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Applied and Environmental Microbiology, April 2004, p. 1964-1972, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.1964-1972.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Phenotypic and Genotypic Characteristics and Epidemiological Significance of ctx+ Strains of Vibrio cholerae Isolated from Seafood in Malaysia

Chien-Hsien Chen,1 Toshio Shimada,2,{dagger} Nasreldin Elhadi,3,{ddagger} Son Radu,3 and Mitsuaki Nishibuchi4*

Graduate School of Medicine,1 Center for Southeast Asian Studies, Kyoto University, Sakyo-ku, Kyoto 606-8501,4 Laboratory of Enteric Infection, National Institute of Infectious Diseases, Shinjyuku-ku, Tokyo, Japan,2 Department of Biotechnology, Faculty of Food Science and Biotechnology, University Putra Malaysia, Serdang, Selangor, Malaysia3

Received 6 October 2003/ Accepted 23 December 2003


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ABSTRACT
 
Of 97 strains of Vibrio cholerae isolated from various seafoods in Malaysia in 1998 and 1999, 20 strains carried the ctx gene and produced cholera toxin. Fourteen, one, and five of these toxigenic strains belonged to the O139, O1 Ogawa, and rough serotypes, respectively. The rough strains had the rfb gene of the O1 serotype. The toxigenic strains varied in their biochemical characteristics, the amount of cholera toxin produced, their antibiograms, and the presence or absence of the pTLC plasmid sequence. DNA fingerprinting analysis by arbitrarily primed PCR, ribotyping, and a pulsed-field gel electrophoresis method classified the toxigenic strains into 3, 7, and 10 types, respectively. The relatedness of these toxigenic strains to clinical strains isolated in other countries and from international travelers was examined by using a dendrogram constructed from the pulsed-field gel electrophoresis profiles. The results of the examination of the antibiogram and the possession of the toxin-linked cryptic plasmid were consistent with the dendrogram-based relatedness: the O139 strains isolated from Malaysian seafoods could be separated into two groups that appear to have been introduced from the Bengal area independently. The rough strains of Malaysian seafood origin formed one group and belonged to a cluster unique to the Thailand-Malaysia-Laos region, and this group may have persisted in this area for a long period. The single O1 Ogawa strain detected in Malaysian seafood appears to have an origin and route of introduction different from those of the O139 and the rough strains.


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INTRODUCTION
 
Vibrio cholerae strains that carry the ctx genes in the CTX genetic element can produce cholera toxin (CT), and these strains are termed toxigenic strains. Toxigenic strains are responsible for cholera epidemics. Water is recognized as the most important vehicle for cholera transmission. In addition, outbreaks of food-borne cholera have been noted quite often in the past 30 years; seafood, including molluscan shellfish, crustaceans, and finfish, are most often incriminated in food-borne cholera cases in many countries (1, 35). However, the ctx genes are rarely detected in V. cholerae strains isolated from environmental samples, including seafood, that are not implicated in outbreaks. V. cholerae strains belonging to the O1 and O139 serotypes almost exclusively carry the ctx genes, and the O serotype is often used as a marker for the toxigenic strains. However, there are atypical environmental strains that possess the ctx genes. For example, a DNA probe study carried out in Japan revealed that 26.6% of the V. cholerae O1 strains isolated from imported seafood and none of the V. cholerae O1 strains isolated from the natural water carried the ctxA gene (28). On the other hand, a DNA probe study showed that a small percentage of environmental strains of V. cholerae non-O1 had the ctx gene (31).

Outbreaks of cholera due to the El Tor biotype of the O1 serotype occur periodically in Malaysia (26, 40), and this poses a public health problem if the seafood is contaminated with the toxigenic strains of V. cholerae. In our previous study on the distribution of pathogenic Vibrio species in fresh seafood marketed in Malaysia between July 1998 and June 1999, we isolated 97 strains of V. cholerae from various seafoods (10a); 1 strain belonged to the O1 serotype, and 14 belonged to the O139 serotype. Therefore, in this study we examined the distribution of the ctx gene in the V. cholerae strains isolated from Malaysian seafood and characterized the toxigenic strains in detail. We examined phenotypic characteristics, including CT production and antibiotic sensitivity, of the strains. To confirm the characteristics and the subtypes of the toxigenic strains, we analyzed these strains for the gene sequences that are associated with toxigenic strains.

