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Previous Article | Next Article 
Journal of Clinical Microbiology, April 1999, p. 1173-1177, Vol. 37, No. 4
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Identification of Vibrio
parahaemolyticus Strains at the Species Level by PCR Targeted to
the toxR Gene
Yung Bu
Kim,1,
Jun
Okuda,1
Chiho
Matsumoto,1
Naoki
Takahashi,2
Satoru
Hashimoto,2 and
Mitsuaki
Nishibuchi1,*
Center for Southeast Asian Studies, Kyoto
University, Yoshida, Sakyo-ku, Kyoto,1 and
Kansai Airport Quarantine Station, Tajiri, Sennan,
Osaka,2 Japan
Received 10 August 1998/Returned for modification 14 October
1998/Accepted 21 December 1998
 |
ABSTRACT |
The DNA colony hybridization test with the polynucleotide probe for
Vibrio parahaemolyticus toxR gene was performed. All 373 strains of V. parahaemolyticus gave positive results, and
the strains belonging to four other Vibrio species
including Vibrio alginolyticus gave weakly positive
results, suggesting that toxR sequence variation may
reflect the phylogenetic relationships of Vibrio species.
We then established a toxR-targeted PCR protocol for the
specific detection of V. parahaemolyticus.
| |
TEXT |
Vibrio parahaemolyticus
is a marine bacterium, and some strains can cause gastroenteritis in
humans through the consumption of contaminated seafood. Molecular
epidemiological studies revealed a strong correlation between the
possession of particular hemolysin genes (tdh,
trh, or both) and the ability to cause disease, supporting the fact that these genes are important virulence genes (4, 16). However, a small portion of clinical strains carried neither of the virulence genes (4, 16). Therefore, the isolation of the organism in the clinical setting and from food samples suspected
of being the source of infection, followed by identification of the
isolated strains, is still the standard procedure in investigations of
gastroenteritis due to V. parahaemolyticus. If a PCR
method allows detection of the nucleotide sequence specific to
V. parahaemolyticus, it can facilitate identification
of the organism.
The genes encoding two hemolysins designated TLH and 
-VPH of
V. parahaemolyticus were examined by Taniguchi et al.
(18, 19). These genes appear to be fairly specific to
V. parahaemolyticus. Lee et al. (6, 7)
cloned a 0.76-kb nucleotide sequence of unknown function and claimed
that the nucleotide sequence is specific to V. parahaemolyticus by hybridization and PCR methods (6, 7). However, only limited numbers of strains were examined in
those studies, and the relationships of the hemolysin genes and the
0.76-kb sequence with the phylogeny of V. parahaemolyticus are not known. For identification purposes, it is
ideal to use a nucleotide sequence that is well conserved and that
reflects the phylogenetic relationship. rRNA sequences are often used
for this purpose. However, the rRNA sequence homologies between
V. parahaemolyticus and related species are so high
that the rRNA sequence does not appear to be suitable for the purpose
described above. For example, the 16S rRNA sequences of V. parahaemolyticus and Vibrio alginolyticus are >99%
identical (5, 15). The gyrB gene encodes the B
subunit of DNA gyrase, which is essential for DNA replication. The
homology of the gyrB sequences between V. parahaemolyticus and V. alginolyticus is 86.8%
(20). For this reason, a PCR procedure targeting the
gyrB gene was recently developed for the specific detection
of V. parahaemolyticus in shrimp (20).
The toxR gene was first discovered as the regulatory gene of
the cholera toxin operon, but it was later shown to be involved in the
regulation of many other genes in Vibrio cholerae (1, 9). We subsequently found the toxR gene in
V. parahaemolyticus and demonstrated its regulatory
function (8). The toxR gene sequences have also
been cloned from Vibrio fisheri and at least two other
species of Vibrio, and their sequences have been analyzed (13, 14). Therefore, the toxR gene appears to be
well conserved among Vibrio species. The degree of homology
of the toxR gene between V. parahaemolyticus
and V. cholerae (52% identity) is much lower than that
of the rRNA gene (91 to 92% identity) (5, 8). We therefore
investigated in this study whether the toxR gene sequence
can be used to develop a PCR method for the specific identification
V. parahaemolyticus.
Phenotypic characteristics of bacterial strains.
The bacterial
strains used in this study are listed in Table
1. Clinical strains listed as
V. parahaemolyticus, listed as having an unknown
identification (non-V. parahaemolyticus), and identified as known Vibrio species (11 strains; see below)
were isolated from patients at hospitals in India, Bangladesh, and Korea and at quarantine stations in Japan during the period between 1977 and 1997 (11, 12). The nonclinical strains were
isolated from seawater in Korea in the summer of 1996. The seawater
samples were plated, after enrichment in alkaline peptone water, onto thiosulfate-citrate-bile salts-sucrose agar (Eiken Chemical, Co., Ltd.,
Tokyo, Japan), and blue-green colonies were selected. All clinical
and nonclinical strains examined in this study were tested for the
characteristics listed in Table 2 by
standard procedures (3) except that the NaCl concentration
of the test medium was adjusted to 1.5%.
