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Applied and Environmental Microbiology, April 1999, p. 1483-1490, Vol. 65, No. 4
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Cloning and Nucleotide Sequence Analysis of gyrB of
Bacillus cereus, B. thuringiensis,
B. mycoides, and B. anthracis and
Their Application to the Detection of B. cereus in
Rice
Shoichi
Yamada,1
Eiji
Ohashi,1
Norio
Agata,2 and
Kasthuri
Venkateswaran1,*
Central Research Laboratory, Nippon Suisan
Kaisha, Ltd., Hachioji City, Tokyo 192,1 and
Nagoya City Public Health Research Institute, Mizuho, Nagoya
467,2 Japan
Received 25 September 1998/Accepted 5 January 1999
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ABSTRACT |
As 16S rRNA sequence analysis has proven inadequate for the
differentiation of Bacillus cereus from closely
related species, we employed the gyrase B gene (gyrB)
as a molecular diagnostic marker. The gyrB genes of
B. cereus JCM 2152T,
Bacillus thuringiensis IAM 12077T,
Bacillus mycoides ATCC 6462T, and
Bacillus anthracis Pasteur #2H were cloned and
sequenced. Oligonucleotide PCR primer sets were designed from within
gyrB sequences of the respective bacteria for the
specific amplification and differentiation of B. cereus, B. thuringiensis, and
B. anthracis. The results from the amplification
of gyrB sequences correlated well with results obtained
with the 16S rDNA-based hybridization study but not with the results of
their phenotypic characterization. Some of the reference strains of
both B. cereus (three serovars) and B. thuringiensis (two serovars) were not positive in PCR
amplification assays with gyrB primers. However, complete
sequencing of 1.2-kb gyrB fragments of these reference
strains showed that these serovars had, in fact, lower homology than
their originally designated species. We developed and tested a
procedure for the specific detection of the target organism in boiled
rice that entailed 15 h of preenrichment followed by PCR
amplification of the B. cereus-specific fragment.
This method enabled us to detect an initial inoculum of 0.24 CFU of B. cereus cells per g of boiled rice food
homogenate without extracting DNA. However, a simple two-step
filtration step is required to remove PCR inhibitory substances.
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INTRODUCTION |
Bacillus cereus is a
gram-positive, spore-forming, facultatively anaerobic bacterium.
Differentiation of B. cereus from its closely
related microorganisms depends upon the absence of toxin crystals (from
B. thuringiensis), hemolytic activity (from
B. anthracis), and nonrhizoid growth and
motility (from B. mycoides). B. cereus produces emetic toxin and enterotoxins (2, 16, 18,
28, 29, 33). Strains of B. thuringiensis are also reported to produce
enterotoxins (1, 3, 14, 23), and molecular characterization
revealed that the enterotoxin-encoding gene isolated from B. thuringiensis was similar to that of B. cereus (3).
The nucleotide sequence of 16S rRNAs of the B. cereus group exhibited very high levels of sequence similarity
(>99%) that were consistent with the close relationships shown by
previous DNA hybridization studies (37). The 16S rRNA
sequences of B. mycoides and B. thuringiensis differed from each other and from the
sequences of B. anthracis and B. cereus by 0 to 9 nucleotides (5). Likewise, Ash and
Collins (4) reported that the 23S rRNA gene sequences of
B. anthracis and an emetic strain B. cereus were found to be identical. The lesser variations noted
in the spacer regions between the 16S and 23S rRNAs do not seem to be sufficient to allow the design of a species-specific oligonucleotide probe for the B. cereus-B.
thuringiensis-B. anthracis group
(9). Specific DNA probes based on variable region VI of 16S
rRNAs of B. cereus and B. thuringiensis were designed by te Giffel et al. (39) and used in hybridization experiments, but screening of isolates with this probe from various sources and outbreaks is necessary to validate their claim. Single-strand conformation polymorphism of the PCR products did not allow species discrimination within the B. cereus group (8).
Virulence factors (22, 35), restriction fragment length
polymorphism (25), pulsed-field gel electrophoresis,
analysis of intergenic spacer regions (20), and the
arbitrary PCR (12, 21) differentiated B. anthracis from B. cereus but failed to
differentiate B. cereus from B. thuringiensis. Because of the indistinguishable
phenotypic and genotypic characteristics of these organisms, Ash
et al. (6) proposed considering B. thuringiensis, B. mycoides, and
B. anthracis as subspecies of B. cereus.
Since no universal probe is available to differentiate B. cereus from other related species, we have studied the
possibility of using gyrB genes that encode the subunit B
protein of DNA gyrase (topoisomerase type II) as targets of
highly specific probes (50). In this study, 1.2-kb fragments
of the gyrB genes of B. cereus, B. anthracis, B. thuringiensis, and B. mycoides were
amplified, cloned, and sequenced. We designed suitable PCR primer sets
that could amplify the gyrB fragments of B. cereus, B. anthracis, and B. thuringiensis to specifically identify
the organism irrespective of its phenotypes, serotypes, and virulence
factors. A protocol for the direct detection of B. cereus from boiled rice without extracting DNA is also described.
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MATERIALS AND METHODS |
Bacterial strains.
