Environmental Microbiology Reports (2010) 2(1), 58–66 doi:10.1111/j.1758-2229.2009.00064.

Prevalence and genetic diversity of pathogenic

populations of Vibrio parahaemolyticus in coastal
waters of Galicia, Spain emi4_064 58..66

Alba Rodriguez-Castro,1 Juan Ansede-Bermejo,1 Introduction

Veronica Blanco-Abad,1 Jose Varela-Pet,2
Oscar Garcia-Martin1 and Jaime Martinez-Urtaza1* Vibrio parahaemolyticus is the leading cause of bacterial
1
Instituto de Acuicultura and 2Instituto de gastroenteritis associated with the consumption of
Investigaciones Tecnológicas, Universidad de Santiago seafood in the USA (Mead et al., 1999), and one of the
de Compostela, Campus Universitario Sur, 15782 major food-borne pathogens in Asia (Joseph et al., 1982).
Santiago de Compostela, Spain. In recent years, infections caused by this organism have
emerged in areas where it had previously been rarely
detected, such as Chile (Gonzalez-Escalona et al., 2005),
Summary
Alaska (McLaughlin et al., 2005) and Spain (Lozano-León
The natural reservoirs and biological characteristics et al., 2003; Martinez-Urtaza et al., 2005).
of pathogenic populations of Vibrio parahaemolyticus Despite the growing importance of V. parahaemolyticus
in marine habitats remain unclear due to difficulties in from an epidemiological perspective, limited advances
obtaining pathogenic strains from the environment. have been made in understanding the ecology of patho-
The distribution and characteristics of pathogenic V. genic populations of this organism in the environment.
parahaemolyticus were investigated over 1 year in Almost all strains isolated from human infections
three coastal environments in Galicia (Spain), includ- characteristically bear the tdh or/and trh haemolysin
ing areas of the major international ports in the genes, which are considered universal virulence markers
region. Vibrio parahaemolyticus was present in 35.3% (Kaysner and DePaola, 2001). However, strains with viru-
of the samples analysed, and 535 strains were iso- lence traits are rarely found in the marine environment,
lated over the period of study. Virulence genes were even in regions where outbreaks caused by V. para-
detected in 94 strains with diverse genetic traits: haemolyticus have been reported (DePaola et al., 2000).
66 trh+/tdh-, 24 trh-/tdh+ and 4 trh+/tdh+. Different Pathogenic specimens have accounted for only 1–3% of
spatial and seasonal patterns were observed in rela- all strains retrieved from environmental sources (DePaola
tion to genetic traits. The trh+/tdh- strains were et al., 1990; 2000; Nishibuchi and Kaper, 1995). Thus, the
detected exclusively in northern areas and prevailed lack of information about the prevalence and characteris-
in the autumn, when seawater is warmer and less tics of pathogenic populations of V. parahaemolyticus in
saline, whereas the trh-/tdh+ strains were found in all the environment seriously limits our understanding of the
three areas throughout winter and spring. Character- pathogenic potential and the dynamics of infection asso-
ization of potentially pathogenic strains from the envi- ciated with this organism.
ronment revealed an unexpectedly diverse array Vibrio strains positive for tdh or trh genes have rarely
of serotypes and pulsed-field gel electrophoresis been detected in the marine environment of Galicia. In a
(PFGE) profiles (pulsotypes) that were unrelated to previous study of the presence of V. parahaemolyticus in
clinical strains of V. parahaemolyticus that are preva- the central areas of the four main rias in Galicia, only two
lent in Spain. The results of the current study provide tdh+ strains from a single sample of mussels were iso-
a novel view of V. parahaemolyticus in Europe, in lated after 3 years of study and the characterization of
which diverse pathogenic groups are constitutive more than 2320 strains (Martinez-Urtaza et al., 2008b).
components of the environmental populations in These results contrast with the epidemiological signifi-
coastal habitats. cance of V. parahaemolyticus in Galicia, where three large
outbreaks linked to different trh-/tdh+ groups of V. para-
haemolyticus have been reported in recent years. The first
two large outbreaks of illness were detected in the south
Received 5 May, 2009; accepted 7 July, 2009. *For correspondence.
E-mail: [email protected]; Tel. (+34) 981 528024; Fax: of Galicia in 1999 and 2000 (Lozano-León et al., 2003),
(+34) 981 547165. and were associated with an endemic clonal group of
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd
 Pathogenic V. parahaemolyticus in Galicia 59

