MICROBIALECOLOGY IN HEALTH AND DISEASE
VOL.
1: 237-243 (1988)
Suppression of Potential Pathogens by a Defined Colonic
Microflora
KENNETH WILSON*?, LILLIAN MOORE$, MAYURIKA PATEL? and PATRICIA PERMOADt
?Infectious Disease Section, Ann Arbor Veterans Administration Medical Center and Department of Internal Medicine,
University of Michigan, Ann Arbor, Michigan USA.
f: Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA.
Received 29 February 1988; revised 30 May 1988
Animals acquiring a microflora for the first time do so through the gradual process of ecologic succession. A defined
microflora was derived by experimentally simulating this process in gnotobiotic mice. Diverse bacterial species were
obtained from ex-germfree mice acquiring a microflora from a conventional mouse. All isolates were characterised to
the genus level and polyacrylamide gel electrophoresis (PAGE) was performed on soluble proteins to differentiate
isolates at the species or subspecies level. Selected isolates were fully identified. Only 21 per cent of taxa had been
previously described. Organisms isolated early in succession tended to ferment more diverse carbohydrates
(mean SD = 10.2f8.6 carbohydrates per isolate) than organisms found in climax stage mouse flora (5.7 f7.0).
Bacterial species which predominated during ecologic succession, but which later comprised only a small part of the
microflora, were important in simulating certain functions of the entire flora.
The defined flora suppressed Escherichia coZi to 106.6colony forming units (CFU) per caecum, a degree of suppression similar to that caused by the entire caecal flora. Pseudomonas aeruginosa was suppressed less by the defined
flora (104'4)than by the entire caecal flora (lO''o). While Clostridium dzjicile was suppressed much less by the defined
flora ( 106.6)than by the entire flora (undetectable), animals colonised with the defined flora were protected from colitis
when challenged with a pathogenic strain of C . dzficile.
KEY WORDS -Gnotobiotics;
Colitis; Clostridium dzjicile; Anaerobes: Colonisation resistance.
INTRODUCTION
The indigenous colonic microflora is known to
limit the population sizes of a wide variety of
potentially pathogenic bacterial species, such as
,~
antibiotic-resistant Escherichia c ~ l i Salmonella
S P . , ~Shigella f E e ~ n e r i , ~Clostridium
~ ~ * ' ~ dificile,2'
Pseudomonas aeruginosa4*' and many other organisms with a pathogenic potential greater than the
indigenous microflora. This function of the colonic
flora is of substantial benefit to the host but has yet
to be fully exploited in clinical practice.
Technology to take advantage of the very significant beneficial effects of the indigenous flora will
probably require the use of defined microfloras consisting of collections of isolates which have been
characterised to some extent. Such collections have
been shown to exert desired functions of the entire
although usually for full expression of a
given function the presence of nearly the entire flora
*Author to whom correspondence should be addressed.
089 1460X/88/040237-07 $05.00
0 1988 by John Wiley & Sons, Ltd.
appears to be necessary. Syed et a1.I' and Freter and
Abrams7 inoculated very large collections of isolates from mouse caeca into gnotobiotic mice and
almost fully reproduced the effect of the entire
caecal flora on caecal size and histology, and on the
population size of streptomycin-resistant E. coli.
The E. coli had been established first in germfree
mice, then allowed to come to equilibrium with the
subsequently inoculated defined floras. The work of
Freter and Abrams7 showed that the predominant
anaerobic flora was important for these functions as
95 anaerobic isolates more closely approximated the
function of the entire flora than did the previous
collection of aerobes and anaerobes reported by
Syed et al.
