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Timestamp: 2019-04-19 17:06:13+00:00

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In spite of the potential risk involved with contamination of seafood with Vibrio parahaemolyticus, there is a lack of information on the occurrence of pathogenic V. parahaemolyticus in Europe. This organism was isolated in 1999 from a large outbreak (64 cases admitted to a single hospital) associated with raw oyster consumption in Galicia, Spain, one of the most important regions in shellfish production worldwide. Two V. parahaemolyticus isolates from the 1999 Galicia outbreak, three additional clinical isolates obtained in the same period from hospitals in Spain, two reference strains from clinical sources, and five Spanish environmental isolates were examined. Seventeen isolates belonging to the pandemic clone isolated in Asia and North America were included in the study for comparison. All isolates were characterized by serotyping, PCR for virulence-related genes, pulsed-field gel electrophoresis (PFGE), and plasmid analysis. Four of the five clinical isolates from hospitals in Spain belonged to serotype O4:K11; the remaining isolate was O4:K untypeable (KUT). All five isolates were positive for V. parahaemolyticus toxR and tlh (species-specific genes) and tdh and negative for trh and group-specific PCR (a PCR method for detection of the pandemic clone). PFGE analysis with NotI and SfiI discriminated the European isolates in two closely related PFGE types included in a homogeneous cluster, clearly differentiated from the Asian and North American isolates. The five environmental isolates belonged to serotypes O2:K28, O2:KUT, O3:K53, O4:KUT, and O8:K22 and were negative for all virulence genes. The five isolates were discriminated into five different PFGE types unrelated to any other isolate included in the study. While the virulence characteristics (tdh positive, trh negative) of the Spanish clinical isolates matched those of the O3:K6 clone from Asia and North America, they were clearly excluded from this clone by group-specific PCR, PFGE, and serotyping. The results of this study suggest that a unique and specific clone could be related to the V. parahaemolyticus infections in Europe.
Vibrio parahaemolyticus is a halophilic bacterium that occurs naturally in estuarine environments worldwide (15). This organism is the leading cause of seafood-associated bacterial gastroenteritis in the United States (23), and it is one of most important food-borne pathogens in Asia, causing approximately half of the food poisoning outbreaks in Taiwan, Japan, and Southeast Asian countries (12). Infections caused by V. parahaemolyticus have increased globally in the last 5 years (8). The main vehicle of infection is raw or partially cooked seafood (12). In Europe, disease caused by V. parahaemolyticus is rarely reported (21). The most important outbreak occurred in France and was related to the consumption of seafood imported from Asia (19). In Spain, eight cases of acute gastroenteritis caused by V. parahaemolyticus associated with fish or shellfish ingestion were reported in 1989 (24). Another outbreak associated with live oyster consumption was detected in 1999 (21).
The major virulence factors of V. parahaemolyticus are the thermostable direct hemolysin and the thermostable direct hemolysin-related hemolysin, encoded by the tdh and trh genes, respectively. Thermostable direct hemolysin causes β-hemolysis of human erythrocytes in agar medium, a reaction known as the Kanagawa phenomenon (11). The association between possession of the tdh gene by a strain and its ability to cause gastroenteritis has been well established (25), and the presence of the tdh and trh genes is routinely used to determine the pathogenicity of V. parahaemolyticus strains (14). A serotyping scheme for V. parahaemolyticus was developed in Japan, based on the use of specific antisera for differentiating 11 O groups and 71 K types (32). In spite of this diversity, relatively few serotypes are commonly associated with human infections. A single serotype (O4:K12) was the most prevalent among clinical V. parahaemolyticus isolates from the U.S. Pacific Coast from 1979 to 1995 (27).
Recently, three serotypes, O3:K6, O4:K68, and O1:K untypeable (KUT), were responsible for a pandemic of V. parahaemolyticus infection (8). The strains belonging to the so-called new O3:K6 clone were first detected in India in 1996 and quickly spread throughout Asia (22, 28) and the United States (8). In 1998, the O3:K6 serotype caused a large V. parahaemolyticus outbreak in the United States associated with oyster consumption (9). Recently isolated serotypes O4:K68, O1:KUT, O1:K25, and O1:K41 were related to the new O3:K6 clone by molecular techniques (18, 22), suggesting that these strains may have diverged from the new O3:K6 clone by alteration of the genes associated with the O and K antigens (22).