serotype O4:K11 (Martinez-Urtaza et al., 2004). A third Results and discussion

outbreak occurred in the north of Galicia in 2004 and was
Detection of V. parahaemolyticus
epidemiologically linked to the port of A Coruña. In con-
trast to the two previous outbreaks, the cases detected in The presence of V. parahaemolyticus was investigated in
A Coruña were associated with strains belonging to the three coastal areas of Galicia (northwest Spain): A Coruña
pandemic clonal complex of V. parahaemolyticus, which and Ferrol, located in the north of Galicia, and Vigo, the
emerged in Asia in 1996 (Okuda et al., 1997). The exog- southernmost ria in Galicia (Fig. 1). In each of the three
enous nature of the strains and the link to the port of A sampling areas, between five and six sites were estab-
Coruña were considered preliminary evidence in support lished, including zones located in the interior of the inter-
of port activity as the most probable source of the out- national ports of Vigo, A Coruña and Ferrol. One litre of
break (Martinez-Urtaza et al., 2005). seawater was collected every 15 days from each sam-
In line with previous epidemiological evidence, we pling site for 1 year and analysed for the presence of V.
designed the current study to detect potential pathogenic parahaemolyticus, according to the ISO8914 Absence/
V. parahaemolyticus strains in coastal areas linked to the Presence method (ISO 8914, 1990). Briefly, 200 ml of
outbreaks reported in Galicia, and therefore theoretically seawater per sample were filtered through a 0.45 mm
most appropriate for the detection of such specimens. The sterile filter. The filters were incubated in sterile flasks
strains obtained throughout the study were characterized containing 200 ml of alkaline peptone water (APW,
and compared with a large collection of strains that were pH 8.5) at 37°C for 18–24 h. After incubation, an aliquot
representative of different regions throughout the world in of the broth was plated onto thiosulfate-citrate-bile
order to evaluate the endemic nature of the strains and salt-sucrose (TCBS) agar and ChromAgar Vibrio and
the potential entrance of foreign bacteria into the marine incubated for 16–18 h at 37°C. All presumptive V. para-
environment of Galicia. haemolyticus colonies on the agar plates were confirmed

Fig. 1. Location of the study region showing the three study areas in Galicia (A Coruña, Ferrol and Vigo).

© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 2, 58–66
60 A. Rodriguez-Castro et al.
Table 1. Incidence of total, trh+ and tdh+ V. parahaemolyticus at different sampling sites in each area throughout the period of study.

Area Sampling sites Samples toxR trh tdh

A Coruña 1 21 6 (28.5%) 1 (4.8%) 1 (4.8%)

2 21 9 (42.8%) 1 (4.8%) 0 (0%)
3 21 4 (19.0%) 0 (0%) 0 (0%)
4 21 6 (28.5%) 1 (4.8%) 2 (9.5%)
5 21 6 (28.5%) 2 (9.5%) 2 (9.5%)
6 21 5 (23.8%) 2 (9.5%) 1 (4.8%)
Total 126 36 (28.5%) 7 (5.5%) 6 (4.7%)
Ferrol 1 20 7 (35%) 3 (15%) 2 (10%)
2 20 10 (50%) 3 (15%) 2 (10%)
3 20 8 (40%) 2 (10%) 4 (20%)
4 20 12 (60%) 5 (25%) 3 (15%)
5 20 11 (55%) 2 (10%) 2 (10%)
Total 100 48 (48%) 15 (15%) 13 (13%)
Vigo 1 21 5 (23.8%) 0 (0%) 2 (9.5%)
2 21 5 (23.8%) 0 (0%) 0 (0%)
3 21 5 (23.8%) 0 (0%) 0 (0%)
4 21 5 (23.8%) 0 (0%) 0 (0%)
5 21 13 (61.9%) 0 (0%) 0 (0%)
Total 105 33 (31.4%) 0 (0%) 2 (1.9%)
Total 331 117 (35.3%) 22 (6.6%) 21 (6.3%)

