International Journal of Food Microbiology 118 (2007) 92 – 96 www.elsevier.com/locate/ijfoodmicro
Seaweeds as a reservoir for diverse Vibrio parahaemolyticus populations in Japan Zahid Hayat Mahmud a,b,⁎, Sucharit Basu Neogi a , Afework Kassu b , Takaomi Wada c , M. Sirajul Islam a , G. Balakrish Nair a , Fusao Ota b b
a Laboratory Sciences Division, International Centre for Diarrhoeal Disease Research, Bangladesh, Mohakhali, Dhaka 1212, Bangladesh Department of Preventive Environment and Nutrition, Graduate School of Health Biosciences Research, The University of Tokushima, Kuramoto-cho, Tokushima 770-8503, Japan c Hiroshima Environment and Health Association, Naka-ku, Hiroshima-shi 730-8631, Japan
Received 21 January 2007; received in revised form 3 May 2007; accepted 9 May 2007
Abstract Gastroenteritis caused by Vibrio parahaemolyticus has recently been associated with foods prepared with seaweeds, but little is known about the bacterium's abundance and diversity among seaweeds in coastal environment. Therefore, we determined its phenotypic and genotypic diversity in relation to its seasonal abundance in seawater and seaweed samples from three areas of Kii Channel, Japan during June 2003 to May 2004. Isolates were obtained by selective enrichment of samples and detection of V. parahaemolyticus by colony hybridization with a speciesspecific probe. A total of 128 isolates comprising 16 from each source in each season were characterized by serotyping and ribotyping. V. parahaemolyticus was more abundant in seaweeds (3762 isolates) than in water samples (2238 isolates). Twenty and 17 serotypes were found among the selected seaweed and seawater isolates, respectively. Cluster analysis revealed 19, 11, 7 and 9 ribotypes during summer, autumn, winter and spring, respectively. Seaweeds supported a diverse V. parahaemolyticus population throughout the year and thus seaweeds are a reservoir for the organism. However, V. parahaemolyticus occurrence had positive correlation with water temperature and its abundance in seaweeds was at least 50 times higher during summer than in winter. © 2007 Elsevier B.V. All rights reserved. Keywords: Vibrio parahaemolyticus; Seaweeds; Serotype; Ribotype; Seasonal abundance
1. Introduction Despite the efforts of the Centers for Disease Control and Prevention (CDC) there has been an estimated 126% increase in the incidence of Vibrio-associated infections in the US between 1996 and 2002 (CDC, 2003). Vibrio parahaemolyticus is a halophilic gram-negative bacterium that can cause gastroenteritis, wound infections, and septicemia (Nair et al., 1985). The organism is ubiquitous in brackish and marine waters and has been isolated from the coastal environment of most continents, including Asia (Barbieri et al., 1999; Islam et al., 2004; Mahmud ⁎ Corresponding author. Laboratory Sciences Division, International Centre for Diarrhoeal Disease Research, Bangladesh, Mohakhali, Dhaka 1212, Bangladesh. Tel.: +880 2 8860523 32x2405; fax: +880 2 8812529. E-mail address: [email protected]
(Z.H. Mahmud). 0168-1605/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2007.05.009
et al., 2006). It was first identified as a causative agent of human gastroenteritis in Japan in 1950 (Joseph et al., 1982). However, V. parahaemolyticus infections associated with contaminated seafoods have occurred throughout the world (Honda and Iida, 1993). Shellfish are the most commonly identified sources. Recently, seaweeds used as foods have been associated with outbreaks of V. parahaemolyticus infection (Vugia et al., 1997). Although the pandemic O3:K6 strain is responsible for many epidemics, there are reports of a wide variety of serovars of V. parahaemolyticus that can cause gastroenteritis (Ansaruzzaman et al., 2005; Mahmud et al., 2006). The recent discovery of horizontal transfer of toxigenic genes among related Vibrio populations (Waldor and Mekalanos, 1994) pointed out the importance of non-toxigenic strains. From the ecological point of view, bacterial genotypic diversity is important for understanding of the evolution, adaptation,
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competitive interactions, etc. of a species, whilst epidemiologists are mainly concerned with the survival, prevalence and spread of pathogenic genotypes. Several methods have been developed to reveal clonal relationships or for tracking of microbial source, including ribotyping, pulsed field gel electrophoresis (PFGE) and various polymerase chain reaction (PCR) based methods. Ribotyping takes advantage of the conserved nature of bacterial ribosomal genes, which are present in the genome in multiple copies. It is considered to be an effective genotyping method because of its excellent reproducibility, good discriminatory power, ease of interpretation, and ready adaptation to automation (Grimont and Grimont, 1986; Koblavi et al., 1990). Seaweed is widely used as a seafood in Japan. To understand the risks of V. parahaemolyticus infection from seaweed, a detailed knowledge of its abundance among seaweeds is essential. However, such information is lacking, and it is unclear whether seaweeds can act as a reservoir for the bacterium throughout the year or only during a certain period. Therefore, we conducted a study to understand the seasonal prevalence and diversity of V. parahaemolyticus among the coastal seaweed and seawater samples from various areas of the Kii Channel in Tokushima Island, Japan during June 2003 to May 2004. The occurrence and characteristics of toxigenic V. parahaemolyticus strains isolated from these samples have already been reported in a recent publication (Mahmud et al., 2006). However, the toxigenic strains represented only 0.3% of our total V. parahaemolyticus isolates. Concurrently, we also determined the abundance and diversity of overall V. parahaemolyticus population from the same sources for better understanding of the general feature of V. parahaemolyticus among coastal seaweeds that we report here. Representative isolates from seawater and seaweed samples of different seasons were serotyped and characterized by ribotyping to determine the extent of their temporal variability.
