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Journal of Clinical Microbiology, November 2000, p. 4145-4151, Vol. 38, No. 11
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Rapid Method for Species-Specific Identification of
Vibrio cholerae Using Primers Targeted to the Gene of Outer
Membrane Protein OmpW
Bisweswar
Nandi,1
Ranjan K.
Nandy,1,2
Sarmishtha
Mukhopadhyay,1
G.
Balakrish
Nair,2
Toshio
Shimada,3 and
Asoke C.
Ghose1,*
Department of Microbiology, Bose Institute,
Calcutta 700 054,1 and National
Institute of Cholera and Enteric Diseases, Calcutta 700 010,2 India, and National Institute of
Infectious Diseases, Tokyo 162, Japan3
Received 9 May 2000/Returned for modification 5 July 2000/Accepted 15 August 2000
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ABSTRACT |
The distribution of genes for an outer membrane protein (OmpW) and
a regulatory protein (ToxR) in Vibrio cholerae and other organisms was studied using respective primers and probes. PCR amplification results showed that all (100%) of the 254 V. cholerae strains tested were positive for ompW and
229 (~98%) of 233 were positive for toxR. None of the 40 strains belonging to other Vibrio species produced
amplicons with either ompW- or toxR-specific primers, while 80 bacterial strains from other genera tested were also
found to be negative by the assay. These studies were extended with
representative number of strains using ompW- and
toxR-specific probes in DNA dot blot assay. While the
V. cholerae strains reacted with ompW probe,
only one (V. mimicus) out of 60 other bacterial strains
tested showed weak recognition. In contrast, several strains belonging
to other Vibrio species (e.g., V. mimicus,
V. splendidus, V. alginolyticus, V. fluvialis, V. proteolyticus, V. aestuarianus, V. salmonicida, V. furnissii, and V. parahaemolyticus) showed weak to
strong reactivity to the toxR probe. Restriction fragment length polymorphism analysis and nucleotide sequence data revealed that
the ompW sequence is highly conserved among V. cholerae strains belonging to different biotypes and/or
serogroups. All of these results suggest that the ompW gene
can be targeted for the species-specific identification of V. cholerae strains. The scope of this study was further extended
through the development of a one-step multiplex PCR assay for the
simultaneous amplification of ompW and ctxA genes which should be of considerable value in the screening of both
toxigenic and nontoxigenic V. cholerae strains of clinical as well as environmental origin.
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INTRODUCTION |
The diarrheal disease cholera in the
epidemic form is caused by the organism Vibrio cholerae
belonging to the O1 or O139 serogroup (10). V. cholerae organisms belonging to non-O1/non-O139 serogroups, which
can be isolated in abundance from aquatic or estuarine sources, cause
sporadic cases or limited outbreaks of diarrhea in humans (17). Identification of V. cholerae is usually
achieved through a series of biochemical tests after their growth and
isolation on a selective plating medium e.g., TCBS agar
(25). The process, however, is laborious and time-consuming
and may be quite expensive for a laboratory handling a large number of
clinical and/or environmental samples. Further, close relatedness among
V. cholerae and certain other members of the
Vibrio spp. (e.g., V. mimicus) or
Aeromonas spp. with respect to their biochemical properties
has often made unambiguous identification of the organism quite
difficult. Although the commercial (or otherwise) availability of O1
and/or O139 antisera has considerably helped in the identification of
epidemic causing strains of V. cholerae, it may not be true
for the non-O1/non-O139 strains which, until recently, were known to
range from the O2 to O138 and from the O140 to O193 serogroups
(32). The problem has been accentuated recently as the
non-O1/non-O139 strains of diverse serogroups have been implicated as
the causative agents of a large number of diarrheal cases in the Indian
subcontinent (23, 28) and elsewhere (2, 4). As a
matter of fact, the majority of these strains do not contain virulence
markers such as cholera toxin, toxin-coregulated pilus (TCP), etc.
(10, 17), that are known to be associated with the
pathogenic strains of V. cholerae O1 or O139, thereby making
their identification more difficult.
