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Applied and Environmental Microbiology, August 2000, p. 3506-3514, Vol. 66, No. 8
Center for Southeast Asian Studies, Kyoto
University, Yoshida, Sakyo-ku,1 and
Department of Microbiology, Kyoto Pharmaceutical
University, Yamashina-ku,3 Kyoto, and
Faculty of Applied Biological Science, Hiroshima
University, Kagamiyama, Higashi-hiroshima,
Hiroshima,2 Japan
Received 27 March 2000/Accepted 13 June 2000
Isolation of Vibrio hollisae strains, particularly from
the environment, is rare. This may be due, in part, to the difficulty encountered when using conventional biochemical tests to identify the
microorganism. In this study, we evaluated whether two particular genes
may be useful for the identification of V. hollisae. The two genes are presumed to be conserved among the bacterial species (gyrB) or among the species of the genus Vibrio
(toxR). A portion of the gyrB sequence of
V. hollisae was cloned by PCR using a set of degenerate
primers. The sequence showed 80% identity with the corresponding
Vibrio parahaemolyticus gyrB sequence. The toxR gene of V. hollisae was cloned utilizing a htpG
gene probe derived from the V. parahaemolyticus htpG gene,
which is known to be linked to the toxR gene in V. hollisae. The coding sequence of the cloned V. hollisae
toxR gene had 59% identity with the V. parahaemolyticus toxR coding sequence. The results of DNA colony hybridization tests using the DNA probes derived from the two genes of V. hollisae indicated that these gene sequences could be utilized
for differentiation of V. hollisae from other
Vibrio species and from microorganisms found in marine
fish. PCR methods targeting the two gene sequences were established.
Both PCR methods were shown to specifically detect the respective
target sequences of V. hollisae but not other organisms. A
strain of V. hollisae added at a concentration of 1 to
102 CFU/ml to alkaline peptone water containing a seafood
sample could be detected by a 4-h enrichment incubation in alkaline
peptone water at 37°C followed by quick DNA extraction with an
extraction kit and 35-cycle PCR specific for the V. hollisae
toxR gene. We conclude that screening of seafood samples by this
35-cycle, V. hollisae toxR-specific PCR, followed by
isolation on a differential medium and identification by the above
htpG- and toxR-targeted PCR methods, can be
useful for isolation from the environment and identification of
V. hollisae.
Vibrio hollisae is a
halophilic bacterium that was first described in 1982 by Hickman and
collaborators (6). In the majority of cases reported thus
far, V. hollisae has been isolated from clinical sources
(1, 2, 5, 6, 13, 15, 18). Individuals who had no underlying
illness and consumed seafood such as raw shellfish were infected in
many cases. To our knowledge, there are only two reports on the
isolation of V. hollisae from environmental samples, i.e.,
from a coastal fish and an oyster (9, 19).
The exact pathogenic mechanism of V. hollisae is not known.
However, this organism has been shown to produce a hemolysin similar to
that of the thermostable direct hemolysin of Vibrio
parahaemolyticus (19, 32), which is considered a major
enterotoxic factor of the organism (20). The gene encoding
thermostable direct hemolysin (tdh) is distributed in a
minor population of environmental strains of V. parahaemolyticus and rare strains of Vibrio mimicus and Vibrio cholerae (non-O1 strains) isolated from diarrheal
patients (23). Unlike the findings with these three
Vibrio species, all clinical and environmental strains of
V. hollisae that others and a member of our group have
examined so far carried the tdh gene (19, 21,
22). A hypothesis on past horizontal transfer of the
tdh gene among Vibrio species was proposed
(23). V. hollisae may have served as a source of
the tdh gene in interspecies horizontal transfer between
Vibrio species in the marine environment (22, 23). However, few environmental strains of V. hollisae
were available for examination. This is a critical point of the
hypothesis. To examine the hypothesis and to assess the public health
significance of V. hollisae in the environment, it is
imperative to isolate more strains of V. hollisae from the
environment and examine them for the presence or absence of the
tdh gene.
