods, while most previously described strains
of Bacteroides pneumosintes were tiny cocco-bacilli. Two
of the present isolates, strains S and 4A, showed an alcohol
peak in the ether extract of the PYG broth in the gas
chromatograms. The three isolates were lost during
subculturing.

One isolate, strain 3A, was tentatively identified as
Bacteroides ruminicola ss. brevis in morphological and
biochemical reactions. The succinic acid peak produced.by
this isolate in PYG broth was relatively small in comparison
to the succinic acid peak produced by previously tested
strains.

Strains 9B3l and TB are designated Bacteroides sp.1
and sp.2 respectively. These isolates appear to be dif-
ferent from any of the bacteroides species described to
date. Further biochemical and cultural tests need to be
done to establish their identity as new species or sub-

species of existing species.

Eubacterium

 

The genus Eubacterium includes the anaerobic, Gram-
positive, non-sporulating bacilli in chains and clumps.
Eubacterium varies in its acid end products produced in
PYG broth. While some species produce butyric and other
acids or acetic and formic acids as major products, other
species do not produce any major acids. All isolates were
subjected to a heat test for spore production and found to

be negative.

 

44

Strain G was tentatively identified as Eubacterium
aerofaciens. This organism, unlike the previously tested
strains, did not ferment mannose and did reduce nitrate.

Two bacteria, strains R and TA, similar to Eubacterium
contortum, were isolated. Nitrate was reduced by both
strains, while conventional strains of Eubacterium contortum
were known to reduce nitrate. One isolate, strain TA, did
not ferment maltose.

One intestinal isolate described as Eubacterium tenue,
strain 9B23, was found to be stimulated by the addition of
heme to the PYG broth. Biochemical characteristics of this
isolate are similar to those of the previously tested
strains except that lactose was fermented by the new

isolate.

Fusobacterium

 

Fusobacterium represents the anaerobic, Gram-negative
bacilli that produce butyric acid as a major product in PYG
broth. The bacteroides species that produce major amounts
of butyric acid also produce traces of isovaleric and iso-
butyric acids and in this respect they differ from Fuso-
bacterium. Many species of the genus have pointed ends.

Strains 9B34 and D' were tentatively identified as
species of the genus Fusobacterium. Both isolates produced
major amounts of butyric acid in PYG broth. These isolates
appear to be different from any fusobacterium species
described to date. One isolate, strain 9B34, was a large

blunt end bacillus. This organism was fermentative,

45
stimulated by the addition of heme, and did not grow in the
presence of bile. The second isolate, strain D', was lost
during subculturing. The tapered-end bacillus was non-
fermentative, produced indol, and grew in the presence of

bile.

Lactobacillus

 

The genus Lactobacillus includes anaerobic and micro-
aerophilic, Gram-positive bacilli in chains. All species
produce lactic acid as the sole major acid end product in
PYG broth. Several strains similar to Lactobacillus
fermentum were isolated. Strain 10B24, which is typical
of this group, became aerotolerant after repeated subculture.
Most of the biochemical reactions were in accord with the
previously tested strains. However, maltose was not fer-

mented by strain 10B24, while rhamnose was fermented.

Peptostreptococcus

 

The genus Peptostreptococcus represents the anaerobic
and microaerophilic, Gram-positive cocci in chains. Several
isolates were tentatively identified as Peptostreptococcus
intermedius. Strain J, which is typical of this group,
became aerotolerant after repeated subculture. The bio-
chemical reactions of these and previously described strains
were similar. A moderate amount of propionic acid was pro-

duced in the PYG broth by strain J.

DISCUSSION

In an investigation of the oxygen sensitive bacteria
of the mucosal epithelium of the large bowel of mice,

11

Savage, McAllister, and Davis (36) found 10 viable

organisms per gram of cecum or colon. Moore, Cato, and

10 11

Holdeman (29) reported 10 to 10 organisms per gram of

intestinal contents in various mammals. In the current

10 anaerobic bacteria per gram of intestinal

investigation, 10
contents were observed in the large intestine of laboratory
mice.