10 g portion of each sample was ground and mixed with 90 ml sterile normal saline. The seawater samples and the seaweed homogenates were enriched in alkaline peptone water according to the most probable number (MPN) culture (3-tube, 3-dilution) method (APHA, 1985) followed by streaking on plates of thiosulfate–citrate–bilesalt–sucrose (TCBS) agar (Franklin Lakes, NJ, USA) and CHROMagar Vibrio (CV) agar (CHROMagar, Paris, France) which were incubated for 18 h at 37 °C. Presumptive V. parahaemolyticus were transferred following patch inoculation technique onto Luria Bertani (LB) agar plates supplemented with 2% NaCl and were grown for 18 h at 37 °C for blot preparation. 2.2. Probe preparation and colony blot hybridization A synthetic oligonucleotide probe for the tlh gene, (5′-AAA GCG GAT TAT GCA GAA GCA CTG-3′, 904–927 bp of tlh) was used (McCarthy et al., 1999). Purified oligonucleotide was radioactively labelled with 32P to act as the probe, then colony hybridization was carried out at 40 °C followed by washing at
Table 1 V. parahaemolyticus abundance and serotypes in 24 samples of each of the water and seaweed obtained from Kii Channel, Japan in each of the four seasons Seasons Source
Numbers (MPN/ 100 ml)
No. of Serotypes a isolates
Median Mode Summer Seawater 418
N1100 and 1072 400
2. Materials and methods 2.1. Sample collection, processing and selective culture of V. parahaemolyticus Field samplings were carried out in three coastal areas (Komatsushima, Tokushima and Naruto) of the Kii Channel (Mahmud et al., 2006) at 45 days interval. In each area, samples were collected from 4 different sites according the criteria of the American Public Health Society (APHA, 1985). Subsurface seawaters from a depth of 0.5 m were collected at locations 50 m from the shore. Seaweed samples were collected during high tide from submerged seaweed beds using a sterile knife. For each sample, three subsamples were collected randomly and pooled. Samplings were conducted twice during each of summer, autumn, winter and spring to obtain 96 samples of each of seawater and seaweeds. Surface water temperature, pH and salinity were determined during sample collection using portable field meters (salinity meter, Sankyo Pharmacy Co. Ltd, Japan and pH meter, Horiba, Japan). Seaweeds were washed in sterile normal saline (0.9% w/v of NaCl) to remove bacteria in the surrounding seawater, then a
Autumn Seawater 265
21 and 3
21 and 3
KUT, untypable for the K antigen.