The paucity of the isolation of V. hollisae from clinical
specimens may be due in part to poor growth of this bacterium on selective isolation media for enteric pathogens such as
thiosulfate-citrate-bile salts-sucrose agar and MacConkey agar (1,
2, 5, 7). It is also biochemically inert, so definitive
identification by conventional biochemical tests requires NaCl added to
a final concentration of 1% (2, 6, 7) and some clinical
strains fail to be identified by the API 20E system (1, 2).
It is more difficult to isolate V. hollisae from the marine
environment and identify it because the environmental samples contain
many nonpathogenic Vibrio species and related but
unestablished organisms. In addition, optimum culture conditions for
isolation and identification may not be the same for clinical and
environmental strains. The public health significance of V. hollisae may be underestimated due to the paucity of reports of
isolation from both clinical and environmental sources. If there were a
better method for isolation and identification of V. hollisae, it would be useful for isolation and subsequent study of
this organism. In this study, we examined whether identification of
V. hollisae by PCR is possible. For this purpose, the ideal
situation was to target the nucleotide sequence that is conserved and
that reflects phylogenetic relationship. The sequences of rRNA genes
are usually utilized for this purpose, but the interspecies homology
values of rRNA genes of Vibrio species are so high that
these gene sequences are not suitable (11, 27).
Recently, the gyrB gene encoding the B subunit of DNA
gyrase was utilized for identification of V. parahaemolyticus by PCR (28). Members of our group and
others demonstrated that the toxR gene sequence is also
useful for the identification of V. parahaemolyticus by PCR
(10). We presumed that the toxR gene is a
global regulatory gene possessed by the members of the genus Vibrio because the toxR gene was detected in
V. cholerae, V. parahaemolyticus, Vibrio fisheri, and Vibrio vulnificus and the
gene sequences were analyzed (12, 14, 16, 26). We
therefore cloned and sequenced the gyrB and toxR
genes of V. hollisae and demonstrated that these gene
sequences can be utilized for identification of V. hollisae by PCR. We then evaluated whether the established PCR method could be
applied directly to seafood samples to facilitate screening of V. hollisae-bearing samples.
Bacterial strains.
The bacterial strains used in this study
are listed in Table 1. The bacterial
strains belonging to various species of the genus Vibrio
were from our laboratory stock strains or strains supplied by other
workers for this study. The gyrB gene and the toxR gene of V. hollisae were cloned from
clinical strains 89A 1962 and 525-82 (1, 22), respectively.
A spontaneous streptomycin-resistant mutant of V. hollisae
525-82, a clinical strain, was obtained by plating a concentrated
overnight culture of the wild-type strain onto Luria-Bertani (LB) agar
containing 50 µg of streptomycin per ml. The mutant strain was
designated 525-82SmR.
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Analysis of gyrB and toxR
Gene Sequences of Vibrio hollisae and Development of
gyrB- and toxR-Targeted PCR Methods for Isolation
of V. hollisae from the Environment and Its
Identification
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Results of the tests for the gyrB and
toxR genes
gyrB sequence determination. The gyrB sequence was amplified by PCR using the PCR primers UP-1 and UP-2r designed by Yamamoto and Harayama (31). The 20-µl reaction mixture contained 6 µl of purified cellular DNA of V. hollisae 89A 1962 (0.175 µg/ml), 2 µl of each of the PCR primers (10 µM) 2 µl of 10× buffer containing 20 mM MgCl2 (ExTaq buffer; Takara, Shiga, Japan), 1 µl (0.5 U) of Taq polymerase (ExTaq; Takara), 2 µl of each deoxynucleotide triphosphate (2.5 mM), and 5 µl of distilled water. The amplification conditions were 30 cycles of amplification consisting of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 2 min, followed by one cycle of 72°C for 7 min. Amplification was performed in a Genius thermal cycler (model FGENO5TY; Techne Ltd., Oxford, Cambridge, England). The amplicon of 1.3 kb was cloned into a vector plasmid, pGEM-T Easy (Promega Corp., Madison, Wis.), using E. coli MC1061 as the host. The nucleotide sequences of both strands of the cloned fragment were determined with the ABI-PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit and an ABI-PRISM 310 genetic analyzer (Applied Biosystems Division, Perkin-Elmer, Foster City, Calif.)