In spite of the great number of studies on the occurrence of pathogenic V. parahaemolyticus in marine environments and in human infections worldwide, very little concerning these subjects exists for Europe. V. parahaemolyticus has frequently been found in coastal waters and seafood in Europe (2). However, to date these investigations have only sought to detect or enumerate V. parahaemolyticus, without any further characterization. V. parahaemolyticus was isolated in 1999 from a large outbreak in Vigo, Spain (Galicia region of Spain), associated with raw oyster consumption (21). This was a period when a newly emerged O3:K6 clone of V. parahaemolyticus was making a pandemic spread from Asia to North America. The outbreak in Vigo was similar to a 1998 outbreak in Galveston Bay, Texas (9). Both were extraordinarily large outbreaks linked to shellfish harvested near important international seaports. These similarities suggested that the O3:K6 clone may have spread to Spain.
To examine this hypothesis, V. parahaemolyticus isolates from the 1999 Galicia outbreak, isolates from hospitals in Spain, and strain collections in Europe were characterized and compared to isolates belonging to the pandemic clone isolated in Asia and North America.
Bacterial isolates.A total of 29 V. parahaemolyticus isolates from Europe, Asia, and the United States were included in the study (Table 1). The European panel included five clinical isolates from Spain and two reference strains from two clinical cases in the United Kingdom. Two isolates originated from an outbreak associated with the consumption of raw oysters in Galicia in 1999 (21). Two additional isolates were obtained from the Centro Nacional de Microbiología (Spain) and originated from two cases of gastroenteritis in Madrid and Tarragona in 1998 and 1999. The remaining isolate was obtained from a patient with gastroenteritis in Madrid in 2002. The reference strains of V. parahaemolyticus isolated in the United Kingdom were strain NCTC 11344, isolated from a patient with enteritis (10), and strain ATCC 43996 isolated from cockles involved with fatal food poisoning in Cornwall (United Kingdom) (4). Additionally, 13 clinical isolates from Asia (one from Japan, one from Laos, one from India, three from Bangladesh, one from Taiwan, one from Korea, and five from Thailand) (18, 22), one clinical isolate from the United States (22), one isolate from an international traveler obtained in Thailand (26), and one environmental isolate from seawater in Japan (17) were included in the study. The reference strain ATCC 17802, isolated from a patient with food poisoning in Japan, was also investigated.
Five isolates from environmental sources from mollusk-harvesting areas located in the northwest of Spain were included for comparison in the present study. These isolates were obtained with the American Public Health Association three-tube most-probable-number method (14) from samples of marine sediments and shellfish (one of sediment, two of mussels, one of oysters, and one of cockles).
Serotyping.Lipopolysaccharide (O) and capsular (K) serotypes were determined with specific commercial antisera (Denka; Seiken Corp., Tokyo, Japan) as previously described (31).
PCR confirmation of isolates and detection of hemolysin genes.Presumptive identification of the isolates was confirmed by the presence of the V. parahaemolyticus species-specific genes toxR and tlh. The presence of the V. parahaemolyticus toxR gene was investigated by PCR as described previously (16). Additionally, the presence of tlh, tdh, and trh was determined by multiplex PCR according to the procedure described by Bej et al. (5).
Group-specific PCR.A PCR method to specifically detect the toxRS sequence of the new O3:K6 clone in the V. parahaemolyticus isolates was performed as described previously (22).