From a direct microscopic clump count made from the

10

intestinal contents of mice, 4.4 x 10 anaerobes per gram

of intestinal contents were observed. The mean cultural

count on the other hand was 3.3 x 1010

organisms per gram
of ingesta. In this investigation, the direct microscopic
count, which provides information about the efficiency of a
particular cultural method, was in proximity to the respect-
ive cultural counts. Generally, direct microscopic counts
are higher than actual colony counts since some of the
intestinal flora of the mouse, such as spiral-shaped
organisms, are seen in smears but are rarely cultured (18,36).
The results of this study are similar to the findings
of Gordon and Dubos (l8) and Lee, Gordon, and Dubos (22) on

the total microbial morphotypes present in the Gram-stained

46

47
smears of the mouse intestinal contents. Although.strictly
anaerobic, pointed end, Gram-variable bacilli predominate
in microscopic smears of the mouse intestinal contents,
microaerophilic to anaerobic cocci, bacilli, diphtheroids,
and spiral-shaped organisms are also present.

In the investigation of Gordon and Dubos (18), the ceca
of previously germ-free mice, which were colonized with
isolates of normal mouse intestinal flora (lactobacilli,
enterococci, anaerobic streptococci, slow lactose fermenting
coliform, and two strains of bacteroides), were cultured on
preereduced rumen fluid-glucose-cellobiose agar plates in
an aerobic chamber. Cigar-shaped, pointed-end rods, and
thin, medium, and large tapered rods, some with visible
flagella, were isolated. Other isolates from these mice
were medium-length bacilli with subterminal spores, doughnut-
shaped rods, and spiral organisms. In the present study,
medium-length, tapered bacilli were isolated, with the pre-
dominant rods being pleomorphic cocco-bacillary, short,
medium, and large rods. Diphtheroids, in clusters and chains,
and cocci in chains were also isolated. The strains of mice
used in the two investigations were not identical in intes-
tinal flora initially. While Gordon and Dubos (18) used
previously germ-free mice with a defined intestinal flora,
the current study employed commercially obtained laboratory
mice with a more complex initial intestinal flora.

Lee, Gordon, and Dubos (22) enumerated bacteria in the
intestine of specific pathogen free mice by the anaerobic

chamber method. Lactobacilli, coliform, enterococci,

48

bacteroides, and tapered-end rods were identified respectively
by pre-reduced media. The specific biochemical tests con-
ducted in this study were not described. In the current
investigation, the roll-tube method of Hungate (21) was
used for primary isolation of the intestinal anaerobes. The
isolates were subcultured on pre-reduced media to perform an
extensive battery of biochemical tests previously mentioned.
Gas chromatography was used to determine the acid end products
produced in peptone-yeast extract-glucose (PYG) broth. Many
anaerobes can be tentatively identified into genera by their
acid end products in PYG broth. The results of the present
study show that intestinal contents of laboratory mice con-
sist of Bacteroides, Eubacterium, Fusobacterium, Lactobacillus,
and Peptostreptococcus. Bacteroides species appear to be
the predominant group of.anaerobes. Lee et al. (22) reported
isolating bacteroides but Lee et al. (22) nor Gordon and
Dubos (18) established the numbers.of bacteroides species in
the mouse intestine.

The results of the current investigation are in agree-
ment with those of Gordon and Dubos (18) and Lee et al. (22)
on the population levels reached by the genera. Lacto-
bacillus and Peptostreptococcus reached maximum population
levels of 109 organisms per gram of contents. Bacteroides,

Eubacterium, and Fusobacterium were isolated from the 10-9

11 dilutions of the mouse intestinal contents.

through 10-
Some of the present isolates of the mouse intestinal
contents cannot be speciated because they appear to be

different from all previously described species. Further

49
work is in progress to establish the identity of these iso-
lates. Bacteroides fragilis ss. thetaiotaomicron, Eubacterium
aerofaciens, Eubacterium contortum, Lactobacillus fermentum,
and Peptostreptococcus intermedius, isolated in current
investigation, have been previously found in the gastroin-
testinal tract of rodents (20). Other organisms isolated
in the present study were Bacteroides furcosus, Bacteroides
pneumosintes, Bacteroides ruminicola ss. brevis, and Eubac-
terium tenue. Where speciation was not possible, biochemical
and morphologic characteristics were used to assign the
isolates to respective genera.

The major objective of this study was to categorize the
genera and, where possible, the species of the intestinal
anaerobic flora of laboratory mice. This would help estab-
lish a basis for a better understanding of the intestinal
flora of the normal healthy mouse. Perhaps the role of these
organisms in pathological processes of the mouse can be

understood.

SUMMARY

The intestinal contents of laboratory mice were cul-
tured anaerobically by the roll-tube method of Hungate (21).
Pre-reduced anaerobically sterilized media were used to
perform all biochemical tests and gas chromatography was
used to analyze the acid end products produced by the
anaerobes in peptone-yeast extract-glucose broth. Gram
reaction, morphology, spore formation, gas production, and
specific biochemical tests were criteria employed in this
investigation to identify the anaerobes from the intestine

1 . . .
0 V1able anaerobic organ1sms

of mice. In this study, 10
per gram of intestinal contents were observed.