toxR sequence determination. The approach used in cloning the V. hollisae toxR gene was a design based on the possibility that the toxR gene and the htpG gene encoding a heat shock protein were located contiguously in the chromosome (discussed below). First, a htpG-bearing restriction fragment of cellular DNA of V. hollisae 525-82 was identified and cloned using the V. parahaemolyticus htpG-specific gene probe as follows. The KpnI digest of cellular DNA of V. hollisae 525-82 was size fractionated using agarose gel electrophoresis, and 5.9-kb DNA fragments were cloned into KpnI-cleaved pUC118 (29) in E. coli MC1061. The transformants containing the recombinant plasmid, pKON20, were detected by the DNA colony blot hybridization method under stringent conditions (21) with the V. parahaemolyticus htpG-specific probe.
Next, a toxR-bearing subfragment within the insert was sought. A restriction endonuclease map of the insert of pKON20 was constructed. The pKON20 was digested with appropriate restriction enzymes and was then examined by Southern blot hybridization with the DNA probe specific to the V. parahaemolyticus toxR gene under reduced-stringency conditions (with 31% formamide) as described previously (24). A 2.7-kb PstI-ScaI DNA fragment, one of the subfragments that gave a weak hybridization signal, was subcloned into the PstI-HincII sites of pUC119 vector plasmid (29), resulting in pKON22. The nucleotide sequence of the insert was determined as described above.DNA probes. One of the recombinant plasmids that were confirmed to carry the cloned V. hollisae gyrB sequence (explained below) was named pVAR1 and used for preparation of the V. hollisae gyrB-specific DNA probe. NotI recognition sites flanked the insertion sites in pVAR1. Therefore, pVAR1 was digested with NotI enzyme and the 1.3-kb insert was isolated and used as V. hollisae gyrB-specific probe DNA. To prepare a V. parahaemolyticus htpG-specific DNA probe, a 1.5-kb HindIII fragment internal to the V. parahaemolyticus htpG gene was isolated from a recombinant plasmid, pKTN170 (M. Nishibuchi, J. Okuda, and K. Kumagai, unpublished data.), and used as the probe DNA. The V. parahaemolyticus toxR-specific probe DNA which is internal to the V. parahaemolyticus toxR coding region was prepared by isolating a 687-bp DNA fragment from pKTN118 as described previously (14). The V. hollisae toxR-specific probe DNA was 349-bp amplicons prepared by PCR using pKON22 as the template and primers HT-F1 and HT-R2 (described below). The composition of the PCR reaction mixture and the amplification conditions were the same as those described below for the V. hollisae toxR-specific PCR. The probe DNAs were labeled by the random priming method with 32P-labeled dCTP (4).
DNA colony hybridization test. The strains belonging to V. hollisae, V. cholerae, V. parahaemolyticus, Vibrio fluvialis, Vibrio furnisii, Vibrio damsela, Vibrio metchnikovii, V. mimicus, V. vulnificus, Vibrio alginolyticus, Vibrio anguillarum, and E. coli and those of unknown identification were grown overnight at 25°C using LB agar containing 1% NaCl. The other strains belonging to various species of the genus Vibrio were grown to reasonable colony sizes on marine agar 2216 (Difco Laboratories, Detroit, Mich.) at 25°C. DNA colony blots were prepared as described previously (21). The DNA colony hybridization test was performed under high-stringency conditions (in a solution containing 50% formamide) as described previously (21).
V. hollisae gyrB-specific PCR. The strains belonging to V. hollisae, V. cholerae, V. parahaemolyticus, V. fluvialis, V. furnisii, V. damsela, V. metschnikovii, V. mimicus, V. vulnificus, V. alginolyticus, and E. coli were grown in LB broth containing 1% NaCl at 37°C with shaking (160 rpm) overnight. V. anguillarum PT-87050 and the strains of unknown identification were grown in LB broth containing 1% NaCl at 25°C with shaking (160 rpm) overnight. The other strains belonging to various species of the genus Vibrio were grown to good turbidity in marine broth 2216 (Difco Laboratories) at 25°C with shaking (160 rpm). One milliliter of the broth 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 diluted supernatant was used as the template for PCR amplification.
