Beagle Dogs Bacterial Association in the Gastrointestinal Tract of - Applied and Environmental Microbiology
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1977, P. 194-206 Vol. 34, No. 2
Copyright C 1977 American Society for Microbiology Printed in U.S.A.
Bacterial Association in the Gastrointestinal Tract of
Beagle Dogs
C. P. DAVIS,'* D. CLEVEN, E. BALISH, AND C. E. YALE
Departments of Surgery and Medical Microbiology, University of Wisconsin Center for Health Sciences,
Madison, Wisconsin 53706
Received for publication 3 March 1977
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Nine male beagle dogs, housed in either a conventional or locked environment
for 2.5 years, were killed, and the bacterial flora present in various regions of
each gastrointestinal tract was assessed by culture techniques, light microscopy,
and scanning electron microscopy. All dogs possessed a complex microflora in
their colons; in almost every dog anaerobes predominated. The highest number
of bacteria cultured was 1010/g (dry weight) oftissue and contents; highest counts
obtained with a Petroff-Hauser counting chamber were 1010/ml (wet weight).
Although there was a consistency in the detectable genera, there were also
noticeable differences in the flora of dogs housed under different environmental
conditions. These differences included qualitative and quantitative changes in
the flora as well as alterations in the distribution and localization of microorga-
nisms along the gastrointestinal tract and in the crypts of Lieberkuhn. No
bacterial layers were detected on the surfaces of stomach or proximal bowel in
any of the dogs. Dogs housed in a conventional, open, environment had bacteria
that occurred in layers on their ceca and colons and in their crypts of Lieber-
kuhn; however, dogs housed under "locked" environmental conditions did not
possess them or had them less frequently. Dogs removed from the locked
environment and kept (30 days) in conventional housing conditions were the
only ones with detectable segmented filamentous microbes in their ilea. This
study shows that the microbial flora does not simplify when dogs are housed in a
locked environment. Indeed, it may increase in complexity and cause alterations
in the bacterial flora that is associated closely with gastrointestinal epithelial
cells and crypts of Lieberkuhn.
Studies of the microbial flora of murine spe- berkuhn (10, 20). Also, there are very little data
cies have shown that the adult gastrointestinal available on the impact of altered environ-
(GI) tract contains many microbial species that ments on the GI tract flora. However, recent
live in relatively stable populations that are studies by Holdeman et al. (13) and Tannock
localized in specific regions of the GI tract (20- and Savage (24) indicate that stress (psycholog-
22). In the murine species, some microbial pop- ical, environmental, or dietary) may cause
ulations are known to associate intimately with changes in the flora.
epithelial cells by either direct attachment to The aims of this investigation were to deter-
epithelial cell membranes (8, 9, 11, 12) or layer mine if the GI tracts of adult, male, beagle dogs
formation (6, 20, 23). possessed localized bacterial populations and to
In contrast to what is known about bacterial ascertain if different housing conditions would
localization in the GI tract of murine species, significantly alter their GI flora.
little is known about the bacterial attachment
and layering in the GI tracts of higher mam- MATERIALS AND METHODS
mals. Most of the available information on the Animals and housing conditions. Nine male,
microbial flora of higher mammals has been purebred, beagle dogs, 9 to 12 months old, were
obtained by culturing fecal samples or lumenal obtained from a closed colony at the Argonne Na-
aspirations, which have not yielded any data on tional Laboratory, Argonne, Ill. Two of the dogs
bacterial populations that might be intimately (group 1) were housed in the conventional (open)
associated with the GI surface or crypts of Lie- dog-holding facilities at the University of Wiscon-
sin. Seven dogs were housed and maintained in a
' Present address: Department of Microbiology, Univer- "locked environment" that consisted of stainless-
sity of Texas Medical Branch, Galveston, TX 77550. steel housing units that are designed to maintain
194VOL. 34, 1977 BACTERIA IN BEAGLE GI TRACTS 195
germ-free dogs. The latter system has been de- lished by the Virginia Polytechnic Institute Anaer-
scribed in detail elsewhere (3). The dogs in the obe Laboratory (14). Volatile and nonvolatile fatty
locked environment (group 3) were fed sterile water acids were identified (14) with a gas chromatograph
and diet, and all air entering the unit was filter (Dohrmann, Mt. View, Calif.). Biochemical tests
sterilized. All entries into and out of the unit were were done by a modification of the Minitek proce-
made under sterile conditions. In essence, the dure described previously (5). Other media (litmus
locked-environment dogs were treated in a manner milk, chopped meat, gelatin, and egg yolk agar)
that prevented outside bacteriological contamina- were prereduced and inoculated with bacteria har-
tion for 2.5 years (3). All nine dogs were fed a steam- vested from A II agar. All biochemical tests were
sterilized diet (Purina Dog Meal, Ralston Purina done in the glove box at 35 to 37°C. All morphologi-
Co., St. Louis, Mo.) and water ad libitum. After 2.5 cally dissimilar colony-forming units (CFUs) were
years, four dogs were removed from the locked envi- counted on plates yielding 1 to about 300 colonies
ronment and placed in regular (open) dog-holding and inoculated onto A II agar. Total counts of indi-
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facilities for 1 month (group 2). During the latter 1- vidual CFUs of both aerobes and anaerobes were
month transition period, two of the dogs were fed the tabulated. References to CFUs of specific genera
sterilized diet, whereas the other two dogs were fed represent CFUs per gram (dry weight) of tissue and
the same diet, but unsterilized, ad libitum. All contents. Stomachs and proximal small bowels were
group 1 and group 2 dogs (after removal from the not examined by microbiological methods. Direct
locked environment) were given nonsterilized water counts of bacteria were made from 100-fold dilutions
ad libitum. All dogs were killed by an overdose of of ileal, cecal, and colonic tissue homogenates with
phenobarbital. We then examined and compared the phase optics and a counting chamber (Petroff-
microbial flora of dogs housed under conventional Hauser, Philadelphia, Pa.).