Microorganisms included in this study
were purchased from various culture collections or were gifts from the
Nagoya Public Health Research Institute and Mie University, Nagoya,
Japan. Strains isolated from various aquatic environments and foods are
also included. All microorganisms were grown in nutrient broth (Nissui, Tokyo, Japan) at 35°C for 24 h before use. Presumptive colonies appearing on the NGKG agar (Nissui) were picked, and biochemical characterization of the isolates was carried out as described elsewhere
(28, 38, 45).
DNA isolation.
Chromosomal DNA of overnight-grown cultures
was extracted by phenol-chloroform solvents and ethanol precipitation
(36). The dried DNA was then dissolved in Tris-EDTA (TE)
buffer (pH 7.5) and used as the DNA template. The purity of the DNA was
checked by agarose gel electrophoresis, and the DNA concentration was measured with a spectrophotometer.
Cloning and sequencing of the gyrB gene.
Primers
(UP-1 and UP-2r) within the known DNA sequence (50) were
added to the PCR mixture at a concentration of 1 µM, and the solution
was subjected to 30 cycles of PCR (43). The amplified gyrB fragments from B. cereus JCM
2152T, B. thuringiensis IAM
1077T, B. anthracis (Pasteur #2H),
and B. mycoides ATCC 6462T were cloned in
pGEM-ZF+ (Promega, Madison, Wis.) by conventional
recombinant methods (36). Expansion of the probes was
carried out as documented previously (36). After ligation of
the PCR fragments into the vector, Escherichia coli cells
were transformed with the ligation mixture by calcium chloride-mediated
transformation. After transformation, the transformants were cultured
under conditions that promote growth. Plasmids were recovered from a
transformant by lysis and purification by an alkaline method. The
purified intact plasmid was then utilized as a probe. The identity of
the fragment was verified by sequencing from both ends by the dideoxy
chain termination method with a Sequenase DNA sequencing kit (U.S.
Biochemical Corp., Cleveland, Ohio) and with an ABI 373A automatic
sequencer as described by the manufacturer (Perkin-Elmer Corp., Foster
City, Calif.). DNA sequences were determined from both strands by
extension from vector-specific (T7 and SP6 primers from
pGEM-ZF+) priming sites and by primer walking.
Oligonucleotide primers.
Various oligonucleotide primers
based on the gyrB nucleotide sequence data of the
Bacillus species were synthesized according to the
instructions of the manufacturer (Beckman, Fullerton, Calif.).
PCR assay.
Whole bacterial cells without extracting DNA were
used as templates. In this case, the freshly grown cells (overnight
incubation at 35°C) on nutrient agar plates were used. If the
bacterial cells were grown in liquid medium, the bacterial cells were
harvested by centrifugation and washed once with phosphate-buffered
saline (PBS; 0.1 M, pH 7.5), and appropriate counts of bacterial cells were used. In some cases, purified DNA was used as a template for PCR amplification.
PCR assays were performed in a DNA Thermal Cycler (Perkin-Elmer).
Reaction volumes of 100 µl contained 100 ng of genomic DNA, deoxynucleoside triphosphates at a concentration of 200 µM each, and
primers at 1 µM each in reaction buffer (Tris-HCl, 100 mM; MgCl2, 15 mM; KCl, 500 mM; pH 8.3). The 1.2-kb
gyrB gene was amplified as described elsewhere
(50). The amplification of various Bacillus species-specific (B. cereus, 365 bp; B. thuringiensis, 368 bp; and B. anthracis, 245 bp) fragments was performed by using PCR for 30 cycles, each consisting of 1 min at 94°C, 1.5 min at 58°C, and 2.5 min at 72°C, with a final extension step at 72°C for 7 min.
After DNA amplification, PCR fragments were analyzed by submarine gel
electrophoresis, stained, and visualized under UV illumination (44). Suitable molecular size markers were included in each gel.
Hybridization with 16S rDNA probes.
Hybridizations were
performed with 16S rDNA probes that are specific to B. cereus (TTA AGA ACT TGC TCT TAT) and B. thuringiensis (TTG AGA GCT TGC TCT CAA) as described
by te Giffel et al. (39). The purified 16S fragment was
transferred onto a nylon membrane (Hybond-N; Amersham) by Southern
blotting. The blotted membranes were neutralized in 0.2 M Tris-HCl (pH
7.5) in 0.3 M NaCl-0.03 M sodium citrate and air dried.
Prehybridization and hybridization were performed in 0.5 M sodium
dodecyl sulfate and 1% bovine serum albumin. After 30 min of
prehybridization at 45°C, the commercially available probe, which
had been 3' end DIG labeled (Boehringer Mannheim, Foster City, Calif.),
was added, and signals were detected according to the manufacturer's
instruction with DIG DNA labeling kits (Boehringer Mannheim).
PCR assay sensitivity for the detection of artificially
contaminated B. cereus in boiled rice.
A 25-g
sample of boiled rice was homogenized for 1 min with a homogenizer
(SH-001; Elmex, Tokyo, Japan) in 225 ml of nutrient broth to produce a
uniform food homogenate for all experiments. B. cereus JCM 2152T was grown in nutrient broth
overnight at 35°C and serially diluted with the food homogenate as
the diluent to final concentrations ranging from 0 to 109
CFU per g. These artificially contaminated food homogenate microcosms (10 ml each) were incubated at 35°C. Subsampling (1 ml) was carried out after incubations of 0, 6, and 15 h (overnight); cells were then centrifuged (4°C; 10,000 × g for 10 min) and
resuspended in 1 ml of sterile PBS.