The nucleotide sequence of a part of the V. hollisae gyrB gene determined in this study (described below) was compared with the corresponding V. parahaemolyticus gyrB sequence. The regions that were not well conserved between the two sequences were selected. The V. hollisae gyrB sequences of these regions were examined by a computer program for designing PCR primers (Oligo 4.05; National Bioscience, Inc., Plymouth, Minn.) Pairs of two forward and two reverse primers thus designed were evaluated. The forward primers were HG-F1 (5'-GCTCTGTCGGAAAAACTTGA-3' [positions 100 to 119]) and HG-F2 (5'-CAGATTTATCGCATGGGTGT-3' [positions 154 to 173]), and the reverse primers were HG-R1 (5'-TTGCATCGATACTTCTACTG-3' [positions 501 to 482]) and HG-R2 (5'-ATGCTCAAAATGGAACACAG-3' [positions 462 to 443]). The positions indicated above correspond to those of the gyrB sequence deposited in the DDJB, EMBL, and GenBank databases (see below). The PCR reaction mixture consisted of 2 µl of DNA template (supernatant of the boiled culture diluted 1:10), 1 µl of 2.5 mM deoxynucleotide triphosphate, 5 µl of each of the primers (2 µM) 0.1 µl of Taq polymerase (Taq DNA polymerase in storage buffer A [5 U/µl] Promega Corp.) 2 µl of 10× buffer (thermophilic DNA polymerase 10× buffer, magnesium free; Promega Corp.), 1.2 µl of 25 mM MgCl2, and 3.7 µl of distilled water. The amplification conditions were 30 cycles of amplification consisting of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min, followed by one cycle of 72°C for 7 min. Amplification was performed in a Genius thermal cycler (model FGENO5TY). Ten microliters of the reaction mixture was resolved by electrophoresis in 2% agarose gel to detect the amplicon of the expected size.V. hollisae toxR-specific PCR. The supernatant of the boiled culture of the test strain was obtained and diluted as described for the V. hollisae gyrB-specific PCR above, and the diluted supernatant was used as the DNA template. The broth culture obtained in the spike experiment (described below) was boiled, diluted (1:10), and used as the DNA template in the same manner unless otherwise specified.
The nucleotide sequence of the V. hollisae toxR gene determined in this study (described below) was compared with those of the V. parahaemolyticus toxR, V. cholerae toxR, and V. fischeri toxR genes (14, 16, 26). Three forward primers and two reverse primers specific to the V. hollisae toxR sequence were selected from the regions not conserved among the toxR sequences of four species. The forward primers were HT-F1 (5'-GAGACCGCAGCGAAAACGGAG-3' [positions 391 to 411]), HT-F2 (5'-AACGGAGCCTGAGCTTCTTTCC-3' [positions 405 to 426]), and HT-F3 (5'-CTGCCCAGACACTCCCTCTTC-3' [positions 434 to 454]). The reverse primers were HT-R1 (5'-GCTGCCATTGAGAAATGCGTGC-3' [positions 685 to 664]) and HT-R2 (5'-CTCTTTCCTTACCATAGAAACCG-3' [positions 739 to 717]). The positions indicated above correspond to those of the toxR sequence deposited in the DDJB, EMBL, and GenBank databases (see below). The PCR reaction mixture consisted of 10 µl of DNA template (diluted supernatant of the boiled culture [1:10]), 5 µl of 10× buffer containing 20 mM MgCl2 (ExTaq buffer; Takara), 4 µl of 2.5 mM deoxynucleotide triphosphate, 1 µl of each primer (20 µM) 0.25 µl of Taq polymerase (ExTaq, Takara), and 28.75 µl of distilled water. The amplification conditions were, unless otherwise specified, 24 cycles of amplification consisting of denaturation at 95°C for 1 min, annealing at 62°C for 1.5 min, and extension at 72°C for 1.5 min, followed by one cycle of 72°C for 5 min. Amplification was performed in a DNA Thermal Cycler 480 (Perkin-Elmer Co.). Ten microliters of the reaction mixture was resolved by electrophoresis in 2% agarose gel to detect the amplicon of the expected size.Spike experiments. A 40-g seafood sample was put into 400 ml of alkaline-peptone water (APW; 1% peptone, pH 8.6), the mixture was incubated at 37°C for 5 to 10 min and homogenized by vigorous shaking for 2 min, and 30-ml aliquots of the homogenate were placed in sterile containers. V. hollisae 525-82SmR grown in LB broth to the mid-log phase was added to the aliquoted homogenate at the concentrations of 0, 10, 102, 103, 104, 105, and 106 CFU/ml, and the homogenate was mixed well. Samples for PCR assay (1 ml) and for plate count (1 ml) were taken immediately as zero-hour samples, and the rest were incubated at 37°C without shaking. Samples for the PCR assay and plate count were taken again after 4 h of incubation, and then the rest were incubated at 15°C. The samples for plate count were also obtained 4.5 h later from the samples incubated at 15°C. Unless otherwise specified, the sample for PCR assay was subjected to DNA extraction by the boiling method described above for the V. hollisae toxR-specific PCR.