Attempts to identify V. cholerae strains on the basis of
their 16S rRNA sequences have not been successful so far due to the lack of appreciable differences between these sequences occurring in
V. cholerae and other members of Vibrionaceae
family (13, 24). Other approaches were directed toward the
identification of toxigenic strains of V. cholerae through
the use of cholera toxin gene (ctxAB) probes or appropriate
primers for the amplification of toxin genes by PCR assay
(21). A multiplex PCR has also been developed to identify
epidemic-causing strains of V. cholerae containing the
ctxA and TCP protein subunit (tcpA) genes
(11). V. cholerae strains belonging to the O1 or
O139 serogroups could be detected by using probes or primers designed
from their rfb regions responsible for "O" antigen
biosynthesis (1, 7). However, none of these methods are
applicable for the identification of all V. cholerae
strains. Recently, a PCR based method targeted to the toxR
gene was developed for the species-specific identification of V. parahaemolyticus (12). No information, however, is
available in the literature regarding the identification of V. cholerae using toxR primers, although toxR
genes (sharing sequence homology) are distributed among certain other
Vibrio species, including V. cholerae (12,
15, 18, 22).
In the present study, we have evaluated the use of toxR
primers and/or probes for the identification of V. cholerae
strains. The study is extended using primers and/or probes targeted to a gene encoding an outer membrane protein OmpW of V. cholerae whose nucleotide sequence was first reported by
Jalajakumari and Manning (8). We demonstrate that although
the toxR primers are sensitive and specific for V. cholerae strains, the primers targeted to ompW are
better suited for the purpose due to the unique presence of the gene
with conserved sequence in V. cholerae. Finally, a
multiplex-PCR assay has been developed for the detection of
toxigenic strains of V. cholerae through simultaneous
amplification of ompW and ctxA genes.
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MATERIALS AND METHODS |
Bacterial strains.
A total of 254 V. cholerae
strains isolated from both clinical and environmental sources were
included in this study. The majority of these were isolated locally
over the years and kept in our collection. Others were standard or type
(American Type Culture Collection [ATCC]) strains which had been
initially isolated and used by various groups of workers around the
world and made available to us. Organisms were grown on a selective
medium (TCBS agar) and subsequently characterized by standard
biochemical procedures (25). V. cholerae strains
were subjected to serogroup analysis using O serogroup-specific
antisera. Of the 254 V. cholerae strains included in this
study, 36 and 22 strains belonged to serogroups O1 and O139,
respectively. Two of these O1 strains had ATCC designations. A total of
176 strains belonged to non-O1/non-O139 serogroups on the basis of
agglutination reactions using O2 to O141 specific antisera. Of these,
96 non-O1/non-O139 strains were, in fact, reference strains used to
raise O-specific antisera (29), while two others were ATCC
type strains. Of the remaining 20 V. cholerae strains which
could not be typed by the existing O1 to O141 antisera, 16 belonged to
the rough variety and were agglutinable by the rough antiserum. The
remaining four strains were classified under O untypeable category.
A total of 40 strains belonging to other Vibrio species were
also included in the study (Table 1). Of
these, 17 were type strains with ATCC numbers. The sources of the
others are indicated in Table 1. Other bacteria included in this study
were Aeromonas spp., Escherichia coli,
Shigella spp., Salmonella spp.,
Pseudomonas spp., Klebsiella spp., and
Staphylococcus aureus.
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TABLE 1.
Summarized results of PCR and DNA dot blot analyses of
strains belonging to Vibrio spp. using primers or probes
for ompW and toxR genes
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PCR assay.
Amplification of the target gene was carried out
by PCR assay using bacterial cell lysate as the source of template DNA.
Briefly, bacterial cells were grown overnight at 37°C on Luria agar
(LA) plates. For strains belonging to certain Vibrio
species, the LA medium was supplemented with 3% NaCl. Next, isolated
colonies were picked up and mixed with 100 µl of normal saline, and
bacterial cells were pelleted by centrifugation. The cell pellet was
resuspended in 100 µl of double-distilled water and boiled for 10 min. Cell debris was removed by centrifugation, and the supernatant
containing the template DNA was taken into a fresh microfuge tube for
PCR assay.
Four different primers were designed and synthesized on the basis of
the available nucleotide sequence data of
ompW
(
8).
The primer sequences are indicated in Table
2. Three combinations
of
ompW
primer pairs (1-2, 1-4, and 2-3) were used to generate
amplicons of
three different sizes in separate PCR tubes. Primers
used for the
amplification of the
toxR gene are also indicated
in Table
2. The sense primer corresponds to nucleotides 1 to
18 of the
toxR gene, while the antisense primer was complementary
to
nucleotides 865 to 884 of the
toxR gene (
6,
15).
The primers
used for the amplification of
ctxA are listed in
Table
2.