PFGE.Pulsed-field gel electrophoresis (PFGE) was performed according to the One-Day (24 to 28 h) Standardized Laboratory Protocol for Molecular Subtyping of Non-typhoidal Salmonella by PFGE (Pulse-Net; Centers for Disease Control, Atlanta, Ga.) (6). A single colony of each isolate was streaked onto tryptic soy agar (TSA) supplemented with 2% NaCl and incubated overnight at 37°C. With a cotton swab, a portion of the growth on the agar plate was transferred to 2 ml of cell suspension buffer (100 mM Tris, 100 mM EDTA, pH 8.0), and the cell concentration was adjusted to 0.48 to 0.52 in a Dade Microscan turbidity meter (Dade Behring). Immediately, 400 μl of adjusted cell suspension was transferred to 1.5-ml microcentrifuge tubes containing 20 μl of proteinase K (20 mg/ml) and subsequently mixed with 400 μl of melted 1% SeaKem Gold (Cambrex, East Rutherford, N.J.)-1% sodium dodecyl sulfate-agarose prepared with TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0), and pipetted into disposable plug molds. Three plugs were transferred to 50-ml polypropylene screw-tubes with 5 ml of cell lysis buffer (50 mM Tris, 50 mM EDTA, pH 8.0, 1% Sarcosyl) and 25 μl of proteinase K (20 mg/ml) and incubated at 54°C in a shaker water bath for 2 h with agitation.
Thereafter, the plugs were washed twice with 15 ml of sterile water and three more times with TE buffer at 50°C for 15 min. Chromosomal DNA was digested with 30 U of NotI (Promega, Southampton, United Kingdom) at 37°C for 4 h or with 20 U of SfiI (Promega) at 50°C for 4 h. PFGE was performed on a CHEF DRIII system (Bio-Rad, Hercules, Calif.) in 0.5× Tris-Borate-EDTA (TBE) extended-range buffer (Bio-Rad) with recirculation at 14°C. DNA macrorestriction fragments were resolved on 1% SeaKem Gold agarose (Cambrex) in 0.5× TBE buffer. DNA from Salmonella Braenderup H9812 restricted with 50 U of XbaI (Promega) at 37°C for 2 h was used as a size marker. Pulse times were ramped from 2 to 40 s during an 18-h run at 6.0 V/cm. Macrorestriction patterns were compared with the use of BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium). Different profiles were designated with the letter N (NotI types) or S (SfiI types) in accordance with the restriction patterns. A difference of at least one restriction fragment in the patterns was considered the criterion for discriminating between clones.
Plasmid analysis.Plasmid DNA was isolated by the alkaline lysis method as described previously (13). Samples were analyzed by electrophoresis in 1× Tris-borate-EDTA buffer at 150 V for 4.5 h on 0.8% agarose gels with recirculation at 20°C. Plasmid-containing Escherichia coli strain 39R861 and a supercoiled DNA ladder (Gibco-BRL, Paisley, United Kingdom) were used as size markers. Plasmids were compared by the use of BioNumerics software. The molecular weights of the plasmids were calculated by comparison with external markers, and images were normalized accordingly.
Table 1 describes the source, serotype, virulence genes, and genetic characteristics of the isolates investigated. All V. parahaemolyticus clinical isolates obtained from hospitals located in different parts of Spain belonged to serotype O4:K11 with the exception of the isolate obtained in 2002, which was O4:KUT. All five isolates were toxR, tlh, and tdh positive and trh and group-specific PCR negative. The United Kingdom reference strains belonged to serotypes O3:K7 and O3:K4 and presented virulence attributes identical to those of the Spanish clinical isolates. Environmental isolates from the northwest of Spain belonged to serotypes O2:K28, O2:KUT, O3:K53, O4:KUT, and O8:K22 and were negative for the virulence genes.
PFGE analysis of NotI and SfiI restriction patterns revealed a similar discriminatory power. Each of the methods allowed differentiations of the 29 isolates into 18 restriction types.