Mixtures of selected homogenate and 525-82SmR after 4 h of enrichment incubation at 37°C were also used to compare the DNA extraction methods for PCR temperate preparation. Triplicate 1-ml samples were prepared for each test. One of the samples was treated by the boiling method described above. The remaining two samples were centrifuged (14,000 rpm) on the tabletop centrifuge (model 5415C, Eppendorf) for 5 min, the pellet was obtained and suspended in 0.1 ml of saline, and DNA was extracted and dissolved in 0.1 ml of distilled water. DNA was extracted by a method based on chaotropic extraction with NaI followed by ethanol precipitation using a DNA extraction kit (code no. 295-50201; Wako Pure Chemical Industries, Ltd., Osaka, Japan) or by chaotropic extraction followed by DNA absorption onto silica-coated magnetic beads (MagExtractor-Genome-; TOYOBO Co. Ltd., Osaka, Japan). These DNA extractions were performed according to the manufacturers' specifications. PCR amplification with primer pair HT-F3 and HT-R2 was carried out as described for the V. hollisae toxR-specific PCR except that the total reaction volume was reduced to 20 µl (the volume of each component was reduced accordingly), Taq polymerase (ExTaq; Takara) was replaced by Taq DNA polymerase in storage buffer A (Promega Corp.), and not only 24 but also 35 thermal cycles were employed. The sample for plate count was diluted appropriately in sterile saline and 0.1 ml of the sample was inoculated in triplicate onto the isolation medium (19) and onto the same medium with 100 µg of streptomycin per ml. Purple colonies on the agar medium were counted after overnight incubation at 37°C. The average colony count per sample was determined. When needed, representative purple colonies (up to 20 colonies per plate) were examined by V. hollisae toxR-specific PCR as described above.Nucleotide sequence accession numbers. The nucleotide sequence data of the gyrB and toxR genes of V. hollisae reported in this paper will appear in the DDJB, EMBL, and GenBank nucleotide sequence databases with the accession numbers AB027462 and AB027503, respectively.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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gyrB gene sequence analysis. We were able to amplify a selected region of the V. hollisae gyrB gene by applying a set of degenerate, universal PCR primers to the cellular DNA of V. hollisae 89A 1962. The amplified sequence was 1259 bp long and fairly homologous with the corresponding sequences of the reported gyrB genes of gram-negative bacteria (62 to 80% identity values). The V. parahaemolyticus gyrB sequence exhibited the highest overall identity value. The results indicated that the cloned sequence is the expected region of the V. hollisae gyrB gene.
toxR gene sequence analysis. A 2.7-kb PstI-ScaI fragment was cloned from the cellular DNA of V. hollisae 525-82 as described in Materials and Methods, and the nucleotide sequence was determined. The sequence contained an open reading frame of 292 amino acids, and this open reading frame was identical in length with that of the V. parahaemolyticus toxR coding sequence (14). The two nucleotide sequences shared 59% nucleotide sequence identity. The identity value was almost comparable to that between the V. cholerae toxR gene and the V. parahaemolyticus toxR gene. Deduced amino acid sequences of V. parahaemolyticus ToxR and V. cholerae ToxR shared a region of high homology (80% identity) at the N terminus, which was presumed to be a DNA binding domain, although the identity over the entire sequence was 55% (14). The identities for the putative DNA binding domain region (91 residues) and the entire coding region between the amino acid sequences deduced from the V. hollisae open reading frame and from the V. parahaemolyticus toxR gene were 68 and 47%, respectively (data not shown). We therefore concluded that the open reading frame of V. hollisae 525-82 is the coding region of the V. hollisae toxR gene.
