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Appl Environ Microbiol, April 1998, p. 1338-1343, Vol. 64, No. 4
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A Novel Sensitive Bioassay for Detection of
Bacillus cereus Emetic Toxin and Related Depsipeptide
Ionophores
M. A.
Andersson,1,*
R.
Mikkola,1
J.
Helin,2
M. C.
Andersson,3 and
M.
Salkinoja-Salonen1
Department of Applied Chemistry and
Microbiology,1
Institute of
Biotechnology,2 and
Department of
Clinical Sciences, Animal Reproduction,
Saarentaus,3 University of Helsinki FIN 000140, Finland
Received 24 November 1997/Accepted 3 February 1998
 |
ABSTRACT |
Of the toxins produced by Bacillus cereus, the emetic
toxin is likely the most dangerous but, due to the lack of a suitable assay, the least well known. In this paper, a new, sensitive, inexpensive, and rapid bioassay for detection of the emetic toxin of
B. cereus is described. The assay is based on the loss of
motility of boar spermatozoa upon 24 h of exposure to extracts of
emetic B. cereus strains or contaminated food. The
paralyzed spermatozoa exhibited swollen mitochondria, but no depletion
of cellular ATP or damage to plasma membrane integrity was observed.
Analysis of the purified toxin by electrospray tandem mass spectrometry showed that it was a dodecadepsipeptide with a mass fragmentation pattern similar to that described for cereulide. The 50% effective concentration of the purified toxin to boar spermatozoa was 0.5 ng of
purified toxin ml of extended boar semen
1. This amount
corresponds to 104 to 105 CFU of B. cereus cells. No toxicity was detected for 27 other B. cereus strains up to 108 CFU ml1. The
detection limit for food was 3 g of rice containing
106 to 107 CFU of emetic B. cereus
per gram. Effects similar to those provoked by emetic B. cereus toxin were also induced in boar spermatozoa by valinomycin
and gramicidin at 2 and 3 ng ml of extended boar semen1,
respectively. The symptoms provoked by the toxin in spermatozoa indicated that B. cereus emetic toxin was acting as a
membrane channel-forming ionophore, damaging mitochondria and blocking the oxidative phosphorylation required for the motility of boar spermatozoa.
| |
INTRODUCTION |
Bacillus cereus is known
to cause clinical infections, food poisoning, and toxin-induced
diarrheagenic and emetic syndromes (8). Even fatal cases of
infections (3, 17, 19) and emetic-type food poisoning
(15) have been reported recently. B. cereus is
ubiquitous in nature but has also been isolated from environments where
it may represent a serious health hazard, e.g., infant feed
(20), hospital linen (6), and gunpowder
(14). B. cereus strains have been shown to
produce seven different toxins (11). Of these toxins, the
emetic toxin is very likely the most dangerous but, due to the lack of
a suitable assay, the least well known.
The documented biological activities of the emetic toxin, cereulide,
described by Agata et al. (1) and Isobe et al.
(12) are emesis in primates (22, 24) and swelling
of mitochondria in HEp-2 cells (21) and in hepatocytes of a
fatally food-poisoned patient (15). Even though the
structure and mitochondrial toxicity of cereulide were recently
elucidated, there has been no rapid detection method for the toxin from
bacteria, from food poisoning outbreaks, or from contaminated food. In
this paper, we describe an inexpensive, sensitive, rapid, and
reproducible bioassay for detection of the emetic mitochondrial toxin,
by all criteria identical to cereulide. The assay is based on the loss
of motility of boar spermatozoa.
| |
MATERIALS AND METHODS |
B. cereus strains used in this study.
Strains
F-47, F-528, F-3453, F-4426, and F-5881 were from the Public Health
Laboratory Service, London, United Kingdom; strain 4810/72 was from
Meijerierna Service AB, Lund, Sweden; strain NC 7401 was from the
Nagoya City Public Health Institute, Nagoya, Japan; strains IH41385 and
IH41064 were from the National Public Health Laboratory, Helsinki,
Finland; and all other strains used were from our own collection.
Media and reagents.