The O serotypes of V. cholerae strains are usually determined by agglutination tests with specific antisera, but O1 and O139 strains, in particular, can also be identified by PCR methods targeted to the rfb gene, which is involved in lipopolysaccharide synthesis (13). This genetic method is useful because the rough variants of toxigenic strains (R strains) that are probably derived from O1 and O139 strains were isolated from patients with cholera-like diarrhea (29). The ctx genes are located on a genetic element, termed the CTX genetic element, that is in the genome of a filamentous bacteriophage (designated phage CTX{Phi}) (41). The toxigenic strains also produce another important virulence factor, the toxin-coregulated pilus (TCP), that is needed for intestinal colonization. Interestingly, the tcp gene cluster encoding the TCP exists on a 40-kb DNA segment termed a vibrio pathogenicity island (VPI) (19), and the VPI is encoded on a lysogenic filamentous phage VPI{Phi} and the TCP was reported to serve as a receptor for CTX{Phi} (20). The tcp gene encoding the TCP is usually unique to toxigenic strains but is rare in ctx-negative strains. However, tcpA-positive strains of non-O1, non-O139 serotypes were detected among strains isolated from environmental and clinical sources (34). PCR methods targeted to the tcpA gene can differentiate the tcpA sequences of El Tor and the classical biotypes (21). Rubin et al. (36) reported on a plasmid named toxin-linked cryptic plasmid (pTLC). According to their report, all toxigenic strains of V. cholerae O1 and O139 carried the nucleotide sequence of this plasmid, but nontoxigenic strains did not. This plasmid can exist as both covalently closed circular DNA and tandemly duplicated chromosomally integrated DNA, and the chromosomally integrated form of pTLC is located adjacent to the CTX prophage. pTLC was proposed to play some role in acquisition or replication of phage CTX{Phi} (36). The gene cluster encoding RTX (repeat in toxin), a cytotoxin, is composed of the rtxA, rtxB, rtxC, and rtxD genes, and this cluster is physically linked to the CTX element (24). Chow et al. (8) reported that all clinical and environmental stains of V. cholerae except for classical O1 strains carried the rtxA and rtxC genes and claimed that detection of the rtx genes in O1 strains can differentiate their biotypes.

In this study we examined the Malaysian seafood-borne toxigenic strains and other toxigenic strains isolated in Malaysia for the genetic characteristics described above and also for their DNA fingerprints to examine the relatedness among these strains. We also compared these strains and the toxigenic strains isolated in neighboring countries and from international travelers to infer the significance of the Malaysian seafood-borne toxigenic strains in international epidemiology.


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MATERIALS AND METHODS
 
Bacterial strains.
In our previous study, V. cholerae strains of seafood origin were isolated from various seafood samples collected at wet markets in various locations in Malaysia between July 1998 and June 1999 (10a). El Tor O1 Ogawa strains isolated from patients in Malaysia between 1998 and 1999 were provided by M. Ravichandran of the Universiti Sains Malaysia. An El Tor O1 Inaba strain isolated from a patient during an outbreak in Sarawak, Malaysia, in 1998 was provided by Patrick Benjamin Guda Miri of the General Hospital, Miri, Sarawak. Toxigenic O139 strains isolated from seawater in Malaysia in 1996 were described previously (38). Other V. cholerae O1 Ogawa strains isolated from international travelers arriving in Japan from various Asian countries between 1982 and 1998 were supplied by Osaka Airport Quarantine Station, Kansai Airport Quarantine Station, and Nagoya Airport Quarantine Station, Japan. O139 and R strains isolated from patients in Bangladesh and India between 1992 and 1997 were supplied by G. Barakrish Nair of the International Centre for Diarrhoeal Diseases Research, Dhaka, Bangladesh. Classical strains of V. cholerae (569B and NIH41) were from our laboratory stock cultures.