SfiI-PFGE grouped the Spanish isolates from clinical sources in a unique cluster (Fig. 1) with two SfiI types, S12 and S13. Both types presented similar restriction patterns differing only in the position of one band (similarity, >94%). The S12 type included two isolates from Galicia and Tarragona, while the S13 type was found in the isolates from Galicia and Ma-drid. The United Kingdom reference strain NTCC 11344 isolated from a clinical case presented a closely related type (S14) and was also included in the same cluster (similarity > 90%). Asian and American clinical isolates were mostly grouped in a unique cluster (S01 to S06, similarity > 85%) that included nine of the ten O3:K6 isolates investigated and five isolates belonging to serotypes O1:KUT, O1:K25, O4:K68 (two isolates), and O4:KUT. The O1:KUT and O3:K6 isolates from Bangladesh showed identical restriction patterns (S02). The O1:K25 isolate from Thailand, together with three O3:K6 isolates from Thailand, the United States, and Taiwan, were of type S03, while the three O4 isolates were differentiated from the rest of the O3:K6 isolates and belonged to types S04 and S05. The O3:K6 isolate from 1985 (type S09) was separated from the recent O3:K6 isolates. Environmental isolates and the reference strains ATCC 43996 and ATCC 17802 showed a variety of distinct and unrelated restriction patterns.
Representative SfiI restriction patterns of the 29 V. parahaemolyticus isolates included in the study. The dendrogram was generated by Bionumeric software, showing the relationships between isolates. The numbers at the top of the figure indicate molecular sizes in kilobase pairs.
Clustering of NotI restriction patterns was similar to those revealed by SfiI. NotI-PFGE distinguished 18 types (Fig. 2). The five clinical isolates from Spain were discriminated in a single cluster (similarity > 97%) with NotI types N04 and N05, including two isolates from Galicia and Tarragona and three isolates from Galicia and Madrid, respectively. Reference strain NTCC 11344, of type N06, showed a close relationship with the Spanish clinical isolates (similarity > 92%) and was included in the same cluster. Clinical isolates from Asia and North America were grouped in a unique cluster with six NotI types with mostly one-band differences among them. The O3:K6 strain from 1985 was clearly segregated from the more recent O3:K6 isolates. Environmental isolates and the reference strains ATCC 43996 and ATCC 17802 showed a variety of distinct restriction patterns.
Dendrogram generated by Bionumeric software, showing the relationship of fingerprints (NotI PFGE, or N types) for 29 V. parahaemolyticus isolates. The numbers at the top of the figure indicate molecular sizes in kilobase pairs.
Due to the high degree of clonality within serotypes, plasmid analysis was used to try to increase the intraserotype discriminatory power. Of the 29 isolates, 15 were free of plasmids, while the rest of the isolates contained from one to six plasmids (Table 1). The two Spanish clinical isolates from Vigo and Tarragona with identical NotI and SfiI restriction pattern showed the same plasmid pattern, with a unique plasmid of approximately 3.5 kb. The other three clinical isolates from Spain showed different and unrelated plasmid patterns.
Serotype O4:K11 is the most prevalent among clinical V. parahaemolyticus isolates from Spain. This serotype was detected in hospitals on the Mediterranean and the Atlantic coasts of Spain during a period of more than 2 years. PFGE analysis of these isolates showed a high degree of clonality with the two enzymes used. However, two different types were differentiated within the same serotype. Two isolates from the same outbreak isolated in the same hospital showed slightly different restriction patterns and plasmid profiles, and these were matched to PFGE and plasmid profiles found on epidemiologically unrelated isolates originating from hospitals located thousands of kilometers away. The five isolates were clearly discriminated from the other O4 serotypes included in the study from Asia and only showed a close relationship with the European strain NTCC 11344 serotype O3:K7, isolated from a patient with enteritis in the United Kingdom. The second United Kingdom isolate (ATCC 43996), obtained from a food-borne outbreak associated with cockle consumption, presented a distinct PFGE profile closer to that of the environmental than the clinical isolates. The results of group-specific PCR and PFGE analysis distinguished the Spanish clinical isolates from the isolates belonging to the pandemic clone isolated in Asia and North America, discarding the hypothesis of the possible pandemic origin of the Spanish isolates.
Isolates from Asia and the United States belonging to serotypes O3:K6, O1:KUT, and O4:K68 showed a high degree of clonality; this is consistent with previous studies (3, 7, 8, 29, 33) and supports the hypothesis of a related origin of these three new serotypes (22). However, a combination of NotI and SfiI PFGE showed slight differences between the restriction patterns from isolates belonging to the different serotypes. Our PFGE results demonstrate a similar level of clonality among the five recent clinical isolates from Spain and the 1978 clinical isolate from the United Kingdom, supporting the hypothesis of the related origin of the European clone with similarly diverse serotypes dating back over 30 years.
