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Applied and Environmental Microbiology, October 1999, p. 4637-4645, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Toxigenic Strains of Bacillus licheniformis Related to
Food Poisoning
M. S.
Salkinoja-Salonen,1,*
R.
Vuorio,1
M. A.
Andersson,1
P.
Kämpfer,2
M. C.
Andersson,3
T.
Honkanen-Buzalski,4 and
A. C.
Scoging5
Department of Applied Chemistry and
Microbiology1 and Animal Reproduction,
Department of Clinical Sciences, Saarentaus,3
FIN-00014 University of Helsinki, and Department of Food
Microbiology, National Veterinary and Food Research Institute
(EELA), 00231 Helsinki,4 Finland;
Institut für Angewandte Mikrobiologie, Justus-Liebig
Universität, D-35390 Giessen, Germany2;
and Food Hygiene Laboratory, Central Public Health
Laboratory, Public Health Laboratory Service, London NW9 5HT, United
Kingdom5
Received 18 November 1998/Accepted 5 May 1999
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ABSTRACT |
Toxin-producing isolates of Bacillus licheniformis were
obtained from foods involved in food poisoning incidents, from raw milk, and from industrially produced baby food. The toxin detection method, based on the inhibition of boar spermatozoan
motility, has been shown previously to be a sensitive assay for the
emetic toxin of Bacillus cereus, cereulide. Cell extracts
of the toxigenic B. licheniformis isolates inhibited sperm
motility, damaged cell membrane integrity, depleted cellular
ATP, and swelled the acrosome, but no mitochondrial damage was
observed. The responsible agent from the B. licheniformis
isolates was partially purified. It showed
physicochemical properties similar to those of cereulide, despite having very different biological activity. The
toxic agent was nonproteinaceous; soluble in 50 and 100%
methanol; and insensitive to heat, protease, and acid or alkali and of
a molecular mass smaller than 10,000 g mol
1. The toxic
B. licheniformis isolates inhibited growth of
Corynebacterium renale DSM 20688T, but not all
inhibitory isolates were sperm toxic. The food
poisoning-related isolates were beta-hemolytic, grew anaerobically
and at 55°C but not at 10°C, and were nondistinguishable from the
type strain of B. licheniformis, DSM 13T, by a
broad spectrum of biochemical tests. Ribotyping revealed more
diversity; the toxin producers were divided among four ribotypes when
cut with PvuII and among six when cut with
EcoRI, but many of the ribotypes also contained
nontoxigenic isolates. When ribotyped with PvuII, most
toxin-producing isolates shared bands at 2.8 ± 0.2, 4.9 ± 0.3, and 11.7 ± 0.5 or 13.1 ± 0.8 kb.
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INTRODUCTION |
Bacillus licheniformis,
Bacillus subtilis, and Bacillus pumilus comprise
the subtilis group, which has been associated with a range
of clinical conditions, food spoilage such as ropy bread, and incidents
of food-borne gastroenteritis (27). B. licheniformis has also been associated with septicemia,
peritonitis, ophthalmitis, and food poisoning in humans, as well as
with bovine toxemia and abortions (14, 28). B. licheniformis is a common contaminant of dairy products
(7).
Most food poisoning incidents attributed to Bacillus species
are associated with Bacillus cereus, but the relevance of
the subtilis group as food poisoning organisms is being
increasingly recognized. B. cereus toxins have been well
documented (12), but involvement of toxins produced by
B. licheniformis has not yet been demonstrated. Food-borne
B. licheniformis outbreaks are predominantly associated with
cooked meats and vegetables (20, 24, 26). We report here on
toxin-producing isolates of B. licheniformis obtained from
foods involved in food poisoning incidents, from raw milk, and from
industrially produced baby food.
(A preliminary report on this work has been presented previously
[29].)
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MATERIALS AND METHODS |
Bacterial cultures.