The identification of all environmental and clinical strains of V. cholerae isolated in Malaysia was confirmed by detailed standard biochemical test (23) and by detection of the toxR gene, which is specific to V. cholerae (see below). The O serotypes of these strains were determined by agglutination tests with specific polyclonal antisera as described previously (37). The Inaba and Ogawa serotypes of O1 strains were differentiated by using a commercial latex agglutination kit (V. cholerae AD; Denka Seiken Co., Tokyo, Japan).

Detection of CT production.
Test strains were grown and CT production was determined by an agglutination method with the VET-RPLA Seiken kit according to the specifications of the manufacturer (Denka Seiken Co. Ltd., Tokyo, Japan). Briefly, the test organism was grown in Syncase medium (12) or AKI medium (17) with shaking (110 rpm) at 30°C for 18 to 20 h. The culture was centrifuged at 750 x g for 20 min. Twofold serial dilutions of the culture supernatant were prepared, and an agglutination test with latex particles coated with anti-CT antibody was performed in a V-bottom 96-well plate. The reciprocal of the highest dilution that gave a positive reaction was defined as the CT titer.

Antibiotic susceptibility.
The antibiotic susceptibilities of test strains were determined with Mueller-Hinton agar without added NaCl by the disk diffusion method of Bauer et al. (5) with the following antibiotics at the indicated total amount per disk: ampicillin, 10 µg; chloramphenicol (CM), 30 µg; ciprofloxacin, 5 µg; gentamicin, 10 µg; kanamycin, 30 µg; nalidixic acid, 30 µg; norfloxacin, 10 µg; streptomycin (SM), 10 µg; co-trimoxazole (SXT), composed of sulfamethoxazole at 23.75 µg and trimethoprim at 1.25 µg; and tetracycline (TC), 30 µg. The results defined as intermediate or resistant in the method of Bauer et al. were recorded as resistant in this study.

Detection of gene sequences by PCR.
The presence or absence of the following gene sequences was determined by PCR methods reported previously: ctx (13), rfb (13), toxR (13), tcpA (21), pTLC (4), and rtxA and rtxC (8). The ctx, rfb, and toxR sequences were detected with the multiplex PCR protocol (13), and other gene sequences were detected individually. To prepare the PCR template, the test strain was grown in Luria-Bertani broth medium containing 1% NaCl at 37°C with shaking (160 rpm) overnight. One milliliter of the culture was boiled for 10 min and immediately transferred onto ice. The supernatant was then obtained by centrifugation (10,000 rpm) on a tabletop centrifuge (model 5415C; Eppendorf, Hamburg, Germany) for 10 min at room temperature. The supernatant was diluted 10-fold with distilled water and used as a template solution for PCR. Purified total DNA was prepared from the overnight Luria-Bertani broth culture as described previously (32). The purified total DNA was dissolved in distilled water at 200 ng/ml, and this DNA solution was used as another DNA template. Both the boiled supernatant DNA and the purified DNA solution were tested for all test strains in all PCR tests. Since the results obtained by using the two template preparations were identical, the kind of DNA template used is not specified in Results.

DNA colony blot hybridization test.
DNA colony blots were prepared, and DNA colony hybridization was performed under stringent conditions (in a solution containing 50% formamide with washing at 65°C) as described previously (33). The probe DNA for detection of the pTLC sequence was prepared by the PCR method (4) with total DNA of V. cholerae 569B as the template. The 2.2-kb amplicons were purified by gel electrophoresis and used as the probe DNA for the pTLC sequence. The probe DNA was labeled by using the random priming method with 32P-labeled dCTP (25).