O1:K33, O2:K28, O1: KUT, O2:KUT, O3:KUT, O4:K34, O4:K42, O4: KUT. O6:KUT, O10: KUT, O11:K22, O11: KUT O1:KUT, O2:K28, O2: KUT, O3:K45, O3:K57, O3:K58, O3:KUT, O4: K29, O4:KUT, O5:KUT, O8:K41, O10:K52, O11: KUT O1:K33, O2:K28, O3: K45, O3:KUT, O4:K4, O11:K22, O4:K29, O4: KUT, O5:KUT, O6:KUT, O10:KUT, O11:KUT O1:K33, O1:KUT, O3: K57, O3:K45, O3:KUT, O4:K4, O4:K12, O4: KUT, O5:KUT, O7:KUT, O 1 0 : K 2 9 , O 1 0 : K U T, O11:K22, O11:KUT O3:KUT, O4:K29, O4: KUT, O10:KUT O3:KUT, O4:KUT, O10: KUT, O11:K49 O4:K29, O3:KUT, O10: KUT, O11:K49, O11: KUT O3:K29, O3:KUT, O4: K29, O10:KUT, O11: K49, O11:KUT
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60 °C and detection as described previously (Mahmud et al., 2006). 2.3. Selection of isolates and serotyping In each of the summer, autumn, winter and spring 32 confirmed V. parahaemolyticus isolates were randomly selected for serotyping and ribotyping. An equal number of isolates was selected from seawater and seaweed sources. The selected isolates were serotyped using V. parahaemolyticus antisera (Denka Seiken, Tokyo, Japan) following the manufacturer's instructions. 2.4. Preparation of 16S rRNA gene probe and ribotyping Chromosomal DNAs of the 128 selected isolates were extracted using the method of Murray and Thompson (1980) with modifications described by Chowdhury et al. (2000). A universal 16S rRNA gene-specific PCR was conducted using 5′-GGA TTA GAT ACC CTG GTA GTC C-3′ (forward) and 5′TCG TTG CGG GAC TTA ACC CAA C-3′ (reverse) primers (Talukder et al., 2002) followed by gel electrophoresis of the product and purification using GenElute™ Minus EtBr Spin Columns (Sigma, St. Louis, USA). The purified amplicon was labelled using the digoxigenin (DIG)-dUTP DNA labelling Kit (Boehringer Mannheim, Mannheim, Germany). Initially, several restriction enzymes (HindIII, EcoRI and BglI) were used to digest the chromosomal DNA of some representative isolates. HindIII, (restriction site 5′-A↓AGCTT-3′) which gave the greatest differentiation, was then finally employed for all 128 isolates. Ribotyping was performed using the 16S rDNA probe following the procedures described elsewhere (Mahmud et al., 2006). The size of each band in the gels was determined and the data were coded as 0 (negative) or 1 (positive). Relatedness among the ribotypes was examined by hierarchical cluster
analysis and dendograms were produced using SPSS software (version 10.0, SPSS Inc., Chicago, IL, USA). The patterns with dissimilarity values larger than 10 were arbitrarily grouped as different types. 3. Results 3.1. Isolation and abundance of V. parahaemolyticus V. parahaemolyticus was isolated from all the samples of water and seaweeds during summer and autumn when water temperatures were between 20 and 29 °C. Its numbers were N150 MPN/100 ml in all the samples, and 53 of the 96 samples yielded numbers N1100 MPN/100 ml. In contrast, during winter and spring when water temperatures were between 10 and 18 °C, the numbers were b50 MPN/100 ml in 66 out of 96 samples, and between 51 and 250 MPN/100 ml in 8 samples, with no V. parahaemolyticus being detected at the level of b3 MPN/100 ml in 22 samples. The abundances in samples from different areas of the Kii Channel were not significantly different (P N 0.05). In the winter the bacterium was recovered from 54% of water and 83% of seaweed samples. The isolation rate increased during spring to 75% for water and 96% for seaweed samples. The abundances of V. parahaemolyticus including the median and mode of the MPN values per 100 ml sample in each season for each source is shown in Table 1. However, there were no conspicuous seasonal deviations in water pH (ranged between 7.8 and 8.7) in the three coastal areas. The salinity also did not show any seasonal difference (ranged between 21.7 and 29.4 practical salinity unit, PSU) except lower value during spring (ranged between 13.6 and 20 PSU). A total of 5101 isolates were obtained during summer and autumn, but only 899 isolates were isolated during winter and spring (Table 1). The isolation rate for samples of seaweeds of the species Ulva, Fucus, Laminaria, Porphyra, etc. was higher than that for seawater samples.
Fig. 1. Dendrogram showing the ribotypes of the selected V. parahaemolyticus isolates based on the squared Euclidean distance and average linkage clustering method. Isolates with dissimilarity values N10 were arbitrarily grouped into different types (A to S). Abbreviated Sum, Aut, Win and Spr represent summer, autumn, winter and spring, respectively, beneath which the total number of ribotypes in respective seasons (within brackets) are shown. Water and seaweed sources are abbreviated as ‘Wat’ and ‘SW’ respectively and the numbers in adjacent rows represent prevalence of each ribotype out of 16 representatives for each source in each season.