Suitable controls such as buffer, media, PCR mixtures, and
B. cereus DNA were employed to check any
false-positive or false-negative reactions. Appropriate dilutions of
test sample prepared at various intervals in nutrient broth were spread
plated onto standard plate count agar (Nissui) for total viable counts
and on NGKG agar (Nissui) for the enumeration of the B. cereus population.
To remove any PCR-inhibitory substances from food, samples drawn from
the microcosms were subjected to two-step filtration
as described
previously (
44), with some modifications. Briefly,
a
400-µl sample of a sterile-PBS-washed sample was passed through
a
5-µm-diameter Ultrafree filter tube (UFC3 0GV; Millipore, Bedford,
Mass.) and centrifuged (4°C; 10,000 ×
g for 10 min).
The 5-µm-diameter-tube
filtrate was then passed through
0.2-µm-diameter Ultrafree centrifuge
tube (SE3P009E4; Millipore) and
centrifuged (4°C; 10,000 ×
g for
10 min) to remove
bacteria. The material trapped on the 0.2-µm
(pore size) filter was
then resuspended in 50 µl of sterile PBS
and boiled before 10 µl of
supernatant was used as a template
for the PCR
assay.
Nucleotide sequence accession numbers.
The nucleotide
sequence data reported here will appear in the GenBank nucleotide
sequence database with the indicated accession numbers: B. cereus JCM 2152T (AF090330), B. thuringiensis IAM 12077T (AF090331),
B. anthracis Pasteur #2H (AF090333), and
B. mycoides ATCC 6462T (AF090332).
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RESULTS |
gyrB sequence of Bacillus species.
Complete sequences of the 1.2-kb gyrB fragments of
B. cereus JCM 2152T, B. thuringiensis IAM 12077T, B. anthracis Pasteur #2H, and B. mycoides ATCC 6462T were determined and aligned
(Fig. 1). The percentages
of similarity (Table 1) and numbers of
base-pair substitutions (Table 2) in gyrB nucleotide sequences and translated protein sequences
for all four of these Bacillus species are given. Likewise,
the percentages of similarity and numbers of base-pair substitutions in
16S rDNA nucleotide sequences are depicted in Table
3. The similarity in the gyrB
sequences of B. cereus and B. thuringiensis was 90.2% versus 99.6% for the 16S
rDNA sequences. Alignment of the amino acid sequences of the
gyrB proteins (Fig. 2)
translated from the nucleotide sequences showed that only 2 of the 121 substitutions caused amino acid substitutions. The amino acid sequence
similarity between the gyrase subunit B proteins of B. cereus and B. thuringiensis was
96.8% (Table 1). The frequency of base substitutions in the published
16S rRNA was lower than that in gyrB. For example, between the sequences of B. cereus and B. thuringiensis 121 base substitutions were observed in
gyrB, while only 5 base substitutions were observed in 16S
rRNA. It is interesting to note that no substitution was observed
between the sequences of B. cereus ATCC
14579T and B. anthracis serotype
Sterne.
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FIG. 1.
Nucleotide sequence alignment of gyrB of
various Bacillus species. Nucleotides identical to those of
B. cereus are indicated by dots. Nucleotide
positions for various primers appear on a black field. Primers BC1 and
BC2r are B. cereus specific; primers BT1 and BT2r
are B. thuringiensis specific; and primers
BA1 and BA2r are B. anthracis specific.
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FIG. 2.
Amino acid sequence alignment of the gyrB
products of various Bacillus species. Amino acids identical
to those of B. cereus are indicated by dots.
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Designing B. cereus-, B. thuringiensis-, and B. anthracis-specific PCR primer sets.
Oligonucleotide primers that are universal and specific to various
Bacillus species were synthesized based on the nucleotide sequences of gyrB (Table 4). A
forward primer with a nucleotide position of 175 to 195 (BC1) and an
antisense primer with a position of 519 to 539 were synthesized (BC2r).
When these primers were used to generate 365-bp PCR products,
B. cereus could be differentiated from
B. anthracis and type strains of B. thuringiensis IAM 12077T and B. mycoides ATCC 6462T. Likewise, a 368-bp fragment
specific to B. thuringiensis (made by using
BC1 and BT2r) and a 245-bp amplicon specific to B. anthracis (made by using BA1 and BA2r) were amplified with
the appropriate primer sets.
Specificity of PCR primers in the detection of Bacillus
species.
B. cereus, B. thuringiensis, B. anthracis,
and B. mycoides strains received from various culture
collections and identified as different serogroups or isolated from
numerous food and environmental specimens were tested with
gyrB primer sets specific for these Bacillus species.
PCR results in the differentiation of these
Bacillus species
for the type strains and various serogroups are given in Table
5. Type strains of all four
Bacillus species showed species-specific
positive
amplification. However,
B. cereus-specific signal
was
not observed for some
B. cereus serotypes (H5,
H6, H7, H16, and
H17). In addition, amplification of 365-bp
fragment that is specific
to
B. cereus was noted
for
B. thuringiensis serotypes kurstaki
(HD-1) and aizawai. When tested with various PCR primer sets that
were
established in this study, serotypes H5, H6, H7, and H17
showed a
positive signal for
B. anthracis, and serotype
H16 exhibited
an amplicon specific to
B. thuringiensis.
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TABLE 5.