Ribotyping.
The rRNA gene restriction patterns (ribotypes) of the test strains were analyzed as described by Faruque et al. (11). Briefly, 5-µg aliquots of total DNA were digested with BglI and electrophoresed in a 0.8% agarose gel, and the DNA fragments were blotted onto a nylon membrane (Hybond; Amersham Pharmacia Biotech, Little Chalfont, England). The DNA blot was hybridized with the radiolabeled rRNA gene probe (the 7.5-kb BamHI fragment of pKK3535), and then the autoradiograph was obtained and analyzed.

AP-PCR.
Arbitrarily primed PCR (AP-PCR) was carried out as described previously (22), but only two primers, designated primer 2 and primer 4, which were expected to yield a high resolution (22), were used in this study.

PFGE.
Intact genomic DNA was prepared by a modification of the method described by Kondo et al. (22). Test strains were grown in 5 ml of Luria-Bertani broth medium containing 1% NaCl at 37°C with shaking (160 rpm) for 6 to 7 h. A 500-µl culture was centrifuged (3,000 x g), and the bacterial cells harvested were washed with TSE buffer (10 mM Tris, 20 mM NaCl, 50 mM EDTA) once and then suspended in 100 µl of the same buffer. Agarose plugs were prepared by mixing equal volumes of the cell suspension and a 2% solution of melted low-melting-point agarose (Bethesda Research Laboratories, Gaithersburg, Md.). Cells in the plugs were lysed by incubation in lysozyme buffer (10 mM Tris, 50 mM NaCl, 0.2% sodium deoxycholate, 0.5% sodium lauroyl sarcosine, 1 mg of lysozyme per ml, and 20 µg of RNase per ml) at 37°C overnight, followed by treatment with proteinase K (100 mM EDTA, 0.2% sodium deoxycholate, 1% sodium lauroyl sarcosine, 1 mg of proteinase K per ml) at 50°C for 24 to 28 h. The plug was then washed once in TE buffer (20 mM Tris, 50 mM EDTA [pH 7.0]) containing 1 mM phenylmethylsulfonyl fluoride for 30 min, twice in distilled water for 30 min, and finally twice in TE buffer for 30 min at room temperature. The washed plug was stored in TE buffer at 4°C until digestion with NotI. Prior to digestion with NotI, the stored plugs were washed in 0.1x TE buffer at room temperature for 1 h, and then the plugs were equilibrated in the reaction buffer for NotI for 1 h at room temperature. Thirty units of NotI was added to the 300-µl reaction buffer containing the plug and incubated at 37°C for 16 h. After digestion, the plug was washed once with distilled water and then kept in TE buffer at 4°C until analysis by pulsed-field gel electrophoresis (PFGE).

The digested DNA fragments were separated in 1% agarose (pulse field-certified agarose; Bio-Rad Laboratories, Hercules, Calif.) by using 0.5x Tris-borate-EDTA buffer on a CHEF-DRIII system (Bio-Rad Laboratories). Electrophoresis was performed at 6 V/cm and at a field angle of 120° at 14°C. The pulse times were 1 to 7 s for 8 h, 3 to 18 s for 9 h, and then 3 to 28 s for 11 h. Following electrophoresis, the gel was stained with 1 µg of ethidium bromide per ml for 30 min, destained in distilled water for 30 min, and photographed with a UV transilluminator. According to the proposed method of Tenover et al. (39), different designations (e.g., A, B, etc.) were given to the PFGE patterns if they differed by more than six DNA bands. The PFGE patterns that differed from a designated type by one to six DNA bands were classified as subgroups within the designated pattern, and subgroup designations (e.g., A1, A2, etc.) were given accordingly. A dendrogram was then constructed from the PFGE profiles according to an unweighted pair group method with arithmetic-average clustering analysis by using Dendron software version 3.0 (Stollect Inc., Oakdale, La.).


