IV. Results
II. REVIEW OF LITERATURE
Page 4
Vibrio parahaemolyticus is a Gram negative, facultative anaerobic, straight or curved,
halophilic bacterium, autochthonous to water and sediments of marine and estuarine ecosystem.
It is considered as emerging food-borne pathogen responsible for gastroenteritis. The incidence
of gastroenteritis due to V. parahaemolyticus was first reported in Japan during 1950’s (Fujino et
al., 1951; 1953) and since then, cases have been reported from several parts of the world.
2 .1. Discovery of V. parahaemolyticus
The V. parahaemolyticus was discovered in Japan during 1950s as a cause of diarrhea
and its credit goes to Dr. Fujino. This organism was first isolated form the source of semi
processed Japanese anchovy, Engraulis japonicus “shirasu” during an outbreak of gastroenteritis
in Osaka, Japan (Fujino et al., 1951; Fujino et al., 1953). The classification of V.
parahaemolyticus proposed immediately after its discovery and was assigned to the genus
Pasturella and species parahaemolytica. The identified organism showed motility similar to
Vibrio cholerae (Fujino et al., 1953). Baumann and Baumann (1973) suggested that this
organism should be placed in the genus Benekea because of its ability to hydrolyse chitin.
Taxonomic studies of Colwell (1970) and DNA homology studies by Citarella and Colwell
(1970) helped unambiguous classification of this organism as a separate species within the genus
Vibrio. Subsequently, several researchers performed extensive studies on this species and
concluded that it belongs to the genus Vibrio and should be classified as V. parahaemolyticus
(Sakazaki, 1963a, 1963b; Zen-Yoji et al., 1965; Sakazaki et al., 1968; Fujino et al., 1974).
Similar organism was identified from gastroenteritis outbreaks from India in 1970.
Studies undertaken at the Cholera Research Centre, Calcutta during 1970s indicate that about 5-
10 % of the gastroenteritis cases admitted annually at Infectious Disease Hospital, Calcutta were
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due to V. parahaemolyticus infection. Now this pathogen is a common cause of food-borne
illnesses in many Asian countries, including China, Japan and Taiwan, and is recognized as the
leading cause of human gastroenteritis associated with seafood consumption.
2. 2. Characteristics of V. parahaemolyticus
V. parahaemolyticus is a Gram negative, facultative anaerobic, straight or curved
bacterium of marine origin. They have a mandatory requirement of sodium chloride for their
growth. The guanine-cytosine (G+C) base composition for this organism is 46%. V.
parahaemolyticus can be differentiated from other species by several physiological and
nutritional characteristics. These include swarming on nutrient agar media, negative for acetoin
or diacetyl production, lacking for arginine dehydrolase, oxidase positive, unable to produce gas
from glucose, lacking in the utilization of sucrose and cellobiose. V. parahaemolyticus produces
round blue-green colonies on thiosulfate-citrate-bile salt-sucrose (TCBS) agar and may not grow
on Modified Cellobiose-Polymyxin Colistin (mCPC) agar. The minimum assay required for the
identification of V. parahaemolyticus includes observation of a motile Gram negative rod,
production of acid from glucose but no gas, positive growth at 1 % to 8 % NaCl, positive for
utilization of lysine and ornithine, reaction on TSI should be alkaline slant, acid butt, no gas and
H2S. FDA Bacteriological analytical manual advocates enrichment and enumeration carried out
in alkaline peptone water followed by streaking on TCBS and mCPC agar results in better
isolation and detection of V. parahaemolyticus from samples.
2. 3. Geographical distribution of V. parahaemolyticus
V. parahaemolyticus is a halophilic bacterium natural inhabitant of marine environments
of coastal areas and estuaries world wide. Its distribution restricted to Japan till 1960’s but later it
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has been widely isolated from various parts of the world. Ecological studies have revealed the
presence of V. parahaemolyticus in Hong Kong (Chan et al., 1989), Taiwan, Korea (Chun et al.,
1974) Singapore, Hawaii, Germany, Vietnam (Neumann et al., 1972), United States (DePaola et
al., 2003), India (Chatterjee et al., 1970) and other parts of the world. This human pathogen
occurs naturally in the marine environments and frequently isolated from wide variety of
seafoods including oyster, clam, shrimp, codfish, sardine, mackerel, flounder, , octopus, crab,
lobster, crawfish and scallop (Liston, 1990).