Specificity of gyrB PCR primers in the
differentiation of B. cereus, B. thuringiensis, B. anthracis,
and B. mycoides
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The 1.2-kb
gyrB nucleotide sequences of
B. cereus serotypes H2, H6, and H16 and
B. thuringiensis serotype kurstaki (HD-1)
were
determined. By comparing the
gyrB nucleotide sequences of
these strains along with type strain sequences, both H2 and kurstaki
(HD-1) showed high similarity with
B. cereus JCM
2152
T (98.4%). Likewise, a high similarity value was
noted between
B. anthracis and H6
(97.9%), as well as between
B. thuringiensis and H16 (93.0%).
A total of 104
Bacillus strains isolated from various
environments, including food and clinical sources, consisting of
various
phenotypes, serotypes, and toxigenic properties were tested for
PCR assay (Table
6).
B. cereus isolates obtained from IFO and
IAM culture collections
were identified perfectly as
B. cereus.
However,
among 50
B. cereus strains isolated from foods, 4 and
2% of the strains produced amplicons specific to
B. thuringiensis and
B. anthracis, respectively. Likewise, 6 of 20
B. cereus isolates
from various environmental sources were
identified as
B. anthracis.
Eight of
ten environmental isolates that were identified as
B. thuringiensis on the basis of crystal protein were
recognized
as
B. cereus. One of nine
B. mycoides isolates obtained from the
ATCC showed a positive signal
for
B. cereus.
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TABLE 6.
Differentiation of B. cereus,
B. thuringiensis, B. anthracis, and B. mycoides wild strains by
using gyrB PCR primers
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Comparison of gyrB PCR and 16S rDNA hybridization
techniques in the differentiation of Bacillus species.
Specific DNA probes based on variable region VI of 16S rRNAs of
B. cereus and B. thuringiensis were reported to be useful for
differentiating these species (39). Type strains, serotypes, and wild-type strains that were identified as Bacillus
species on the basis of biochemical and serological
classifications were subjected to Southern hybridization by using 16S
rDNA probes and gyrB PCR with various Bacillus
species primer sets (Table 7). Among the type strains tested, the hybridization signal showed good
correlation with its species by gyrB PCR except for
B. mycoides ATCC 6462T, where a positive
signal was noted when B. cereus-specific 16S rDNA
probes were used. te Giffel et al. (39) also reported such a
false-positive signal for B. mycoides. On the other
hand, gyrB PCR did not produce any false-positive amplicon
and was able to differentiate B. cereus from
B. mycoides. In contrast, serotypes kurstaki and
aizawai of B. thuringiensis were identified
as B. cereus by conceding positive signals for
B. cereus in both hybridization and PCR techniques.
This indicated that B. thuringiensis
serotype kurstaki and aizawai are, in fact, B. cereus and not B. thuringiensis. However, B. cereus serotype H16 showed a
positive signal for B. cereus in hybridization
experiments but showed an amplicon specific for B. thuringiensis by the gyrB PCR technique. To
clarify these findings, we tried to procure H16 serotype from other
culture collections, but we did not succeed in acquiring the strain.
Also, B. cereus serotypes H5, H6, H7, and H17,
which showed positive amplification for B. anthracis, did not yield any B. cereus-specific hybridization signal. Complete sequencing of
1.2-kb gyrB fragments of these strains supported our claim
that these strains were indeed misidentified.
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TABLE 7.
Comparison of gyrB PCR and 16S rDNA
hybridization probe techniques in the differentiation of B. cereus and B. thuringiensis
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Sensitivity of the PCR primers in the detection of
Bacillus species.
To evaluate the sensitivity of the
PCR assay, a dilution series of genomic DNA from various
Bacillus species was prepared in TE buffer, and aliquots
were used as templates for PCR amplification. Aliquots containing a
picogram level of genomic DNA were successfully detected after
amplification with primer-specific PCR primer sets. A dilution series
of freshly cultured cells of Bacillus species showed
that the primer set employed in this study amplified species-specific PCR fragments when 2 to 5 CFU of bacterial cell per reaction tube was used.
Detection of B. cereus in the artificially
contaminated boiled rice.
The sensitivity of the PCR assay for
detecting artificially contaminated B. cereus in
cooked boiled rice is presented in Table 8. Absence of B. cereus-like organisms in the test sample was confirmed by both
the conventional enrichment method and by PCR assay. When the food
homogenate was incubated for 15 h in nutrient broth at 37°C, an
initial inoculum of 0.24 CFU of B. cereus per g of
food homogenate amplified the desired PCR product (Fig.
3). At time zero, 2.4 × 104 CFU of B. cereus per g of
boiled-rice homogenate did not yield any PCR products. The
detection of B. cereus directly from the food
sample was possible by the combination of a 15-h enrichment period in nutrient broth and PCR assay even without DNA extraction.
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TABLE 8.
Influence of preenrichment incubation time and bacterial
population in the amplification of PCR product specific
to B. cereus
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FIG. 3.