by the presence of the species-specific Vp-toxR gene, highest incidence of tdh+ samples (20%) occurred at site
according to the method of Kim and colleagues (1999). 3. The presence of V. parahaemolyticus was less uni-
Confirmed V. parahaemolyticus colonies were examined formly distributed in the ria of Vigo, with incidences
for the presence of the pathogenicity-related tdh and trh ranging from 23.8% to 61.9%. The highest incidence of
genes by PCR, as previously described by Tada and total V. parahaemolyticus was observed in the innermost
colleagues (1992) (Appendix S1). sample site in Vigo, where salinity was low. No trh+
Vibrio parahaemolyticus was detected in 117 of 331 samples were detected in the ria de Vigo, and only two
(35.3%) seawater samples analysed over the period of tdh+ samples were detected at site 1 in the external
the study (Table 1). The incidence of V. parahaemolyticus area of the ria. The incidence of V. parahaemolyticus in
was significantly higher in Ferrol (48%) than in A Coruña samples from A Coruña ranged from 19% to 42.8%. The
and Vigo, where the incidence of positive samples was highest incidence of trh+ samples occurred at sites 5
similar (28.5% and 31.4% respectively). Pathogenic tdh+ and 6 (9.5%), whereas tdh+ samples were primarily iso-
and trh+ strains were detected in 21 (6.3%) and 22 (6.6%) lated from sample sites 4 and 5 (9.5%). Similar patterns
samples respectively. A significantly higher percentage of of distribution were observed for the presence of strains
tdh+ and trh+ strains was observed in Ferrol (13% and with pathogenicity-related genes, with the highest pro-
15% of seawater samples respectively), as compared portion of trh+ strains at site 6 in A Coruña (37.7%) and
with 6 (4.7%) tdh+ samples and 7 (5.5%) trh+ samples site 2 in Ferrol (34.4%), whereas the highest values for
from A Coruña. No trh+ strains were found in the ria of tdh+ strains were obtained at site 3 in Ferrol (29.7%).
Vigo, and tdh+ V. parahaemolyticus was present in only The presence of potentially pathogenic strains in these
two samples from this region (1.9%). areas denotes an important shift in the population struc-
The geographical distribution of total and pathogenic ture of V. parahaemolyticus from a previous report of the
V. parahaemolyticus in each area over the period of marine environment in Galicia, in which the presence of
study was analysed by Geographical Information only a single tdh+ strain was found after examining thou-
System (GIS) software (ArcGIS version 9.1) and the sands of environmental strains (Martinez-Urtaza et al.,
Spatial Analyst extension by Environmental Systems 2008b). An extraordinarily low presence of tdh+ and trh+
Research Institute, using the vector data source strains in the environment has been also reported for
BCN200 (Base Cartografica, Numerical scale 1:200 000) other European countries. In studies carried out in France
from the National Geographic Institute of Spain (Figs 1 and Norway (Hervio-Heath et al., 2002; Robert-Pillot
and 2). The incidence of total V. parahaemolyticus in the et al., 2003; Ellingsen et al., 2008), no tdh+ strains were
different sample sites within the ria of Ferrol ranged from detected after the characterization of 306 and 330 strains
35% to 60%, and the percentage increased towards the respectively, whereas trh+ strains represented 5% and
internal areas of the ria (Table 1). The highest incidence 2.1%, respectively, of V. parahaemolyticus examined.
of trh+ samples (25%) occurred at site 4, whereas the This is also true for the USA and Japan, where the pro-

© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 2, 58–66
 Pathogenic V. parahaemolyticus in Galicia 61