conditions (group 1), locked-environment conditions Sample preparation for microscopy. Pieces of tis-
(group 3), and transitory conditions, in which dogs sue, taken from areas directly adjacent to those used
were removed from the locked environment and put for CFU determination, were either immediately
into a conventional environment (group 2). placed in cacodylate-buffered glutaraldehyde (at
Tissue sample locations. Two adjacent pieces of 4°C) or put serosal-side down into a vial, which was
tissue from the cardiac region of the stomach, proxi- immediately immersed in liquid nitrogen. This fro-
mal small bowel, distal ileum, cecum, and colon zen sample was subsequently cleaved with a razor
were obtained from dogs by first clamping the width blade, sectioned in a cryostat, and stained with a
of the GI tracts with hemostats and then cutting, tissue Gram stain while another piece of the cleaved
with sterile instruments, tissue pieces (about 1 to 2 sample was fixed in glutaraldehyde, as above. This
cm2) from the GI tract. Upper small bowel tissues technique was used to preserve delicate surface
were removed from a region about 20 cm proximal to structures that are often removed by immediate fix-
the ileocecal valve. Colonic tissues were taken from ation in glutaraldehyde (4). All samples fixed in
a region approximately 20 cm distal to the ileocecal glutaraldehyde were postfixed in osmium tetroxide,
valve. dehydrated in an ethanol and amyl acetate series,
Microbiological methods. For the growth of strict critical-point dried, metal coated, and examined
anaerobes, separate pieces of ilea, ceca, and colons with a scanning electron microscope (SEM). Details
were immediately placed into tubes of prereduced of the above procedure have already been described
transport broth (2) and, within 30 min, passed into (4, 18).
an anaerobic glove box (1, 2) through a rapid-entry A Zeiss Universal microscope was used for all
port, and homogenized in a blender. Pooled samples light micrography and either a JEOL or a JEM U3
of either ileal, cecal, or colonic tissues in the groups SEM, operated at 20 kV, was used for all SEM.
1 and 2 dogs housed together and the two group 3
dogs housed together were homogenized. Serial 10- RESULTS
fold dilutions were made in Trypticase soy broth (5). Genera and species of bacteria. Table 1 lists
Then, 0.1 ml of each dilution (10-' to 10-9) was the genera and species of microorganisms culti-
plated onto prereduced A II agar (2), and the plates vated from all of the dogs. Eighty-four species
were incubated in a transparent, plastic incubator
especially designed for the glove box (2a). The latter of bacteria representing 27 genera and five ge-
dilution tubes, after removal from the glove box nera of fungi were isolated. This count also
through the air lock, were used to inoculate en- includes those organisms only partially identi-
riched and differential media for the detection and fied. Although the quantitative and qualitative
identification of aerobic and microaerophilic bac- composition of the microbial flora differed from
teria. The media and culture conditions were as sample to sample, similarities of microbial colo-
described elsewhere (3, 5) except that S.F. agar was nization were observed when the most numer-
not used. ous bacteria and their localization sites were
All media used to grow the anaerobic bacteria
were prereduced in the glove box at least 48 h prior
shown (Tables 1 and 2). The highest numbers
to inoculation (2). The anaerobic bacteria were iden- (109 to 1010/g [dry weight]) of viable bacteria
tified by their Gram reaction, biochemical tests, and were from anaerobic genera (Bacteroides, Bifi-
volatile and nonvolatile fatty acids produced in PYG dobacterium, Peptostreptococcus, Eubacter-
broth according to the identification protocol estab- ium, Clostridium, and Peptococcus) and mi-TABLE 1. Microorganisms isolated from ileal, cecal, and colonic homogenates of beagle cjogsa
Highest CFUb Occurrence in dog
Species groupsc
No. detected Location
Streptococcus
S. bovis 3 x 10O0 Colon 1, 2
S. acidominimus 3 x 1O"° Colon 1
S. mitis 1 x 109 Colon 1, 2, 3
Streptococcus species 2 2 x 108 Cecum 1
S. salivarius 4 x 107 Cecum 1, 2
S. dysgalactiae 3 x 107 Ileum 2
S. intermedius 3 x 107 Colon 2
S. faecium 6 x 106 Colon 2, 3
Streptococcus species ld 5 x 106 Colon 1, 2, 3
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S. agalactiae 6 x 104 Ileum 2
S. faecalis 5 x 104 Ileum 2, 3
Streptococcus species 3 1 X 102 Ileum 2
Lactobacillus acidophilus 2 x 1010 Colon 1, 2, 3
L. casei subsp. rhamnosus 8 x 108 Cecum 3
L. leichmannii 5 x 108 Colon 2, 3
L. plantarum 4 x 108 Colon 3
L. fermentum 3 x 105 Colon 3
L. lactis 3 x 105 Colon 2, 3
L. crispatus 3 x 106 Colon 2
L. casei subsp. casei 2 x 106 Colon 2
L. minutus 3 x 105 Ileum 3
L. helveticus 1 X 104 Ileum 3
L. salivarius subsp. salivarius 3 x 103 Ileum 2
Bifidobacterium infantis lactentis 2 x 1010 Colon 2, 3
B. adolescentis 3 x 107 Cecum 2
Bacteroides vulgatus 1 x 1010 Colon 1, 2, 3
B. distasonis 1 x 1010 Colon 1, 2, 3
B. corrodens 1 x 1010 Colon 2, 3
B. amylophilus 9 x 109 Colon 3
B. capillosus 1 x 109 Colon 3
Bacteroides species 1 x 109 Colon 1, 2
B. furcosus 2 x 108 Cecum 3
Eubacterium ventriosum 6 x 109 Colon 1
E. ruminantium 6 x 109 Colon 1
Eubacterium species 1 5 x 109 Colon 1, 2, 3