Detection of B. cereus in
artificially contaminated cooked rice by the BC-1 and BC-2 primers
(365-bp product). B. cereus cells grown
overnight in nutrient broth were serially diluted in cooked rice
homogenate (see details in Materials and Methods) to obtain appropriate
dilutions. Lanes: M, 100-bp DNA ladder; 1 to 6, initial inocula of
2.4 × 104 (lane 1), 2.4 × 103 (lane
2), 2.4 × 102 (lane 3), 2.4 × 101
(lane 4), 2.4 (lane 5), and 0.24 (lane 6) CFU of B. cereus added per g of rice homogenate and incubated overnight
at 35°C; 7, cooked rice homogenate that was not spiked with
B. cereus (negative control) incubated overnight at
35°C; and 8, 2.4 × 104 CFU of B. cereus cells per ml prepared in PCR mixture (positive
control).
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DISCUSSION |
Enterotoxigenic B. cereus, which causes acute
gastroenteritis after the consumption of contaminated foods, has been
known to produce the emetic and the diarrheal toxin types
(18). B. thuringiensis can be
distinguished from B. cereus only by the production of toxin crystals and can be detected by simple
microscopy or by hybridization with specific probes for the delta
endotoxin gene (10). However, this character is plasmid
borne and transmissible to B. cereus by conjugation
(13, 15). B. cereus strains can grow at
temperatures between 4 and 37°C (40, 42), and
psychrotrophic B. cereus strains can produce
enterotoxin both aerobically and anaerobically (17). Good
diagnostic tools are thus required to ensure the hygienic quality of
susceptible food items. Although some PCR methods are developed to
detect B. cereus targeting the toxigenic properties
(30), the production of B. cereus-type enterotoxins in Bacillus species, including B. thuringiensis (23), raised doubts about the
validity of these diagnostic probes in specifically identifying
B. cereus. Thus, identifying B. cereus irrespective of its virulence factors is necessary from
the viewpoint of public health. We therefore wanted to develop a rapid
and reliable method for differentiating B. cereus
from B. thuringiensis.
As 16S rDNA sequences have very high similarity between B. cereus and B. thuringiensis, we
were looking for appropriate molecular taxonomic markers. Yamamoto and
Harayama (50) suggested that genes that are not spread
horizontally among different bacterial species may be used to trace the
evolutionary record of host bacteria. Based on the information that the
average substitution rate for 16S rRNA is 1% per 50 million years and
that the rate for synonymous sites of protein-coding DNA is 0.7 to
0.8% per million years (31), Yamamoto and Harayama
(50) proposed the gyrB gene as a substitute for
16S rRNA as a molecular taxonomic marker for bacterial species. The gyrB gene is essential for DNA replication, a
housekeeping activity; it is also single copied and has conserved
regions for the development of PCR primers. We have designed PCR
primers by exploiting differences in gyrB genes to
differentiate V. parahaemolyticus from V. alginolyticus, a pair that exhibited 99.7% similarity in their
16S rDNA nucleotide sequences (43). Likewise, the similarity of 1.2-kb gyrB sequences of B. cereus
and B. thuringiensis was not high (90.7%);
thus, we were able to design a suitable PCR primer set for
differentiating these two organisms by amplifying a B. cereus-specific 365-bp amplicon. The type strains of both B. cereus and B. thuringiensis responded specifically with the developed PCR primer set by giving a positive signal for B. cereus and a negative signal for B. thuringiensis. Also, these PCR primers, which are
specific for B. cereus, did not yield any amplicon
for the B. anthracis strain and the
B. mycoides type strain.
The gyrB PCR primer set designed in this study detected 2 to
5 CFU of B. cereus cells per reaction tube or
correspondingly low levels (10 pg) of extracted DNA. The sensitivity
accords with that described for PCR with other bacteria, being between
1 and 20 CFU (27, 32, 41, 47) or between 1 and 100 pg
for DNA extracted from the bacterial population (24,
49). Increased sensitivity may be achieved by Southern
blot analyses as reported earlier (7). The gyrB
primer set recognized all strains identified as B. cereus and its group by conventional methods, but it did not
recognize the other bacteria tested. In addition, some serotypes that
were designated in culture collections as B. cereus
or B. thuringiensis were differentiated and
their species were identified. Specialized laboratories use
elaborate techniques for confirming the B. cereus group and their strain identity, techniques such as
toxin antigen detection, detection of virulent plasmids, the use of
crystal protein, etc. Since the gyrB PCR result is in
agreement with 16S rDNA-based hybridization probe technique, this
simple PCR method is thus a powerful tool for the confirmation and
differentiation of the B. cereus species in
clinical, food, and environmental samples.
Two major limitations to using PCR as a diagnostic tool are that
false-positive reactions can occur from DNA contamination and that
false-negative reactions can occur from a number of substances found in
samples that inhibit PCR (44, 47). We have included suitable
negative and positive controls to overcome these limitations when and
where necessary. Sensitivity of detection in food samples has, however,
been low because only small samples (10 to 100 µl) can be analyzed,
since many sorts of food contain substances that are PCR inhibitory.
Such PCR-inhibitory substances were reported in many clinical samples,
such as urine, blood, sputum, fecal specimens, food, and environmental
samples (26, 46, 48). However, with bacteria in food, the
sensitivity of the PCR was far lower than that with bacteria in saline.
