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Applied and Environmental Microbiology, September 2001, p. 3819-3823, Vol. 67, No. 9
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.9.3819-3823.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Fate and Dissemination of Bacillus subtilis Spores
in a Murine Model
Tran Thu
Hoa,1
Le Hong
Duc,2
Rachele
Isticato,3
Loredana
Baccigalupi,3
Ezio
Ricca,3
Pham Hung
Van,1 and
Simon M.
Cutting2,*
Laboratory of Microbiology, Ho Chi Minh
University of Medicine and Pharmacy, Ho Chi Minh City,
Vietnam1; School of Biological
Sciences, Royal Holloway University of London, Egham, Surrey TW20
0EX, United Kingdom2; and
Section of Microbiology, Department of General and
Environmental Physiology, University Federico II, 80134 Naples,
Italy3
Received 27 February 2001/Accepted 27 May 2001
 |
ABSTRACT |
Bacterial spores are being consumed as probiotics, although little
is known about their efficacy or mode of action. As a first step in
characterizing spore probiotics, we have studied the persistence and
dissemination of Bacillus subtilis spores given orally
to mice. Our results have shown that spores do not appear to
disseminate across the mucosal surfaces. However, we found that the
number of spores excreted in the feces of mice was, in some
experiments, larger than the original inoculum. This was an intriguing
result and might be explained by germination of a proportion of the
spore inoculum in the intestinal tract, followed by limited rounds of cell growth and then sporulation again. This result raises the interesting question of whether it is the spore or the germinated spore
that contributes to the probiotic effect of bacterial spores.
| |
INTRODUCTION |
The gram-positive soil
microorganism Bacillus subtilis has been studied
extensively, primarily as a model with which to study cell
differentiation and for exploitation in the biotechnology industry.
While some Bacillus species are pathogenic (e.g.,
B. anthracis and some B. cereus strains),
B. subtilis has, at most, been associated with opportunistic
infections of immunocompromised patients (6, 11,
17). For these reasons, it has received relatively little
clinical interest. Bacillus spores, though, are currently
available as probiotics and as competitive exclusion agents (CE
agents). Probiotics are live bacterial supplements which can enhance
the normal intestinal flora, while CE agents are bacteria which can
suppress infection and may contain undefined mixtures of more than one
bacterial species (7, 8, 19). Ingestion of significant
quantities of spores is thought to restore the normal microbial flora
following extensive antibiotic usage or illness (13). How
this occurs is unclear but could include competitive exclusion of
pathogens, whether by immunostimulation or competition for adhesion
sites. Spore probiotics are primarily used by humans as an
over-the-counter supplement for oral bacteriotherapy and
bacterioprophylaxis of mild gastrointestinal disorders, many of which
lead to diarrhea (13). In the livestock and poultry industries, probiotics containing Bacillus spores are used
extensively; an example is Biogrow (Provita Eurotech Ltd., County
Tyrone, Northern Ireland), which contains a mixture of B. subtilis and B. licheniformis spores. With the recent
ban on the use of antibiotics as growth promoters in Denmark, the use
of probiotics or CE agents as antibiotic alternatives seems likely to increase.
The validity of spores as probiotics or CE agents was recently
demonstrated by showing that oral inoculation of 1-day-old chicks with
2.5 × 108 B. subtilis spores
suppressed all aspects of infection when chicks were challenged with
Escherichia coli 078:K80 (12). One dogma regarding the use of spores as probiotics or CE agents is their mode of
action, which presumably must be substantially different from that of
the other, better known bacterial supplements, such as
Lactobacillus spp., which exist only in the vegetative
state. As part of a study to investigate the mode of action of spore probiotics, we have characterized a number of commercially available products for human use (9, 10). Surprisingly, almost all of these were found to carry mislabeled species, raising serious questions about the regulatory procedures in force to control the use
of probiotics or CE agents. In this work we have addressed the question
of what happens to spores taken orally. Using a murine model, we show
that spores do not disseminate in significant numbers beyond the
gastrointestinal tract. However, we show that spores can persist in the
gastrointestinal tract and may germinate despite the anaerobic environment.
| |
MATERIALS AND METHODS |
Preparation of spores.