The genera isolated were Bacteroides, Eubacterium,
Fusobacterium, Lactobacillus, and Peptostreptococcus. The
species isolated included Bacteroides fragilis ss. thetaio-
taomicron, Bacteroides furcosus, Bacteroides pneumosintes,
Bacteroides ruminicola ss. brevis, Eubacterium aerofaciens,

Eubacterium contortum, Eubacterium tenue, Lactobacillus

fermentum, and Peptostreptococcus intermedius.

50

BIBL IOGRAPHY

10.

11.

BIBLIOGRAPHY

Aalbaek, B. 1971. Sphaerophorus necrophorus: A
study of 23 strains. Acta Vet. Scand. 12: 344-364.

Aranki, A., S. Syed, E. Kenney, and R. Freter. 1969.

.Isolation of anaerobic bacteria from human gingiva and

mouse caecum by means of a simplified glove box pro-
cedure. Appl. Microbiol..l7: 568-576.

Barnes, E., and H. Goldberg. 1962. The isolation of
anaerobic Gram-negative bacteria from poultry reared
with and without antibiotic supplements. J. Appl.
Bacteriol. 25: 94-106.

Barnes, E., and C. Impey. 1968. Anaerobic Gram-
negative non-sporing bacteria from caeca of poultry.
J. Appl. Bacteriol. 31: 530-541.

Barnes, E., and C. Impey. 1970. .The isolation and
properties of the predominant anaerobic bacteria in
the caeca of chickens and turkeys. Br. Poult. Sci.
11: 467-481.

Barry, V., H. Conalty, J. Denney, and F. Winder. 1956.
Peroxide formation in bacteriological media. Nature.
178: 596-597.

Biberstein, E., Night, and K. England. 1968. Bac-
teroides melaninogenicus in diseases of domestic
animals. J. Am. Vet. Med. Assoc. 153: 1045-1049.

Caldwell, D. R., and M. P. Bryant. 1966. Medium
without rumen fluid for nonselective enumeration and

isolation of rumen bacteria. Appl. Microbiol. 14:
794-801.

Callow, A. 1923. On catalase in bacteria and its
relation to anaerobiosis. J. Path. Bact. 26: 320-325.

Collee, J., B. Watt, E. Fowler, and R. Brown. 1972.
An evaluation of the Gaspak System in the culture of
anaerobic bacteria. J. Appl. Bacteriol. 35: 71-82.

Dowell, V., and T. Hawkins. 1967. Isolation and

identification of anaerobic bacteria from clinical
materials. CDC. Atlanta, Georgia.

51

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

52

Drasar, B. S. 1967. Cultivation of anaerobic intestinal
bacteria. J. Path. Bact..94: 417-427.

Dubos, R., and R. Schaedler. 1960. The effect of the
intestinal flora on the growth rate of mice and on their

susceptibility to experimental infections. J. Exp. Med.,
111: 407-417.

Dubos, R., and R. Schaedler. 1962. The effect of diet
on the fecal bacterial flora of mice and their resistance
to infection. J. Exp. Med. 115: 1161-1171.

Dubos, R., R. Schaedler, R. Costello, and P. Hoet. 1965.
Indigenous, normal, and autochthonous flora of the gas-
trointestinal tract. J. Exp. Med. 122: 67-75.

Finegold, S. M. 1968.- Infection due to anaerobes.
Med. Times 96: 174-187.

Foo, M., and A. Lee. 1972. Immunological response of
mice to members of the autochthonous intestinal micro-
flora. Infec. Immun. 6: 525-532.

Gordon, J., and R. Dubos. 1970. The anaerobic bacterial
flora of the mouse cecum. J. Exp. Med. 132: 251-260.

Hall, I. C.. 1929. A review of the development and

application of physical and chemical principles in the
cultivation of obligately anaerobic bacteria. J. Bacteriol.
17: 255-301.

Holdeman, L. V., and W. E. C. Moore (eds.). 1972.
Anaerobe Laboratory Manual. VPI. Anaerobe Laboratory,
Blacksburg, Virginia 24060.

Hungate, R. E. 1950. -The anaerobic mesophilic cellu-
lolytic bacteria. Bacteriol. Rev. 14: l-49.

Lee, A., J. Gordon, and R. Dubos. 1968. Enumeration of
oxygen sensitive bacteria usually in the intestine of
healthy mice. Nature 220: 1137-1139.