Boar semen lots were obtained from the
Artificial Insemination Center (AI Cooperative, Kaarina, Finland) and
from the Department of Animal Reproduction (University of Helsinki).
The microbial media were from Difco, and gramicidin (a mixture of A, B,
C, and D), polymyxin B sulfate, surfactin, ionomycin,
N,N-dihexylcarbodiimide, 2,4-dinitrophenol, and
proteinase K were from Sigma (St. Louis, Mo.). Valinomycin was from
Fluka (Buchs, Switzerland). Anatoxin A and nodularin were gifts of
Kaarina Sivonen (University of Helsinki). The microconcentrator
membranes were from Amicon Inc., Beverly, Mass. Other chemicals were
from local suppliers and were of analytical quality.
Extraction of emetic toxin from pure cultures and directly from
food. (i) Cell extracts.
Bacteria were grown on tryptic soy agar
plates at 28°C for 10 days to stabilize the growth phase and obtain
mainly sporulated and lysed cells, as observed by phase-contrast
microscopy. Colonies were scraped from the agar and suspended in water
at 100 mg ml1. Cell extracts were prepared by sonication
and freeze-thaw cycles of this suspension. The extracts were filtered
(0.2-µm pores), and the permeate, diluted in dimethyl sulfoxide
(DMSO), was tested for toxicity.
(ii) Methanol extraction of the toxin from pure cultures.
Five hundred milligrams of cells was collected from tryptic soy agar
plates, grown at 28°C for 10 days, and extracted with 100 ml of 100%
methanol, as described by Andersson et al. (5). The
evaporated residue, diluted in DMSO, was tested for toxicity.
(iii) Methanol extraction of the toxin directly from food.
Boiled rice (60 g, 150 ml) was experimentally infected with
105 to 106 CFU of B. cereus or
Bacillus mycoides strains g1. The rice was
kept at room temperature for 2 days. The rice was examined by
phase-contrast microscopy and found to contain many spores and lysed
cells. The rice was reheated to ca. 80°C (30 min) on day 2 and
incubated for two more days at room temperature. On day 4, the rice was
extracted with 1 volume of methanol, the extract was evaporated, and
the residue, diluted in DSMO, was tested for sperm toxicity.
Biological analyses.
A spermatozoon toxicity test was
performed according to protocols described earlier (5)
except that the boar semen was diluted to 12% with a commercial semen
extender (MR-A; Kubus, S.A., Madrid, Spain) to a density of 30 × 106 to 50 × 106 sperm cells
ml1. Sperm motility (the amount of motile versus
nonmotile sperm cells) was subjectively estimated by phase-contrast
microscopy by using a heated stage (37°C), and the exact percentage
of highly motile sperm cells was objectively measured with a
computerized sperm motility analyzer (4) (HTM-S, version
7.2; Hamilton-Thorn Research, Danvers, Mass.).
Plasma membrane integrity was assayed with a combination of two
fluorescent stains, calcein-AM and ethidium homodimer-1 (Molecular Probes, Inc., Eugene, Oreg.), as described by Januskauskas and Rodriguez-Martinez (13). Cellular ATP content was measured
by a bioluminescense technique (13) with a Bio-Orbit 1235 luminometer and an ATP Biomass kit 1243-118 (Bio-Orbit, Turku,
Finland). Morphology of sperm mitochondria was analyzed with a
transmission electron microscope, as described elsewhere
(5). Osmolality measurements of extended boar semen were
done with an automatic micro-osmometer (Herman Roebeling Messtechnik,
Berlin, Germany). Analyses were done in duplicate and the results were
averaged.
Purification and analysis of the sperm toxin of B. cereus 4810/72.
An extract of B. cereus 4810/72,
prepared by sonication and freeze-thaw cycles of the harvested cells,
was precipitated with ammonium sulfate (70%) and centrifuged. The
precipitate was dissolved in 90% ethanol, and, after centrifugation,
the supernatant was evaporated to dryness under N2. The
chloroform-soluble part of the residue was collected, evaporated, and
dissolved in 60:40 methanol-water. This solution was injected into a
Sep-Pak C18 cartridge (Waters Co., Milford, Mass.) and
washed with 80:20 methanol-water. The final elution was done with
methanol.