PCR amplification of the target DNA was carried out in a thermal cycler
(Perkin-Elmer) using 200-µl PCR tubes with a reaction
mixture volume
of 25 µl. Each of the reaction mixtures contained
3 µl of template
DNA (lysate), 2.5 µl of each primer (10 pmol/µl),
2.5 µl of 2.5 mM deoxynucleoside triphosphates, 0.3 µl (5 U/µl)
of
Taq
DNA polymerase (Takara Shuzo Co., Ltd.), 2.5 µl of 10×
reaction
buffer containing 20 mM MgCl
2 (Extaq; Takara), and 11.8
µl of distilled water. The reaction mixture was subjected to an
amplification of 30 cycles, each of which consisted of three steps
in
the following order: denaturation of template DNA at 94°C for
30 s, annealing of the template DNA at 64°C for 30 s, and extension
of the primers at 72°C for 30 s. Before initiation of the first
cycle, the reaction mixture was heated at 94°C for 5 min to allow
complete denaturation of the template. PCR products, thus obtained,
were electrophoresed through 1.5% (wt/vol) agarose gel to resolve
the
amplified products which were visualized under UV light after
ethidium
bromide
staining.
A multiplex PCR assay was carried out by the simultaneous addition of
primer pairs for
ompW (primers 1 and 2, Table
2) and
ctxA (primers 7 and 8, Table
2) in the same reaction
mixture.
In initial experiments, the
ctxA primer
concentration was varied
between 1.0 and 0.15 pmol/µl, keeping the
ompW primer concentration
fixed either at 1.0 or at 1.2 pmol/µl in the final reaction mixture
of 25 µl. Optimum results
were obtained with primer concentrations
of 1.2 and 0.25 pmol/µl for
ompW and
ctxA, respectively. Other
conditions for
PCR amplification remained as described
earlier.
DNA dot blot assay.
Genomic DNA of bacterial strains was
isolated using a miniscale preparation method (31) with
minor modifications. For this, organisms were grown overnight at 37°C
in 5 ml of Luria broth (supplemented with 3% NaCl to support the
growth of certain noncholera vibrios). Next, 1.5 ml of the bacterial
culture was centrifuged, and the pellet thus obtained was resuspended
in 567 µl of TE buffer (10 mM Tris-HCl containing 1 mM EDTA; pH 8.0)
followed by the addition of 30 µl of 10% (wt/vol) sodium dodecyl
sulfate and 3 µl of a proteinase K solution (20 µg/µl) (Sigma
Chemical Co.). The mixture was incubated at 50°C for 90 min to obtain
a clear lysate. Next, 100 µl of 5 M NaCl and 100 µl of 10%
(wt/vol) cetyltrimethylammonium bromide (CTAB) in 0.7 M NaCl were added
to the lysate and kept at 65°C for 10 min. Thereafter, the DNA
material was extracted by adding an equal volume of a mixture of
chloroform-isoamyl alcohol (24:1). After centrifugation, the aqueous
phase containing nucleic acids was collected and reextracted with
phenol-chloroform-isoamyl alcohol (25:24:1) to remove proteinaceous
material. The aqueous phase was transferred to a fresh 1.5-ml microfuge
tube, and DNA was precipitated by the addition of 0.6 volume of
isopropanol at room temperature. The precipitate was collected by
centrifugation, washed with 70% (vol/vol) ethanol, and finally
reconstituted in 100 µl of TE buffer. The concentration of DNA was
measured spectrophotometrically (26).
Purified bacterial DNA preparations were used for dot blot assay using
ompW and/or
toxR probes. The probes were prepared
by
PCR amplification of target genes
ompW and/or
toxR using primer
pairs 1 and 2 (for
ompW) and 5 and 6 (for
toxR) (Table
2). Appropriate
amplicons thus
obtained were purified by using the QIAQuick purification
kit (Qiagen)
and labeled with horseradish peroxidase by the glutaraldehyde
conjugation method using the Direct Nucleic Acid Labeling Kit
ECL
(Amersham Lifesciences). Briefly, 50 ng of DNA sample (amplicon)
was
taken in 10 µl of water and denatured by heating for 10 min
in a
boiling water bath, followed by immediate chilling. The cooled
DNA was
mixed with equal volumes of the labeling reagent and glutaraldehyde
solution (supplied with the kit). Following incubation at 37°C
for 20 min, the labeled probe was taken in hybridization buffer
(5 ml) and
used. For the dot blot assay, the target DNA was denatured
by boiling
in a water bath for 10 min and spotted onto a nylon
membrane presoaked
with 2 N NaOH and 2× SSC (30 mM trisodium citrate
plus 0.3 M NaCl)
buffer. The spotted DNA was linked to the membrane
by UV irradiation,
prehybridized at 42°C for 1 h, and hybridized
with the labeled
probe (10 ng/ml) for 14 h at 42°C. After hybridization,
the
membrane was washed thoroughly with primary (twice at 42°C)
and
secondary (twice at room temperature) wash buffers under highly
stringent conditions. Next, the membrane was exposed to the detection
solution and autoradiographed using X-ray
film.