E. lentum 1 x 109 Colon 3
E. contortum 3 x 108 Colon 3
E. cellulosolvens 2 x 108 Ileum 2
E. aerofaciens 6 x 107 Cecum 2
Clostridium
Clostridium species 1 5 x 109 Colon 1, 2, 3
Clostridium species 2 2 x 109 Colon 1, 2, 3
C. inulinum 2 x 109 Ileum 2
C. irregularis 1 x 109 Colon 3
C. cochlearium 9 x 108 Colon 3
C. perfringens 3 x 108 Cecum 3
C. histolyticum 3 x 107 Cecum 3
Clostridium species 4 3 x 107 Ileum 2
Clostridium species 3 2 x 107 Cecum 2
Peptostreptococcus
P. magnus 6 x 109 Colon 2, 3
P. micros 5 x 109 Colon 1, 2
P. productus 3 x 108 Cecum 2
Peptostreptococcus species 1 6 x 107 Cecum 2
P. parvulus 3 x 107 Cecum 3
P. anaerobius 3 x 107 Ileum 1
Peptococcus
P. constellatus 3 x 109 Colon 3
196TABLE 1.-Continued
Highest CFUb Occurrence in dog
Species groupsc
No. detected Location group5
P. prevotii 3 x 108 Colon 1
P. magnus 6 x 107 Ileum 2
Fusobacterium
F. necrogenes 3 x 101 Cecum 3
F. varium 3 x 108 Cecum 3
F. gonidiaformans 3 x 107 Cecum 3
Gaffkya anaerobica 3 x 108 Cecum 3
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Veillonella parvula 2 x 108 Cecum 3
Acidaminococcus fermentans 2 x 108 Cecum 2
Corynebacterium
C. pseudotuberculosis 3 x 107 Ileum 1, 3
Corynebacterium species 1 3 x 106 Ileum 1, 3
Corynebacterium species 2 3 x 105 Ileum 3
C. ovis 1 x 105 Ileum 3
Ruminococcus albus 3 x 107 Cecum 2
Propionibacterium acnes 7 x 107 Colon 2
Proteus mirabilis 5 x 106 Cecum 1, 3
Escherichia coli 4 x 106 Colon 1, 2, 3
Klebsiella pneumoniae 3 x 106 Colon 3
Enterobacter cloacae 1 x 106 Cecum 2, 3
Staphylococcus aureus 2 x 105 Ileum 1, 2, 3
Bacillus
B. coagulans 1 x 105 Ileum 1
B. pantothenticus 3 x 104 Ileum 1
Anaerobiospirillum succiniciproducens 1 x 105 Colon 3
Moraxella (group M-5) 6 x 104 Ileum 2
Micrococcus
M. cryophiles 5 x 104 Ileum 3
M. varians 1 x 104 Ileum 3
Micrococcus species 4 x 103 Ileum 3
Othersd
Gram-negative rod 6 x 106 Cecum 3
Gram variable 2 x 105 Ileum 2
Coccobacillus
Alternaria species 1 x 105 Colon 1
Rhodotorula rubra 9 x 103 Ileum 3
Cladosporium species 6 x 103 Ileum 3
Unidentified fungus 4 x 103 Colon 3
Trichosporon cutaneum 3 x 103 Ileum 3
a Ileal, cecal, and colonic tissues and contents were homogenized with a blender in an anerobic glove box
to quantitate anaerobes. Homogenates were removed from the glove box and plated onto selective media for
197198 DAVIS ET AL. APPL. ENVIRON. MICROBIOL.
TABLE 1. -Continued
aerobes, facultative anaerobes, and fungi.
b CFU per gram of tissue and contents (dry weight).
c Indicates which dog groups (1, conventionally housed; 2, transitionally housed; 3, housed in a locked
environment) possessed detectable microbes in their GI tracts.
d Those organisms listed as either unknown, species, or other were either unidentifiable by our methods
or did not correspond to any known species or subspecies.
TABLE 2. Viable and microscopic counts of bacteria in the ilea, ceca, and colons of beagle dogs
Region of Environmental conditionsa
tract Counting method
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counted Group 1 Group 2 Group 3
Ileum Cultureb 1.2 x 108 3.1 x 107, 4.4 x 109 1.4 x 106, 4.0 x 108
Microscopicc NDd 4.0 x 107, 7.6 x 109 1.1 x 109
Cecum Culture 3 x 107 4.5 x 108 1.9 x 109 2.4 x 109, 7.7 x 109
Microscopic ND 6.2 x 107 1.5 x 109 6.0 x 109
Colon Culture 8.6 x 1010 1.9 x108 , 9.6 x 109 3.4 x 101', 4.6 x 1010
Microscopic ND 3.0 x 1010, 1.8 x 1010 4.4 x 10"0
a Three different environmental conditions were used: group 1 dogs were housed in a conventional dog-
holding facility; group 2 dogs were housed in a locked environment (sterile food, water, and air) for about 2.5
years and then removed to a conventional environment for 1 month; and group 3 dogs were housed in the
locked environment for 2.5 years.
b Total CFU of all bacteria obtained per gram (dry weight) of tissue and lumenal contents from a single
dog or two dogs in the same group.
c
Direct counts of undiluted or 100-fold dilutions of bacteria, observed with phase optics, were counted in a
Petroff-Hauser counting chamber.
d ND, Not done.
croaerophilic to anaerobic Lactobacillus species ally housed dogs (group 1) possessed only two
(Table 1). The highest number of facultative genera of microbes (Bacteroides and Strepto-
anaerobes was almost always streptococci. Mi- coccus) in their ceca, whereas the dogs in
croscopic observation of diluted contents indi- groups 2 and 3 possessed 8 and 13 different
cated that ceca and ilea usually possessed about genera, respectively (Table 4). The ceca of con-
one to three logs less bacteria than did the colon ventionally housed dogs also had lower total
(Table 2). When total microscopic counts were CFUs of anaerobes (one to two logs) and lower
compared with total CFUs, the data suggested total CFUs of aerobes (about one-half to three
that most of the bacteria viewed with phase logs) (Table 4) then did the ceca of dogs in
optics were viable (Table 2). groups 2 and 3. Four different microbial species
The ilea of all three groups of dogs possessed were isolated from the ceca of conventional
a heterogeneous microbial flora. The ileal an- dogs, whereas isolated dogs of group 3 had 25
aerobes also showed a wider variation in viable different species, and group 2 dogs had 18 differ-
bacteria (103 to 109 [Table 3]) than did anaer- ent species present in their ceca.