2. 4. Distribution of V. parahaemolyticus in marine environments
The survival of V. parahaemolyticus is influenced by many environmental factors such as
temperature, salinity, tidal influence and plankton biomass. These environmental factors made
the organism much more prevalent in the coastal and estuarine waters around the globe. Higher
densities of V. parahaemolyticus with faecal pollution and their presence in the near surface
waters suggest a relationship to light (Watkins and Cabelli, 1985). In marine environment there
is a noticeable interaction between V. parahaemolyticus and sediment, water, and zooplankton. It
was found to be essential in the natural estuarine ecosystem for the active survival of the
organism (Kaneko and Colwell, 1978). In Chesapeake Bay oysters, V. parahaemolyticus
densities varied seasonally and were found to be positively correlated with water temperature,
turbidity, and dissolved oxygen (Parveen et al., 2008).
According to the study conducted on occurrence of V. parahaemolyticus in Oregon oyster
growing environments between November 2002 and October 2003 a positive correlation
between V. parahaemolyticus in seawater and water temperatures was observed with the highest
populations of V. parahaemolyticus in water being detected in the summer months (Duan and
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Su, 2005). Temperature and seawater salinity are the two important environmental factors that
are known to influence the levels of Vibrio spp. in coastal environments (Tamplin et al., 2001).
Hence, in marine environment the growth of V. parahaemolyticus is favoured by warmer
temperatures (Kaneko and Colwell 1978; DePaola et al., 2003a).
Schwarz and Colwell (1974) examined V. parahaemolyticus for their ability to survive
and grow at deep ocean hydrostatic pressures but were unable to grow at hydrostatic pressure
which is high in the deep oceans. Martinez-Urtaza et al. (2008) showed that salinity is the
primary factor governing the temporal and spatial distribution of V. parahaemolyticus. They
found a strong correlation between presence of V. parahaemolyticus and reduced water salinity.
In tropical countries, the occurrence of the organism correlated with the rainy and dry seasons;
the lowest numbers were found in rainy months and the highest numbers in the dry season
(Deepanjali et al., 2005). Seasonal variations in abundance of V. parahaemolyticus have been
confirmed by several authors. The abundance of V. parahaemolyticus in marine environment is
governed generally by several biotic and abiotic factors (Venkateswaran, 2006).
2. 5. Prevalence of V. parahaemolyticus in fish and shellfish
The degree of V. parahaemolyticus load in fish and shellfish is known to relate to the
water temperatures. Therefore, it is more likely to detect V. parahaemolyticus in fish harvested in
the spring and the summer than in the winter (DePaola et al., 2000). Reports showed that the
density of V. parahaemolyticus in contaminated oysters is usually lower than 103 CFU/g at
harvest, but it could exceed 103 CFU/g in oysters harvested from warmer seawater (DePaola et
al., 2000). In India, prevalence of V. parahaemolyticus in fish and shellfish has been reported by
several workers (Karunasagar and Mohankumar, 1980; Venugopal et al., 1984; Karunasagar et
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al., 1986; Pradeep and Lakshmanaperumalswamy, 1986; Dileep et al., 2003; Deepanjali et al.,
2005; Bhaskar and Setty, 2006; Raghunath et al., 2006; 2008a; b, 2009; Chowdhury et al., 2013;
Collin et al., 2013). Sani et al. (2008) reported the incidence of V. parahaemolyticus in frozen
and unprocessed Penaeus monodon as well as in their culture environment in Malaysia. It was
reported that Vibrio spp. dominates in the marine and brackishwater fish (Al-Harbi et al., 2005).