MS analyses.
Electrospray (ESI) mass spectra were collected
with an API300 triple quadrupole mass spectrometer (MS) (Perkin-Elmer
Sciex Instruments, Thornhill, Ontario, Canada). Samples in 50%
methanol-water with 0.5% acetic acid were injected directly into the
electrospray chamber with a syringe pump (Harvard Apparatus, South
Natick, Mass.) at a flow rate of 3 µl min1. The
instrument was calibrated with a polypropyleneglycol mixture supplied
by the instrument manufacturer. MS/MS spectra were acquired by causing
collisions between precursor ions and nitrogen collision gas at
acceleration voltages of 50 V. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS was performed in the delayed-extraction mode with a BIFLEX MS (Bruker-Franzen Analytik, Bremen, Germany) with a 337-nm nitrogen laser. A thin-layer matrix preparation was used, according to the method of Vorm et al.
(26).
| |
RESULTS |
Extracts from emetic B. cereus cells inhibited motility
and disrupted mitochondria of boar spermatozoa.
B.
cereus 4810/72 and NC 7401, documented producers of emetic toxin
(2, 24), were tested for toxicity toward boar spermatozoa. Cell extracts of both strains inhibited sperm motility within 1 day of
exposure at a dosage of 2 mg (wet weight) of bacteria ml of extended
boar semen1. Similarly prepared extracts of cells of
B. cereus ATCC 14579 (type strain), B. mycoides
ATCC 6462 (type strain), and Bacillus licheniformis DSM 13 (type strain) had no effect on sperm motility even after 5 days of
exposure (Table 1). Sperm motility
subjectively estimated by phase-contrast microscopy was confirmed by
the percentage of highly motile sperm cells objectively measured with
the Hamilton-Thorn sperm analyzer. The amount of cell extract applied
to the extended semen did not affect osmolality or pH. The sperm cells,
exposed to extracts of emetic B. cereus strains, exhibited
dose-dependent frequencies of swollen mitochondria visible with a
transmission electron microscope (Fig.
1A), indicating that the filtrate
contained a mitochondrial toxin. No mitochondrial swelling was observed in sperm cells exposed to extracts of B. cereus ATCC
14579T or B. mycoides ATCC 6462T
(Fig. 1B).
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FIG. 1.
Thin sections of the middle piece of a boar spermatozoon
exposed to cell extracts of B. cereus 4810/72 and ATCC
14579T for 7 days. (A) Mitochondrial damage in the middle
piece of a spermatozoon exposed to cell extracts of 2 mg (wet weight)
of cells ml1 of strain 4810/72. The frequency of
similarly swollen mitochondria was 75% to over 90% after exposure to
extracts from 4 mg (wet weight) of cells ml1 and <20%
after exposure to extracts from 0.5 mg (wet weight) of cells
ml1 (not shown). (B) Middle piece of a spermatozoon
exposed to cell extracts of B. cereus ATCC
14579T (2 mg [wet weight] ml1).
Mitochondria of ordinary size with intact membranes are seen. A
spermatozoon exposed to the same extract for 25 days exhibited no
change of morphology, and those of unexposed spermatozoa were identical
to that shown in panel B. Bars, 200 nm.
|
|
We tested 32 strains of
B. cereus of different origins for
sperm toxicity. The results in Table
2
show that cell extracts
of five strains, F-5881, F-47, F-4426, 4810/72,
and NC 7401, caused
the loss of motility of boar spermatozoa at
extremely low concentrations,
equivalent to 50 ng (dry weight) of
B. cereus cells (equivalent
to 10
4 CFU) per ml
of diluted boar semen. Extracts of the other 27 strains
of
B. cereus strains had no effect on the motility of spermatozoa
even
at 10,000-fold-higher concentrations (
10
8 CFU
ml
1).