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RESULTS AND DISCUSSION
 
Detection of toxigenic strains among the seafood isolates.
In our previous study (10a), we identified by biochemical tests 97 strains of V. cholerae among Vibrio strains that were isolated from seafood samples collected at various locations in Malaysia in 1998 and 1999. In this study, we detected the V. cholerae-specific toxR sequence in the 97 strains by the PCR method and thus confirmed that these 97 strains are V. cholerae. We then examined whether these strains carry the ctxA gene by the PCR method. Twenty strains gave positive results, where 1 of the ctx-positive strains belonged to the O1 Ogawa serotype and 14 belonged to the O139 serotype. These toxigenic strains had the rfb genes corresponding to their O serotypes (Table 1). The O serotypes of five of the ctx-positive strains could not be determined, and they were found to be R strains by the agglutination test with the rough-strain-specific antiserum. These strains carried the rfb gene of the O1 serotype (Table 1). The R strains carrying the ctx gene and the rfb gene of the O1 serotype were previously isolated from patients with cholera-like diarrhea, and these strains lacked the side chains of the lipopolysaccharide (29). Therefore, the five R strains found in this study are likely to be the lipopolysaccharide variants of O1 strains, and they are potentially of public health significance. The 20 toxigenic strains were isolated from various kinds of seafoods collected at various locations in Malaysia (Table 1). This result and the isolation of toxigenic O139 strains from seawater in Malaysia in 1996 (38) suggest that the aquatic environment may serve as the reservoir for toxigenic V. cholerae in Malaysia.


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  		TABLE 1. Phenotypic and genotypic characteristics that varied among toxigenic V. cholerae strains isolated from seafood in Malaysia between July 1998 and June 1999

Characteristics of the toxigenic strains of seafood origin.
The 20 toxigenic strains isolated from Malaysian seafood varied in some phenotypic characteristics other than the O serotype. These phenotypic characteristics included four biochemical characteristics (Table 1), the antibiogram (Table 1), and the level of CT production. We confirmed that all 20 toxigenic strains produced CT, but the level of CT produced varied from strain to strain. CT was produced at the same or higher levels in AKI medium than in Syncase medium, except for one R strain. This result agreed fairly well with reports that AKI medium better supported CT production from El Tor and O139 strains and from some but not all R strains (17, 29). Only one of 20 toxigenic strains isolated from Malaysian seafood belonged to the O1 serotype, whereas most strains isolated in clinical laboratories in Malaysia belong to the O1 serotype. We do not know the reason for this difference in the serotype. Examination of the isolated strains of V. cholerae for the ctx and rfb genes contributed at least in part to better recovery of toxigenic strains, since five toxigenic strains belonged to the R group. These strains escaped screening by the conventional O serotyping method in our earlier study (10a).

We also analyzed the 20 toxigenic strains for the presence or absence of the gene sequences associated with the toxigenic strains (explained in the introduction) by using PCR methods. The tcpA gene sequence of the El Tor biotype and the rtxA and rtxC gene sequences were detected in all 20 strains (not shown in Table 1). However, the pTLC sequence was absent in all R strains, which is unusual for toxigenic strains of V. cholerae (Table 1). The absence of the pTLC sequence in these strains was confirmed by using the DNA colony blot hybridization test with a DNA probe. Differences in the O serotype and the varying phenotypic and genotypic results even among strains belonging to the same serogroup suggest that at least some of these toxigenic strains were derived from different clones.

Molecular typing of Malaysian toxigenic strains by DNA fingerprinting methods.
We next compared the 20 toxigenic strains isolated from Malaysian seafood by various DNA fingerprinting methods to examine their relatedness at the molecular genetic level. Other available toxigenic strains isolated in Malaysia were also included for comparison. These included four O139 strains isolated from seawater in 1996 and four O1 strains isolated from patients between 1998 and 1999. These 28 Malaysian toxigenic strains were differentiated into 3 and 2 types by the AP-PCR method with two primers, designated primer 2 and primer 4, respectively (Table 2); into 7 types (profiles a to g) by the ribotyping method (Table 2); and into 10 types by the PFGE method (Table 2; Fig. 1, profiles A1 to A5, B, C1 to C3, and D1). These results indicate that the PFGE method was the most discriminatory. This result is in agreement with the reports by other workers that the PFGE analysis of NotI-digested DNA had a high discriminating power for V. cholerae O1 strains (7, 10, 22). When the entire DNA fingerprinting results are combined, the discriminating power can be further enhanced. Even under this condition, the 5 R strains of seafood origin were indistinguishable from each other, and 6 of the 14 O139 strains of seafood origin (ribotype C, PFGE type C1) were indistinguishable from 3 of the 4 O139 strains of seawater origin (Table 2). The latter result suggests that these O139 strains may be prevalent in the natural environment of Malaysia.