With bacteria in food, the lower detection limit was higher than the
number of CFU per unit volume of food, a result which is usually found
with processed food. Previous studies suggested the extraction of DNA (27) or the application of chemicals (11). A
simple two-step filtration procedure that we reported previously
successfully removed PCR-inhibitory products during this study
(44). However, a simple preenrichment in a nonspecific
medium was necessary to permit the proliferation of target bacteria. A
similar phenomenon was documented for the detection of V. parahaemolyticus from shrimp (43).
The findings reported here describe a rapid, sensitive, specific, and
reliable method for the detection of B. cereus in
boiled rice. The fact that this technique allows detection of the
genetic potential and permits differentiation from related species may make it useful as both a screening test and a confirmatory test. The
data provided from this test could yield additional
information that will be useful to epidemiological studies.
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ACKNOWLEDGMENTS |
We are grateful to M. Satake for encouragement, I. Uchida for
providing DNA of B. anthracis, and I. Sugahara
for various Bacillus strains. We also thank Y. Kamijoh, T. Kurusu, K. Hanai, and Y. Hara for their technical assistance.
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FOOTNOTES |
*
Corresponding author. Present address: Environmental
Engineering Sciences, California Institute of Technology, Mail Code
138-78, 1200 E. California Ave., Pasadena, CA 91125. Phone: (626)
395-2994. Fax: (626) 395-2940. E-mail:
kjvenkat{at}cco.caltech.edu.
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REFERENCES |
| 1.
|
Abdel-Hameed, A., and R. Landen.
1994.
Studies on Bacillus thuringiensis strains isolated from Swedish soils: insect toxicity and production of B. cereus diarrhoeal-type enterotoxin.
World J. Microbiol. Biotechnol.
10:406-409.
|
| 2.
|
Agata, N.,
M. Ohta,
Y. Arakawa, and M. Mori.
1995.
The bceT gene of Bacillus cereus encodes an enterotoxic protein.
Microbiology
141:983-988[Abstract/Free Full Text].
|
| 3.
|
Asano, S. I.,
Y. Nukumizu,
H. Bando,
T. Iizuka, and T. Yamamoto.
1997.
Cloning of novel enterotoxin genes from Bacillus cereus and Bacillus thuringiensis.
Appl. Environ. Microbiol.
63:1054-1057[Abstract].
|
| 4.
|
Ash, C., and M. D. Collins.
1992.
Comparative analysis of 23S ribosomal RNA gene sequences of Bacillus anthracis and emetic Bacillus cereus determined by PCR-direct sequencing.
FEMS Microbiol. Lett.
73:75-80[Medline].
|
| 5.
|
Ash, C.,
J. A. E. Farrow,
M. Dorsch,
E. Stackebrandt, and M. D. Collins.
1991.
Comparative analysis of Bacillus anthracis, Bacillus cereus, and related species on the basis of reverse transcriptase sequencing of 16S rRNA.
Int. J. Syst. Bacteriol.
41:343-346[Abstract/Free Full Text].
|
| 6.
|
Ash, C.,
J. A. E. Farrow,
S. Wallbanks, and M. D. Collins.
1991.
Phylogenetic heterogeneity of the genus Bacillus revealed by comparative analysis of small-subunit-ribosomal RNA sequences.
Lett. Appl. Microbiol.
13:202-206.
|
| 7.
|
Bej, A. K.,
R. J. Steffan,
J. Dicesare,
L. Haff, and R. M. Atlas.
1990.
Detection of coliform bacteria in water by polymerase chain reaction and gene probes.
Appl. Environ. Microbiol.
56:307-314[Abstract/Free Full Text].
|
| 8.
|
Borin, S.,
D. Daffonchio, and C. Sorlini.
1997.
Single strand conformation polymorphism analysis of PCR-tDNA fingerprinting to address the identification of Bacillus species.
FEMS Microbiol. Lett.
157:87-93[Medline].
|
| 9.
|
Bourque, S. N.,
J. R. Valero,
M. C. Lavoie, and R. C. Levesque.
1995.
Comparative analysis of the 16S to 23S ribosomal intergenic spacer sequences of Bacillus thuringiensis strains and subspecies and of closely related species.
Appl. Environ. Microbiol.
61:1623-1626[Abstract].
|
| 10.
|
Bourque, S. N.,
J. R. Valero,
J. Mercier,
M. C. Lavoie, and R. C. Levesque.
1993.
Multiplex polymerase chain reaction for detection and differentiation of the microbial insecticide Bacillus thuringiensis.
Appl. Environ. Microbiol.
59:523-527[Abstract/Free Full Text].
|
| 11.
|
Brakstad, O. G.,
K. Aasbakk, and J. A. Maeland.
1992.
Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene.
J. Clin. Microbiol.
30:1654-1660[Abstract/Free Full Text].
|
| 12.
|
Brousseau, R.,
A. Saint-Onge,
G. Prefontaine,
L. Masson, and J. Cabana.
1993.
Arbitrary primer polymerase chain reaction, a powerful method to identify Bacillus thuringiensis serovars and strains.
Appl. Environ. Microbiol.
59:114-119[Abstract/Free Full Text].
|
| 13.
|
Damgaard, P. H.
1995.
Diarrhoeal enterotoxin production by strains of Bacillus thuringiensis isolated from commercial Bacillus thuringiensis-based insecticides.
FEMS Immunol. Med. Microbiol.