Restriction fragment length polymorphism (RFLP) analysis of
ompW PCR amplicons.
The 588-bp PCR amplicons of
ompW obtained from representative V. cholerae
strains using primers 1 and 2 (Table 2) were purified by the Gel
Extraction Purification kit (Qiagen) and subsequently digested with the
three restriction enzymes HindIII, NdeI, and HpaI (Gennei) in separate reactions. Digested materials were
run on a 2% (wt/vol) agarose gel, stained with ethidium bromide, and viewed under UV light.
Sequencing of ompW amplicons.
The nucleotide
sequence of ompW amplicons obtained by PCR using the primer
pair 1 and 2 (Table 2) from V. cholerae strains was
determined using an automated DNA sequencer (Perkin-Elmer 310).
| |
RESULTS |
PCR assay using ompW and toxR primers.
PCR amplification of ompW gene of V. cholerae
using three combinations of primers (primers 1 and 2, primers 1 and 4, and primers 2 and 3; Table 2) yielded amplicons of 588, 304, and 336 bp, respectively. Initial experiments were carried out with about 50 V. cholerae and other strains. Representative data are shown in Fig. 1. It should be noted that the
bacterial strains other than V. cholerae did not produce any
amplified product under the experimental conditions used.
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FIG. 1.
PCR amplification results obtained with bacterial
strains using ompW-specific primer pairs 1 and 2 (A), 1 and
4 (B), and 2 and 3 (C). The bacterial strains used were V. cholerae O1 classical (lane 2), O1 El Tor (lane 3), O139 (lane 4),
rough (lane 5), and non-O1/non-O139 (lanes 6 and 7). Other bacteria
used were V. parahaemolyticus (lane 8), V. mimicus (lane 9), V. anguillarum (lane 10), V. alginolyticus (lane 11), V. furnissii (lane 12),
Aeromonas spp. (lane 13), and enteroaggregative E. coli (lane 14). Lane 1 represents marker DNA of known molecular
weights. The amplicon sizes are indicated by arrows.
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Subsequent experiments were carried out with the
ompW primer
pair 1 and 2 because this produced an amplicon (588 bp) which
differed
considerably in size from the
ctxA amplicon (301 bp)
generated with the primers 7 and 8 (Table
2). PCR amplification
data
obtained with 254 strains of
V. cholerae, 40 strains of
other
Vibrio spp., and 80 bacterial strains from other
genera are presented
in a summarized form (Table
3). While all
V. cholerae
strains
were found to be positive by the
ompW-based PCR
assay, other organisms
tested, including those belonging to other
Vibrio species, were
found to be negative. When these
strains were subjected to PCR
using
toxR primers, only 4 of
233
V. cholerae strains tested were
found to be negative.
All four strains belonged to typeable non-O1/non-O139
serogroups.
Noncholera vibrios and other bacterial species failed
to yield any
toxR amplicon when tested under comparable conditions.
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TABLE 3.
Summarized PCR results obtained with V. cholerae and other bacteria using ompW and
toxR-specific primers
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DNA dot blot analysis using ompW and toxR
probe.
Strains belonging to different bacterial species were
subjected to DNA dot blot analysis using ompW and
toxR probes. The results presented in Table 1 show that all
of the 18 V. cholerae strains tested produced positive
signals with both of the probes. In contrast, of 40 strains belonging
to other Vibrio species tested, only 1 V. mimicus
strain gave a weak signal against the ompW probe, while 10 strains, e.g., V. mimicus (two strains), V. splendidus, V. alginolyticus, V. fluvialis,
V. proteolyticus, V. aestuarianus, V. salmonicida, V. furnissii, and V. parahaemolyticus gave weak to strong signals with toxR
probe (Table 1, Fig. 2). Twenty strains belonging to other bacterial species (e.g., Aeromonas spp.,
E. coli, Shigella spp., Salmonella
spp., Klebsiella spp. Pseudomonas spp., and
S. aureus) were found to be negative for both
ompW and toxR genes in the dot blot assay (data
not shown).
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FIG. 2.