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  		TABLE 2. DNA fingerprinting profiles of toxigenic strains of V. cholerae isolated in Malaysia



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  		FIG. 1. PFGE profiles of representative toxigenic strains of V. cholerae. The profile designations indicated at the top of the gel photograph were the results obtained with the following strains: A1, VCF3; A2, VCF111; A3, VC38; A4, VC94; A5, VC362; A6, ALO45; B, M12; C1, VCF22; C2, VCF66; C3; VCF75; D1, VCF38; D2, AJ34644; D3, AM3351; E1, MDO-2; E2, MO579; E3, SG-25; F, VO1; G, Arg-03; H, KX-C1; I, KX-C5; J, KX-C17; K, Vc162; L, NIH41; M, AQ1005; N, AQ1030; O, MO99, P, SG-15; Q, AQ1033; and R, AQ1053. Positions of representative bands of molecular weight markers (DNA size standard maker [lambda ladder; Bio-Rad]) run alongside are indicated.

Since the PFGE analysis method had the highest resolution, the results of the PFGE analysis were chosen for epidemiological analysis of the toxigenic strains isolated from seafood and the related strains from Malaysia. The relationship among the 10 PFGE profiles (Table 2, A1 to D1) was first analyzed, and the dendrogram shown in Fig. 2A was obtained. The profile of an O1 Inaba strain was slightly closer to those of O139 strains than to those of R and O1 Ogawa strains. Our PFGE analysis indicates that current O1 strains and O139 strains can form different clusters, although O139 strains were proposed to have diverged from an O1 El Tor strain (27). Jiang et al. (18) reached a similar conclusion when they examined various strains of V. cholerae. The close relationship of the R strains to O1 Ogawa strains that we examined in our study suggests the possibility that these R strains were derived from an O1 Ogawa strain(s) by mutation in the somatic antigen.



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  		FIG. 2. Dendrograms constructed from the PFGE profiles. (A) Comparison of the toxigenic strains of serotypes O139, O1, and R isolated in Malaysia. (B) Comparison of the toxigenic O139 strains isolated in Malaysia and other countries and from international travelers. (C) Comparison of the toxigenic O1 and R strains isolated in Malaysia and other countries and from international travelers. More detailed information on the PFGE profiles and the stains is given in Table 2. SAB, scale for similarity coefficient.

Significance of the Malaysian toxigenic strains in international epidemiology.
In order to evaluate the significance of toxigenic strains isolated from Malaysian seafood in international epidemiology, the PFGE profiles of O139, O1, and R strains that were isolated from clinical sources from other countries and from international travelers were determined and included in the comparative analysis. In addition, the possession of the pTLC gene and the antibiogram, which were shown to be useful in differentiating the toxigenic Malaysian strains, were also employed for the comparison. The results are summarized in Table 3. The additional PFGE profiles exhibited by some of the newly included strains (D2 to D3, E1 to E3, and F to R) are shown in Fig. 1.


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  		TABLE 3. PFGE profiles and selected characteristics of toxigenic strains of V. cholerae O139, R, and O1 studied