There are many reports of defined microfloras
inducing either complete or partial resistance
to colonisation by potentially pathogenic bacteria.3v5,'5 , 2 0 However, in most of these examples,
the microflora has antagonised the pathogen
only if the microflora has been introduced into
238
K. WILSON ET AL.
experimental animals before the pathogen. Under Bacterial Isolates
natural conditions, it would be difficult to assure
C. dificile strain 49A was originally isolated from
this sequence of events. Another drawback to relying entirely on colonisation is that the population a hamster with caecitis, is toxigenic and causes
size of a potential pathogen measured shortly after colitis in hamsters, but not in mice. C . dificile strain
challenge of an experimental animal may be lower VPI 10463 is hypertoxigenic and causes colitis in
than the population size attained later once equilib- both hamsters and mice.” E. coli strain C25 is a
rium has been e~ tab lish ed .~For
.’~these reasons, the streptomycin resistant mutant of an isolate from
florae have been studied at equilibrium in this human faeces and has been used in several previous
studies of the colonic m i c r ~ f l o r a . ” ~ . ~
The
. ’ ~strain
report.
We recently reported efforts to expand the obser- of P . aeruginosa (provided by Dr. Paula Jones of
vations of Syed et a1.” and of Freter and Abrams’ M.D. Anderson Hospital, Houston, Texas) was an
by studying the ability of the predominant anaero- isolate from the blood of a neutropenic patient.
Because C . dificile has no effect on the population
bic flora of hamsters to suppress C . dificile.”
Collections of as many as 150 isolates were obtained size of E. coli,20 and the defined microflora conon a medium able to recover from hamster caeca tained other strains of E. coli anyway, C . dificile and
50-100 per cent of the organisms counted microsco- E. coli C25 were inoculated together into germfree
pically. When introduced into gnotobiotic mice, mice. However it was found that C . dificile and E.
these isolates only slightly suppressed C . diBcile. coli suppressed P . aeruginosa (see below), so P . aeruAn effort was made to diversify the collection of ginosa was studied separately.
Four sets of isolates were obtained during
isolates as follows: A smaller collection of such isolates (N = 100) combined with 67 isolates obtained ecologic succession, and will be referred to as the S
from a continuous flow culture during ecologic (succession) flora. The S flora was obtained by
succession (i.e. prior to establishment of equilibrium placing a conventional mouse in an isolator with
among the various bacterial populations derived germfree mice. The conventional mouse was caged
from hamster caeca) suppressed both C . dificile and above the germfree mice such that its faecal pellets
E. coli significantlymore than had the collections of fell into the cage containing the germfree animals.
isolates taken only from the predominant flora at As the formerly germfree mice acquired the conventional mouse’s flora, one mouse was removed from
equilibrium.
The present study followed a similar experimental the isolator on days 2, 5, 8 and 10 after association
approach and was undertaken to gain more detailed with the conventional mouse and sacrificed by CO,
information about the composition of defined anaesthesia. Caeca were homogenised in 30 ml of
microfloras obtained using our methodology, and pre-reduced trypticase soy broth. Serial 10-fold
to establish whether or not crossing species lines dilutions were seeded on modified Bryant medium
(introducing hamster flora into gnotobiotic mice) 10 and Aranki-Freter medium’, and incubated inhad affected results in the previous study. In side an anaerobic chamber for 5 d at 37°C in plastic
addition, the scope of the study was expanded to bags containing palladium-coated aluminium pellook at suppression of P . aeruginosa as well as of lets. Microscopic counts of caecal homogenates
C . dificile and E. coli, and to find whether or not were performed in a Petroff-Hawser chamber. Colthe defined microflora would actually prevent the onies were sub-cultured only if the viable count was
greater than 50 per cent of the microscopic count.
development of antibiotic-associated colitis.
Sufficient colonies were taken to sample all colony
types. Finally, the conventional mouse used to
MATERIALS AND METHODS
donate the flora was sacrificed, and its caecal contents were cultured only on M 10, as Aranki-Freter
Animals
medium was found consistently to recover less than
Germfree CD- 1 mice, originally obtained from 50 per cent of the microscopic count from such mice.
Charles River Breeding Laboratories, Wilmington, This collection of isolates from the predominant
MA, were maintained and bred in germfree Trexler- caecal flora of the conventional mouse will be
type polyvinyl germfree isolators. Conventional termed the C (climax stage) flora. Isolates from the
Balb mice were obtained from Dr. William Murphy C flora were sub-cultured at random from the
in the Department of Microbiology and Immu- primary isolation medium. All isolates were subcultured in broth consisting of modified Bryant
nology at the University of Michigan.