Examination of Vibrio strains by V. hollisae
gyrB and V. hollisae toxR probes.
DNA
fragments internal to the V. hollisae gyrB and
toxR genes were employed as the probe DNAs in DNA colony
hybridization tests to examine the distribution of homologous sequences
in the strains belonging to 33 species of genus Vibrio,
and 74 strains of unknown identification (Table 1). The strains of
unknown identification were isolated from coastal fishes using the
medium designed for isolation of V. hollisae.
However, the results of the tests for selected
biochemical characteristics, which appear to be important for identification of V. hollisae strains
(6, 8), indicated that these strains are likely to be
distinct from V. hollisae (Table
2). V. hollisae strains
exhibited strong hybridization signals with the V. hollisae
gyrB-specific probe, whereas most strains belonging to other
Vibrio spp. and E. coli MC1061 showed distinctly weaker hybridization signals and the other strains of Vibrio spp. exhibited no reaction. The representative
hybridization signals are shown in Fig.
1. On the other hand, only V. hollisae strains demonstrated hybridization signals with the
V. hollisae toxR-specific probe. No other test strains,
including the presumed non-V. hollisae strains of fish
origin, showed any sign of hybridization with this probe. The
representative hybridization patterns are presented in Fig.
2.
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Examination of Vibrio strains by V. hollisae
gyrB- and V. hollisae toxR-targeted PCR.
The strains listed in Table 1 were examined by the PCR methods that
were designed for specific detection of V. hollisae. Two forward primers (HG-F1 and HG-F2) and two reverse primers (HG-R1
and HG-R2) were designed for detection of the V. hollisae gyrB gene as described in Materials and Methods. Four pairs of the
primers were tested using the amplification conditions described in
Materials and Methods. Three primer pairs (HG-F1 and HG-R2, HG-F2 and HG-R1, and HG-F2 and HG-R2) yielded the results
shown in Table 1, and thus the PCR using any of these primer pairs was
judged to be specific to the V. hollisae gyrB gene.
Amplicon patterns of selected strains are shown in Fig.
3. The V. hollisae gyrB
gene in 3 of 11 clinical strains of V. hollisae could
not be detected when the primer pair HG-F1 and HG-R1 was employed under the conditions described in Materials and Methods, although a
false-positive result was not obtained with any of the test strains
listed in Table 1 (data not shown in Table 1). We did not pursue
further the optimal amplification conditions for the primer pair HG-F1
and HG-R1.
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X 174 phage DNA
digested with HaeIII); 2 to 9, V. hollisae
strains 89A 1961, 89A 1960, 89A 4206, 91A 2120, 93A 5688, SJ 90, FO 93, and RIMD 2221011, respectively; 10, V. anguillarum
PT-87050; 11, V. cholerae O1 NIH41; 12, V. fluvialis RIMD 2220002; 13, V. furnisii RIMD
2223001; 14, V. parahaemolyticus WP1; 15, V. vulnificus RIMD 2219009; 16, V. alginolyticus AM2;
17, V. damsela RIMD 2222001. The position of the
specific amplicons (363 bp) is indicated by the solid triangle.
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Detection by V. hollisae toxR-specific PCR and
isolation of V. hollisae added to seafood
samples.