Corynebacterium renale DSM
20688T, B. licheniformis DSM 13T,
Bacillus amyloliquefaciens DSM 7T, B. pumilus DSM 27T, and B. cereus DSM
31T were obtained from the German Collection of
Microorganisms and Cell Cultures, Braunschweig, Germany, and B. subtilis ATCC 6051T was obtained from the American
Type Culture Collection, Manassas, Va. The emetic-toxin-producing
strains of B. cereus, 4810/72, NC7401, and F-5881, are
described elsewhere (3). The bacteria were cultured for
toxicity assays on blood or brain heart infusion (Difco Laboratories,
Detroit, Mich.) or in Trypticase soy agar (LAB M, Bury, England) plates
or Trypticase soy broth. Temperature tolerance was tested at 10°C
(with B. cereus DSM 31T as a positive control)
and at 55°C (±0.5°C) in liquid medium on a shaking incubator (150 rpm) and at 28, 37, and 55°C (±0.5°C) on agar plates.
Biological analyses.
The following phenotypic traits were
assayed as described by Smibert and Krieg (25): hydrolysis
of lecithin, of starch, and of casein and lysozyme resistance (7 days,
25°C); hemolysis (bovine blood agar plates, read after 24 and 72 h at 37°C); and anaerobic growth (read after 1 and 7 days, 37°C).
The brain heart infusion agar plates were preincubated in anaerobic
chambers before use. Lipase activity was assayed with modified Sierra
lipolysis agar containing peptone (25) (10 g), NaCl (5 g),
CaCl2 (0.1 g), beef extract (3 g), ferrous citrate (0.2 g),
and agar (15 g) per liter. After autoclaving, 0.5 ml of Victoria Blue B
(stock, 0.1 g/150 ml) and 0.1 ml of Tween 80 were added per 10 ml of
the medium. Microconcentrator membranes were obtained from Amicon Ltd.,
Stonehouse, United Kingdom.
Physiological tests.
The food poisoning isolates (coded F)
of B. licheniformis were identified by 25 phenotypic and
biochemical tests as described in reference 10. Good
anaerobic growth and utilization of propionate were used to distinguish
the strains from B. subtilis. All B. licheniformis isolates were characterized by using API 50 CH
cassettes (bioMérieux, Marcy l'Etoile, France), read after 24 and 48 h at 37°C with Bacillus identification profile
database API Lab+ (version 2.1) and with a battery of 87 physiological
tests, as described previously (17). The reaction profiles
of these tests were compared with a database (16).
Toxicity tests.
Bacteria were grown on tryptic soy agar
plates for 10 days at 28°C to obtain mainly sporulated and lysed
cells, verified by phase-contrast microscopy. Colonies were scraped
from the agar and suspended in aqua destillata to 100 mg
ml1. The suspension was treated by repeated freeze-thaw
cycles and filtered (0.2-µm pore size). The permeate was diluted in
dimethyl sulfoxide and tested for toxicity by using the same
concentration of dimethyl sulfoxide as the blank.
The motility inhibition of boar spermatozoa by the cell extracts was
tested as described for the emetic toxin of B. cereus (3). Of each bacterial strain, two to five independently
prepared extracts were tested. The sperm motility inhibition by the
extracts was given as the concentration required to block motility of
50% of the cells (see Table 1) or by indicating the percentage of motile cells (see Table 3). Three microscopic fields of 102
spermatozoa (magnification, ×200) were analyzed for motility with a
Hamilton-Thorne sperm analyzer (HTM-S, version 7.2; Hamilton-Thorne Research, Danvers, Mass.) as described in reference
3. The results were confirmed by subjective
estimation of motility by phase-contrast microscopy (5 to 10 fields;
magnification, ×200).
The cell membrane-damaging capacity of the bacterial extracts was
measured by selective staining with ethidium homodimer and calcein AM,
carried out as described in reference 15. Damage to
acrosomes was recorded by light microscopy as described elsewhere (30). Mitochondrial damage was documented by transmission
electron microscopy of spermatozoan thin sections as described
elsewhere (3). ATP loss from the spermatozoan was assayed as
described elsewhere (3).