It is also reported that the organism was found to be associated with mortalities in Iberian
toothcarp (Aphanius iberus) with the signs centering on external haemorrhages, and tail rot
(Austin and Austin, 2007) and also recovered from diseased milkfish (Chanos chanos) in the
Philippines (Austin and Austin, 2007). V. parahaemolyticus is also considered as well-
recognized pathogen of invertebrates such as shrimp (Jayasree et al., 2006; Cai et al., 2007).
2. 6. Association with planktonic organisms
The presence of protozoa allows survival, replication and distribution of some species of
pathogenic bacteria in the natural environment (Kaneko and Colwell, 1975). Vibrio spp. has the
ability to survive and multiply within biofilm of amoebae, as it offers protection in adverse
conditions (Barker and Brown, 1994). It is proved that V. parahaemolyticus is not a native
microflora of freshwater ecosystem but their association with freshwater plankton could play a
role in the dissemination of infection (Sarkar, et al., 1983).
Seasonal increments of Vibrio numbers have been shown in relation to zooplankton on
which the organisms adsorb. This will play an important role in the cycling of elements in the
marine environment and from which they are released during the mineralization process (Kaneko
and Colwell 1973, 1975). V. parahaemolyticus produces a chitinase enzyme and that may play a
major role in the recycling of chitinous materials in the marine environment. Zooplankton
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possesses exoskeleton composed of chitin and often carries high numbers of V.
parahaemolyticus (Kaneko and Colwell, 1973, 1975).
2. 7. Pathogenic V. parahaemolyticus in the environment
Occurrence of V. parahaemolyticus has been reported from many parts of the world, with
more than 90 % of clinical isolates but less than 1-2 % of environmental isolates capable of
producing TDH. Only a very small proportion of the V. parahaemolyticus isolates in the
environment are known to be virulent and carry virulence genes tdh and/or trh, whereas, most
clinical isolates are virulent. V. parahaemolyticus strains isolated on the West coast of the United
States demonstrated urease activity (Janda et al., 1988). Increased incidences of gastroenteritis
caused by V. parahaemolyticus serotype O3:K6 have been reported in many countries since
1996. Enumeration of pathogenic V. parahaemolyticus recorded a higher ratio of pathogenic
strains to total V. parahaemolyticus strains in oysters from the west (Kelly and Stroh, 1989;
DePoala et al., 2003). In contrast to the strains of US coast wherein the strains harboured almost
both the trh and tdh genes, the isolates from Japan recorded an incidence of 87 % strains being
positive for tdh and only 7.8 % were trh positive (Suzuki et al., 1994). Similar results were
recorded from Thailand where majority of the strain possessed the tdh genes while only 6 % had
both tdh and trh genes and only 2 % had only trh gene (Suthienkul et al., 1995). A perfect
correlation of the trh strains with urease was deduced from both the studies.
Prevalence of pathogenic V. parahaemolyticus from India was reported by Karuanasagar
et al. (1990), Dileep et al. (2003) and Deepanjali et al. (2005) along the coast of Southern India
and by Ghosh and Sehgal (1998) in the Andaman Sea. Cabrera-Garcia et al. (2004) reported the
presence of tdh-carrying V. parahaemolyticus from environmental samples from Mexico for the
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first time. Robert-Pillot et al. (2004) isolated both tdh and trh-carrying V. parahaemolyticus
isolates from raw shellfish collected in two French coastal areas and from seafood imported into
France. Hara-Kudo et al. (2003) reported tdh gene in 33 of 329 seafood samples (10.0%). The
levels of tdh-positive V. parahaemolyticus ranged from
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in Japanese waters, thus responsible for outbreaks of V. parahaemolyticus infection due to
seaweeds.