Fig. 2. Geographical Information System (GIS) maps showing the spatial distribution of total, trh+ and tdh+ V. parahaemolyticus among
sampling sites in the three study areas in Galicia. Note the different values associated with the colour scales in the maps.

portion of strains with pathogenic traits from major envi- identified in environmental sources (Baffone et al.,
ronmental areas has rarely exceeded 3% (DePaola et al., 2008; Caburlotto et al., 2008). The distinct geographical
1990; 2000; 2003a; Vuddhakul et al., 2000; Mahmud distribution of trh+ and tdh+ populations suggests a
et al., 2006; Vongxay et al., 2008). temperature-mediated affinity of tdh+ and trh+ populations
The presence of trh+ V. parahaemolyticus in most of the that might affect their distribution along European coasts,
areas investigated along the north coast of Galicia (A with trh+ specimens dominating in the coldest waters, and
Coruña and Ferrol) contrasts with the total absence of tdh+ V. parahaemolyticus distributed in the warmer waters
strains in the ria of Vigo. No trh+ strains were found in the to the south. Furthermore, in this context, the coexistence
rias located in the south of Galicia in a previous 3-year of tdh+ and trh+ populations along the Galician coast
survey (Martinez-Urtaza et al., 2008b). The dominance of suggests the existence of a transitional area character-
trh+ strains in the north of Galicia is consistent with pre- ized by a moderate climate between the warm waters of
vious observations in Northern Europe, where reduced the Mediterranean Sea and the cold waters of Northern
numbers of trh+ strains were recovered in studies carried Europe. A similar biogeographical range is widely
out in the English Channel and along the Atlantic coast of observed in many other marine species of zooplankton,
France (Hervio-Heath et al., 2002; Robert-Pillot et al., such as the copepods Temora stylifera and Oncaea medi-
2003), and in Norway (Bauer et al., 2006; Ellingsen et al., terranea (Razouls et al., 2005–2009) and mollusks, such
2008). By comparison, no trh+ strains were recovered as the Mediterranean mussel Mytilus galloprovincialis
from the Mediterranean Sea, where only tdh+ strains were (Seed, 1992).

© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 2, 58–66
62 A. Rodriguez-Castro et al.

Seasonality during the summer months in many parts of the world

(Daniels et al., 2000; Gonzalez-Escalona et al., 2005;
Vibrio parahaemolyticus was detected throughout the
McLaughlin et al., 2005; Martinez-Urtaza et al., 2008a).
year, although the highest incidence (58.7%) and number
DePaola and colleagues (2003a) reported a predomi-
of strains (272) were found in autumn (Fig. 3). A similar
nance of tdh+ strains in the winter in the Gulf Coast of the
pattern was observed for trh+ samples, with the highest
USA, and attributed it to an increase in the ratio of tdh+
incidence in November (31.2%). The presence of tdh+
strains in oysters during the winter due to a reduction in
populations showed a different seasonal pattern, with an
the total V. parahaemolyticus population, rather than to a
overall maximum incidence in February (25%), and an
real rise in strains with pathogenicity-related genes over
extraordinarily high rate of occurrence in Ferrol, where
the winter. However, both absolute and relative numbers
tdh+ samples reached 60% in this month. The highest
of tdh+ strains were higher in Galicia in winter. The factors
incidence of tdh+ samples in A Coruña was observed in
favouring the extraordinary presence of tdh+ populations
June (16.6%), while only two tdh+ samples were detected
in the coolest months of the year remain unclear, since no
in the ria of Vigo in February and November. The highest
unusual environmental conditions were observed.
proportion of trh+ strains occurred in the autumn months,
when 85% of trh+ strains were isolated. In contrast, 75%
of tdh+ strains were isolated in winter and spring. No tdh+
Statistical analysis
strains were detected in the summer, although 20% of the
samples were positive for V. parahaemolyticus throughout The association between environmental factors (air
this period. This observation conflicts with the previously temperature, wind, hours of sunshine, rainfall, seawater
described preference for warm waters, and with the temperature and salinity) and presence of V. para-
overall dominance of V. parahaemolyticus infections haemolyticus was analysed by logistic regression analy-

Fig. 3. Overall incidence of total, trh+ and tdh+ V. parahaemolyticus in the three study areas (A), and in Ferrol (B), A Coruña (C) and Vigo
(D), and variations of salinity and seawater temperature throughout the period of study. No salinity data were available for A Coruña.

© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 2, 58–66
 Pathogenic V. parahaemolyticus in Galicia 63

sis. The presence of V. parahaemolyticus in each area group were detected over the entire period of the study,
was significantly associated with salinity (P < 0.05) and with the exception of summer, when neither trh+ nor tdh+
temperature (P < 0.01), with negative and positive corre- strains were detected. Only one O3:K4 strain isolated
lations. None of the other parameters correlated signifi- from Vigo showed a restriction pattern related to the domi-
cantly with the presence of the bacterium. In the northern nant clone. An additional pulsotype (PT34) grouped three
areas of the rias of Ferrol and A Coruña, the presence of strains isolated from Ferrol in winter. The remaining five
V. parahaemolyticus was positively and strongly influ- trh-/tdh+ strains had diverse serotypes and unrelated
enced by atmospheric temperature in the days prior to restriction patterns. Two strains with serotypes O2:K28
sample collection. and O2:KUT shared an exclusive PFGE pattern that was
only detected in samples from the ria of Vigo. All trh-/tdh+
strains were negative in the Group-Specific PCR (GS-
Strain characterization
PCR) assay, which was designed for the detection of
A total of 1351 presumptive strains were selected from pandemic clone-specific nucleotides in the toxRS operon
the seawater samples. Vibrio parahaemolyticus was con- of V. parahaemolyticus (Matsumoto et al., 2000).
firmed by the presence of the species-specific Vp-toxR The four trh+/tdh+ strains that were recovered during
gene, which was found in 535 strains. Virulence genes the study were from the ria of Ferrol and showed distinct
were detected in 94 strains (17.6%) with the following serotypes and unrelated genetic profiles. To our knowl-
distribution: 24 (4.5%) contained the tdh gene, 66 (12.3%) edge, these strains represent the first such environmental
contained the trh gene and 4 (0.8%) contained trh and strains with these particular characteristics reported in
tdh. Europe.
All of the tdh+ and trh+ strains were subjected to sero- A comparison of all the tdh+ and trh+ strains obtained
typing by agglutination tests with specific antisera accord- during this study with representative pathogenic V. para-
ing to the manufacturer’s instructions (Denka-Seiken, haemolyticus strains isolated from different areas of the
Tokyo, Japan), and to pulsed-field gel electrophoresis world (Peru, Chile, Taiwan, India, Korea, Laos, Japan, the
(PFGE) analysis, as previously described (Martinez- USA, Bangladesh, Thailand and the UK) showed that
Urtaza et al., 2004). We observed a highly diverse array of the strains from Galicia had PFGE patterns that were
serotypes and genetic profiles among the 66 trh+/tdh- unrelated to the pathogenic strains from other geographi-
strains (Fig. 4). Serotype analysis revealed that there cal areas (Figs S1 and S2).
were 16 different serogroups, with the predominant sero- Vibrio parahaemolyticus infections in Spain, and
types belonging to serogroups O1 (16 strains), O5 (15 throughout Europe, have been associated exclusively
strains) and O10 (13 strains). The most frequent sero- with tdh+ strains (Martinez-Urtaza et al., 2004; 2005), and
types were O1:KUT and O5:K17, comprising 12 strains no illnesses linked to trh+ strains have been reported to
each, followed by O10:KUT, which was identified in eight date. This situation clearly contrasts with the dominance
strains. Of the 33 trh+/tdh- strains from A Coruña, 11 of trh+ populations in the marine environment. Further-
strains belonging to serotypes O10:KUT, O10:K71 and more, the serological and genetic characteristics of the
O1:KUT shared identical PFGE patterns (PT8), whereas tdh+ environmental strains were not related to the O4:K11
another seven strains with serotype O5:K17 belonged to clinical clone dominant in clinical sources in Spain
pulsotype PT26. The remaining 15 strains belonged to (Martinez-Urtaza et al., 2004). This apparent incongruity
seven different pulsotypes. The 33 trh+/tdh- strains between strains with virulence traits prevailing in the
obtained from the ria of Ferrol showed extraordinary environment and those isolated from humans infections
diversity, with 12 different serotypes distributed over 20 provides a novel context for this pathogen in Galicia
pulsotypes. The predominant pulsotype was PT6, which that requires further investigation.
included seven strains belonging to serogroup O1 Few comprehensive studies have been carried out
(O1:KUT, O1:K1 and O1:K56). The remaining 26 trh+/tdh- worldwide to investigate the characteristics and the
strains from the ria of Ferrol also presented highly diverse genetic relationships among potential pathogenic strains
serotypes and unrelated restriction patterns. Only trh+/ of V. parahaemolyticus in the environment, and therefore,
tdh- strains of the pulsotypes PT8 and PT26 were found few comparisons can be made with the current results.
simultaneously in A Coruña and Ferrol. Most information about the presence and characteristics
In contrast to the trh+/tdh- strains, the 24 trh-/tdh+ of pathogenic V. parahaemolyticus in the environment
strains were highly uniform (Fig. 4). A dominant and comes from the USA (DePaola et al., 2003b; McLaughlin
homogeneous genetic group, containing the pulsotypes et al., 2005; Johnson et al., 2008), where the dominant
PT1, PT2 and PT3, was identified among trh-/tdh+ strains genotype reported for the marine environments of the
obtained from A Coruña and Ferrol, with 16 of the strains Pacific and Gulf coasts of the USA is trh+/tdh+. These
belonging to serotype O4:K63. Strains included in this strains have shown a great diversity of serotypes

© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 2, 58–66
64 A. Rodriguez-Castro et al.

Fig. 4. Circular unrooted dendrogram generated by Bionumerics software and drawn using the online iTOL software package, showing the
genetic relationships among trh+/tdh-, trh-/tdh+ and trh+/tdh+ strains of V. parahaemolyticus in relation to season and site of isolation. From
inner to outer, rings respectively indicate presence of pathogenicity-related genes, area of origin and season of isolation for each strain
positive for trh or tdh gene.

(DePaola et al., 2003b; Gonzalez-Escalona et al., 2005; although the number of strains characterized and the
Johnson et al., 2008) similar to those found for the trh+/ duration of the study may have limited the ability to infer
tdh- strains in Galicia. Very few trh-/tdh+ or trh+/tdh- any general trend.
strains have been detected in any of these previous The results of the current study provide an unexpected,
studies. The most comprehensive study to date was novel view of the composition and dynamics of V. para-
carried out in the Gulf of Mexico (Johnson et al., 2008) haemolyticus populations in Galicia, and demonstrate that
where the authors reported an absence of spatial or tem- trh+ and tdh+ populations constitute part of the normal
poral patterns among potentially pathogenic strains, flora in the marine environment. The presence of identical

© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 2, 58–66
 Pathogenic V. parahaemolyticus in Galicia 65