C. renale DSM 20688
T growth inhibition
(
4) was read after 2 days at 28°C from two to three
replicate Trypticase soy agar
plates with wells each holding 150 to 200 µl of the cell extract
of the isolate to be
tested.
Ribotyping.
Ribotyping was performed with a robotized
instrument as described in reference 31. The
B. licheniformis strains were prepared and analyzed
similarly to the B. cereus strains (23). In
short, the DNA was restriction endonuclease cut with EcoRI
or PvuII and hybridized to phosphorescently labeled
Escherichia coli whole ribosomal operon. Fragment sizes were
determined with the GelCompar program (version 4.0; Applied Maths BVBA,
Kortrijk, Belgium) from the ribotypes produced by the RiboPrinter
(Qualicon, Wilmington, Del.) with DNA molecular size markers
(1.1, 2.2, 3.2, 6.5, 9.6, and 48 kb) in every third lane.
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RESULTS |
Detection of toxin-producing B. licheniformis
isolates.
In total, 210 B. licheniformis isolates
involved in food poisoning or suspected food poisoning and 29 strains
and isolates originating from veterinary samples, food packaging
material, air, and industrial contaminants were studied for toxicity by two tests, the spermatozoan motility test and the C. renale
DSM 20688T growth inhibition test. Thirteen strains were
found to be positive in one or both toxicity tests. Isolates toxic by
both tests (n = 10) originated from incident-associated
food or clinical specimens from Finland and the United Kingdom over a
period of >10 years, mainly from cases where B. licheniformis was isolated from food in high numbers
(104 to 105 CFU g1). In addition,
toxic isolates were obtained from the udder of a cow that had
clinically recovered from severe mastitis. Table 1 is a compilation of the properties of
the 13 positive and 9 nontoxic strains.
Table
1 shows that crude cell extracts (filtered through
0.2-µm-pore-size filters) prepared from 10
B. licheniformis isolates
inhibited boar spermatozoan motility when
spermatozoa were exposed
to the extracts. This protocol has been shown
to be a sensitive
indicator for the presence (in nanograms per
milliliter) of the
emetic toxin of
B. cereus (
3).
The 10 toxic isolates included
two isolates (553/1 and 553/2)
cultured from infant feed formula
following an infant fatality (Table
1). Cell extracts prepared
from these 10 isolates also inhibited
growth of
C. renale DSM
20688
T. The type strain
of
B. licheniformis, DSM 13
T, was not toxic by
either test. The type strains of
B. subtilis (ATCC 6051
T) and
B. pumilus (DSM
27
T) were also tested and found to be nontoxic to sperm
cells.
Two
B. licheniformis isolates (575U/5 and 575E/P) of eight
tested from unused infant feed packages of the same brand as that
connected to the fatal case blocked sperm motility and inhibited
C. renale (Table
1; the six nontoxic isolates are not
shown).
Toxic strains were also isolated from a fecal specimen of a
hospital
patient with acute-phase food poisoning symptoms (F287/91) and
from food poisoning cases connected with curry rice and fast foods
(F2943/92, F5520/96, and F231/97). Two of three isolates from
milk
(each from a separate quarter of the udder) of a postmastitic
cow were
inhibitory to
C. renale DSM 20688
T (Table
1; the
nontoxic isolate is not shown) and blocked boar
spermatozoan motility.
Even though all sperm-toxic isolates inhibited
growth of
C. renale, the reverse was not true. However, the sperm
cells were
exposed to extracts corresponding to 2 to 4 mg (wet
weight) of
B. licheniformis cells ml
1, whereas the amount used in
the
C. renale test corresponded to
20 to 40 mg (wet weight)
of
B. licheniformis cells per well in
the agar plates. The
C. renale test, using a higher amount of
the agent, may thus
have detected weak toxin producers which were
undetectable in the sperm
test. High doses could not be tested
with sperm cells because of
nonspecific interference by the crude
bacterial extracts. The results
thus do not exclude the possibility
that both effects may have been
caused by the same toxic agent.