The phenotypic characteristics of these 494 strains were examined in
two steps. The following characteristics were examined in the initial
screening step. The strains that showed an alkaline top, an acidic
bottom, and no H2S production in the TSI reaction and that
gave positive results in the lysine decarboxylase, indole, and motility
tests were selected. These strains were further examined for the other
characteristics listed in Table 2. Three hundred sixty-five strains
that gave the results listed in Table 2 were identified as
V. parahaemolyticus. Seven, two, two, and one strain were identified as Vibrio mimicus, Vibrio
furnisii, V. alginolyticus, and V. cholerae non-O1 and non-O139, respectively, and are included in
Table 1 as such. The other strains could not be assigned to the species listed above from the characteristics examined. These strains were not characterized further and are thus included in Table 1
as strains with unknown identifications.
The test strains that were not selected in the initial
screening step were not characterized further and,
except for eight strains, are included in Table 1 with the strains with
unknown identifications. The eight exceptional strains were negative by the lysine decarboxylase test (two strains) or the indole test (six
strains). However, these strains carried the toxR gene
(described below), and therefore, the other characteristics of these
strains were examined. These strains had the characteristics listed in Table 2 and carried the V. parahaemolyticus gyrB gene
(described below). Therefore, these strains were identified as
V. parahaemolyticus and are included in Table 1 as such.
Most of the strains belonging to other Vibrio species
and other genera were our laboratory stock strains or strains
supplied by other workers for this study, and their phenotypic
characteristics were not examined.
DNA colony hybridization.
We examined 373 strains of
V. parahaemolyticus and 290 strains belonging
to non-V. parahaemolyticus species by the DNA colony hybridization test with the 678-bp V. parahaemolyticus
toxR gene probe as described previously (8). Marine
Agar 2216 (Difco Laboratories, Detroit, Mich.) and an incubation
temperature of 25°C were used in place of Luria-Bertani (LB) agar and
an incubation temperature of 37°C to grow the standard strains
belonging to 26 species of the genus Vibrio (those with
specific strain numbers in Table 1). The results are summarized in
Table 1. All clinical and nonclinical strains of V. parahaemolyticus gave clearly positive results. Of the
non-V. parahaemolyticus strains, those belonging to
four Vibrio species including V. alginolyticus gave positive results, although the
hybridization signals were weaker than those for the V. parahaemolyticus strains. The other non-V.
parahaemolyticus strains yielded negative results (Table 1). The
hybridization signals of representative strains of Vibrio
species (reference strains; Table 3) are
presented in Fig. 1. The results indicate that the V. parahaemolyticus toxR sequence is perfectly
conserved among V. parahaemolyticus strains and that
some other Vibrio species carry nucleotide sequences that
are fairly homologous to that of the V. parahaemolyticus toxR gene. These non-V.
parahaemolyticus species are phylogenetically closely
related to V. parahaemolyticus (5, 15).
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FIG. 1.
DNA colony hybridization test with the V. parahaemolyticus toxR gene probe for reference strains of
Vibrio species. (A) Location of the inoculated strains. The
numbers correspond to those of the reference strains listed in Table 3.
Strain 24 was Escherichia coli HB101 (a negative control).
(B) Hybridization signals of the test strains detected on X-ray film.
|
|
The presence or absence of the tdh, trh1, and
trh2 genes in the test strain was also examined by the DNA
colony hybridization test with polynucleotide probes as described
previously (10). Of the 363 clinical strains of
V. parahaemolyticus, 14 strains (4%) carried
neither the tdh, trh1, nor trh2 gene.
These results encouraged us to develop a PCR protocol that allows
the specific detection of the V. parahaemolyticus
toxR sequence.
PCR.
To develop a PCR method specific for the V. parahaemolyticus toxR gene, various oligonucleotide primer
sets were tested with the reference strains. These strains
included 14 strains of V. parahaemolyticus and 14 strains of non-V. parahaemolyticus species (Table
3). The primer sequences were selected from the regions not conserved
between the V. parahaemolyticus toxR and the
V. cholerae toxR sequences (8). Five forward
primers and two reverse primers (data not shown) were selected, and all
10 combinations of these primers were tested. The dilution of the
boiled culture supernatant, the annealing temperature, and the
numbers of amplification cycles of the PCR were varied and the results
were compared. Amplicons of the expected sizes were also detected in
strains of V. alginolyticus and V. vulnificus when various PCR primer sets were tested with the
reference strains under low annealing temperatures (data not shown).