Each of various seafood samples was homogenized in APW,
and the homogenate was not spiked or was artificially contaminated with
V. hollisae 525-82SmR at 10, 102, 103, 104, 105, and
106 CFU/ml. The sensitivity of the V. hollisae toxR-specific PCR method for detecting
525-82SmR by direct examination of the inoculated
homogenate before and after 4 h of enrichment incubation at 37°C
was determined. The PCR samples were examined by PCR employing not only
24 but also 35 thermal cycles. All seafood samples without
525-82SmR yielded negative results. The lowest initial
levels of 525-82SmR that gave a positive PCR result are
shown in Table 3. The detection limits of
the 24-cycle PCR were 105 CFU/ml or above and
103 CFU/ml or above, respectively, before and after the
enrichment incubation. The detection limits of 35-cycle PCR ranged
between 103 and 106 CFU/ml and between 10 and 104 CFU/ml, respectively, before and after the
enrichment incubation.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We analyzed the gyrB homologue of V. hollisae in this study. Although the entire V. hollisae gyrB gene sequence was not analyzed, high homologies between the determined sequence and reported gyrB sequences of gram-negative bacteria allowed us to conclude that the cloned amplicon was the expected region of the V. hollisae gyrB gene. The 73 to 80% identity that was detected between the V. hollisae gyrB sequence and the corresponding regions of V. parahaemolyticus, V. alginolyticus, and E. coli (data not shown) can explain the weak reactions observed in the DNA colony hybridization test. These and the hybridization results listed in Table 1 suggest that the divergence in the gyrB sequence between V. hollisae strains and other test strains would be in the range of approximately 20 to 27% for weakly positive strains and more for negative strains. The divergence was sufficient for differentiation of V. hollisae from other organisms by the PCR method (Table 1 and Fig. 3).
As was reported for V. cholerae (25), the htpG gene was located immediately upstream of the toxR gene in V. parahaemolyticus (M. Nishibuchi, J. Okuda, and K. Kumagai, unpublished data). We therefore presumed that the toxR-htpG gene arrangement is universal in vibrios. The homology between the V. cholerae htpG gene and the V. parahaemolyticus htpG gene was considerably higher than the homology between the toxR genes of the two species (14; M. Nishibuchi, J. Okuda, and K. Kumagai, unpublished data). Our attempt to utilize the V. parahaemolyticus htpG gene probe for cloning the V. hollisae toxR gene was successful. The result supports our hypothesis on the general toxR-htpG gene arrangement. This approach would be useful for cloning the toxR genes of other vibrios.
The overall nucleotide sequence identity between V. hollisae and V. parahaemolyticus was much lower for the toxR gene (59%) than for the gyrB gene (80%). In addition, the conserved region encoding the putative DNA binding domain of ToxR was not included in the V. hollisae toxR gene probe. The sequences of the PCR primers for V. hollisae toxR detection were selected from within the probe sequence. This explains why both the DNA probe method and the PCR method were shown to be successful for specific detection of the V. hollisae toxR gene (Table 1; Fig. 2 and 4).
The primary purpose of this study was to develop easy-to-perform and reliable genetic methods for identification of V. hollisae among the strains isolated from the marine environment. The test strains included not only the standard strains of Vibrio spp. but also organisms presumed to be non-V. hollisae isolated from marine fishes. The PCR results served to confirm the presumptive classification of the fish isolates by brief biochemical characterization. No discrepancy was seen between the results obtained by two PCR methods targeting the two different genes (Table 1). We conclude therefore that both PCR methods are equally useful for specific detection of V. hollisae. PCR methods targeted to the gyrB and toxR genes were shown to be useful for identification of V. parahaemolyticus in previous studies (10, 28) and of V. hollisae in this study. The same approach would be applicable to establishment of the PCR-based identification method for other species of the genus Vibrio.