12:245-250[Medline].
|
| 14.
|
Damgaard, P. H.,
P. E. Granum,
J. Bresciani,
M. V. Torregrossa,
J. Eilenberg, and L. Valentino.
1997.
Characterization of Bacillus thuringiensis isolated from infections in burn wounds.
FEMS Immunol. Med. Microbiol.
18:47-53[Medline].
|
| 15.
|
Gonzales, J. M., Jr.,
B. J. Brown, and B. C. Carlton.
1982.
Transfer of Bacillus thuringiensis plasmids coding for delta-endotoxin among strains of Bacillus thuringiensis and Bacillus cereus.
Proc. Natl. Acad. Sci. USA
79:6951-6955[Abstract/Free Full Text].
|
| 16.
|
Granum, P. E.,
A. Andersson,
C. Gayther,
M. te Giffel,
H. Larsen,
T. Lund, and K. O'Sullivan.
1996.
Evidence for a further enterotoxin complex produced by Bacillus cereus.
FEMS Microbiol. Lett.
141:145-149[Medline].
|
| 17.
|
Granum, P. E.,
S. Brynestad, and J. M. Kramer.
1993.
Analysis of enterotoxin production by Bacillus cereus from dairy products, food poisoning incidents and non-gastrointestinal infections.
Int. J. Food Microbiol.
17:269-279[Medline].
|
| 18.
|
Granum, P. E., and T. Lund.
1997.
Bacillus cereus and its food poisoning toxins.
FEMS Microbiol. Lett.
157:223-228[Medline].
|
| 19.
|
Granum, P. E.,
S. Brynestad,
K. O'Sullivan, and H. Nissen.
1993.
Enterotoxin from Bacillus cereus: production and biochemical characterization.
Neth. Milk Dairy J.
47:63-70.
|
| 20.
|
Harrell, L. J.,
G. L. Andersen, and K. H. Wilson.
1995.
Genetic variability of Bacillus anthracis and related species.
J. Clin. Microbiol.
33:1847-1850[Abstract].
|
| 21.
|
Henderson, I.,
C. J. Duggleby, and P. C. Turnbull.
1994.
Differentiation of Bacillus anthracis from other Bacillus cereus group bacteria with the PCR.
Int. J. Syst. Bacteriol.
44:99-105[Abstract/Free Full Text].
|
| 22.
|
Henderson, I.,
D. Yu, and P. C. Turnbull.
1995.
Differentiation of Bacillus anthracis and other `Bacillus cereus group' bacteria using IS231-derived sequences.
FEMS Microbiol. Lett.
128:113-118[Medline].
|
| 23.
|
Jackson, S. G.,
R. B. Goodbrand,
R. Ahmed, and S. Kasatiya.
1995.
Bacillus cereus and Bacillus thuringiensis isolated in a gasteroenteritis outbreak investigation.
Lett. Appl. Microbiol.
21:103-105[Medline].
|
| 24.
|
Johnson, W. M.,
S. D. Tyler,
E. P. Ewan,
F. E. Ashton,
D. R. Pollard, and K. R. Rozee.
1991.
Detection of genes for enterotoxins, exfoliative toxins, and toxic shock syndrome toxin 1 in Staphylococcus aureus by the polymerase chain reaction.
J. Clin. Microbiol.
29:426-430[Abstract/Free Full Text].
|
| 25.
|
Keim, P.,
A. Kalif,
J. Schupp,
K. Hill,
S. E. Travis,
K. Richmond,
D. M. Adair,
M. Hugh-Jones,
C. R. Kuske, and P. Jackson.
1997.
Molecular evolution and diversity in Bacillus anthracis as detected by amplified fragment length polymorphism markers.
J. Bacteriol.
179:818-824[Abstract/Free Full Text].
|
| 26.
|
Kiehn, T. E., and D. Armstrong.
1990.
Changes in the spectrum of organisms causing bacteremia and fungemia in immunocompromised patients due to venous access devices.
Eur. J. Clin. Microbiol.
9:869-872.
|
| 27.
|
Lebech, A. M.,
P. Hindersson,
J. Vuust, and K. Hansen.
1991.
Comparison of in vitro culture and polymerase chain reaction for detection of Borrelia burgdorferi in tissue from experimentally infected animals.
J. Clin. Microbiol.
29:731-737[Abstract/Free Full Text].
|
| 28.
|
Lund, T., and P. E. Granum.
1996.
Characterisation of a non-haemolytic enterotoxin complex from Bacillus cereus isolated after a foodborne outbreak.
FEMS Microbiol. Lett.
141:151-156[Medline].
|
| 29.
|
Lund, T., and P. E. Granum.
1997.
Comparison of biological effect of the two different enterotoxin complexes isolated from three different strains of Bacillus cereus.
Microbiology
143:3329-3336[Abstract/Free Full Text].
|
| 30.
|
Mäntynen, V., and K. Lindström.
1998.
A rapid PCR-based DNA test for enterotoxic Bacillus cereus.
Appl. Environ. Microbiol.
64:1634-1639[Abstract/Free Full Text].
|
| 31.
|
Ochman, H., and A. C. Wilson.
1987.
Evolution in bacteria: evidence for a universal substitution rate in cellular genomes.
J. Mol. Evol.
26:74-86[Medline].
|
| 32.
|
Olive, D. M.
1989.
Detection of enterotoxigenic Escherichia coli after polymerase chain reaction amplification with a thermostable DNA polymerase.