2. 8. Epidemiology
Since the first identification of V. parahaemolyticus as the etiological agent of human
gastroenteritis, it has been implicated as a major cause of foodborne illness around the globe. In
Japan, 70 % of food poisoning cases are due to this organism (Sakazaki et al., 1968) and it
reduced to 47 % in 1990s (Lee et al., 1992). In 2002, it has been reported to account for 20 to 30
% of food poisoning cases in Japan (Alam et al., 2002). For the last 20 years V.
parahaemolyticus has been the most common Vibrio spp. isolated from cases of human
gastroenteritis. In 1971, the first case of V. parahaemolyticus outbreaks outside Japan was
reported from East coast of United States. Between 1973 and 1998, 40 outbreaks of V.
parahaemolyticus infections were reported to the Centers for Disease Control and Prevention,
USA (CDC), and these outbreaks included 11000 illnesses (Daniels et al., 2000a). In 1998, V.
parahaemolyticus in oysters and clams harvested from the Long Island Sound caused an
outbreak in Connecticut, New Jersey, and New York, and the implicated serotype (O3:K6) was
recognized as pathogenic strain in Asia, but had not previously been isolated in the United States
(Posnick et al., 2000).
In India, the incidence of V. parahaemolyticus infection was first reported during 1970’s
by Chatterjee and co-workers (Chatterjee et al., 1970). After that several workers reported
isolation and distribution of V. parahaemolyticus in and around Calcutta (Sakazaki et al., 1971;
De et al., 1977). The incidence of gastroenteritis at the Christian Medical College, Vellore,
reported by Lalitha et al. (1983) was supposed to be mainly due to cross contamination of the
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food. Lall et al. (1979) reported the occurrence of environmental strain of V. parahaemolyticus
showing positive Kanagawa phenomenon from Port Blair. In 1996, there was a sudden increase
in the incidence of V. parahaemolyticus infection in Calcutta due to the emergence of pandemic
clone of V. parahaemolyticus belonging to O3:K6 serotype (Okuda et al., 1997), incidence of
diarrhoea due to this pathogen remains high showing pandemic potential of this serovar. It
spread to United States in 1998 and recently to Chile, where it has caused hundreds of infections.
The O3:K6 and O4:K68 strains from Calcutta and Bangkok carried the tdh gene but not the trh
gene and molecular studies revealed that there is a higher similarity between those two serovars
thus indicating genetic relationship. This pandemic clone exhibits unique DNA fingerprinting
pattern in an arbitrarily primed PCR assay (AP-PCR) (Chowdhury et al., 2000a, 2004). Some
studies have shown that V. parahaemolyticus strains belonging to several other serotypes have an
equal potential to cause outbreaks and are genetically highly similar to new O3:K6 strains.
Parvathi et al. (2006) reported a dominance of trh+ V. parahaemolyticus over tdh strains in
estuarine environments of Mangalore.
2. 9. Pandemic clone of V. parahaemolyticus
Till mid 1990s, V. parahaemolyticus gastroenteritis were considered sporadic with
occasional local outbreaks involving diverse serotypes. Analysis of strains isolated during active
surveillance of diarrheal aetiologies among hospitalised patients in Calcutta, India in 1996
revealed that 50-80 % of infections recorded after February 1996 belonged to O3:K6 serotype
(Okuda et al., 1997). Subsequently, infections caused by this serovar were detected in different
parts of the world and the outbreaks were termed “pandemic”. But as pointed out by
Nair et al.(2007), in the epidemiological sense, there was no outbreak that could fit the Webster
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dictionary definition of pandemic- “occurring over wide geographical areas and afflicting an
exceptionally high proportion of population”. Though outbreaks have been reported over wide
geographical area, it has not affected exceptionally high proportion of the population and
mortality has been low. However, the term pandemic continues to be used in literature. Further,
the outbreaks are not caused by a single strain of O3:K6 serotype, other serovariants have been
involved, but the isolates share certain genetic and phenotypic characters that support the view
that there is clonality in the isolates. All the isolates in what is termed as the pandemic group
carry tdh but not trh gene, are urease negative and belong to the same biochemical phenotype
according to biochemical fingerprinting by Pheneplate system (Php-48; Pheneplate microplate
Techniques, Stockholm, Sweden), while non pandemic isolates are heterogenous (Rahman et al.,
2006). Currently, more than 20 serovariants including O3:K6, O4:K68, O1:K25, O1: KUT
(untypable) and others are known to belong to the pandemic clone (Chen et al., 2011).