obes isolated from either the cecum (107 to 109 The dogs, regardless of housing conditions,
[Table 4]) or the colon (108 to 1010 [Table 5]). The generally showed a similar colonization pattern
total number of aerobic and facultative bacteria in their colons by the various genera of anaer-
in the ilea (105 to 107 [Table 3]) of the dogs was obes. Dogs removed from the locked environ-
comparable to those found in their ceca (105 to ment and then placed into a conventional envi-
108 [Table 4]), but less numerous than those ronment (group 2) showed a decrease of 0.5 to 1
aerobes and facultative anaerobes found in the log in the total number of colonic anaerobes
colons (107 to 1010 [Table 5]). Whereas 13 species (Table 5). In addition, the dogs housed under
of bacteria were isolated from the ilea of con- conventional conditions (group 1) had a popula-
ventional dogs (group 1), 21 and 20 species were tion of Streptococcus bovis, S. acidominus, and
isolated from the ilea of group 2 and group 3 S. mitis that was 1 to 2 logs higher (109 to 1010)
dogs, respectively. In addition, fungi (three than that found in the colons of other group 2
genera) were mainly isolated from group 3 and 3 dogs (Table 5). The dogs in confinement
dogs. (group 3) had 26 different microbial species in
There was a remarkable difference in the their colon. The dogs in transition (i.e., group
microbial flora in the ceca of dogs housed con- 2) had 22 different species of bacteria in their
ventionally (group 1) when compared with the colon, whereas conventional (group 1) dogs had
two other groups of dogs (Table 4). Convention- 16 different species in the colonic contents.VOL. 34, 1977 BACTERIA IN BEAGLE GI TRACTS 199
TABLE 3. Predominant microbes isolated from the ilea of beagle dogs housed in conventional or locked
environments a
Predominant microbes isolated from:
Genera
Group 1 dogs Group 2 dogs Group 3 dogs
Anaerobic and Bacteroides (1)b 107c Clostridium (4) 107-9 Bacteroides (1) 107
facultative Peptostreptococcus (1) 107 Eubacterium (2) 108 Clostridium (2) 105-6
Peptococcus (1) 107 Eubacterium (2) 1067
Bifidobacterium (1) 108 Bifidobacterium (1) 101
Lactobacillus (3) 103-7 Lactobacillus (4) 104-5
Total anaerobes 1.2 x l0"d 6.0 x 103, 4.4 x 109 4.0 x 108, 9.0 x 105
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Aerobic and Streptococcus (6) 1046 Streptococcus (6) 102-7 Streptococcus (4) 104-5
facultative Staphylococcus (1) 105 Staphylococcus (1) 105 Staphylococcus (1) 105
Corynebacterium (2) 1067 Moraxcella (1) 104 Corynebacterium (1) 105
Bacillus (2) 104-5 Enterobacter (1) 102 Micrococcus (3) 1034
Escherichia (1) 104 Klebsiella (1) 105
Rhodotorula (1) 103
Cladosporium (1) 103
Unidentified fungus (1) 103
Total aerobes 6.6 x 107 4.2 x 105, 3.1 x 107 6.5 x 105, 1.4 x 106
a
See footnote a, Table 2.
b Parentheses indicate the number of species found in the genus.
c Indicates the total population number(s) of the species within the genus.
d Indicates the total number of all genera (two numbers
indicate that two different samples were
examined).
TABLE 4. Predominant microbes isolated from the ceca of beagle dogs housed in conventional or locked
environments a
Predominant microbes isolated from:
Genera
Group 1 dogs Group 2 dogs Group 3 dogs
Anaerobic and Bacteroides (l)b 107c Bacteroides (3) 107-8 Bacteroides (4) 1019
facultative Peptostreptococcus (2) 107- Peptostreptococcus (1) 107
Clostridium (4) 1068 Clostridium (6) 107-
Eubacterium (2) 107-8 Eubacterium (1) 106
Bifidobacterium (2) 107-8 Lactobacillus (1) 106
Lactobacillus (1) 107 Fusobacterium (3) 107-8
Veillonella (1) 108
Peptococcus (1) 108
Total anaerobes 3 X 107d 4.5 x 108, 1.9 x 109 2.4 x 109, 7.7 x 109
Aerobic and Streptococcus (3) 104-5 Streptococcus (3) 1067 Streptococcus (3) 108
facultative Escherichia (1) 105 Proteus (1) 106
Enterobacter (1) 106
Klebsiella (1) 105
Alternaria (1) 105
Total aerobes 6.5 x 105 1.0 X 106, 3.1 x 107 2.2 x 108, 8.0 x 108
a-d See footnote
a, Table 2 and footnotes b-d, Table 3.
Tables 3, 4, and 5 indicate that the dogs Fusobacterium and Veillonella species (10' to
housed in the locked environments possessed a 108) in their ceca (Table 4), whereas the conven-
more complex microbial flora (22 genera and 52 tional (group 1) and transitional (group 2) dogs
species) in their ilea, ceca, and colons than the did not possess detectable CFUs of the latter
conventionally housed group (12 genera and 26 two genera at any site sampled. There were
species). The conventionally housed dogs other minor genera differences in the three
(group 1) did not have detectable Bifidobacter- groups of dogs, but these differences were found
ium species in any region of the GI tract. The in the minor components (103 to 106) of the
latter genus was a predominant member of the aerobic-facultative microflora. For example,
microbial flora (10" to 10'0) of the dogs in groups Staphylococcus aureus, which was found in the
2 and 3. Additionally, group 3 dogs harbored ilea of all dogs, was not found in the ceca of any200 DAVIS ET AL. APPL. ENVIRON. MICROBIOL.
TABLE 5. Predominant microbes isolated from the colons of beagle dogs housed in conventional or locked
environments I
Predominant microbes isolated from:
Genera
Group 1 dogs Group 2 dogs Group 3 dogs
Anaerobic and mi- Bacteroides (3)b 1O>'0 Bacteroides (4) 1074 Bacteroides (3) 101
croaerophillic Peptostreptococcus (1) 109 Peptostreptococcus (2) 109 Peptostreptococcus (1) 109
Peptococcus (1) 108 Clostridium (2) 108 Peptococcus (1) 109
Clostridium (2) 109 Eubacterium (1) 109 Clostridium (3) 107-8
Eubacterium (3) 109 Lactobacillus (5) 10 Eubacterium (3) 109
Lactobacillus (1) 1010 Bifidobacterium (1) 109 Lactobacillus (3) 107
Bifidobacterium (1) 1010
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Anaerobiospirillum (1) 105
Total anaerobes 8.6 x lOlod 1.9 x 108, 9.6 x 109 3.4 x 10"', 4.6 x 1010
Aerobic and facul- Streptococcus (3) 10910"0 Streptococcus (4) 108 Streptococcus (5) 101
tative Escherichia (1) 105 Escherichia (1) 106 Escherichia (1) 105
Proteus (1) 104 Enterobacter (1) 106 Proteus (1) 105
Staphylococcus (1) 104 Enterobacter (1) 105
Klebsiella (1) 105
Unidentified fungus (1) 103
Total aerobes 6.0 x 1010 5.6 x 107, 1.5 x 108 3.0 x 108, 8.0 x 108
a See footnote a, Table 2.
b Parentheses indicate the number of species found in the genus.