Extracts prepared from the type strain
of
B. cereus (DSM 31
T) strongly inhibited growth
of
C. renale at amounts equivalent
to 20 to 40 mg of
B. cereus cells per
well.
Description of the toxigenic B. licheniformis
isolates.
All toxic B. licheniformis isolates
grew anaerobically; were lecithinase negative, lipolytic (i.e., they
hydrolyzed Tween 80; isolates coded F and 123/3 were not tested),
and lysozyme sensitive; and hydrolyzed starch and casein (123/3 was not
tested). The 10 sperm-toxic strains grew at 55 but not at 10°C in
Trypticase soy broth. Twelve of 17 strains inhibitory to C. renale (Table 1) were beta-hemolytic, and 9 of the 10 sperm-toxic isolates were (the exception was F231/97) also
beta-hemolytic. Four of the 15 beta-hemolytic isolates were not
inhibitory to C. renale and not sperm toxic. The type strain
of B. licheniformis (DSM 13T) was nonhemolytic
and nontoxic. The growth inhibition of C. renale and/or
toxicity to boar spermatozoa was thus not due to the production of
beta-hemolysins by the B. licheniformis isolates.
All isolates listed in Table
1 could use propionate as the sole carbon
source, the characteristic which distinguishes
B. licheniformis from
B. subtilis and
B. pumilus. The sperm-toxic
and nontoxic strains of
B. licheniformis were compared by using
87 biochemical traits. The 21
B. licheniformis isolates presented
in Table
1 were
characterized as follows. The results presented
for the 21 isolates are
identical to those obtained for the type
strain DSM 13
T.
Type strain DSM 13
T and all isolates assimilated
N-acetyl-
D-glucosamine,
L-arabinose,
p-arbutin,
D-cellobiose,
D-fructose,
D-galactose, gluconate,
D-glucose,
D-mannose,
D-maltose,
-
D-melibiose,
L-rhamnose,
D-ribose, sucrose,
salicin,
D-trehalose,
D-xylose,
i-inositol, maltitol,
D-mannitol,
D-sorbitol, acetate, propionate,
cis-aconitate,
trans-aconitate,
4-aminobutyrate,
citrate, fumarate,
DL-3-hydroxybutyrate,
DL-lactate,
oxoglutarate, pyruvate,
L-alanine,
L-aspartate,
L-ornithine, and
L-proline. Type strain DSM 13
T and all isolates
did not assimilate adonitol, putrescine, adipate,
azelate, glutarate,
itaconate,
L-malate, mesaconate, suberate,
-alanine,
L-histidine,
L-leucine,
L-phenylalanine,
L-serine,
L-tryptophan, 3-hydroxybenzoate, 4-hydroxybenzoate,
and phenylacetate.
All isolates (including type strain DSM
13
T) did not hydrolyze esculin,
para-nitrophenyl-
-
D-galactopyranoside,
para-nitrophenyl-
-
D-glucopyranoside,
para-nitrophenyl-
-
D-glucopyranoside,
bis-
para-nitrophenyl-phosphate, and
L-glutamyl-
-3-carboxy-
para-nitroanilide.
Type strain DSM 13
T but none of the isolated
hydrolyzed
para-nitrophenyl-
-
D-glucuronide,
para-nitrophenyl-phenyl-phosphonate,
para-nitrophenyl-phosphorylcholine,
2-deoxythymidine-5'-
para-nitrophenyl-phosphate,
L-alanine-
para-nitroanilide,
and
L-proline-
para-nitroanilide. The results
confirmed their identity
as
B. licheniformis, with a Willcox
probability of
P > 0.99 (
17).