These results supported our hypothesis that the toxR gene is
well conserved in Vibrio species and that variation in the toxR sequences may reflect the phylogenetic relationship of
Vibrio species.
After extensive efforts to optimize the PCR conditions, the following
method was shown to allow the specific detection of the V. parahaemolyticus toxR gene. The test strain was grown in LB broth
containing 1% NaCl at 37°C with shaking (160 rpm) overnight. One
milliliter of the culture was boiled for 5 min, and the supernatant was
obtained by centrifugation (13,000 rpm) on a tabletop centrifuge (Centrifuge 5415C; Eppendorf, Hamburg, Germany) at room temperature. The supernatant was diluted 10-fold in distilled water. The PCR conditions were as follows. The reaction mixture consisted of 3 µl of
the template (supernatant of the boiled culture diluted 1:10), 5 µl
of 10× buffer containing 20 mM MgCl2 (Ex Taq buffer; Takara, Tokyo, Japan), 0.25 µl of Taq polymerase (Ex Taq;
Takara), 4 µl of 2.5 mM deoxynucleoside triphosphate, 2 µl of each
primer (10 pmol/µl), and 33.75 µl of distilled water. The
amplification conditions were 20 cycles of amplification consisting of
denaturation at 94°C for 1 min, annealing at 63°C for 1.5 min, and
extension at 72°C for 1.5 min. The primer sequences were
5'-GTCTTCTGACGCAATCGTTG-3' (forward) and
5'-ATACGAGTGGTTGCTGTCATG-3' (reverse). The
sequences of the forward and reverse primers correspond to base
positions 609 to 629 and 956 to 958, respectively, of the previously
described V. parahaemolyticus toxR sequence
(8). Ten microliters of the reaction mixture was mixed with
2 µl of the dye solution (0.07% bromophenol blue, 20% Ficoll), and
the mixture was resolved by electrophoresis in 2% agarose. The 368-bp
amplicons of the reference strains obtained by this method are shown in
Fig. 2A and B. The culture condition
described above allowed enough bacterial growth so that only 20 cycles
of amplification was enough to achieve clearly visible amplicon bands.
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FIG. 2.
PCR assay detects the toxR gene of
V. parahaemolyticus. The reference strains are
described in Table 3. (A) Reference strains of V. parahaemolyticus; lane 1,  X174 phage DNA digested with
HaeIII (molecular weight markers); lanes 2 to 15, reference
strains 1 to 14 (Table 3), respectively. (B) Reference strains of
non-V. parahaemolyticus species; lane 1, X174 phage
DNA digested with HaeIII; lane 2, reference strain
V. parahaemolyticus 13 (a positive control); lanes 3 to
16, reference strains 15 to 28 (non-V. parahaemolyticus
species), respectively. (C) Comparison of selected strains of
V. parahaemolyticus and all test strains of
V. vulnificus; lane 1, X174 phage DNA digested with
HaeIII; lanes 2 to 6, V. parahaemolyticus
strains (positive controls); lanes 7 to 17, test strains of
V. vulnificus.
|
|
The PCR protocol established with the reference strains was applied to
the examination of all test strains (Table 1). All strains of
V. parahaemolyticus gave only specific amplicons,
whereas non-V. parahaemolyticus strains did not.
Nonspecific amplicons were generated by only 5 of 11 strains of
V. vulnificus. These nonspecific amplicons were larger
and less evident than the specific amplicons and thus were
distinguishable from the specific amplicons (Fig. 2C, lanes 7 to 11).
The isolation of V. vulnificus from patients with
diarrhea appears to be a very rare event, but this possibility must not
be ruled out (2). The nonspecific amplicons of some strains
of V. vulnificus can be distinguished from the specific
amplicon if the positive control is included in the gel electrophoresis
analysis. Therefore, we do not think that it is necessary to run
additional tests to exclude the possibility that the amplicon resulted
from amplification of the V. vulnificus nucleotide
sequence. However, if desired, such additional tests are available.
This can be a simple biochemical test, for example, growth in 8% NaCl
(3). Alternatively, the modification of the PCR protocol
described above, in which the PCR primer set is substituted by the
primer set 5'-AGCCCGCTTTCTTCAGACTC-3' and
5'-AACGAGTCTTCTGCATGGTG-3' or the primer set
5'-CGCTTTCTTCAGACTCAAGC-3' and
5'-AACGAGTCTTCTGCATGGTG-3', can be used; all strains of
V. parahaemolyticus produced amplicons of 399 or 394 bp, respectively; none of the V. vulnificus strains produced any amplicons; some strains of V. alginolyticus, however, produced the amplicons (data not shown).