I
Indicates the total population number(s) ofthe species within the genus per gram (dry weight) oftissue and contents.
d Indicates the total number (per gram [dry weight] of tissue and contents) of all genera (two numbers indicate that two
different samples were examined).
of the dogs and was only found in the colons of surfaces, but the bacteria occurred singly or in
group 2 dogs, and a Veillonella species was only a small microcolony (Fig. 3). No population of
isolated from the ceca of group 3 dogs (Table 4). segmented filamentous bacteria was observed
One consistent finding was that Streptococcus to associate with the proximal small bowel epi-
was usually the most numerous of the faculta- thelium in any of the dogs studied (Table 6).
tive and aerobic genera, whereas the most nu- Dogs housed either in the conventional or
merous of the anaerobic genera varied from locked environments possessed no bacterial
sample to sample. populations that attached or layered to the dis-
Although many genera were consistently iso- tal ileum. Distal ilea in three out of four dogs
lated from ilea, ceca, and colons of these three from group 2, however, had segmented filamen-
groups of dogs, the species of these genera did tous organisms attached to their ileal epithelial
not segregate into any discernible groups ac- cells (Table 6). The attachment sites and seg-
cording to the dog housing conditons. Two spe- ments of the microorganisms were easily ob-
cies, however, did occur in every dog examined: servable in SEM examination of the dogs in
Streptococcus mitis and a Eubacterium species. group 2 (Fig. 4a). Both frozen and glutaralde-
This Eubacterium was unique because it pro- hyde-fixed specimens indicated that the seg-
duced long filaments and spontaneously lysed mented filamentous microbes were located un-
after 24 h of incubation. derneath a mucin layer (Fig. 4a and b).
Microscopy: light and scanning. In all of the Ceca from the three dog groups showed varia-
dog stomachs, the cardiac epithelium was usu- bility in the possession of a bacterial layer on
ally covered with a thick layer of mucin (Fig. their cecal surfaces. Conventionally housed
1), and bacteria were often undetectable. To dogs showed almost no bacteria on the epithe-
find any bacteria on the stomach epithelium, lial surface; similarly, two dogs from each ofthe
many fields had to be observed with specimens other two groups of dogs (2 and 3) housed under
prepared for both light microscopy and SEM. different conditions showed either no bacteria
Close examination of the stomach surface on the cecal epithelium or, at the most, a few
showed that two of the nine dogs possessed a regions with layering bacteria. In those ceca
bacterium that had unusually tight helical coils that lacked a layer of bacteria on a large por-
and bipolar flagella (Fig. 2). Unlike the murine tion of their cecal epithelium, a layer of mucin
stomach, no bacterial population layered or at- was present. Mucin predominated in the ceca of
tached to the stomach epithelium of the dog dogs housed conventionally (Fig. 5), which cor-
(Table 6). responded with a relatively lower (107 versus
The proximal small bowel contained a sparse 108 to 9) bacterial count (Tables 2 and 5). Ceca
bacterial population. Bacteria could occasion- with a bacterial layer (five out of nine ceca
ally be found in the GI lumen adjacent to villus [Table 5]) showed a predominantly heteroge-Downloaded from http://aem.asm.org/ on March 17, 2021 by guest
FIG. 1. SEM of the cardiac region of a dog stomach covered with a layer of mucin. Arrows show where
mucin layer has been split (probably a drying artifact) to reveal the underlying epithelium. x45.
FIG. 2. An unusual helical coiled bacterium with bipolar flagella (arrows) on a dog stomach. xl0,650.
FIG. 3. Proximal small bowel villi from a dog that shows mucin strands (double arrows) and a single bac-
terium (arrow) adjacent to the villi. x275.
FIG. 4. Segmented filamentous microbes, found only in dogs in transition from a locked to a conventional
environment (group 2 dogs), attached directly to distal ileal epithelial cells. Double arrows (a) indicate the
individual segments ofthese organisms and the single arrows show their attachment site. x920. (b) shows the
mucin that covers the microbes in the distal ileum. x960.
201202 DAVIS ET AL. APPL. ENVIRON. MICROBIOL.
TABLE 6. Layering and attachment of microbes in the GI tracts of beagle dogs housed in conventional or
locked environments a
Environment Microscopy Stomach Proximal ileum Distal ileum Cecum Colon
Group 1 Light 0/2b 0/2 0/2 0/2 2/2
SEM 0/2 0/2 0/2 0/2 2/2
Group 2 Light 0/4 0/4 3/4 3/4 4/4
SEM 0/4 0/4 3/4 2/4 2/4
Group 3 Light 0/3 0/3 0/3 1/3 1/3
SEM 0/3 0/3 0/3 2/3 2/3
Total 0.9 0/9 3/9 5/9 8/9
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a
See footnote a, Table 2.
bNumber of dogs with layering or attaching microorganisms per total number of dogs examined by either
light microscopy or SEM.
nous bacterial population on the cecal epithe- DISCUSSION
lium. The cecal layer, when it was observed, Almost all previous studies of the microbial
consisted mainly of gram-positive rods and flora of dogs have been limited to an elucidation
cocci and some gram-negative (mainly rods) of the fecal or nasopharyngeal flora (3, 22).