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3.2. Serotyping Among the 128 selected isolates, 24 serotypes could be differentiated of which 17 and 20 were found in isolates from water and seaweed, respectively, 3 being common (O3:KUT, O10:KUT and O4:K29) in all seasons (Table 1). Common serotypes in both types of sample were: during summer, types O1:KUT, O2:KUT, O3:KUT, O4:KUT, O11:KUT, and O2:K28; during autumn, types O3:KUT, O5:KUT, O10:KUT, O11:KUT, O4:K4, O11:K22, O1:K33, and O3:K45; during winter, types O3:KUT, O4:KUT, and O10:KUT; and during spring, types O3: KUT, O10:KUT, O11:KUT, O4:K29, and O11:K49. 3.3. Ribotyping A ribotype was considered ‘unique’ when the band pattern of one strain differed by one or more bands from those of all other strains. The numbers of unique pattern were 30, 19, 9 and 20 for isolates obtained in summer, autumn, winter and spring, respectively. Of the ribotypes identified by cluster analysis among isolates obtained in summer, autumn, winter and spring (Fig. 1) 4, 4, 3 and 5, respectively, were common. One type (A) was predominant among isolates obtained during the winter. 4. Discussion Cultivable V. parahaemolyticus in samples from the Kii Channel showed high seasonal fluctuations, in accordance with previous studies of other coasts (Cook et al., 2002; DePaola et al., 2003). The seasonal variations in water pH were trivial while the relatively low water salinity during spring could be linked to the increased flow of freshwater into the channel during this season. The higher abundance of V. parahaemolyticus during summer and autumn than during winter clearly indicates the bacterium's preferential growth at higher temperature. In spring, with the rise in temperature, the frequency of V. parahaemolyticus also increased. During winter, this bacterium can survive in a viable but non-culturable (VBNC) state that allows it to withstand adverse conditions (Wong and Wang, 2004). During the more favourable summer conditions, some VBNC bacteria may again become culturable (Roszak and Colwell, 1987). Populations of V. parahaemolyticus may also be regulated by the lysis due to the presence of Bdellovibrio at temperatures as low as 5 °C, but not at higher temperatures (Miyamoto and Kuroda, 1975). V. parahaemolyticus growth during summer increases the probability of toxigenic strains being present in seaweeds as well as other seafoods (Croci et al., 2001; Pillot et al., 2005). The persistence of some serotypes throughout the year might be linked to their greater resistance power and/or their high numbers. The scenario of greater resistance is also reflected in the predomination of one specific ribotype among the selected winter isolates. However, although different serotypes were common in both water and seaweed samples, some were only abundant in seaweeds indicating affinity of some strains to seaweeds. The higher rate of isolation of V. parahaemolyticus from seaweed than from water indicates that seaweed can act as
a reservoir for the bacterium. It is possible that when conditions are adverse, seaweeds can provide better conditions than seawater for survival of the organism. The selected 32 isolates for each season had equal representation from both seawater and seaweed sources, and represented 20% of the bacterium's lowest isolation number during winter. Identification of 24 serotypes out of 128 isolates implies that V. parahaemolyticus had high phenotypic diversity along the Kii Channel. We have previously reported the existence of toxigenic V. parahaemolyticus including the pandemic O3:K6 strains in this channel (Mahmud et al., 2006). The fractions of toxigenic serotypes among coastal V. parahaemolyticus populations is generally very low (Pillot et al., 2005). The greater number of serotypes obtained from seaweeds than from seawater may be related to higher abundance of V. parahaemolyticus in seaweed. Similarly, the higher phenotypic and genetic diversity of this bacterium during warmer months correlates with its greater abundance in those seasons. The differentiation into 19 ribotypes and 30 unique patterns among 32 isolates clearly reveals the bacterium's high genetic diversity during summer. The cluster analysis also suggests a wide spreading of the ribotypes in both water columns and seaweeds indicating high genetic heterogeneity among V. parahaemolyticus. There are also reports of high genetic diversity among Vibrio species from other coastal locations (Thompson et al., 2005). Interestingly, seaweed isolates could be differentiated into higher serotypes but lower ribotypes than seawater isolates. Higher serotypes indicate the bacterium's diverse external responses involving its outer body-covering during association with seaweeds. Persistence of large cultivable V. parahaemolyticus population in seaweed samples despite washing with normal saline (as adopted in our methodology) indicates its intimate association which can be facilitated by the bacterium's invasive capability to penetrate epithelial membrane (Akeda et al., 1997). Galactans of agars and carrageenans derived from seaweeds are common foods for many bacterial species (Michel et al., 2006). The major constituents of the seaweed extracts include L-fucose, D-mannose, D-galactose, and D-glucuronic acid which can be utilized by V. parahaemolyticus (Sarker et al., 1994; Sakai et al., 2002). A recent study reveals that the asexual zoospore, produced in the seaweed leaves during spring and early summer, can be interacted by the bacterial N-acylhomoserine lactone (AHL) quorum sensing signal molecules influencing bacterial attachment (including Vibrio species) on the seaweed surfaces (Tait et al., 2005). The majority of V. parahaemolyticus isolates in the Kii Channel were untypable for the K antigen, a phenomenon also observed in previous studies (Nair et al., 1985). Because of high variability in the K antigen, 71 antisera have been developed so far in comparison to 11 antisera for the O antigen (Iguchi et al., 1995). Due to adaptive environmental responses, the recombination rate via mutation, horizontal gene transfer, etc. in the gene for the capsular K antigen is probably higher than that of the somatic O antigen. However, the large number of serotypes and ribotypes detected among the few representative isolates clearly indicates high phenotypic and genetic diversity of V. parahaemolyticus along the Kii Channel.