No
biochemical difference was detected between the isolates that
were
toxic and those that were nontoxic to sperm cells or
C. renale DSM 20688
T: all biochemical reactions were the
same as those of the type
strain
B. licheniformis DSM
13
T.
Ribotype patterns of toxic and nontoxic B. licheniformis strains.
The 21 isolates and the type strain
of B. licheniformis in Table 1 were ribotyped by using
EcoRI and PvuII, and the multiband patterns
obtained are shown in Fig. 1. Ten
distinct ribotype patterns were obtained with EcoRI, and 11 were obtained with PvuII. In the ribotype patterns obtained
with PvuII (Fig. 1A), the sperm-toxic isolates clustered in
four groups, in three (Fig. 1, lanes A, B, and E) of which the fragment
patterns were closely similar. Two of these ribotypes (A and B) also
contained nontoxic isolates. The three related ribotype patterns shared
bands at 2.7 ± 0.2, 5.0 ± 0.2, and 11.6 ± 0.1 kb
(Table 2). The type strain B. licheniformis DSM 13T (nontoxic) has ribotype A
together with another nontoxic strain and two toxic strains (lane A,
Fig. 1). The fourth toxic strain ribotype (lane H in Fig. 1) shared the
bands at 2.7 and 5.0 kb but had, in addition, four larger fragments (13 to 35 kb). When cut with EcoRI, one ribotype pattern
contained the type strain DSM 13T and seven other isolates,
of which five were toxic (Fig. 1B, lane A). The other sperm-toxic
isolates were scattered among six EcoRI ribotype patterns.
The results thus show that among the toxic strains there was
considerable diversity in ribotypes, indicating that the toxic isolates
were not a clone. However, the PvuII ribotypes containing
toxic isolates shared bands of similar sizes (2.5, 5.0, 7 to 8, 11.6, or 13.6 kb [Table 2]). The fragment sizes obtained by the unweighted
pair group method with averages algorithm and a commercial computer
program for 22 isolates analyzed with an automated ribotyper over a
period of 1 year showed standard deviations of approximately 5% for
fragments of <10 kb and 5 to 10% for fragments of 10 to 36 kb.
Considering this reproducibility, an automated search for fragments of
specific sizes may be useful as a preliminary screening method for
toxic strains.
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FIG. 1.
Ribotyping of 25 isolates and strains of B. licheniformis of different origins, with PvuII (A) or
EcoRI (B) and hybridization with labeled whole ribosomal
operon of E. coli. The patterns obtained from B. amyloliquefaciens DSM 7T, B. cereus DSM
31T, and B. subtilis ATCC 6051T are
also shown. Strains indicated as being of the same ribotype exhibited
patterns with a similarity value of >0.95. (A) Lane A, DSM
13T, 553/2 (toxic), 575E/P (toxic), and TSP29a; lane B,
553/1 (toxic), F287/91 (toxic), F231/97 (toxic), Hulta 53/97, and Hulta
54/97 (toxic); lane E, F2943/92 (toxic); lane H, 575U/5 (toxic),
F2667/94, F5520/96 (toxic), Hulta 52/97 (toxic), and TSP19; lanes C, D,
F, G, I, J, and K, no toxic isolates; lanes L, M, and N, reference
strains B. amyloliquefaciens DSM 7T, B. subtilis ATCC 6051T, and B. cereus DSM
31T, respectively. (B) Lane A, DSM 13T, 553/1
(toxic), 575U/5 (toxic), F281/91 (toxic), F5734/93, TSP29a, and Hulta
52/97 (toxic); lane C, F9229/95, Hulta 53/97, and Hulta 54/97 (toxic);
lane D, F2943/92 (toxic), F2896/95, and F231/97 (toxic); lane E, 575E/P
(toxic); lane F, 553/2 (toxic); lane I, F5520/96 (toxic); lanes B, G,
H, and J, no toxic isolates; lanes K, L, and M, reference strains
B. amyloliquefaciens DSM 7T, B. subtilis ATCC 6051T, and B. cereus DSM
31T, respectively.