bacteria (Fig. 6 and 7). Studies of the bacterial flora located in and on
Layers of bacteria, composed primarily of the surface of canine GI tissues usually re-
gram-positive rods and cocci, were most promi- ported only the total counts of genera cultivated
nent on colonic epithelium of the dogs from on selective media; identification of species, es-
groups 2 and 3 (see Fig. 7 for similar results pecially of the anaerobic genera, was rarely
from the cecum). Dogs of group 1 and occasion- attempted (3, 22). This study detailed the ge-
ally those of groups 2 and 3 (in some regions of nera and species, as far as they could be identi-
the colon in dogs of the latter two groups) mani- fied with our methods, in the distal ileum,
fested a distinct microbial population that con- cecum, or colon of the dogs. It indicated that
sisted of gram-negative spiral- and rod-shaped bacteria did not localize on the epithelial sur-
microbes. This layer, which was distinct from face of dog stomach or proximal small bowel but
the layer shown in Fig. 7, of bacteria separated could localize in the distal ileum, cecum, and
the bulk of the lumenal flora from the colonic colon. Total counts of anaerobes were about 1 to
epithelial cells (Fig. 8) and was often difficult to 2 logs higher than were the total counts of
see unless the tissue-gram-stained sections aerobes, except in the conventionally housed
were viewed with phase optics (cf. Fig. 9 with dogs, whose colon counts yielded approximately
Fig. 8). SEM observation of the latter microbes, the same numbers (1010) of anaerobic and facul-
which were most numerous in the colons of tative bacteria. Such high numbers of aerobic
group 1 dogs, indicated that the population was and anaerobic bacteria have been reported pre-
heterogenous with respect to the rods and spi- viously in dog feces (3), although the anaerobes
rals present (Fig. 10a and b). were usually about 1 log higher than were the
All of the dogs with either a predominantly aerobes (108 to 9 versus 109 to 10).
gram-positive or a gram-negative layer in We observed a wide variation in isolatable
either the cecum or the colon possessed spiral- bacterial species from dog to dog regardless of
and rod-shaped bacteria in their crypts of Lie- housing conditions. However, an examination
berkuhn. Some crypts had only spiral- or rod- of the predominant genera isolated from all
shaped bacteria, whereas other crypts had both three groups of dogs showed only one case
types present (Fig. 9). Dogs were also able to where a major difference in the number of ge-
have bacteria in the crypts without a discerni- nera was detected. This difference in genera
ble layer of bacteria on their epithelial cells. was observed in the ceca of the conventionally
The density of this microbial population associ- housed group 1 dogs where only two genera,
ated with crypts varied from crypt to crypt in Bacteroides and Streptococcus, were detected.
the cecum and colon of each animal. Some The latter observation was the most obvious
crypts possessed large numbers of bacteria part of a general trend that suggested that
(Fig. 11), whereas other crypts had few or none. conventionally housed dogs possessed a less
Conventionally housed dogs (group 1) had eas- complex flora, in terms of genera and species
ily detectable bacteria within their crypts; dogs present, than either of the other groups of dogs
in group 2 had only a few bacteria in their (cf. Tables 3, 4, and 5) that were housed in a
crypts, and only one of the three dogs from locked environment.
group 3 showed any bacteria in its crypts of A study on the fecal flora of man in locked
Lieberkuhn. environments showed that his fecal flora doesDownloaded from http://aem.asm.org/ on March 17, 2021 by guest
M , -
-
FIG. 5. An example of the thick layer of mucin (m) that covers the dog cecum and overlays most ofthe cecal
epithelium (e). Bacteria were difficult or impossible to locate in such ceca (in the mucin or on the epithelium)
in contrast to other dog ceca that possessed a layer of bacteria (see Fig. 6). x95.
FIG. 6. Light micrograph of a frozen section of the gram-positive layer of bacteria on a dog's colonic
epithelium (e) stained with a tissue Gram stain. This type oflayer was also seen on the cecum. Arrows indicate
some of the gram-negative bacteria that also could be observed in this layer. x1,320.
FIG. 7. SEM of the gram-positive layer of bacteria found on a dog's cecal surface. Note the predominance of
rods, cocci, and coccobacilli, and compare this figure with Fig. 10. Arrow indicates the epithelial cell surface.
x3,500.
FIG. 8. Light micrograph of a layer (shown by bars) that separates the colonic epithelium (lower left side)
from the lumenal contents (right side). A weakly staining gram-negative (spiral- and rod-shaped) bacterial
population inhabits this layer. The latter bacteria are best viewed with light microscopy at a high magnifica-
tion and with phase optics (see Fig. 9). x195.
FIG. 9. Light micrograph of a frozen section of a dog colon (tissue Gram stained) that shows spiral- and
rod-shaped bacteria in the layer of mucin. x2,365.
203204 DAVIS ET AL. APPL. ENVIRON. MICROBIOL.
not markedly change with a short-term (about
2 months) confinement; however, subtle altera-
tions can take place (13). Other studies on con-
finement suggest that the microbial and fungal
flora increase after short- (16 days) or long-term
(12 months) confinement (17, 25, 26). Our study
suggests that in dogs housed in a locked envi-
ronment for over 2 years, the colonic flora does
not markedly change, but some other changes,
such as an increase in the diversity of genera
and species present, may occur in the cecal and
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ileal flora. The group 2 dogs had a flora inter-
mediate in complexity between groups 1 and 3
(cf. Tables 3 through 5), which suggests that
upon removal ofthe dogs from a locked environ-
ment, the flora begins to simplify and thus
resembles the flora in conventionally housed
animals.
Most fungi were detected in those dogs kept
in the locked environment (group 3). Results
from our study should not be construed to mean
that because organisms (both bacterial and fun-
gal) were not cultured they were not present.
Our results simply mean that the organisms
were not detected by our culture techniques;
they could be present in such low numbers (1 to
103) that many culture samples and highly se-
lective media would be needed to detect them.
Another source of error may be due to selection
of strains to study on the basis of colony counts;
bacterial species that have colonies visibly
identical to colonies of strains isolated may be-
long to different species. Also, only 1 composite
sample from each site in dogs of group 1 were
studied. In addition, AII medium, although one
of the best enriched nonselective media we
have used for growing anaerobes, probably did
not allow us to culture all of the bacteria pres-
ent. For example, morphologically distinct mi-
crobes (spirals, types a, b, and c, Fig. 10) were
never cultured. Thus, SEM results show that
not all the bacteria present can, as yet, be
cultured.