Our ultimate goal is to isolate V. hollisae strains from the environment. Our previous approach was inoculation of the seafood samples onto the isolation medium (Vh-M) followed by conventional biochemical tests of the isolated strains (19). The PCR methods established in this study can replace the standard biochemical tests and facilitate the identification process. We also evaluated whether the screening process can be further facilitated by direct application of the V. hollisae toxR-specific PCR method to the seafood samples. Our idea was to screen V. hollisae-bearing seafood samples before attempting isolation of the candidate strains on Vh-M. It would obviate plating of the V. hollisae-negative enrichment culture and subsequent screening and identification and greatly reduce the workload. It is essential, then, that this direct PCR method have a high sensitivity. In addition, the V. hollisae strain in the sample has to be maintained in the enrichment broth so that the strain can be recovered after examination by PCR. The sensitivities of the V. hollisae toxR-specific PCR with 24 and 35 thermal cycles for LB broth cultures of six selected V. hollisae strains were 105 CFU/ml (40 CFU/PCR reaction) and 103 to 104 CFU/ml (0.4 to 4 CFU/PCR reaction), respectively, (data not shown). Equal sensitivities were observed in the direct PCR detection of V. hollisae 525-82SmR in some seafood homogenates before enrichment culture, but sensitivities were lower for some seafood samples (Table 3), suggesting that some seafood homogenates inhibit the PCR reaction to some degrees. Enrichment culture of the seafood sample in APW for 4 h and examination by the 35-cycle PCR allowed detection of 10 or 102 CFU/ml 525-82SmR (initial level) in seafood homogenates other than octopus and shrimp homogenates. Since none of the seafood homogenates inhibited the growth of 525-82SmR in APW (Table 3), the low sensitivities of PCR observed with the octopus and shrimp homogenates were attributable to these tissues inhibiting the PCR reaction. The effect of potential PCR inhibitors in these samples could be removed by employing the DNA extraction kit based on chaotropic extraction followed by absorption onto silica-coated magnetic beads (Table 4, method 3). Thus, inclusion of this DNA extraction method allowed detection of 1 to 102 CFU/ml 525-82SmR (initial level) regardless of the kind of seafood sample.
We chose a short-term enrichment because preliminary experiments suggested that non-V. hollisae organisms in some seafood samples can considerably overgrow if enrichment is carried out for longer than 4 h. Twenty-five percent or more of the purple colonies on Vh-M, which resulted from inoculation of 10 to 105 CFU/ml of 525-82SmR in seafood homogenates, apparently contained 525-82SmR after the 4-h enrichment incubation (Table 3). The reason for the result that the number of purple colonies on Vh-M supplemented with SM exceeded that on Vh-M (the ratios in parentheses in Table 3 became more than 1) is not known but it may be partly due to growth inhibition among the organisms, including non-purple colonies, on Vh-M. Approximately 4.5 h, including the chaotropic and silica-magnetic bead DNA extraction step, was needed to obtain the PCR result. After the 4.5-h lag, the number of V. hollisae 525-82SmR CFU recoverable from the enrichment broth was reduced only by about one log unit and were still ca. 103 CFU/ml or more if the enrichment culture was maintained at 15°C during PCR examination (data not shown). The results suggest that screening of the purple colonies recovered from the 35-cycle PCR-positive enrichment culture for V. hollisae may not require intensive search of the purple colonies. From these results, we conclude that a 4-h enrichment incubation of seafood samples in APW, followed by the chaotropic and silica-magnetic bead DNA extraction and examination with the 35-cycle V. hollisae toxR-specific PCR is a sensitive screening method. V. hollisae cells can be recovered from the enrichment culture onto the screening medium after detection of V. hollisae-positive sample by PCR. The colonies can then be examined by the PCR methods for the V. hollisae toxR and V. hollisae gyrB genes, as described above. We propose that this approach can be an effective method to isolate V. hollisae from the environment.
V. hollisae may be present as viable but nonculturable cells in the environment. Such cells may be observed by the fluorescent antibody staining method if a V. hollisae-specific antibody becomes available. Alternatively, the methods shown to be useful to resuscitate viable but nonculturable cells of vibrios (17, 30) can be included in the above enrichment step. This approach may be helpful in recovering V. hollisae in the viable but nonculturable form and will be included in our future project.
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ACKNOWLEDGMENTS |
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This research was supported in part by the COE program on "Making Regions: Proto-Areas, Transformations and New Formations in Asia and Africa," by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan, and by the "Research for the Future" program of The Japan Society for the Promotion of Science (JSPS-RFTF97L00706).
V. Vuddhakul and J. Okuda contributed equally to this study.
We thank the Research Center for Emerging Infectious Diseases, Osaka University, and Satoru Matsuoka of Ehime Prefectural Fish Disease Control Center for providing bacterial strains. We are grateful to Yohko Takeda for technical assistance.
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FOOTNOTES |
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* 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}mb.med.kyoto-u.ac.jp.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, August 2000, p. 3506-3514, Vol. 66, No. 8
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