Spores were prepared from large
(200-ml) cultures as described previously (15) using
strain SC1712, which carries cat and erm
insertions, encoding resistance to chloramphenicol and erythromycin, respectively (note that neither insertion interfered with cell growth
or spore formation). Strain SC1712 was derived from prototrophic, wild-type (Spo+) strain PY79 of B. subtilis (20). The spore suspensions were then heated
at 65°C for 1 h to kill any residual, nonsporulated cells, and
aliquots (0.5 ml) were frozen at 
80°C. The number of CFU per
milliliter of frozen aliquots was determined and, if necessary, spore
suspensions were concentrated further on the day of use (by
centrifugation) to produce 108 to
109 CFU/ml.
Fecal counts.
For experiment 1, outbred mice (female, 6 to 8 weeks old) were obtained from the Pasteur Institute, Ho Chi Minh City,
Vietnam. For the remaining experiments, pathogen-free female BALB/c
mice (6 to 8 weeks old) were purchased from Harlan UK (Oxon, United Kingdom).
Spores (0.2 ml of spore suspension) were administered by intragastric
gavage to mice anesthetized by inhalation of halothane. Animals
were housed individually in cages with gridded floors. Total feces were
collected at various time points and processed immediately for
experiment 1 (see below); however, for experiments 2 and 3, total fecal
collections were frozen at 80°C until the time of processing. To
determine the number of spores in feces, samples were suspended in 10 to 30 ml of phosphate-buffered saline (PBS) at 65°C. Sterile glass
beads (2 mm; 3 ml) were added, and the suspension was incubated at
65°C for 1 h with frequent vortexing until there was little
remaining residual solid matter. Serial dilutions were then made with
PBS (65°C), plated on Difco sporulation medium plates containing
chloramphenicol (5 µg/ml) and erythromycin (1 µg/ml), and incubated
at 37°C for 2 days. B. subtilis colonies of strain SC1712
were identified by their colony morphology. Spore counts were
extrapolated for the total weight of feces collected. In experiment 1, to determine the total number of B. subtilis viable units,
including spores and vegetative cells, the same procedure was used,
except that feces were suspended in PBS at room temperature and no heat
treatment was applied.
Dissemination experiments.
Groups of four mice (6-week-old
female BALB/c; Harlan UK) were dosed orally (0.2 ml) with purified
spore suspensions (1.83 × 108/ml). As
controls, we inoculated groups of four mice with PBS alone. Groups of
mice were sacrificed at 12, 86, and 158 h and dissected for
selected organs and tissues. Organs were weighed by difference, rinsed
with 2 ml of PBS, suspended in PBS (1 ml), and homogenized with glass
beads (1 mm) using a Beadbeator (Biospec Inc.) with 10-s cycles and
intermittent cooling on ice (10 s) until tissues were completely
broken. Homogenized suspensions were heat treated (65°C, 45 min),
serially diluted in PBS, and plated for determination of CFU per
milliliter. Heat treatment was used to reveal the number of spores
present in organs or tissues.
| |
RESULTS AND DISCUSSION |
Fate of spores in mice inoculated orally.
As an indicator of
the fate and longevity of spores administered orally, we gave a fixed
dose of spores of a multiply drug-resistant spo+ strain, SC1712
(Cmr Ermr), to mice and
measured the number of spores excreted or, in experiment 1, the number
of spores and vegetative cells excreted. In experiment 1 we used
outbred mice, and in experiments 2 and 3 we used inbred BALB/c mice. In
experiment 1 a large inoculum, 1.75 × 1011 spores, was given, while in experiments 2 and 3, lower doses, 5.97 × 108 and 2.0 × 109 spores, respectively, were given.