The spermatozoon-paralyzing agent in the
B. cereus extracts
was insensitive to heating at 100°C (20 min), treatment with acid
(pH
2 [with HCl for 30 min]) or alkali (pH 12 [with NaOH for 30
min]), or the action of proteinase K (100 µg ml
1, pH
7, 3 h at 37°C). The toxic agent was filterable through
the
microconcentrator membranes, with a nominal cutoff of >100,000
g
mol
1, and of >10,000 g mol
1 as a methanol
extract, but not as an extract in water or DMSO.
The agent paralyzing
the sperm cells was thus heat stable and
nonpolar, resistant to
inactivation by heat, acid, alkali, or
protease, and of an apparent
molecular size smaller than 10,000
g mol
1.
Isolation, purification, and analysis of the sperm toxin from
B. cereus 4810/72.
An extract of B. cereus
4810/72 was precipitated with ammonium sulfate, dissolved in
chloroform, purified by a single step of Sep-Pak C18
sorption, eluted with methanol, and analyzed by ESI-MS. Four peaks were
obtained with m/z of 1,153.85, 1,170.71, 1,175.71, and 1,191.51 (Fig. 2A). These
were tentatively identified as the protonated, ammonium, sodium, and
potassium adducts, respectively, of a single molecular species.
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FIG. 2.
Mass fragmentation of the purified sperm toxin of
B. cereus 4810/72. (A) m/z of the
molecular ions as determined by ESI-MS. (B) ESI-MS/MS fragmentation of
the protonated ion, m/z of 1,153.85, shown in
panel A. Peak numbers correspond to the fragment ions assigned in Table
3. Peaks marked with asterisks represent loss of water.
|
|
Figure
2B shows the ESI-MS/MS spectrum of the ion with an
m/
z of 1,153.85 (protonated adduct [Fig.
2A]).
The assignments of
the fragment ions are shown in Table
3. The mass values of all
fragments
matched within 0.35 mass units those expected from the
published
dodecadepsipeptide structure of cereulide (
12). In
particular, the prominent tetrapeptide cleavages with
m/
z of 384.43
(peak 14 [Fig.
2B]) and 412.43 (peak 15) from the [M+H]
+ match exactly with
(Val)
1(OLeu)
1(Ala)
1(OVal)
1
and
CO(Val)
1(OLeu)
1(Ala)
1(OVal)
1,
respectively. Cereulide consists of three of these tetrapeptide
units
forming a cyclic structure. The expected neutral loss of
water, 18, was
also observed (Fig.
2B). The putative ammonium
adduct,
m/
z of 1,170.71, gave in MS/MS an analogous
fragmentation
pattern, as well as the [M+H]
+ ion through
loss of ammonia, thus confirming the assignment.
The data given above, as well as further details to be described
elsewhere, indicate that the mitochondrion-toxic, boar sperm-paralyzing
agent purified from the emetic strain
B. cereus 4810/72 is a
cyclic
dodecadepsipeptide with a structure identical to that reported
for cereulide (
2,
12).
Biological properties of the purified sperm-toxic agent from
B. cereus 4810/72 compared to other toxins and
chemicals.
The sperm-toxic agent isolated as described above from
the extract of B. cereus 4810/72 cells is referred to below
as the sperm toxin. Yield of the sperm toxin from a 10-day-old culture of B. cereus 4810/72 was 240 ng (wet weight) mg of
cells1 (or 3 fg cell1), as determined by
high-performance liquid chromatography and verified by MALDI-TOF MS.