Lee, A., and E. Gemmell. 1971. Changes in the mouse
intestine microflora during weaning: Role of volatile
fatty acids. Infec. Immun. 5: 1-7.

Lee, A., J. Gordon, C. Lee, and R. Dubos. 1971. The
mouse intestinal microflora with emphasis on the strict
anaerobes. J. Exp. Med. 133: 339-352.

Martin, M., and J. Crawford. 1971. Practical methods
for isolation of anaerobic bacteria in the clinical
laboratory. Appl. Microbiol. 22: 1168-1171.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

53

McMinn, M., and J. Crawford. 1970. Recovery of
anaerobic microorganisms from clinical specimens in
pre-reduced media versus recovery by routine clinical
laboratory methods. Appl. Microbiol. 19: 207-213.

Moore, W. E. C. 1966. Techniques for routine culture
of fastidious anaerobes. Int. J. Syst. Bacteriol. 16:
173-190.

Moore, W., and W..Gross. 1968. Liver granulomas of
turkeys; causative agents and mechanisms of infection.
Avian Dis. 12: 417-422.

Moore, W., E. Cato, and L. Holdeman. 1969. Anaerobic
bacteria of the gastrointestinal flora and their occur-
rences in clinical infections. J. Infect. Dis. 119:
641-649.

Moore, W., and L. Holdeman. 1972. Identification of
anaerobic bacteria. Amer. J. Clin. Nutr. 25: 1306-1313.

Reed, G. B., and J. H. Orr. 1943. Cultivation of
anaerobes and oxidation reduction potential. J. Bac-
teriol. 45: 309-320.

Rosenblatt, J., A. Fallow, and S. Finegold. 1973.
Comparison of methods for isolation of anaerobic bac-

teria from clinical specimens. Appl. Microbiol. 25:
77-85.

Savage, D., R. Dubos, and R. Schaedler. 1967. The
gastrointestinal epithelium and its autochthonous
bacterial flora. J. Exp. Med. 127: 67-76.

Savage, D., and R. Dubos. 1968. Alterations in the
mouse cecum and its flora produced by antibacterial
drugs. J. Exp. Med. 128: 97-110.

Savage, D., and J. McAllister. 1971. Cecal enlarge-
ment and microbial flora in suckling mice given anti-
bacterial drugs. Infec. Immun. 3: 342-349.

Savage, D., J. McAllister, and C. Davis. 1971.
Anaerobic bacteria on the mucosal epithelium of the
murine large bowel. Infec. Immun. 4: 492-502.

Schaedler, R., and R. Dubos. 1962. The fecal flora
of various strains of mice. Its bearing on their
susceptibility to endotoxins. J. Exp. Med. 115:
1149-1160.

Schaedler, R., R. Dubos, and R. Costello. 1965. The
development of the bacterial flora in the gastroin-
testinal tract of mice. J. Exp. Med. 122: 59-66.

 

39.

40.

41.

42.

43.

44.

45.

46.

54

Schaedler, R., R. Dubos, and R. Costello. 1965.
Association of germ-free mice with bacteria isolated
from normal mice. J. Exp. Med. 122: 77-82.

Shapton, D., and R. Board. 1971. Isolation of
Anaerobes. Technical Series 5. Academic Press.
London, New York.

Simon, P., and P. Stonell. 1969. Diseases of animals
associated with Sphaerophorus necrophorus: Character-
istics of the organisms. Vet. Bull. 39: 311-315.

Socransky, S., and R. Gibbons. 1965. Required role
of Bacteroides melaninogenicus in mixed anaerobic
infections. J. Infec. Dis. 115: 247-253.

Smith, L., and L. Holdeman. 1968. The Pathogenic
Anaerobic Bacteria. Charles L. Thomas Publishers,
Springfield, Ill.

Strociak, M., M. Desrosiers, and J. Truant. 1971.
Anaerobic organisms isolated from clinical specimens
and their spectra to chemotherapeutic agents. Mich.
Academician 3: 63-70.

Spears, R., and R. Freter. 1967. Improved isolation
of anaerobic bacteria from the mouse cecum by maintain-
ing strict anaerobiosis. Proc. Soc. Exp. Biol. Med.
124: 903-908.

Spiers, M. 1971. Classification system of the Bacteroides
group. Med. Lab. Tech. 28: 360-366.

l N STATE UNIVERSITY LIBRARIES

I||||| ”III! I

3084 9263

 

”'I'I‘IIIII‘III II

1293