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Effects of toxic B. licheniformis extracts on boar
spermatozoa.
The responses, shown as four different viability
parameters, of boar spermatozoa to cell extracts prepared from nine
sperm-toxic isolates of B. licheniformis (Table 1) are
compiled in Table 3. None of the extracts
was cytolytic toward spermatozoa. All nine isolates inhibited motility
of the exposed spermatozoa and damaged cell membrane integrity (Fig.
2), depleted the cellular ATP content
(Table 3), and swelled the acrosome (Fig.
3). None of these extracts swelled the
mitochondria as observed by transmission electron microscopy (Fig.
4). Extracts prepared from the type strains of B. licheniformis, B. subtilis,
B. pumilus, Bacillus mycoides, and B. cereus caused none of the effects exhibited by the strains of
B. licheniformis (Table 3). Extracts prepared from three
emetic-toxin-producing strains of B. cereus (4810/72, NC7401, and F-5881) were tested in the same assay; all inhibited spermatozoan motility at extremely low concentrations
(corresponding to <0.002 mg [wet weight] of cells
ml1) and swelled the mitochondria in the sperm tail but
had no effect on the membrane integrity, cell ATP content, or the
acrosome (Table 3). The extracts of each of the three emetic B. cereus strains had no effect on the growth of C. renale
at doses corresponding to 20 to 40 mg (wet weight) of cells per well in
the agar plate.
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TABLE 3.
Effects of cell extracts from food- and food
poisoning-related isolates of B. licheniformis and from
reference strains on boar spermatozoaa
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FIG. 2.
Fluorescence micrographs of boar spermatozoa stained for
determination of viability after being exposed to cell extracts of
different B. licheniformis strains. (A) Sperm cells (5 × 106) in 1 ml of extended (with commercial extender) boar
semen were exposed to cell extract from 4 mg (wet weight) of B. licheniformis DSM 13T cells. Over 80% of the
spermatozoa showed intact cell membranes. Similar results were obtained
with spermatozoa exposed to the negative control (staining green). (B)
Effect of extract prepared from 2 mg (wet weight) of cells of B. licheniformis 553/2. Fifty percent of the sperm cells lost
integrity in the cell membrane (staining orange). (C) Sperm cells were
exposed to extract prepared from 4 mg (wet weight) of the same isolate
as in panel B. Seventy percent of sperm cells lost cell membrane
integrity (red). Magnification in all panels, ×2,000 (i.e., sperm head
dimensions are 2 to 3 by 3 to 5 µm).
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FIG. 3.
Light micrographs of Giemsa stained boar spermatozoa
exposed to cell extracts of B. cereus and toxic and nontoxic
strains of B. licheniformis. (A and B) Extended boar semen
(1 ml) exposed to extract from 4 mg (wet weight) of cells of the emetic
B. cereus strain 4810/72 (A) or the (nontoxic) B. licheniformis type strain DSM 13T (B). Over 90% of
the exposed sperm cells showed heavily staining dark intact acrosomes,
similar to spermatozoa exposed to negative control extract (data not
shown). (C) Extended boar semen (2 ml) exposed to B. licheniformis 553/2 (extract from 4 mg [wet weight] of cells).
Over 50% of the cells showed lightly staining fused acrosomes or
swollen and disrupted acrosomes. Magnification in all panels, ×2,000
(i.e., sperm head dimensions are 2 to 3 by 3 to 5 µm).
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FIG. 4.
Thin cross sections of the middle segments of boar
spermatozoa exposed for 4 days to cell extracts of B. licheniformis 553/2 and F287/91, the B. licheniformis
type strain DSM 13T, and an emetic toxin producer strain,
B. cereus 4810/72. (A and B) Sperm cells exposed to cell
extracts prepared from 4 mg (wet weight) of isolates 553/2 and F287/91.