Toxicity thresholds of the purified sperm toxin toward boar spermatozoa
were compared to those of selected bacterial toxins
and chemicals
(Table
4). The results revealed the sperm
toxicities
of three materials: the toxin purified from
B. cereus 4810/72,
valinomycin (a depsipeptide and potassium
ionophore [
7]), and
gramicidin (a membrane
channel-forming linear homopeptide, potassium
ionophore, and
protonophore [
7]). Low concentrations (<3 ng
ml
1) of these materials caused loss of sperm motility;
concentrations
of <400 ng ml
1 swelled the mitochondria
but did not deplete the cells of ATP
even at high concentrations (up to
12,500 ng ml
1). Calcimycin A 23187 (a polyether calcium
ionophore [
10]) inhibited
sperm motility at 32 ng
ml
1 and depleted the spermatozoa of ATP at 125 ng
ml
1 but caused no morphological damage to mitochondria
even at concentrations
of 2,000 ng ml
1. Other bioactive
peptides tested for sperm toxicity were the
polyether calcium ionophore
ionomycin (
11), the membrane-active
lipopeptide polymyxin B
sulfate (
10), surfactin (a cyclic lipopeptide
antibiotic
(
25), nodularin (a cyanobacterial hepatotoxin
[
23]),
anatoxin A (a cyanobacterial neurotoxin
[
23]),
N,
N-dihexylcarbodiimide
(an ATPase inhibitor [
7]), and 2,4-dinitrophenol (an
uncoupler
of oxidative phosphorylation [
7]). It was
found that the sperm
cells were relatively insensitive to all of these;
the 50% effective
concentrations (EC
50) for the vitality
parameters shown in Table
4 ranged from 100 to >50,000 ng
ml
1. In conclusion, sperm cell injuries (loss of motility
and swollen
mitochondria) caused by
B. cereus 4810/72 were
similar to those
caused by valinomycin and gramicidin but had little
resemblance
to those caused by the other toxins tested.
Direct detection of sperm-paralyzing toxin from methanol extracts
of contaminated food.
Extracts of rice contaminated with B. cereus 4810/72 or B. cereus F-5881 inhibited sperm
motility at quantities corresponding to 3 g (7.5 ml) of rice
(Table 5). No paralysis of motility was observed for extracts prepared from rice contaminated with B. mycoides ATCC 6462T or B. cereus ATCC
14579T, tested up to quantities corresponding to >60 g
(150 ml) of boiled rice. Table 5 also shows the sperm toxicities of the
inoculant strains of B. cereus and B. mycoides
extracted from the rice. The yields of methanol-soluble substances of
B. cereus F-5881, 4810/72, and ATCC 14579T and
B. mycoides ATCC 6462T were 24, 16, 9, and 13 mg
(dry weight), respectively, per gram (wet weight) of cells. The
respective EC50 of the methanol-soluble substances toward
boar spermatozoa were 0.072 µg (equivalent to 2 × 105 CFU of strain F-5881) ml1, 0.03 µg
(equivalent to 105 CFU of strain 4810/72)
ml1, 17 µg (equivalent to 108 CFU of strain
ATCC 14579T) ml1, and >68 µg (equivalent
to >1010 CFU of strain ATCC 6462T)
ml1 of extended boar semen.
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TABLE 5.
Toxicity to boar spermatozoa of methanol extracts
prepared from boiled rice inoculated with different
Bacillus strains 4 days earlier
|
|
Conclusions.
Methanol extracts prepared from inoculated boiled
rice, as well as from cultures of B. cereus F-5881 and
4810/72, caused the same injuries on exposed spermatozoa as did the
toxin purified from the extract of strain 4810/72. Sperm toxicity of
the extract prepared from boiled rice was thus caused by cereulide
toxin produced by strains F-5881 and 4810/72 in rice. The results show
that the cereulide toxin in food was detectable based on the responses of spermatozoa to a methanol extract prepared directly from the food.
Inhibited motility of the boar spermatozoa was observable within
24 h of exposure of the semen dilution to extracts prepared directly from food.
| |
DISCUSSION |
In this paper, we describe a rapid bioassay for detection of a
sperm toxin produced by B. cereus strains documented as
producers of the emetic toxin. The test was readable after 24 h by
ordinary phase-contrast light microscopy, required no costly reagents, was easy to perform (5), and was highly reproducible. The
average difference between replicate testings was 20% when toxicities towards spermatozoa of three different boars were compared. The EC50 was as low as 0.5 ng of purified toxin per ml of
extended boar semen. This amount of toxin is equivalent to
104 CFU of toxin-producing B. cereus organisms
per ml of extended boar semen. The test thus is >2 orders of magnitude
more sensitive than methods described to date: the monkey
(Maccacis muratta) feeding test (22) and
vacuolization assay of trypsinized HEp-2 cells (human carcinoma of the
larynx) (18).