DNA dot blot hybridization test carried out with
V. cholerae strains using ompW (A) and
toxR (B) gene probes. The test strains used were V. cholerae O1 classical (O395 [blot 1], 569B [blot 2], and ATCC
14035 [blot 3]), O1 El Tor (PG27 [blot 4] and ATCC 39315 [blot
5]), O139 (Arg3 [blot 6] and SG25 [blot 7]), rough ALO46 (blot 8),
non-O1/non-O139 (ATCC 25872 [blot 9], ATCC 25874 [blot 10], V5
[blot 11], and S7 [blot 12]), V. mimicus ATCC 33653 (blot 13), V. tyrogens (blot 14), V. alginolyticus ATCC 17749 (blot 15), V. anguillarum ATCC
19264 (blot 16), V. furnissii ATCC 35016 (blot 17), V. proteolyticus ATCC 15338 (blot 18), V. mimicus (blot
19), V. vulnificus (ATCC 33816 [blot 20] and ATCC 27562 [blot 21]), V. salmonicida ATCC 43839 (blot 22), V. parahaemolyticus (121 [blot 23] and RIMD 2210001 [blot 24]),
V. splendidus ATCC 33125 (blot 25), V. carchariae ATCC 35084 (blot 26), V. aestuarianus ATCC 35048 (blot 27), V. nereis ATCC 25917 (blot 28), V. natriegens ATCC 14048 (blot 29), V. tubiashii ATCC 19109 (blot 30), V. fluvialis ATCC 33809 (blot 31), Aeromonas sp. (blot 32), E. coli ATCC
25922 (blot 33), Shigella sp. (blot 34),
Salmonella sp. (blot 35), and P. aeruginosa ATCC
27853 (blot 36).
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RFLP analysis of ompW amplicons.
The 588-bp
ompW amplicons obtained from different V. cholerae strains using the primer pair 1 and 2 (Table 2) were
digested with the restriction enzymes (HindIII,
NdeI, HpaI). The RFLP patterns presented in Fig.
3 demonstrate identity among the V. cholerae strains with respect to these restriction sites in the
ompW gene.
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FIG. 3.
RFLP analysis of ompW amplicons of different
V. cholerae strains using the restriction enzymes
HindIII (A), NdeI (B), and HpaI
(C). The bacterial strains used were V. cholerae O1
classical (lane 1), O1 El Tor (lane 2), O139 (lane 3), and
non-O1/non-O139 (lanes 4 to 9). Lane 10 represents the uncut
ompW amplicon shown for a comparison. The fragment sizes are
indicated by arrows.
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Nucleotide sequence analysis of ompW amplicons.
The ompW amplicons generated from five V. cholerae strains belonging to different serogroups and/or biotypes
(O1/El Tor, O34, O37, O53, and O139) were subjected to nucleotide
sequence analysis, and the data were compared with each other as well
as with the published sequence data of an O1 classical strain 569B
(8). The results (Table 4)
showed only minimum variation (ranging between 0.2 and 2.2%) in the
ompW sequence among these strains.
Multiplex-PCR assay using ompW and ctxA
primers.
Simultaneous amplification of ompW and
ctxA genes in a given reaction mixture generated amplicons
of both ompW (588 bp) and ctxA (301 bp) in
toxigenic V. cholerae strains belonging to the O1, O139, and
non-O1/non-O139 serogroups (Fig. 4). As
expected, a nontoxigenic strain yielded only the ompW
amplicon (lane 6), while a V. mimicus strain failed to yield
any amplicon under comparable conditions (lane 7).
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FIG. 4.
Multiplex-PCR analysis of V. cholerae strains
using primers 1 and 2 for ompW and primers 7 and 8 for
ctxA. Bacterial strains used were V. cholerae O1
(lane 3), O139 (lane 4), non-O1/non-O139 (lane 5), a nontoxigenic
non-O1/non-O139 (lane 6), and a V. mimicus strain (lane 7).
The positions of the ompW and ctxA amplicons
generated by the use of individual primer pairs are shown in lanes 1 and 2, respectively.
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DISCUSSION |
The toxR gene was shown to be involved in the
regulation and expression of several genes of V. cholerae
(19). Subsequent studies demonstrated the presence of
toxR-related gene sequences in other organisms belonging to
Vibrio spp., although their sequences showed considerable
variations (14, 18, 22). As a matter of fact, the
toxR gene was recently used as a probe for the
species-specific identification of V. parahaemolyticus
(12). Interestingly, this gene probe developed for V. parahaemolyticus failed to detect V. cholerae despite
52% identity in their toxR gene sequences. These results
appear to be somewhat consistent with our data since the
toxR probe for V. cholerae recognized only one,
though weakly, of the seven V. parahaemolyticus strains
tested (Table 1). However, as observed with the V. parahemolyticus toxR probe (12), the toxR
probe for V. cholerae recognized organisms belonging to
certain other Vibrio species with variable level of
reactivities (Fig. 2). Incidentally, the organism V. alginolyticus was the common species recognized by both of the
toxR probes.