Traditionally, molecular typing was employed to distinguish among isolates with identical phenotypic characteristics (mostly serotype), providing information to discriminate isolates beyond the phenotypic level (20). However, clustering analysis of V. parahaemolyticus strains showed a clear disagreement between O:K serotypes and PFGE profiles. Different serotypes from the same area were included in the same cluster, and identical or close serotypes from distant regions or different sources showed a well-differentiated PFGE profile. Serotyping, as a unique epidemiological marker, presented limited application for epidemiological purposes, and additional molecular characterization should be included to establish an accurate relationship between isolates from diverse geographical regions.
Studies to investigate the presence or the number of V. parahaemolyticus in coastal waters have been done frequently in Europe (2). However, there is very little information regarding their virulence characteristics. A recent review of epidemiological information showed that outbreaks of V. parahaemolyticus associated with the consumption of shellfish have not been reported in Europe for the last 30 years (30). Based on this, the risk of infection caused by V. parahaemolyticus in Europe was considered very low (2). However, the real risk of V. parahaemolyticus infection may be underestimated due to deficiencies in the monitoring and investigation of food-borne illness (21). V. parahaemolyticus is not included in the European Network for Epidemiologic Surveillance and Control of Communicable Diseases, and it is also excluded from the microbiological surveillance system for infectious gastroenteritis (2). Monitoring for this pathogen is usually restricted to specific hospitals located in coastal areas with a tradition of shellfish consumption during summer.
In the summer of 1999, an outbreak with three case clusters associated with oyster consumption was detected in Galicia (northwest Spain) and affected 64 persons (21). The analysis of stool samples from the patients revealed the presence of V. parahaemolyticus in all cases. The detection of this outbreak was only possible due to the temporary surveillance for Vibrio spp. introduced in a particular hospital during the summer months. The detection of this outbreak instigated an investigation into the number of cases of V. parahaemolyticus infections registered in hospitals in this region. Between 1997 and 2003, the Microbiological Surveillance System of Galicia recorded 84 cases of V. parahaemolyticus infection. With the exception of two isolates from the 1999 outbreak, no other isolate was stored in the culture collections. The number of registered cases was not regular for these 7 years, with most years showing no cases, possibly due to deficiencies in identification of the etiological agents associated with the infections. In addition, none of these cases was included in the Spanish Epidemiological Bulletin or in any other published document. This lack of information contributes to the underestimation of the real incidence of this pathogen. Control of V. parahaemolyticus is not incorporated in the applicable microbiological requirements for live mollusks included in European Directive 91/492/EEC (1). In addition, this organism is being excluded from the new European regulation on microbiological criteria for foodstuffs (SANCO/4198/2001, revision 6, Draft of Commission Regulation on Microbial Criteria for Foodstuffs, European Community) currently under discussion.
At present, we have limited data concerning the ecology of V. parahaemolyticus in Europe and its real incidence in human illness. The Spanish isolates included in this study represent the strongest link to date associating seafood produced in Europe to human V. parahaemolyticus infections (21). While the virulence characteristics (tdh positive, trh negative) of the Spanish clinical isolates matched those of the O3:K6 clone from Asia and North America, they were clearly excluded from this clone by group-specific PCR, PFGE, and serotyping results. All of the isolates in this study were confirmed as V. parahaemolyticus by PCR to detect tlh and toxR, supporting the equivalency of molecular identification methods that have been used primarily in the United States and Asia, respectively. Future environmental studies and seafood surveys in Europe should emphasize the identification and enumeration of virulent V. parahaemolyticus, while serological and molecular subtyping techniques should be reserved primarily for those possessing virulence genes or clinical isolates.
We thank Aurora Echeita (Centro Nacional de Microbiología, Instituto de Salud Carlos III) for supplying the V. parahaemolyticus isolates used in this work and Alberto Malvar Pintos (SERGAS, Consellería de Sanidade, Xunta de Galicia) for providing the epidemiological information.
Returned for modification 6 June 2004.
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