DEFINED COLONIC MICROFLORA
medium 10 (M10) without agar and sub-cultured
repeatedly until heavy growth was obtained. Isolates were pooled and 0.05 per cent dithiothreitol
added. The suspension was placed in glass vials,
sealed anaerobically, and sterilised externally with 2
per cent peracetic acid in the locks of Trexler-type
polyvinyl germfree isolators. The contents of the
vials were then administered to gnotobiotic mice.
Inoculation of Mice
Animals were kept in sterile Trexler-type polyvinyl isolators with access through a stainless steel
lock sterilised with 2 per cent peracetic acid. The
mice were inoculated with overnight broth cultures
of C . dificile 49A, E. coli, and P . aeruginosa intragastrically through steel feeding needles, and subsequently inoculated with isolates from caecal flora.
The isolates from caecal flora were inoculated into
the gnotobiotic mice in the order in which they
were obtained during ecologic succession. Animals
were first given 0.5 ml of a sterile solution of 7-5 per
cent sodium bicarbonate equilibrated with CO,
to neutralise gastric pH followed by 0.5ml of the
bacterial suspension intragastrically and 1-0ml per
rectum to each animal. Suspensionsofeach group of
isolates were inoculated into mice three times over a
two week period. The defined flora was then allowed
to equilibrate for three weeks before mice were
sacrificed by cervical dislocation for quantitation
of bacterial population sizes and caecal size.
Quantitative Cultures
Population sizes of C . dificile were measured
by sacrificing mice and taking them into an
anaerobic chamber, homogenisingthe caeca in 30 ml
TSB and seeding serial dilutions onto cefoxitincycloserine-fructose agar’ containing 0.05 per cent
sodium cholate. Dilution tubes were then taken
from the anaerobic chamber and inoculated onto
desoxycholate agar containing 100 mg/ml streptomycin or 10mg/ml cefotaxime to estimate the
numbers of E. coli and P . aeruginosa respectively.
IdentiJicationof Isolates
Taxonomy of the anaerobic colonic microflora is
time consuming and difficult. For that reason our
overall approach was to place isolates in a genus and
perform polyacrylamide gel electrophoresis of soluble proteins (PAGE) to “fingerprint” them.I4
Briefly, all isolates were streaked onto the surface of
239
MI0 and/or Aranki-Freter medium, and were incubated in 5 per cent CO, to determine whether or not
they were anaerobic. Smears of bacterial cultures
were Gram-stained using the Koploff modification” and treated with tannic acid and stained
with fuschin to detect flagella. Analysis of bacterial
cultures for non-volatile organic acids and volatile
fatty-acids was performed using standard
methods.” Two-week old cultures were heated to
80°C for 10 minutes to test for spores.
For PAGE, isolates were grown in MI0 broth
until they reached a turbidity equivalent to
McFarland standard #3 or had grown for 5d;
occasionally an isolate did not grow adequately for
further testing in 5 d and exceptions were made in
such cases to allow growth for up to 2 wks. The
duration of growth necessary was reproducible for a
given isolate. The culture was then centrifuged at
7000 x g for 10 min in a refrigerated centrifuge and
the supernatant removed. The pellet was processed
for PAGE by the method of Moore et a l l 4 Glass
beads and tris buffer, pH 7, were added to the pellet,
and the test tube was shaken vigorously on ice to
cause cell breakage. The specimen was recentrifuged
and 35 ml of the supernatant was placed in a well
of a 1.5mm-thick polyacrylamide slab gel and
electrophoresed with 150 V constant voltage at
25°C ambient temperature. Stacking gels were of
4.5 per cent polyacrylamide and resolving gels 8.5
per cent polyacrylamide. The buffer system was
discontinuous and the pH of the resolving gel was
8.8. Gels were stained with Coomassie blue. If
staining with Coomassie blue yielded weak band
patterns, duplicate lanes were stained with Kodak
Kodavue stain. Representative isolates were also
chosen at random and fully identified by means of
standard methods.”
Statistical Analysis
Population sizes were converted to log, values
and student’s t-test used for comparisons.