The dendrograms of the O139 strain group (Fig. 2B) and the O1-R strain group (Fig. 2C) were constructed separately from their PFGE profiles. For O139 strains, the profiles of C1, C2, C3, and F were closely related (Fig. 2B). The strains that exhibited these profiles included 13 of 14 strains isolated from Malaysian seafood between 1998 and 1999, all four strains isolated from Malaysian seawater in 1996, and four strains isolated from patients in India between 1993 and 1995 (Table 3). Five other clinical strains isolated in India between 1993 and 1996 showed the profiles E1, E2, and E3 (Table 3). The E3-E1-E2 profiles formed a cluster, and this cluster was linked to the C1-C3-F-C2 cluster (Fig. 2B). These results suggest that an O139 clone that was prevalent in India in early years (1993 to 1996) may have been introduced into the Malaysian seawater environment before 1996. If so, this clone probably persisted, spread to various parts of Malaysia, and contaminated Malaysian seafood. One O139 strain isolated from Malaysian seafood in 1998, VCF38, showed the profile D1, which formed a cluster with the profiles D2 and D3 (Fig. 2B). These three profiles were also exhibited by the clinical strains isolated in India and Bangladesh between 1994 and 1997 (Table 3). These results suggest that a subclone of O139 that diverged in the Bengal area around 1994 and 1995 may have been introduced into Malaysia independently from the above-described O139 clone.

One feature of O139 strains is resistance to a set of antibiotics; many O139 strains isolated in 1992 and 1993 in the Bengal area were reported to be resistant to sulfamethoxazole, trimethoprim, CM, and SM, and these resistances are encoded by the genes contained in a conjugative transposon termed the SXT element or SXT constin (2, 3, 14, 42). The antibiotic resistance of O139 strains, particularly that associated with the SXT element, can be transferred and partly modified by discrete genetic mechanisms, and thus it can be a good epidemiological marker for O139 strains (14, 30, 43). The antibiograms apparently associated with the SXT element of the O139 strains supported the epidemiological relationship of the O139 strains inferred from the PFGE profiles in our study. The strains belonging to the C1-C3-F-C2 cluster showed resistance to SM and SXT (Table 3). Five of the 20 strains belonging to this cluster were also resistant to CM. Therefore, the resistance to SM, SXT, and CM in the strains belonging to the C1-C3-F-C2 cluster is likely to be encoded on the SXT element. The E3-E1-E2 cluster was linked to the C1-C3-F-C2 cluster (Fig. 2B), and the antibiograms of the strains of the E3-E1-E2 cluster also seem to be associated with the SXT element, except for strain MO579 with the E2 profile. The resistance of strain MO579 to SM but not to SXT may have resulted from a deletion of the genes encoding SXT from the SXT element. The antibiogram of the strain VCF38, isolated from Malaysian seafood, was considerably different from those of the Bangladeshi and Indian strains within the D2-D3-D1 cluster (Table 3). This result does not appear to support the above-described hypothesis on independent introduction of VCF38 into Malaysia. One possible explanation is that VCF38 acquired resistance to SXT and SM in an SXT element-mediated manner recently in Malaysia.