J. Clin. Microbiol.
27:261-265[Abstract/Free Full Text].
|
| 33.
|
Ombui, J. N.,
H. Schmieger,
M. M. Kagiko, and S. M. Arimi.
1997.
Bacillus cereus may produce two or more diarrheal enterotoxins.
FEMS Microbiol. Lett.
149:245-248[Medline].
|
| 34.
|
Patra, G.,
P. Sylvestre,
V. Ramisse,
J. Therasse, and J.-L. Guesdon.
1996.
Specific oligonucleotide primers for rapid identification of Bacillus anthracis strains.
In
P. C. B. Turnbull (ed.), Proceedings of the International Workshop on Anthrax. Salisbury Med. Bull. Spec. Suppl. 87:45-46.
|
| 35.
|
Ramisse, V.,
G. Patra,
H. Garrigue,
J. L. Guesdon, and M. Mock.
1996.
Identification and characterization of Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosomal DNA.
FEMS Microbiol. Lett.
145:9-16[Medline].
|
| 36.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 37.
|
Seki, T.,
C. K. Chung,
H. Mikami, and Y. Oshima.
1978.
Deoxyribonucleic acid homology and taxonomy of the genus Bacillus.
Int. J. Syst. Bacteriol.
28:182-189[Abstract/Free Full Text].
|
| 38.
|
te Giffel, M. C.,
R. R. Beumer,
P. E. Granum, and F. M. Rombouts.
1997.
Isolation and characterisation of Bacillus cereus from pasteurized milk in household refrigerators in The Netherlands.
Int. J. Food Microbiol.
34:307-318[Medline].
|
| 39.
|
te Giffel, M. C.,
R. R. Beumer,
N. Klijn,
A. Wagendorp, and F. M. Rombouts.
1997.
Discrimination between Bacillus cereus and Bacillus thuringiensis using specific DNA probes based on variable regions of 16S rRNA.
FEMS Microbiol. Lett.
146:47-51[Medline].
|
| 40.
|
Väisänen, O. M.,
N. J. Mwaisumo, and M. S. Salkinoja-Salonen.
1991.
Differentiation of dairy strains of the Bacillus cereus group by phage typing, minimum growth temperature, and fatty acid analysis.
J. Appl. Bacteriol.
70:315-324[Medline].
|
| 41.
|
Van Ketel, R. J.,
B. De Wever, and L. Van Alphen.
1990.
Detection of Haemophilus influenzae in cerebrospinal fluids by polymerase chain reaction DNA amplification.
J. Med. Microbiol.
33:271-276[Abstract/Free Full Text].
|
| 42.
|
van Netten, P.,
A. van de Moosdijk,
P. van Hoensel,
D. A. A. Mossel, and I. Perales.
1990.
Psychrotrophic strains of Bacillus cereus producing enterotoxin.
J. Appl. Bacteriol.
69:73-79[Medline].
|
| 43.
|
Venkateswaran, K.,
N. Dohmoto, and S. Harayama.
1998.
Cloning and nucleotide sequence of the gyrB gene of Vibrio parahaemolyticus and its application in detection of this pathogen in shrimp.
Appl. Environ. Microbiol.
64:681-687[Abstract/Free Full Text].
|
| 44.
|
Venkateswaran, K.,
Y. Kamijoh,
E. Ohashi, and H. Nakanishi.
1997.
A simple filtration technique to detect enterohemorrhagic Escherichia coli O157:H7 and its toxins in beef by multiplex PCR.
Appl. Environ. Microbiol.
63:4127-4131[Abstract].
|
| 45.
|
Venkateswaran, K.,
A. Murakoshi, and M. Satake.
1996.
Comparison of commercially available kits with standard methods for the detection of coliforms and Escherichia coli in foods.
Appl. Environ. Microbiol.
62:2236-2243[Abstract].
|
| 46.
|
Virji, M., and J. E. Heckels.
1985.
Role of anti-pilus antibodies in host defense against gonococcal infection studied with monoclonal anti-pilus antibodies.
Infect. Immun.
49:621-628[Abstract/Free Full Text].
|
| 47.
|
Vitanen, A. M.,
T. P. Arstilla,
R. Lahesmaa,
K. Granfors,
M. Skurnik, and P. Toivanen.
1991.
Application of polymerase chain reaction and immunofluorescence techniques to the detection of bacteria in Yersinia-triggered reactive arthritis.
Arthritis Rheum.
34:89-96[Medline].
|
| 48.
|
Warhurst, D. C.,
F. M. A. El Kariem, and M. A. Miles.
1991.
Simplified preparation of malarial blood samples for polymerase chain reaction.
Lancet
337:303-304[Medline].
|
| 49.
|
Wilson, I. G.,
J. E. Cooper, and A. Gilmour.
1991.
Detection of enterotoxigenic Staphylococcus aureus in dried skimmed milk: use of the polymerase chain reaction for amplification and detection of staphylococcal enterotoxin genes entB and entC1 and the thermonuclease gene nuc.
Appl. Environ. Microbiol.
57:1793-1798[Abstract/Free Full Text].
|
| 50.
|
Yamamoto, S., and S. Harayama.
1995.
PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains.
Appl. Environ. Microbiol.
61:1104-1109[Abstract].
|
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Abstract
Introduction