The toxR primers used in this study were found to be quite
specific (~98%) for V. cholerae and were able to
differentiate these from other Vibrio spp. (Table 3). These
results suggest that one or both the primer sequences of V. cholerae toxR are likely to differ from the corresponding
toxR sequences of other vibrios. That this may indeed be the
case is supported by the information, though limited, available on
toxR sequences (14, 18).
The ompW primers showed 100%
specificity for all V. cholerae strains tested (Table 3).
More importantly, the ompW gene probe did not hybridize with
target DNAs of other bacteria except for showing a weak reaction with
only one of six V. mimicus strains examined (Table 1, Fig.
2). This observation and the fact that ompW primers can
differentiate between V. cholerae and V. mimicus strains assume considerable significance in view of the report that
these two groups of organisms share common biochemical properties and
serological markers (5). Therefore, the presence of
ompW in V. cholerae strains, coupled with the
fact that its nucleotide sequence remained practically unchanged among
different V. cholerae strains, makes it a highly suitable
genetic marker for the organism.
A literature survey showed that genes partially homologous to
ompW of V. cholerae are present in certain other
bacteria, e.g., E. coli, Aeromonas spp., etc.
(9, 16). Several functions were proposed for the
OmpW-related proteins in these bacteria, including their pore- or
channel-forming (9) and colicin receptor properties
(20). Although the precise function of the OmpW protein in
V. cholerae is not yet known, it may play a role in the
adherence process, which is likely to facilitate the survival of the
organism within the host or in the environment or both (27).
Preliminary genome data available (30) demonstrate the
presence of two chromosomes in V. cholerae. It is important
to note that while the ompW gene is present in the smaller
chromosome, the toxR gene is located in the larger
chromosome of the organism.
Epidemic-causing strains of V. cholerae belong to the O1 or
O139 serogroups and produce cholera toxin, which is the major contributing factor for profuse diarrhea (cholera gravis)
(10). However, genes related to ctxAB have also
been demonstrated in a number of strains of non-O1/non-O139 V. cholerae (6, 17, 21) that are responsible for diarrheal
episodes in humans, causing a considerable public health problem. The
multiplex PCR described here is likely to facilitate the rapid
detection of toxigenic V. cholerae strains and therefore
play a key role in the cholera surveillance program.
The rRNA nucleotide sequences have provided valuable information for
the identification and taxonomy of different bacterial species.
Unfortunately, the 16S rRNA sequences of different Vibrio spp. show minimal differences, making species-specific identification difficult (13, 24). In a recent study, Chun et al.
(3) were able to design primers based on subtle differences
in the nucleotide sequences of 16S-23S rRNA intergenic spacer regions of V. cholerae and V. mimicus. PCR amplification
using the primer pair was able to generate amplicons, though of
variable sizes (295 to 310 bp), from several V. cholerae
strains tested. The data presented in this study, however, demonstrate
that PCR primers 1 and 2 designed on the basis of ompW
sequence (uniquely present in V. cholerae) generate
amplicons of identical sizes (588 bp) from all V. cholerae
strains, which should provide a very rapid and reliable method for the
species-specific identification of V. cholerae and for their
differentiation from other bacteria. Further, identification of
V. cholerae strains harboring genes for cholera toxin by
one-step multiplex PCR assay (Fig. 4) is likely to enhance the scope of
this method significantly toward the screening of both toxigenic and
nontoxigenic V. cholerae strains of clinical as well as
environmental origin.
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ACKNOWLEDGMENTS |
This study was supported by grants from the Indian Council of
Scientific and Industrial Research (CSIR). The work was also supported,
in part, by the Japan International Cooperation Agency (JICA/NICED,
project 054-1061-EO).
The helpful technical assistance of Prabal Gupta is also acknowledged.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Bose Institute, P-1/12, CIT Scheme VII-M, Calcutta 700 054, India. Phone: 033-337-9416/9544/9219. Fax: 91-33-334-3886. E-mail:
acghosh{at}boseinst.ernet.in.
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Abstract
Introduction