The O1 and R strains isolated from Malaysian seafood showed profiles A1 (R variant) and A2 (O1 Ogawa). When these profiles were compared with the profiles exhibited by O1 and R strains isolated in areas other than Malaysia, the A1 profile was included in a cluster composed of A3, A1, A4, and A5, and this cluster was distantly related to other profiles, including A2 (Fig. 2C). The close relationship among the strains of the A3-A1-A4-A5 cluster was also supported by their similar antibiograms and the absence of the pTLC sequence (Table 3). These results suggest a strong epidemiological association among these strains, although the R strains isolated from Malaysian seafood did not express the O1 antigen gene. All of these strains were isolated in 1998 or 1999 and were resistant to SM, SXT, and TC. Some of these strains were also resistant to CM. The resistance of these strains to SM, SXT, and CM may be encoded on the SXT element. Not only O139 strains but also the El Tor O1 strains that emerged during the O139 outbreak in late 1992 and later were reported to exhibit resistance associated with the SXT element (3, 15, 42). An epidemic or outbreak due to O1 strains resistant to TC is recognized very infrequently. In the last decade, O1 Ogawa strains resistant to TC were isolated from epidemics in southern Thailand in 1997 and 1998 (22) and in Laos in 1998 (16). The strains isolated from an epidemic in southern Thailand in 1998 were included in this study, and they showed the profile A1 (Table 3). Other strains in the A3-A1-A4-A5 cluster were isolated from seafood and patients in Malaysia in 1998 or 1999. Because Laos, Thailand, and Malaysia are geographically connected and since southern Thailand and Malaysia in particular are very close, it is not surprising that O1 Ogawa or R strains belonging to the A3-A1-A4-A5 cluster were resistant to TC. We demonstrated in our previous study that the TC-resistant O1 Ogawa strains were unique to southern Thailand and proposed the possibility that the epidemic was due to strains that had persisted in the environment of southern Thailand (22). It is possible that the O1 Ogawa and R strains belonging to the A1 profile group persisted in the environment of the southern Thailand-Malaysia region and that these strains of this group were responsible for the southern Thailand epidemic and clinical cases in Malaysia in 1999. The absence of the pTLC gene sequence in toxigenic strains of V. cholerae has rarely been reported. Dalsgaard et al. (9) found that toxigenic O141 strains of V. cholerae isolated from clinical cases did not have the pTLC sequence. In addition, the pTLC sequence was absent in toxigenic strains of Vibrio mimicus isolated from clinical sources (6). The O1 Ogawa and R strains belonging to the A3-A1-A4-A5 cluster lacked the pTLC sequence and are unique among the O1 and R strains of V. cholerae. Although the data obtained in this study do not allow us to infer exactly when and from which area these strains were introduced into Malaysia, it is tempting to speculate that these strains of the A3-A1-A4-A5 cluster persisted for a fairly long period in the environment of the Thailand-Malaysia-Laos region.

One O1 Ogawa strain, VCF111, isolated from Malaysian seafood and from an international traveler originating in Thailand in 1992, showed the profile A2 (Table 3). This profile was closely related to profiles J and I, which were shown by the O1 Ogawa strains isolated from international travelers originating in various Southeast Asian countries between 1995 and 1997 (Fig. 2C; Table 3). The antibiograms of the strains of the A2-J-I cluster were considerably different from those of the strains belonging to the A3-A1-A4-A5 cluster (Table 3). These results suggest that VCF111 is not very closely related to the R strains and was introduced into Malaysia from a surrounding country, possibly between 1992 and 1997, through a channel different from that for the R strains.

Conclusion.
The toxigenic strains of V. cholerae isolated from Malaysian seafood in 1998 and 1999 varied in their phenotypic and genetic characteristics. However, the result of the PFGE-dendrogram analysis as well as their antibiograms and possession of the pTLC sequence suggested that these strains could be separated into four groups of possibly separate origins. O139 strains could be divided into two groups that appear to have been introduced from the Bengal area independently. R strains formed one group and belonged to a cluster unique to the Thailand-Malaysia-Laos region, and this group may have persisted in this area for a long period of time. An O1 Ogawa strain seems to have been introduced into Malaysia from a surrounding country independently from the other three groups.


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ACKNOWLEDGMENTS
 
We are grateful to Yohko Takeda for technical assistance. We thank those who kindly supplied V. cholerae strains (indicated in Materials and Methods) and S. M. Faruque for supplying plasmid pKK3535.

This research was supported, in part, by funds from the Ohyama Health Foundation and Yuasa International Foundation; the 4th Advanced Scientific Technology Research Fund from Toyota Motor Corporation; a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan; the U.S.-Japan Cooperative Medical Science Program, Cholera and Related Diarrheal Diseases, Japanese Panel; the COE program "Making Regions: Proto-Areas, Transformations and New Formations in Asia and Africa"; and the "Research for the Future" Program of The Japan Society for the Promotion of Science (JSPS-RFTF97L00706).


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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: nisibuti{at}cseas.kyoto-u.ac.jp. Back

{dagger} Present address: Graduate School of Health Science, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8519, Japan. Back

{ddagger} Present address: Kulliyyah of Pharmacy, International Islamic University Malaysia, Kuantan, Pahang Darul Makmur, Malaysia. Back


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Applied and Environmental Microbiology, April 2004, p. 1964-1972, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.1964-1972.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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