These sperm cells had lost motility and ATP and showed damaged cell
plasma membrane, but the mitochondria were intact. (C) Spermatozoon
exposed to extract of the type strain B. licheniformis DSM
13T (nontoxic). These cells displayed normal motility and
cellular ATP content after exposure, and the figure shows an intact
plasma membrane. (D) Sperm cell exposed to cell extract from 2 mg (wet
weight) of the emetic strain B. cereus 4810/72
(12) ml1. These cells have a morphologically
intact plasma membrane and normal ATP content, but over 90% of the
cells exposed showed no motility, and their mitochondria were swollen
with a disrupted outer membrane. Bars, 200 nm.
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These results show that the sperm-toxic agent(s) produced by the toxic
strains of
B. licheniformis was uniform in action.
The
exerted toxic effect differed in biological activity from
that of the
cereulide produced by the emetic strains of
B. cereus.
It is
interesting that the extracts prepared from the two sperm-toxic
B. licheniformis isolates (Hulta 52/97 and Hulta 54/97)
originating
from raw milk from a postmastitic cow exhibited toxic
responses
in spermatozoan cells quantitatively and qualitatively
similar
to those seen after exposure to the toxic food poisoning
isolates,
including those from an unused package of branded infant feed
(Tables
1 and
3). The effects on the spermatozoan plasma membrane
and
on the acrosomal response to
B. licheniformis
extracts were
dose dependent. The toxic threshold of these effects
was equivalent
to 2 to 4 mg (wet weight) of bacterial cells per
ml of extended
boar semen for all toxic
B. licheniformis
strains (Fig.
2 and
Table
1).
Toxic activities of extracts prepared from the
B. licheniformis isolates 553/1, 553/2, 575U/5, Hulta 52/97,
and Hulta 54/97
were insensitive to heat (100°C for 20 min),
inactivation by pronase
(200 µg ml
1, 3 h),
acid (pH 2 with HCl for 30 min), and alkali (pH 12 with
NaOH for 30 min). The observed heat stability suggests that the
toxin(s) was not an
enzyme. The toxic agent was more soluble in
50 or 100% methanol than
in water. As a solution in 50% (vol/vol)
methanol, the toxic agent was
filterable through microconcentrator
membranes with a nominal cutoff of
10,000 g mol
1 but not as a water extract, indicating a
tendency to hydrophobic
interactions. The 10,000-g
mol
1 filtrates of the toxic extracts exhibited unaltered
toxic effects
on the spermatozoa. These results show that the
B. licheniformis sperm-toxic agent was
nonproteinaceous, heat stable, and nonpolar
and of an apparent mass
smaller than 10,000 g mol
1. The agent inhibiting the
growth of
C. renale also survived the
treatments listed
above, indicating that it was the same or a
similar type of compound as
the agent blocking spermatozoan
motility.
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DISCUSSION |
This paper is to our knowledge the first demonstration of toxins
produced by B. licheniformis isolates associated with human disease. Toxins produced by B. licheniformis were detected
by the boar spermatozoan motility inhibition assay, which has been reported as a sensitive and specific test for detecting
emetic-toxin-producing B. cereus strains (3).
The toxins of B. licheniformis inhibited sperm motility
(Table 1) by interfering with the cellular energy metabolism in a manner different from that shown with the emetic toxin of
B. cereus, a toxin that causes swelling of
mitochondria (3, 21). The toxic threshold of the B. licheniformis extracts was 
100 times higher than those observed
for emetic-toxin-producing B. cereus strains (3).
The occurrence of B. cereus in sensitive foods is regulated
in many countries (in Finland, the limit is 
103 CFU
g1). There is no restriction on B. licheniformis, which often occurs in high numbers in foods (we
observed 104 to 108 CFU g1
[Table 1]). The toxigenicity observed in the present work may thus be
of food poisoning significance. The toxic B. licheniformis extracts induced the acrosome reaction (Table 3 and Fig. 3), a very
likely novel trait among bacterial toxins, and possibly indicating an
impact on the cellular signalling system.