Although no difference in the microflora was
observed in the stomachs or proximal small
bowels of dogs housed under different condi-
tions, changes in the microflora were pro-
nounced when their ileal, cecal, and colonic
tinct spiral-shaped bacteria can be observed: (a)
short and thin spiral; (b) wavy outer-enveloped cov-
ered spiral (see also [B] for types a and b); (c) spiral
(vibrio) with a single polar flagellum; (d) spiral with
bipolar flagella. x3,000. (B) Enlargement of(A) that
shows a and b organisms. x9,600. (C) Rods (e,f),
coccobacilli (g,h), and cocci (i). Note the fine fila-
ments attached to organism h. x9,600.
FIG. 11. Colonic crypt of Lieberkuhn in a light
FIG. 10. Heterogenous population of spiral- and micrograph showing a predominant population of
rod-shaped bacteria found adjacent to a dog's colonic thin, gram-negative rods. The "c"-shaped structures
epithelium (A). At least four morphologically dis- are goblet cells (G) that empty into the crypt. x3,015.VOL. 34, 1977 BACTERIA IN BEAGLE GI TRACTS 205
surfaces were examined by light microscopy or layering of bacteria in the canine GI tract was
SEM. For example, only group 2 dogs, in tran- somewhat different from that described in mu-
sition from the locked environment to the con- rine species (20). In dogs, bacterial populations
ventional, showed segmented filamentous orga- adjacent to the epithelial cells were much more
nisms in their ilea. In addition, the bacterial sparse and contained markedly fewer fusiform-
population that comprised the epithelial cell shaped bacteria than those bacterial popula-
layers differed; the conventionally housed dogs tions found in mice and rats. Additionally, an-
possessed a distinct gram-negative bacterial other type of layer, composed mainly of gram-
layer adjacent to the colonic epithelium, but the positive organisms, was observed in the dogs.
locked-environment dogs showed mainly a In mice, a predominance of gram-positive mi-
gram-positive bacterial layer. Furthermore, ob- crobes adjacent to the large bowel epithelium
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vious differences between group 1 and groups 2 strongly suggests that they have an altered
and 3 were observed at the frequency with microbial flora (19). The occurrence of bacteria
which crypts of Lieberkuhn were populated by (mainly spiral-shaped microbes) has been noted
bacteria; confinement appeared to suppress in the crypts of Lieberkuhn of rat ceca (7) and
crypt populations. Thus, differences in the lay- in the large intestines of randomly bred dogs
ering and localization of the microbial flora (15). Our observations in dogs generally agree
were noted in the three groups of dogs, but the with the latter findings.
reasons for the changes are not clear. If the most numerous bacterial genera and
It is known that either antibiotics, diet, or species from the GI tracts of dogs are compared
environmental stress, or a combination of these with those found in the GI contents of man
factors, can alter the composition, attachment, (W.E.C. Moore, M. Ryser, and L. V. Holde-
and layering of the GI flora (9, 19, 24). Of these man, Abstr. Annu. Meet. Am. Soc. Microbiol.
factors, only the housing environment was var- 1975, DS7, p. 61), several differences and simi-
ied in our study, and flora changes were noted. larities in the type and number of genera pres-
Although each of two dogs (group 2) was fed ent can be found. For example, the most nu-
autoclaved and nonautoclaved Purina Dog merous genera uniformly found in the dog ilea
Meal, their flora was not markedly changed and large bowel are Bacteroides and Streptococ-
from that of the other group 2 dogs. cus (S. bovis and S. acidominimus). Although
The observation that segmented filamentous the latter are among the predominant genera,
organisms were found in group 2 dogs but did they are not the most numerous bacteria in the
not occur in dogs housed under either conven- GI contents of man. Fusobacterium, a predomi-
tional or locked environments is of interest but nant genus found in man, was not as numerous
should be confirmed by further studies. Three in dogs (approximately 1010 `O 11 versus 107 to 8).
samples (one for light microscopy and two for The Clostridium species predominant in man
SEM) were taken from adjacent regions of each are easily identified but those in the dog are not
dog ileum, but such sampling may not detect the (Table 1). Genera that are numerous in both
organisms. For example, it is known that the the GI tract of dogs and man are Lactobacillus,
segmented filamentous microbes colonize non- Bifidobacterium, Eubacterium, Bacteroides,
uniformly the ilea of the murine species; that and Peptostreptococcus. Localization of GI flora
is, the segmented filamentous microbes may be in man, in terms of either bacterial layer for-
found both proximal and distal to a region of mation or bacterial populations in crypts of Lie-
the ileum that possesses no such organisms (8). berkuhn have not, to our knowledge, been re-
Although it is very unlikely that we would miss ported. Our work with dogs suggests that other
by chance segmented filamentous microbes in higher mammals, including man, may possess
five of the nine dogs (groups 1 and 3) and find such localized bacterial populations.
them in three out of the four dogs of group 3, it Our results and those of other investigators
is possible. To our knowledge, no other reports (3, 13, 17) do not support the concept that the
on the occurrence of these segmented filamen- microbial flora of mammals will undergo a dra-
tous bacteria in beagles or other dogs are avail- matic change (simplification to one or two gen-
able. era) if confined to a locked environment and fed
This study indicates that specific regions of sterile food and water for a long time period
the GI tracts of higher mammals such as dogs (16). Indeed, our results indicate that with
can possess populations of bacteria that attach long-term confinement, the flora becomes more
and localize, either in crypts of Lieberkuhn or complex, and its distribution along the GI tract
in layers. Attachment of segmented filamen- changes. We hypothesize that microorganisms
tous microbes to epithelial cells paralleled that that are not predominate members of the mi-
found in murine species (4, 8, 12), but the fre- croflora (transient microflora), but which are
quency of their occurrence in dogs was less. The initially carried into the locked environment by206 DAVIS ET AL. APPL. ENVIRON. MICROBIOL.
any animals, will have to adapt to the environ- sion, attachment, and morphology of segmented, fila-
ment to survive. In addition, the chance for mentous microbes indigenous to the murine gastroin-
testinal tract. Infect. Immun. 10:948-956.
reassociation with the animal in a locked envi- 9. Davis, C. P., and D. C. Savage. 1976. Effect of penicillin
ronment is much greater than in a conven- on the succession, attachment, and morphology of
tional environment. Thus, adaptation to the segmented filamentous microbes in the murine small
locked environment and frequent reassociation bowel. Infect. Immun. 13:180-188.