RESULTS
Composition of Defined Microfloras
Tables 1 to 3 show the results of identification
procedures. The majority (79 per cent) of isolates
did not belong to established species. The composition of both the S flora and the C flora was complex. On the second day of succession 25 isolates
were collected, 15 of which were E. coli, other
aerobic bacilli or S. fuecalis. The ten anaerobic
isolates gave six different band patterns by PAGE;
240
K. WILSON ET AL.
Table 1. Identification of isolates obtained on days 2 and 5
of ecologic succession (S flora). Anaerobes with species
names were identified fully by standard techniques;others
were distinguished on the basis of band patterns on
polyacrylamidegel electrophoresis of soluble proteins
No. times
isolated*
Escherichia coli
Enterobacter
Enterococcus
Aerobic Gram-positive rod
Clostridium M-1
Clostridium M-4
Clostridium ramosum
Clostridium cochleatum
Day 5 of succession
Clostridium ramosurn
Unidentified Fusobacterium
(7 PAGE patterns)
Fusobacteriurn M-2, M-3
Unidentified Bacteroides
(2 PAGE patterns)
Bacteroides M - I , M-2
Clostridium M-I
Clostridium M-5
Eubacterium M-2
Day 8 of succession
No. times
Day 2 of succession
Species
Table 2. Identification of isolates from days 8 and 10 of
succession
4
10
1 each
3
1 each
2
1
2
'Number of times an organism of the taxon in the left-hand
column was identified. For example, there were 10 Fusobacteria
isolated of day 5; these 10 isolates produced a total of 7 PAGE
patterns.
an example of each isolate was identified fully and
all belonged to four species of Clostridia (Table 1).
All of the 26 isolates picked on the fifth day of
succession were anaerobes yielding 17 different
band patterns by PAGE, none of which were yielded
by bacteria present on the second day. Although
Clostridium M-1 was detected on both days, band
patterns for these strains were distinguishable.
Although all 17 isolates that represented the 17
different PAGE patterns were inoculated into
gnotobiotic mice, only nine survived to be fully
identified and were found to belong to eight species
(Table 1). Isolates from the eighth and tenth days
were identified only to the genus level and characterised by PAGE. On day 8 (Table 2), there were one E.
coli, 24 Bacteroides, five Fusobacteria, four Eubacteria and one anaerobic non-fermenter with a polar
flagellum which did not fit into any known genus. Of
Species
isolated
Escherichia coli
(1 PAGE pattern)
Bacteroides sp.
(1 PAGE pattern)
Bacteroides sp.
(1 PAGE pattern)
Bacterodies sp.
(1 PAGE pattern)
Bacteroides sp.
(2 PAGE patterns)
Bacteroides sp.
(6 PAGE patterns)
Fusobacterium sp.
(5 PAGE patterns)
Eubacterium sp.
(1 PAGE pattern)
Genus undetermined
1
6
5
3
2 each
1 each
I each
4
1
Day 10 of succession
Bacteroides sp.
(1 7 PAGE patterns)
Fusobacterium sp.
(1 PAGE pattern)
Fusobacterium sp.
(3 PAGE patterns)
Butyrovibrio sp.
(1 PAGE pattern)
Eubacterium sp.
Clostridium sp.
(2 PAGE patterns)
Genus undetermined
1 each
2
1 each
1
1
1 each
1
the 19 different band patterns found among isolates
from day 8, only two had been detected on a previous
day. On the tenth day there were 17 Bacteroides, one
Butyrovibrio, five Fusobacteria, one Eubacterium,
two Clostridia and one isolate which did not fit into
any genus. Twenty-six separate band patterns, only
one of which had been found previously, were
detected. Among the 69 isolates taken from the C
flora, there were 54 different band patterns; none
had been detected prior to climax stage. Isolates
representing 20 of these band patterns were picked
at random to be identified fully (Table 3).