The cereulide toxin was first isolated from B. cereus NC
7401. We isolated and purified the sperm-toxic agent from a cell extract of B. cereus 4810/72 and found it to be identical to
cereulide described by Agata et al. (1, 2) and Isobe et al.
(12) in heat stability, resistance to protease, inactivation
by acid or alkali, and molecular mass (1,153.85 g mol1
[protonated form]). Fragmentation analyses by ESI-MS/MS showed that
the sperm toxin isolated in the present study was a dodecadepsipeptide with a cleavage pattern identical to that of cereulide.
The sperm toxin was structurally and functionally related to the
commercially available potassium ionophore valinomycin. Valinomycin also caused the loss of motility and swelled the sperm mitochondria, but it did not deplete spermatozoal ATP or damage plasma membrane integrity. Similar toxic effects were observed with sperm cells exposed
to methanol extracts prepared directly from food contaminated with
B. cereus F-5881 and 4810/72. The cereulide toxin was thus extractable directly from food and measurable on the basis of decreased
motility of boar spermatozoa within 24 h, allowing toxin detection
without the time-consuming process of cultivating and isolating
suspected B. cereus strains. The assay utilizes commercially available boar semen and requires no experimental animals. This is the
first rapid and sensitive bioassay to be described for detection of the
emetic toxin of B. cereus.
Spermatozoa are transcriptionally inactive, which makes them
insensitive to toxins affecting synthesis of proteins or nucleic acids
or their regulation (9). This excludes interference by other
microbial toxins, such as the cyanobacterial toxins (23) and
most mycotoxins of importance for foods. However, spermatozoa brilliantly couple external signals to cellular responses such as
motility, which is mainly controlled by ion fluxes (27). This makes spermatozoa sensitive indicators for ion channel-forming toxins like potassium ionophores and protonophores and explains the
decreased motility of boar spermatozoa exposed to valinomycin and
gramicidin as well as their insensitivity to membrane-active agents
without ion channel-forming capacity, such as polymyxin and surfactin
(25).
Motility in boar spermatozoa (meaning progressive and high motility) is
exclusively dependent on oxidative phosphorylation in mitochondria. ATP
production by substrate-level phosphorylation is too slow in boar
spermatozoa to maintain motility (16). The spermatozoa
exposed to cereulide exhibited loss of motility and swollen, disrupted
mitochondria. However, vitality staining and measurements of cellular
ATP content revealed intact plasma membranes and a supply of ATP
explainable by ongoing substrate-level phosphorylation in the
cytoplasm. The sperm cells thus were paralyzed but not lethally
injured. We conclude that the cereulide intoxicates boar spermatozoa by
acting as an ionophore, which leads to loss of motility caused by
blockage of oxidative phosphorylation in the mitochondria. Thus, the
spermatozoa are paralyzed due to mitochondrial damage. This type of
sublethal injury is not detectable with comparable sensitivity in other
eukaryotic cells.
| |
ACKNOWLEDGMENTS |
This work was financially supported by grants from the Ministry
of Agriculture and Forestry (Finland), the Centre of Excellence Fund of
the University of Helsinki, the Technology Development Center of
Finland, and the Academy of Finland.
We express special gratitude to N. Agata (Nagoya City Public Health
Institute, Nagoya, Japan), R. J. Gilbert and A. Scoging (Public
Health Laboratory Service, London, United Kingdom), and A. Christiansson (Meijerierna Service AB, Lund, Sweden) for B. cereus strains. We thank Tuire Koro and Mervi Lindman for
preparing thin sections. Equipment at the laboratory for electron
microscopy of Helsinki University, Biocenter, was at our disposal.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Applied Chemistry and Microbiology, POB 56, Helsinki University FIN
000140, Finland. Phone: 358 9 70859339. Fax: 358 9 7085301. E-mail:
Maria.A.Andersson{at}Helsinki.fi.
| |
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Appl Environ Microbiol, April 1998, p. 1338-1343, Vol. 64, No. 4
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