The 1996 V. parahaemolyticus O3:K6 isolates from Calcutta differs from isolates of the
same serotype isolated prior to 1996. The global regulatory gene toxR involved in the regulation
of many virulence-associated genes is conserved in the members of the genus Vibrio. A Group
Specific PCR (GS PCR) that specifically detects toxRS sequence of the pandemic O3:K6 clone
was established to differentiate it from non pandemic clones of V. parahaemolyticus (Matsumoto
et al., 2000). This PCR was developed on the basis of the differences in the toxRS sequences of
the pandemic and the pre-pandemic O3:K6 isolates. The former were invariably found to differ
from the latter in 7 base regions within the 1,364 bp toxRS region. Also, pandemic strains
representing serovars other than O3:K6, eg: O4:K68 and O1: K untypeable (KUT) serovars give
positive GS-PCR results thus providing additional evidence for the clonality of these isolates
(Chowdhury et al., 2000 a, 2000b; Matsumoto et al., 2000). Isolates of pandemic clone
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belonging to serovars O3:K6, O4:K68 and O1: KUT from different countries show identical
ribotypes and pulse field gel electrophoresis (PFGE) patterns (Wong et al., 2000; Yeung et al.,
2002; Nair et al., 2007). However, exceptional strains of pandemic O3:K6 group that lack the
tdh gene, and exhibit arbitrarily primed PCR (AP-PCR) profiles slightly different from the
typical profiles of the pandemic clone have been reported (Matsumoto et al., 2000). Multilocus
sequence typing (MLST) indicates genetic diversity among pre-pandemic O3:K6 isolates and
clonality among pandemic isolates (Chowdhury et al., 2004b). The presence in the genome of
the pandemic isolates of a filamentous phage f237 is another important discriminating feature
and orf8 of f237 has been used as marker for identification of this clone by PCR (Nasu et al.,
2000). However, some of the O3:K6 strains from Bangladesh isolated between 1997 and 2000
(Bhuiyan et al., 2002) and from Vietnam (Chowdhury et al., 2004a) were negative for orf8. Four
genomic islands (VPI1, VPI4, VPI5 and VPI6) have been found to be unique to pandemic clones
of V. parahaemolyticus (Hurley et al., 2006), but screening of 91 pandemic clone isolates
showed presence of VPI1 in all and VPI5 in 90 isolates (Chao et al., 2009).
Though O3:K6 pandemic clones were detected during active surveillance of diarrhea in
Calcutta in 1996, the first reported isolate of this serotype was from a traveller returning from
Indonesia to Japan in 1995 (Okuda et al., 1997). Detection of toxRS sequences identical to that of
pandemic clones in four tdh negative O3:K6 isolates from Japan obtained between 1983 and
1988 led Okura et al. (2003) to hypothesise that pandemic O3:K6 could have originated from
these non-pathogenic strains by acquisition of tdh gene in the environs of Japan. In India, though
O3:K6 serotype accounted for 50-80 % infections in 1996, its proportion declined subsequently.
Analysis of 258 isolates of pandemic clone during 1996-2004 showed fluctuation with peaks in
1996 and 2004. The 1996 peak was due to O3:K6 serotype and the 2004 peak was due to
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O1:K25 serotype that accounted for most isolates in 2000 and 2004 (Nair et al., 2007). As shown
in Fig. 2. 1, the pandemic clones of V. parahaemolyticus have been reported from Asia, Africa,
Europe, North and South America. The appearance of different serotypes of the pandemic clones
in different regions are summarised in Table 2. 1. The number of V. parahaemolyticus cases in
Japan in 1996 were 5241 which increased to 6786 in 1997, and almost doubled to 12346 cases in
1998 (WHO, 1999). Serotype O4:K8 was the dominant serotype in Japan till 1996 after which
O3:K6 replaced this. In Taiwan the dominance of O3:K6 serotype in V. parahaemolyticus
infections was noted almost at the same period as in India. While this serotype accounted for
only 0.6 % infections in 1995 in Taiwan, it accounted for 50.1 % infections in 1996 and 83.7 %
infections in 1997 (Chiou et al., 2000). Isolation of pandemic clone of V. parahaemolyticus in
Bangladesh, Laos, Vietnam, China, Thailand, Indonesia, Korea and Japan has been reported
subsequently (Nair et al., 2007).