24 1
DEFINED COLONIC MICROFLORA
Table 3. Twenty isolates from the climax stage (C) flora
identified by standard techniques
Species
Fusobacterium M4
Fusobacterium M6
Eubacterium M3
Eubacterium M4
Eubacterium M5
Eubacterium M6
Eubacterium M7
Eubacterium plexicaudatwn
Peptostreptococcus MI
Bacteroides M2
Bacteroides M3
Bacteroides uniformis
Clostridium M3
Clostridium M4
Clostridium M4
Clostridium M5
Clostridium M6
Clostridium M7
Clostridium M8
Clostridium M9
No. of
occurrences*
1
1
2
1
1
2
1
1
1
3
1
1
2
2
1
1
1
2
1
2
Isolates representing 20 of the 54 band patterns found on PAGE
of isolates from climax stage were identified fully.
*The number of times each of these species occurred among the
69 isolates making up the C flora. Some species produced more
than one PAGE pattern.
Metabolic Activities of Isolates
The standard methods used to identify anaerobesI2 include tests for a wide variety of metabolic
reactions. Since bacteria in the colonic ecosystem
are thought to multiply under carbohydrate-limited
conditions, we looked at the relative abilities of
our isolates to utilise carbohydrates as substrates,
and compared organisms isolated early in ecologic
succession with those isolated at climax stage.
There was a great deal of overlap in the number of
different substrates which organisms isolated on
different days were able to utilise. However, at least
as indicated by the in vitro tests used to classify
anaerobes, there was a tendency for isolates from
climax stage to be more specialised in their utilisation of carbohydrates, i s . to be capable of
fermenting fewer carbohydrates than organisms
present early in succession. When isolates from
days 2 and 5 were looked at as a group they were
capable of utilising a mean of 10.2f8-6 (fSD)
carbohydrates, while isolates from climax stage
could utilise only 5.7 f7.0 (t = 1.84,p < 0-05).
Conventionalisationof Mice
Table 4 shows data on the degree to which the S
flora ,C flora and combined S C flora suppressed
C . dificile, E. coli and P . aeruginosa,and normalised
caecal size. Statistically, there was a highly significant trend for the S + C flora to have a more marked
effect than either the S or the C flora in normalising
these measures. The population size of C. dificile in
the presence of the full S + C flora was only one
hundredth its population size in monoassociated
mice (2 log,, decrease, Table 4). Similarly, the S C
flora reduced E. coli and P . aeruginosa to one three
thousandth and one thirty thousandth respectively,
of their original population sizes in monoassociated
animals. The combined flora also decreased caecal
size from about 5 per cent of body weight to
approximately normal. Despite the fact that the
combined flora eliminated 99 per cent of the
C. dificile from the caecum, the effect was
not comparable to that of the entire indigenous
microflora, which is able to totally eliminate this
pathogen.20*2'The effect on the population size
of E. coli, on the other hand, was similar to that of
the entire flora and of previously studied defined
micro flora^.'.^^.^' Interestingly, the combination
of E. coli and C. dificile suppressed P . aeruginosa
in gnotobiotic mice to 5.1k0.1 (log,, CFU per
caecum & standard deviation), a decrease to one
sixteen thousandth of the population found in
monoassociated mice. This effect was nearly equal
to the effect of the S +C flora. Whole mouse flora
suppressed P. aeruginosa to undetectable levels
(<300CFU/caecum) in six of 12 animals; the
remaining six animals harboured a mean of 3.6 f 1* 1.
+
+
Prevention of Colitis
Nineteen germfree mice and six mice harbouring
the S + C flora were challenged with lo8 CFU of C.
dificile strain VPI 10463 and observed for 3 wks.
Eight of the germfree mice died, all within 4 d of
challenge; no mice associated with the S + C flora
died. Both germfree mice and those harbouring the
S C flora (N =6/group) were challenged with VPI
10463 and sacrificed 48 h later for histological
examination of caeca. Sections were fixed in formalin; stained with hematoxylin and eosin; and
examined microscopically by an observer who did
not know the source of any given tissue section.