Our results from dosing of outbred mice (Table
1) showed that
B. subtilis
viable units were first detectable 3 h after inoculation;
the
majority of counts occurred within the first 24 h, although
B. subtilis was still detectable in the 4-day (96-h)
samples.
These results revealed two apparent paradoxes. First, the count for
spores and vegetative cells was almost always lower than
the count for
spores alone. Since heat-treated and untreated feces
were always
prepared at the same time, this result was intriguing.
This phenomenon
has been observed previously (
10), and it has
been
postulated that the heat treatment used to process spores
actually
"activates" the spores and so enhances their germination
and
outgrowth. This notion is consistent with established studies
showing
that "heat activation" is a prerequisite for efficient
and
synchronous germination (
4). In the untreated suspensions
then, a substantial number of spores (up to 2 log units) are unable
to
germinate and therefore produce an apparently lower count.
Thus, the
count for spores more accurately reflects the actual,
or real, number
of viable
units.
We found from preliminary trials that without heat treatment, a
substantial number of different bacterial species (up to five
in
outbred mice) were able to grow on agar plates containing two
antibiotics (chloramphenicol and erythromycin). Despite this
contamination,
we could discern
B. subtilis SC1712 simply
from its colony morphology
(and presence in high numbers at high serial
dilutions). Heat
treatment, though, reduced the background almost
completely and,
in fact, virtually no other bacterial species (i.e.,
spore formers)
were detectable in fecal
samples.
The second intriguing observation was that for two mice (mice 4 and 5),
the total accumulated number of spores excreted over
96 h was
higher (more than three times) than the original inoculum.
One
straightforward explanation is, of course, experimental error,
although
as discussed below this seems unlikely. The other possibility
is that
the counts were in fact real, in which case the only plausible
explanation is that a proportion of the spores had germinated,
undergone one or more rounds of growth and replication, and then
formed
spores. This explanation would at first seem most unlikely,
but before
coming to any firm conclusions, we repeated this experiment
with inbred
BALB/c mice. Two of these experiments are shown in
Tables
2 and
3.
In these experiments, we used smaller inocula
of spores (5.97 × 10
8 and 2.0 × 10
9)
but maintained sampling for 7 days. In experiment 2 (Table
2 and Fig.
1), the results showed that with each
mouse the cumulative
number of spores excreted was larger (by a factor
of 4.40 ± 1.53
[mean and standard error]) than the
initial inoculum. Strikingly,
considerable numbers of spores were still
being detected in the
feces on day 7. In experiment 3 (Table
3), we did
not observe
an increase in the number of spores excreted (0.41 ± 0.18), yet
we did observe substantial numbers of spores in the feces on
day
7 of sampling.
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TABLE 2.
B. subtilis counts in feces from inbred mice
orally inoculated with 5.97 × 108 spores of SC1712 in
experiment 2
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TABLE 3.
B. subtilis counts in feces from inbred mice
orally inoculated with 2.0 × 109 spores of strain
SC1712 in experiment 3
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FIG. 1.
Excretion of spores in feces of mice immunized orally.
Shown is a graphic presentation of the data from experiment 2 (Table 2)
to evaluate the fate and longevity of spores following oral inoculation
of inbred mice. Shown are the cumulative spore counts ( ) and the
temporal spore counts ( ) obtained from feces collected at the
indicated time points after mice were immunized with purified spore
suspensions. Spore counts are averages for the four mice shown in Table
2. Standard error bars are shown. The broken line shows the number of
spores inoculated (5.97 × 108).