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Among the Japanese population the contribution of the seaweed was the highest among the dietary fiber intake followed by vegetables, pulses and fruits (Fukuda et al., 2007). Our results imply that during risk analysis of V. parahaemolyticus borne illness a detailed knowledge of its occurrence and diversity in association with seaweeds and in adjacent waters should be taken into account. We can conclude that coastal seaweeds can act as a reservoir for diverse V. parahaemolyticus populations and so are likely to be more unsafe during summer than winter. Therefore, proper hygienic practice should be maintained during the postharvest handling of seaweeds and adequate cooking is necessary before its consumption. Acknowledgements This study was supported by the Ministry of Education, Government of Japan and the Yakult Ltd., Japan. References Akeda, Y., Nagayama, K., Yamamoto, K., Honda, T., 1997. Invasive phenotype of Vibrio parahaemolyticus. Journal of Infectious Diseases 176, 822–824. Ansaruzzaman, M., Lucas, M., Deen, J.L., Bhuiyan, N.A., Wang, X., Safa, A., Sultana, M., Chowdhury, A., Nair, G.B., Sack, D.A., Seidlin, L., Puri, M., 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. Journal of Clinical Microbiology 43, 2559–2562. APHA, 1985. Laboratory Procedures for the Examination of Seawater and Shellfish, 5th ed. American Public Health Association, Washington, DC. Barbieri, E., Falzano, L., Fiorentini, C., Pianetti, A., Baffone, W., Fabbri, A., Matarrese, P., Casiere, A., Katouli, M., Kuhn, I., Mollby, R., Bruscolini, F., Donelli, G., 1999. Occurrence, diversity, and pathogenicity of halophilic Vibrio spp. and non-O1 Vibrio cholerae from estuarine waters along the Italian Adriatic coast. Applied and Environmental Microbiology 65, 2748–2753. CDC. Centers for Disease Control and Prevention, 2003. Preliminary Food Net Data on the Incidence of Foodborne Illnesses-Selected Sites, United States, 2002. Morbidity and Mortality Weekly Report, vol. 52, pp. 340–343. Chowdhury, N.R., Chakraborty, S., Ramamurthy, T., Nishibuchi, M., Yamasaki, S., Takeda, Y., Nair, G.B., 2000. Molecular evidence of clonal Vibrio parahaemolyticus pandemic strains. Emerging Infectious Diseases 6, 1–8. Cook, D.W., Bowers, J.C., DePaola, A., 2002. Density of total and pathogenic (tdh+) Vibrio parahaemolyticus in Atlantic and Gulf coast molluscan shellfish at harvest. Journal of Food Protection 65, 1873–1880. Croci, L., Serratore, P., Cozzi, L., Stacchini, A., Milandri, S., Suffredini, E., Toti, L., 2001. Detection of Vibrionaceae in mussels and in their seawater growing area. Letters in Applied Microbiology 32, 57–61. DePaola, A., Nordstrom, J.L., Bowers, J.C., Wells, J.G., Cook, D.W., 2003. Seasonal abundance of total and pathogenic V. parahaemolyticus in Alabama oysters. Applied and Environmental Microbiology 69, 1521–1526. Fukuda, S., Saito, H., Nakaji, S., Yamada, M., Ebine, N., Tsushima, E., Oka, E., Kumeta, K., Tsukamoto, T., Tokunaga, S., 2007. Pattern of dietary fiber intake among the Japanese general population. European Journal of Clinical Nutrition 61, 99–103. Grimont, F., Grimont, P.A.D., 1986. Ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools. Annales de l'Institut Pasteur. Microbiology 137B, 165–175. Honda, T., Iida, T., 1993. The pathogenicity of Vibrio parahaemolyticus and the role of thermostable direct haemolysin and related haemolysins. Reviews in Medical Microbiology 4, 106–113.
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