Growth of the test strain in LB broth at 37°C was used in the test
described above. This growth condition was chosen for the organisms of
clinical significance and is not suitable for most nonpathogenic
Vibrio species in the marine environment. To confirm the
specificity of the primer set, the standard strains belonging to 26 species of the genus Vibrio (those with specific strain numbers in Table 1) were also grown to good turbidity in Marine Broth
2216 (Difco Laboratories) at 25°C and were subjected to the PCR
assay. All these strains gave negative results, whereas the
V. parahaemolyticus strains grown under the same
conditions gave positive results.
All 373 strains identified as V. parahaemolyticus
carried the toxR gene, as described above. Of these strains,
47 strains showed atypical biochemical characteristics such as negative
results by tests for lysine or ornithine decarboxylation or indole
production or the Simmons citrate test and positive results
by the rhamnose fermentation test (Table 2). To support the
identification, these rare strains were examined for the presence of
the V. parahaemolyticus gyrB gene by the PCR method.
The PCR for the specific detection of the V. parahaemolyticus gyrB gene was carried out essentially as
described by Venkateswaran et al. (20). The boiled culture supernatant was prepared as described above, diluted to 1:10, and
used as the template solution. We used an annealing temperature of
60°C rather than 58°C, which was specified by Venkateswaran et al.
(20), because in our hands V. alginolyticus
strains gave the amplicons of the expected size at the latter annealing
temperature. All strains tested gave positive results.
In conclusion, the PCR protocol for the V. parahaemolyticus
toxR gene established in this study is specific and rapid
(amplification can be achieved in 2 h). Simultaneous use of this
PCR method and the PCR method for the detection of virulence genes like
tdh and trh (17) would be useful for
the rapid investigation of suspected V. parahaemolyticus strains isolated from clinical specimens
and food samples implicated as sources of infection for patients with cases of food poisoning.
| |
ACKNOWLEDGMENTS |
This research was supported in part by funds from the Ohyama Health
Foundation and Heiwa Nakajima Foundation, by a Grant-in Aid for
Scientific Research from the Ministry of Education, Science, Sports and
Culture of Japan, by the U.S.-Japan Cooperative Medical Science
Program, Cholera and Related Diarrheal Diseases, Japanese Panel, and by
Pusan National University's Overseas Research Program for Professors.
We are grateful to the following individuals in Japan who kindly
supplied bacterial strains: Takeshi Honda of Osaka University, Masanori
Ishibashi of Osaka Prefectural Public Health, Sumio Shinoda of Okayama
University, Tatsunosuke Nakamura of Chiba University, and Toshihiro
Nakai of Hiroshima University. We thank Yohko Takeda for technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Center for
Southeast Asian Studies, Kyoto University, 46 Shimoadachi-cho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan. Phone: 81-75-753-7367. Fax:
81-75-753-7350. E-mail: nishibuc{at}med.kyoto-u.ac.jp.
Present address: Department of Microbiology, College of Medicine,
Pusan National University, Pusan 602-739, Korea.
| |
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Journal of Clinical Microbiology, April 1999, p. 1173-1177, Vol. 37, No. 4
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Ansaruzzaman, M., Lucas, M., Deen, J. L., Bhuiyan, N. A., Wang, X.-Y., Safa, A., Sultana, M., Chowdhury, A., Nair, G. B., Sack, D. A., von Seidlein, L., Puri, M. K., Ali, M., Chaignat, C.-L., Clemens, J. D., Barreto, A.
(2005). Pandemic Serovars (O3:K6 and O4:K68) of Vibrio parahaemolyticus Associated with Diarrhea in Mozambique: Spread of the Pandemic into the African Continent. J. Clin. Microbiol.
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Martinez-Urtaza, J., Lozano-Leon, A., DePaola, A., Ishibashi, M., Shimada, K., Nishibuchi, M., Liebana, E.
(2004). Characterization of Pathogenic Vibrio parahaemolyticus Isolates from Clinical Sources in Spain and Comparison with Asian and North American Pandemic Isolates. J. Clin. Microbiol.
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Williams, T. L., Musser, S. M., Nordstrom, J. L., DePaola, A., Monday, S. R.
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Hara-Kudo, Y., Sugiyama, K., Nishibuchi, M., Chowdhury, A., Yatsuyanagi, J., Ohtomo, Y., Saito, A., Nagano, H., Nishina, T., Nakagawa, H., Konuma, H., Miyahara, M., Kumagai, S.
(2003). Prevalence of Pandemic Thermostable Direct Hemolysin-Producing Vibrio parahaemolyticus O3:K6 in Seafood and the Coastal Environment in Japan. Appl. Environ. Microbiol.
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Bhuiyan, N. A., Ansaruzzaman, M., Kamruzzaman, M., Alam, K., Chowdhury, N. R., Nishibuchi, M., Faruque, S. M., Sack, D. A., Takeda, Y., Nair, G. B.
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