|
|
Since both experiments 2 and 3 were done with inbred mice, we performed
a simple statistical analysis. Analysis of variance
indicated
that the results of experiments 2 and 3 were significantly
different
(
P < 0.01). For experiment 1, where we used outbred
mice, we found that for some mice we observed an increase in spore
counts (mice 4 and 5) and for others we observed a decrease (mice
1, 2, and 3). When mice 1, 2, and 3 from experiment 1 were considered
to be
one group, the average ratio of spore counts to inoculum
was
0.50 ± 0.216. In a comparison with the data of experiment
3 (where spore counts also decreased), a
t test revealed a
P value
of >0.10, demonstrating that there was no
significant difference
in these data. Similarly, for mice 4 and 5 of
experiment 1, a
comparison of the average ratio of spore counts to
inoculum (3.14
± 0.63) with the data of experiment 2 (where an
increase in spore
counts also was observed) revealed a
P
value of >0.10, again showing
no significant difference between spore
count data. While the
sample sizes are small, our analyses appear to
show that the results
obtained are not dependent upon the mouse genetic
background.
A number of factors could severely limit the accuracy of these
experiments. First, between fecal collections, excreted spores
might be
able to germinate in the feces and undergo successive
rounds of growth
and division. Our experimental procedure for
the detection of spores
used a high temperature (65°C; 60 min)
to homogenize fecal matter;
this temperature is sufficient to
kill all vegetative cells. Thus, if
spores had germinated, then
they would have had to resporulate (an 8-h
process) in order to
survive the fecal homogenization. We examined 12-h
fecal samples
containing spores. Spore counts were determined with a
portion
of this sample, after which the feces were stored at room
temperature
for 12 and 24 h and spore counts were determined. We
found that
the spore counts were unchanged (data not shown); therefore,
the
spores do not appear to germinate in feces in detectable
numbers.
A second factor that could introduce error was the fecal extraction
procedure itself. Complete homogenization of feces was
sometimes
difficult to obtain, so it is possible that the actual
spore counts
were higher than those obtained here but not lower.
Another
factor which would decrease, but not increase, spore counts
is fluid
loss during intragastric
inoculation.
Our interpretation which explains the increased spore counts in
experiments 1 and 2 is that a proportion of spores germinate
in the
gut. We base this explanation on the following: (i) the
total number of
spores excreted is, on average, larger than that
inoculated; (ii)
substantial numbers of spores are still present
in the feces 7 days
after the initial inoculation, when we might
otherwise expect to see
complete clearance; (iii) there is a gradual
decline in spore counts
over time; and (iv) if experimental error
were attributed to this
procedure at each step, then the spore
counts should be dramatically
decreased.
We have repeated our experiments using inbred mice in their entirety
several times and have found that an increase in spore
counts is
observed on some occasions but not on others. What is
important,
though, is that an increase is observed. We were initially
skeptical of
these results as well as perplexed but have come
to the conclusion that
on some occasions, spores can germinate
and then resporulate. That
spores do not always germinate in measurable
amounts may in some way
reflect the constitution of the mouse,
that is, the physiological
conditions of the gastrointestinal
tract.
We are now attempting, using the molecular technique of reverse
transcription-PCR, to show definitively that the spores germinate.
However, in the absence of molecular results, is there any work
that
supports the data we have presented? First, and foremost,
there is the
assumption that
B. subtilis is a strict aerobe. However,
given glucose and nitrate as a terminal electron acceptor,
B. subtilis has been shown to grow anaerobically (
14).
It seems
unlikely that spores could germinate in the acidic conditions
of the stomach; however, the upper intestinal tract, being rich
in
nutrients, might reasonably be expected to allow spore germination.
Ligated loop studies have shown that spores can germinate in the
intestine (
13). Moreover, studies on the fate of
B. natto, the
probiotic agent contained in the Japanese product
Natto, have
shown that the number of
B. natto CFU recovered
from the feces
of animals inoculated with this organism was between 246 and 1,133%
larger than the original inoculum (
13). These
studies with weaned
piglets showed that spores could germinate and
multiply to a limited
degree in the upper intestinal tract. If
intestinal colonization
occurs, then it must be very limited, since
most of the inoculum
had transited within 24 h. What is
interesting from our studies
is that 10
3 to
10
4 spores are still detectable 7 days after
inoculation. If spores
can germinate to some limited degree and then
undergo limited
rounds of replication, then these processes may account
for the
continued excretion 7 days later, although these cells must
have
formed
spores.