genetic groups in distant areas during different periods of Cook, D.W. (2003a) Seasonal abundance of total and
the year suggests that these populations are endemic to pathogenic Vibrio parahaemolyticus in Alabama oysters.
the area. In addition, this study also provides a preliminary Appl Environ Microbiol 69: 1521–1526.
DePaola, A., Ulaszek, J., Kaysner, C.A., Tenge, B.J., Nord-
insight into the association between potentially patho-
strom, J.L., Wells, J., et al. (2003b) Molecular, serological,
genic strains and maritime traffic. tdh+ strains were and virulence characteristics of Vibrio parahaemolyticus
detected in the three ports, and mainly concentrated in isolated from environmental, food, and clinical sources in
sampling areas that corresponded to the anchoring of North America and Asia. Appl Environ Microbiol 69: 3999–
ships engaged in long-distance international transport, 4005.
and many of the strains present demonstrated distinct Ellingsen, A.B., Jørgensen, H., Wagley, S., Monshaugen, M.,
genetic profiles unrelated to the dominant clones in the and Rørvik, L.M. (2008) Genetic diversity among Norwe-
gian Vibrio parahaemolyticus. J Appl Microbiol 105: 2195–
region.
2202.
Gonzalez-Escalona, N., Cachicas, V., Acevedo, C., Rioseco,
Acknowledgements M.L., Vergara, J.A., Cabello, F., Romero, J., et al. (2005)
Vibrio parahaemolyticus diarrhea, Chile, 1998 and 2004.
The authors are grateful for the support and assistance of the Emerg Infect Dis 11: 129–131.
Port Authorities at A Coruña, Ferrol and Vigo. We also thank Hervio-Heath, D., Colwell, R.R., Derrien, A., Robert-Pillot, A.,
Mitsuaki Nishibuchi and Romilio Espejo for providing some of Fournier, J.M., and Pommepuy, M. (2002) Occurrence of
the strains included in this study; Joaquin Triñanes (Univer- pathogenic Vibrios in coastal areas of France. J Appl
sity of Santiago de Compostela) and Ana Miranda (Instituto Microbiol 92: 1123–1135.
Oceanografico de Vigo) for advice regarding oceanographic International Organization for Standardization. (1990)
data; Yolanda Pazos (Instituto Tecnolóxico para o Control do Microbiology-General guidance for the detection of Vibrio
Medio Mariño de Galicia) for providing some environmental parahaemolyticus. ISO 8914: 1990. In Annual Report
data; Isabel Mayán Barreiro and Silvia Carlés Gonzalez for 1990, ed. Switzerland: International Organization for
technical assistance; and R.G. Gavilán for help in construct- Standardization.
ing the dendrograms. This study was funded by project ‘AGL Johnson, C.N., Flowers, A.R., Young, V.C., Gonzalez-
2005-07870’ from the Spanish Ministry of Education and Escalona, N., DePaola, A., Noriea, N.F., III, and Grimes,
Science. D.J. (2008) Genetic relatedness among tdh+ and trh+
Vibrio parahaemolyticus cultured from Gulf of Mexico
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hemolysin gene of Vibrio parahaemolyticus: a virulence
gene acquired by a marine bacterium. Infect Immun 63: Fig. S1. Dendrogram comparing the genetic relationship of
2093–2099. PFGE patterns for trh+ strains of V. parahaemolyticus
Okuda, J., Ishibashi, M., Hayakawa, E., Nishino, T., Takeda, obtained in this study with trh+ strains from different parts of
Y., Mukhopadhyay, A.K., Garg, S., et al. (1997) Emergence world. Numbers at the top the figure indicate molecular sizes
of a unique O3:K6 clone of Vibrio parahaemolyticus in in kb.
Calcutta, India, and isolation of strains from the same Fig. S2. Dendrogram showing the PFGE analysis of restric-
clonal group from Southeast Asian travelers arriving in tion patterns for the tdh+ strains of V. parahaemolyticus
Japan. J Clin Microbiol 35: 3150–3155. obtained in this study compared with tdh+ strains from differ-
Razouls, C., Bovée, F., Kouwenberg, J., and Desreumaux, N. ence parts of world. Numbers at the top of the figure indicated
(2005–2009) Diversity and geographic distribution of molecular sizes in kb.
marine planktonic copepods [WWW document]. URL http:// Appendix S1. Complete sampling details and experimental
copepodes.obs-banyuls.fr/en. procedures.
Robert-Pillot, A., Guénolé, A., Lesne, J., Delesmontc, R., Please note: Wiley-Blackwell are not responsible for the
Fourniera, J.M., and Quilici, M.L. (2003) Occurrence of the content or functionality of any supporting materials supplied
tdh and trh genes in Vibrio parahaemolyticus isolates from by the authors. Any queries (other than missing material)
waters and raw shellfish collected in two French coastal should be directed to the corresponding author for the article.

© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 2, 58–66