+
242
K. WILSON ET AL.
Table 4. Effect of defined microfloras on caecal size and population sizes of C. dijicile, E. coli, and P . aeruginosa
Flora inoculated
Caecal
C. dijicile
into mice
size
population
Monoassociated
Sflora(N=11)'
C flora (N = 9)
S+Cflora(N=12)
*
2.5 f0.4%
2.5 f0.5%
4.4 f0.7%
8.6k0.1
7.4 f0.4
7.1 f 0 . 2
6.6 f0.3
E. coli
population
10-1f0-1
7.8 k 0.3
8.7 0.7
6.6 k 0.4
P . aeruginosa
population
8.9 f0.1
Not done
Not done
4.4f0.7
Caecal size is reported as percent of total body weight. Population sizes are presented as the mean of log,, colony forming units per
caecumfstandard deviation. Except for the difference between caecal size in C flora group vs S + C flora group (significant at
p <0.005), p < O W 1 by the Student t-test for all comparisons of S flora or C flora with S + C flora.
*Caecal size for monoassociated mice is not reported here. However, caecal size for mice monoassociated with E. coli' and mice
diassociated with C. dijicile and E. COPhas been reported to be about 5%.
Colitis was graded in severity on a scale of 1 to 4,
where 1 represented a slight increase in the number
of polymorphonuclear neutrophils in the lamina
propria and 4 represented formation of pseudomembranes. Germfree animals had a mean score of
3.5 (range 3 to 4) while animals colonised with the
S C flora had a mean score of 0.3 (range 0 to 1).
This difference was statistically significant with t =
10.26 and p < 0.00 1.
+
DISCUSSION
It has been known for many years that in nature the
colonic microflora does not become stable until it
has gone through the process of ecologic succ e ~ s i o n ' ~ . ' ~In~ 'this
~ . process, as an animal first
begins to acquire a microflora, organisms able to
dominate the colonic ecosystem are suppressed by
other groups of organisms, which are in turn suppressed and so forth. Succession thus progresses
through 'stages', each stage being dominated by a
different group of organisms, and eventually culminates in the establishment of a stable ecosystem
dominated by a wide variety of highly fastidious,
anaerobic species. Previous
has indicated that the process takes 2 to 3 weeks. Although
the process undoubtedly has some features which
occur at random, the overall sequence of events follows a pattern. The organisms dominating very
early in succession were in general easier to classify,
less fastidious (data not shown) and able to utilise
more diverse carbon sources than organisms dominating the climax stage ecosystem. Although highly
'fit' to ultimately dominate the colonic ecosystem, a
large number of isolates from the predominant flora
were relatively inefficient at reducing the size of the
germfree mouse caecum and at suppressing E. coli,
C . dificile and P . aeruginosa. A defined microflora
combining organisms found at various stages of
succession was much more efficient at carrying out
all these functions. In the case of P . aeruginosa, organisms found prior to climax stage were actually
more efficient at suppression than were isolates
dominating the climax stage flora. Thus, the data
presented here support the concept that organisms
dominating the colonic flora early in succession are
important for the overall function of the entire flora,
even though they may occur in relatively small
numbers in the mature flora.
The present study also supports the results of previous work on the composition of the caecal flora of
Taken as a whole all these studies leave no
doubt that the vast majority of isolates from the
colonic flora of rodents cannot be grouped in any
known taxa even when modem methods are used to
attempt classification. In addition, the flora is
clearly highly complex; we identified 120 taxa on the
basis of band patterns on PAGE of soluble proteins.
The impossibility of applying established nomenclature to isolates from the flora of experimental
animals, and the extreme complexity of the caecal
flora are often not fully appreciated. However,
these unavoidable issues have very important implications for the design of studies of interactions
between the indigenous microflora and potential
pathogens.
The effect of the defined microflora on the population size of C . dificile was far less than the effect of
mouse whole flora. This observation suggests that
some bacteria important for the suppression of C.
dzficile were still missing from this defined flora,
243
DEFINED COLONIC MICROFLORA
which was more efficient a t carrying out the other
functions studied. However, despite this shortcoming, the flora prevented colitis in gnotobiotic mice.
As the number of isolates used in these experiments
was unwieldy, an alternative approach to further
develop a defined microflora would probably be
needed if one wished t o better simulate the effect of
the entire microflora on C . dificile.
9.
10.
11.
ACKNOWLEDGEMENTS
This work was supported by the Veterans Administration and NIH grant AM 33967 t o KW. We thank
Nancy Williams for secretarial assistance.
12.
13.
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