Spore formation in
B. subtilis is well studied and occurs
primarily as a response to nutrient depletion (
5,
16). The
decision to sporulate requires other inputs, such as the cell
having
reached the correct stage in replication and pheromonal
information
from other
B. subtilis cells. As such, many cells
will not
normally sporulate, although in the laboratory sporulation
efficiencies can reach 80 to 90%. In the absence of entering the
sporulation life cycle, the vegetative cell will either lyse or
transit
the gastrointestinal tract. Indeed, recent work has shown
B. subtilis to be sensitive to bile salts in the
gastrointestinal
tracts of mice (
18). From our results, we
can only infer that
spore formation is occurring. When the spore
germinates in a nutrient-rich
environment, it will be quickly
translocated to a more hostile
environment, where spore formation may
be a reasonable strategy
for survival. Interestingly, other
spore-forming organisms are
found in the intestinal tract
(
2), and spore formation has
been shown to initiate in the
ileum as part of the life cycle
of the gram-positive bacterium
Metabacterium polyspora in guinea
pigs (
1).
Dissemination of spores.
Although B. subtilis is
considered nonpathogenic, it is important to address the ethical issues
of potential spore dissemination across the mucosal epithelium, since
spores are currently being consumed as probiotics. Accordingly, we
administered spores (SC1712; 1.83 × 108
CFU/ml) to groups of 12 mice by intragastric gavage (4 mice were also
inoculated with PBS as controls; data not shown). Mice were housed in
groups of four in cages with gridded floors to prevent coprophagia.
Four mice from each group were sacrificed at 12 h and at 3.5 and
7.5 days. Selected organs and tissues were dissected, and spore counts
were determined from homogenized sections as described previously
(3). As observed earlier, we found from trial experiments
that we could not determine the total number of B. subtilis
viable units, so we heat treated homogenized sections and counted
spores only. Average spore counts for each group of four mice are shown
in Table 4. We found that oral
immunization produced extremely low levels of dissemination, other than
in the lungs and gut, a result which can be accounted for by the inoculation procedure. It should be noted that the numbers of spores
detected in the mesenteric lymph nodes, liver, and spleen must be
considered insignificant, since previous work has shown that
approximately 50 viable units is the limit of accurate quantification for these organs (3).
These results show that spores do not appear to disseminate
substantially following oral dosing, consistent with the results
of
other studies (
18). Moreover, the persistence that we
observed
from analyses of feces suggests that
B. subtilis is
a transient
resident of the gut. Although our procedure measured only
viable
spores, it is possible that inactive or killed spores were
present
in selected organs. Similarly, if spores did germinate, then we
could not account for the dissemination of vegetative
cells.
In conclusion, our results are important because they address the dogma
of how spore probiotics function. Although the mode
of action remains
unclear, we have presented evidence that spores
may germinate in the
gut. As such, they may function in the same
way as other probiotic
bacteria, such as the lactobacilli, and
possibly exert their probiotic
action by a metabolic effect following
spore germination.
Alternatively, germination of spores may simply
be an incidental
feature of the spore entering a nutrient-rich
environment, and the mode
of action may be unique to the spore
state.
| |
ACKNOWLEDGMENTS |
This work was supported by grants from the Wellcome Trust
(to S.M.C.) and the European Union (to S.M.C. and E.R.).
We thank Pham Thi Nguyen Thuy, Gabriella Casula, and Dana Cohen for
assistance in this project.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: School of
Biological Sciences, Royal Holloway University of London, Egham, Surrey
TW20 0EX, United Kingdom. Phone: 44-1784-443760. Fax: 44-1784-434326. E-mail: s.cutting{at}rhul.ac.uk.
| |
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Applied and Environmental Microbiology, September 2001, p. 3819-3823, Vol. 67, No. 9
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.9.3819-3823.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