V. parahaemolyticus O3:K6 isolates that resembled the pandemic clone in PFGE were
isolated from 28 stool samples of patients involved in an oyster (harvested from Galveston Bay)
associated outbreak of gastroenteritis in Texas, United States in 1998 (Daniels et al., 2000a,
2000b).
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Fig. 2. 1. Dissemination of the O3:K6 isolate of Vibrio parahaemolyticus and its serovariants around the world
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During the same year, V. parahaemolyticus O3:K6 infections associated with
consumption of oysters and clams harvested from Long Island Sound occurred among residents
of Connecticut, New Jersey and New York (CDC, 1999).
A retrospective analysis of isolates of V. parahaemolyticus in Peru showed that the
earliest O3:K6 isolate was seen in 1996. Other serovariants of pandemic clone were also found
in Peru (Gil et al., 2007). In 1998, about 300 cases of V. parahaemolyticus gastroenteritis were
reported from Antofagasta, Chile (Cordova et al., 2002). Most of the tested isolates of this
outbreak belonged to O3:K6 serotype and had molecular features of the pandemic clone
(Gonzales-Escalona et al., 2005). During 2004-2007, there were large outbreaks in Puerto Montt,
southern Chile with a peak in 2005 (about 1500 cases in 2004, 3600 in 2005, 900 in 2006 and
475 in 2007). Even during 2004, a high proportion of the strains were found to be of pandemic
clone. The diversity in strains seemed to increase during later years. Pandemic clones accounted
for 88 % isolates in 2005 and decreased to 66 % in 2007. In 2005, nine different PFGE profiles
were identified and this increased to sixteen profiles in 2007 (Dauros et al., 2011). Though there
was a marginal increase (1153 cases) in 2008, the numbers came down to 441 in 2009. During
2009, 64 % clinical isolates and 24 % shellfish isolates tested belonged to the pandemic clone
(Garcia et al., 2009).
Molecular studies on the V. parahaemolyticus isolates from outbreaks and sporadic cases
that occurred in northeast Brazil during 2001-2002 indicated that the 2001 outbreak involved
O3:KUT serotype and the 2002 outbreak involved O3:K6 serotypes showing characteristics of
pandemic clone (Leal et al., 2008). During a surveillance of diarrhoea in Beira, Mozambique 42
stool samples in 2004 and 16 in 2005 yielded V. parahaemolyticus. In 2004, 32/42 belonged to
O3:K6 serotype, and in 2005, 6/16 belonged to this serotype. Two isolates each during the two
years belonged to O4:K68 serotype. Thus, 72 % of the isolates belonged to pandemic serotype.
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In PFGE studies, these isolates clustered with Asian pandemic clone (Ansaruzzaman et al., 2005,
2008).
Analysis of 13 clinical isolates of V. parahaemolyticus obtained during 1997-2004 in
France showed that five isolates (one isolated during 1997, 1998, 1999 and two during 2003)
belonged to O3:K6 serotype and had molecular features of pandemic clone (Quilici et al., 2005).