Despite differences in biological activity, the sperm-toxic agents from
the isolates of B. licheniformis studied (Table 3) were
similar in many physicochemical properties to cereulide, the emetic
toxin of B. cereus (1, 3). Cereulide is a
dodecadepsipeptide structurally resembling valinomycin (1).
Peptide toxins are nonribosomally produced by a wide range of
microorganisms (18). Also, some strains of B. licheniformis are known to produce peptide antibiotics
(13), such as bacitracin (11, 18) and amoebicins (8, 9, 19), some of which have been used as antimicrobial agents, but none have so far been shown to be associated with food
poisoning. The B. licheniformis toxins reported in this
paper also possessed antimicrobial activity, demonstrated by the
induction of large inhibition zones by cell extracts of many of the
B. licheniformis isolates when introduced onto plate
cultures of the actinomycete C. renale DSM
20688T (Table 1). C. renale has been shown
elsewhere to be susceptible toward human pathogenic
Staphylococcus aureus strains producing staphylococcin BacR1
(4) and to be associated with the production of the
exfoliative toxin B (22). The anticorynebacterial activity of B. licheniformis isolates was caused by an agent(s)
physicochemically similar to that toxic to spermatozoa (both are
resistant to heat, acid, and alkali and soluble in methanol). Sperm
toxin-producing B. licheniformis strains were not
readily differentiated from nontoxigenic strains by only
biochemical and physiological criteria (Table 1; see also above).
Ribotyping revealed great genetic diversity; the toxigenic strains thus
formed no clone. However, the toxic ribotypes were related (shared
bands [Table 2]); only 4 of the 11 PvuII ribotypes
contained toxigenic strains. Automated ribotyping may be useful as a
preliminary screening test for putative toxin producers.
It is interesting that the ribotype of a toxic isolate obtained from
the baby food associated with a fatal food poisoning (553/1) was
identical to that of the isolate obtained from an unopened package of
the same brand (575U/5) and to that of a toxic isolate cultured from
the raw milk of a cow that had apparently recovered from mastitis
(Hulta 52/97). As B. licheniformis is a sporeformer and
likely to survive all industrial processing of milk, such as the
manufacture of milk powder and whey concentrate, such a finding may
indicate a possible route of infection.
These results also indicate genetic diversity among the toxic B. licheniformis isolates: two different toxigenic ribotypes were
isolated from two different quarters of the same udder of a cow, two
different toxigenic ribotypes were isolated from the same batch of baby
food, and two toxigenic strains with different ribotypes were recovered
from an unused package of commercial baby food. It is possible that
genotypically distinguishable different toxigenic isolates might have
been detected also in the other food poisoning cases listed in Table 1,
had more than one isolate been available for study.
Recombinant strains of B. licheniformis are used to produce
industrial enzymes on a large scale, e.g., carbohydrase and protease used in food processing (5, 6). The species is considered safe and has generally recognized-as-safe status with the U.S. Food and
Drug Administration (6). In the light of our findings, the
generally recognized-as-safe status of the species B. licheniformis may require reassessment.
| |
ACKNOWLEDGMENTS |
This work was financially supported by the Academy of Finland
(M.S.S.) and the Centre of Excellence Fund of the University of
Helsinki (M.S.S.). We thank the Institute of Biotechnology of the
University of Helsinki for the use of the electron microscope.
We are grateful to Jyrki Juhanoja for expert technical support in
electron microscopy, to Paula Hyvönen (EELA, Kuopio) for donating
strain 123/3, to Irina Tsitko for help with the GelCompar program, and
to Irmgard Suominen and Camelia Apetroaie for their contributions in
sperm toxicity testing.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Applied Chemistry and Microbiology, P.O. Box 56 (Biocenter), 00014 University of Helsinki, Finland. Phone: 358-9-70859300. Fax:
358-9-70859301. E-mail:
mirja.salkinoja-salonen{at}helsinki.fi.
| |
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Abstract
Introduction