10. Drasar, B. S., and M. J. Hill. 1974. Human intestinal
(i.e., ingestion) could select for those microbes flora. Academic Press Inc., New York.
that are not predominate members of the flora. 11. Erlandsen, S. L., and D. G. Chase. 1974. Morphological
Ultimately, successful competition could lead alterations in the microvillous border of villous epi-
to an increase in the number and variety of thelial cells produced by intestinal microorganisms.
Am. J. Clin. Nutr. 27:1277-1286.
microorganisms in the GI tracts of the animals 12. Erlandsen, S. L., A. Thomas, and G. Wendelschafer.
Downloaded from http://aem.asm.org/ on March 17, 2021 by guest
within the locked environments. Alternatively, 1973. A simple technique for correlating SEM and
a hypothesis that the microenvironments of GI TEM on biological tissue originally embedded in
tracts in animals held in locked environments epoxy resin for TEM, p. 350-365. In 0. Johari and I.
Covin (ed.), Scanning electron microscopy, 1973. ITT
may change with time should also be consid- Research Institute, Chicago.
ered. Such an alteration in the GI tract also 13. Holdeman, L. V., I. J. Good, and W. E. C. Moore. 1976.
could allow other less predominate species to Human fecal flora: variation in bacterial composition
proliferate. within individuals and a possible effect of emotional
stress. Appl. Environ. Microbiol. 31:359-375.
As indicated by our data, whether prolonged 14. Holdeman, L. V., and W. E. C. Moore (ed.). 1972.
contact with such an increase in the numbers Virginia Polytechnic Institute anaerobe laboratory
and variety of microorganisms could pose a manual. Virginia Polytechnic Institute, Blacksburg,
threat to a host is simply not known. Nonethe- Va.
15. Leach, W. D., A. Lee, and R. P. Stubbs. 1973. Localiza-
less, the flora should be monitored carefully tion of bacteria in the gastrointestinal tract: a pos-
when humans are confined in close chambers sible explanation of intestinal spirochaetosis. Infect.
for any considerable length of time. Immun. 7:961-972.
16. Luckey, T. 0. 1966. Potential microbic shock in manned
ACKNOWLEDGMENTS aerospace systems. Aerosp. Med. 37:1223-1228.
17. Puleo, R. J., G. S. Oxborrow, N. D. Fields, C. M.
We would like to thank Bangio Wong and Jim Brown for Herring, and L. S. Smith. 1973. Microbiological pro-
their excellent technical assistance. files of four Apollo spacecraft. Appl. Microbiol.
This study was supported by Public Health Service Medi- 26:838-845.
cal Microbiology and Immunology Training grant A100451- 18. Savage, D. C., and R. Blumershine. 1974. Surface-sur-
04 from the National Institute of Allergy and Infectious face associations in microbial communities populat-
Diseases, NASA grant NGR-50-002-191, and NIH RR 05427. ing epithelial habitats in the murine gastrointestinal
LITERATURE CITED ecosystem: scanning electron microscopy. Infect. Im-
mun. 10:240-250.
1. Aranki, A., and R. Freter. 1972. Use of anaerobic glove 19. Savage, D. C., and R. Dubos. 1968. Alterations in the
boxes for the cultivation of strictly anaerobic bacte- mouse cecum and its flora produced by antibacterial
ria. Am. J. Clin. Nutr. 25:1329-1334. drugs. J. Exp. Med. 128:97-110.
2. Aranki, A., S. Syed, E. Kenney, and R. Freter. 1969. 20. Savage, D. C., R. Dubos, and R. W. Schaedler. 1968.
Isolation of anaerobic bacteria from human gingiva The gastrointestinal epithelium and its autochtho-
and mouse cecum by means of a simplified glove box nous bacterial flora. J. Exp. Med. 127:67-75.
procedure. Appl. Microbiol. 17:568-576. 21. Smith, H. W. 1965. Observations on the flora of the
2a.Balish, E., J. F. Brown, and T. D. Wilkins. 1977. alimentary tract of animals and factors affecting its
Transparent plastic incubator for the anaerobic glove composition. J. Pathol. Bacteriol. 89:95-122.
box. Appl. Environ. Microbiol. 33:525-527. 22. Smith, H. W., and W. E. Crabb. 1961. The fecal bacte-
3. Balish, E., C. Shih, C. E. Yale, and A. Mandel. 1974. rial flora of animals and man: its development in the
Effect of a prolonged stay in a locked environment on young. J. Pathol. Bacteriol. 82:53-66.
the microbial flora in dogs. Aeros. Med. 45:1248-1254. 23. Suegara, N., M. Morotomi, T. Watanabe, Y. Kawai,
4. Davis, C. P. 1976. Preservation of gastrointestinal bac- and M. Mutai. 1975. Behavior of microflora in the rat
teria and their microenvironmental associations in stomach: adhesion of lactobacilli to the keratinized
rats by freezing. Appl. Environ. Microbiol. 31:304- epithelial cells of the rat stomach in vitro. Infect.
312. Immum. 12:173-179.
5. Davis, C. P., D. Cleven, J. Brown, and E. Balish. 1976. 24. Tannock, G. W., and D. C. Savage. 1974. Influences of
Anaerobiospirillum, a new genus of spiral-shaped dietary and environmental stress on microbial popu-
bacteria. Int. J. Syst. Bacteriol. 26:498-504. lations in the murine gastrointestinal tract. Infect.
6. Davis, C. P., J. S. McAllister, and D. C. Savage. 1973. Immun. 9:591-598.
Microbial colonization of the intestinal epithelium in 25. Taylor, G. R., M. R. Henney, and W. I. Ellis. 1973.
suckling mice. Infect. Immun. 7:666-672. Changes in the fungal autoflora of Apollo astronauts.
7. Davis, C. P., D. Mulcahy, A. Takeuchi, and D. C. Appl. Microbiol. 26:804-813.
Savage. 1972. Location and description of spiral- 26. Zaloguyey, S. N., T. G. Utkina, and M. M. Shin Kar-
shaped miroorganisms in the normal rat cecum. In- eva. 1971. The microflora of human integument dur-
fect. Immun. 6:184-192. ing prolonged confinement. Life Sci. Space Res. 9:55-
8. Davis, C. P., and D. C. Savage. 1974. Habitat, succes- 59.You can also read