It is interesting to note that presence of pandemic clone in clinical cases in France came to light
due to retrospective analysis of clinical isolates, and not due to any noticeable outbreak
occurring. Though isolates were obtained as early as 1997, there were only one isolate per year
except in 2003 when there were two. This suggests that though pandemic clones were found in
clinical cases in France, they did not cause any noticeable spike in hospitalizations. This should
be viewed the context that France is one of the largest consumers of bivalve molluscs within the
European Union (Erwan and Paquotte, 1998). In Spain, there were two clinical isolates of V.
parahaemolyticus O3:K6 and one of O3: KUT that showed molecular features of pandemic
clone during an oyster associated outbreak in 2004 (Martinez-Urtaza et al., 2005). One case of V.
parahaemolyticus O3:K6 gastroenteritis was reported from Italy in 2007 and one in 2008
(Ottaviani et al., 2008, 2010). Pandemic clone of V. parahaemolyticus has also been isolated
from Russia (Smolikova et al., 2001).
2. 10. Virulence properties
Many virulence characters are described to play a role in the pathogenicity of V.
parahaemolyticus. The most important virulence property is the production of TDH coded by
gene tdh, this produces well defined clear haemolysis in a specially prepared high salt blood
agar, Wagatsuma agar and the resultant action is termed as Kanagawa phenomenon (Fig. 2. 2).
Isolates of V. parahaemolyticus from clinical samples are able to produce Kanagawa
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phenomenon in Wagatsuma agar (Nishibuchi and Kaper, 1995). Second important pathogenic
marker is the TRH of V. parahaemolyticus coded by the gene trh; this Kanagawa negative strain
was isolated for the first time from patients during gastroenteritis outbreaks in the Republic of
Maldives (Honda et al., 1987a). This shows that both tdh positive and tdh negative but trh
positive isolates of V. parahaemolyticus were strongly associated with human gastroenteritis
(Nishibuchi and Kaper, 1995). TDH and TRH encoded by the tdh and trh genes share about 70
% nucleotide sequence similarity (Nishibuchi et al., 1989).
Fig. 2. 2. Representative photograph showing the haemolytic activity of V.
parahaemolyticus in Wagatsuma agar
2. 11. Haemolysin in V. parahaemolyticus
Many vibrios are pathogenic for humans and/or marine vertebrates and invertebrates,
with the virulence mechanisms reflecting the presence of enterotoxin, haemolysin, cytotoxin,
protease, lipase, phospholipase, siderophore, adhesive factor and/or haemagglutinins (Iida and
Honda, 1997; Shinoda, 1999; Zhang and Austin, 2005). Among these, the haemolysin produced
by the V. parahaemolyticus is an exotoxin that attacks blood cell membranes and causes cell
rupture, lysis of erythrocyte membranes with the complete degradation of hemoglobin. This
phenomenon is known as beta haemolysis, which leads to release of iron in to the outside
environment and that can then be taken up by various siderophores, and is subsequently taken up
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through receptors in the cell membrane (Zhang and Austin, 2005). Hence, expression of
haemolysin in marine vibrios is regulated in iron-limited conditions, which occurs in the host
during infection (Stoebner and Payne, 1988).
2. 11. 1. Thermostable direct haemolysin (TDH)
Major pathogenicity of V. parahaemolyticus has been associated with Kanagawa positive
(KP+) isolates capable of producing beta haemolysin on Wagatsuma agar. It is known that the
Kanagawa phenomenon is due the production of TDH, which is highly heat stable at 100°C up to
10 minutes (Nishibuchi and Kaper, 1995). This haemolysin has wide variety of biological
activities, including haemolytic activity, cardiotoxicity, mouse lethality and enterotoxicity
(Honda and Iida, 1993).
The production of TDH is encoded by the gene tdh which is located usually, but not
exclusively in the chromosome. Five variants of tdh genes viz., tdh1, tdh2, tdh3, tdh4 and tdh5
encoding TDH in V. parahaemolyticus have been reported (Kaper et al., 1984; Baba et al.,
1991). Usually, KP+ strains consisted of 2 copies of tdh i.e., tdh1 and tdh2 genes (Nishibuchi and
Kaper, 1990) and were mainly responsible for haemolytic activity. The nucleotide sequences of
tdh1 and tdh2 were not identical but nonetheless were extremely similar (97.2%). The predicted
amino acid sequences of the mature TDH proteins encoded by tdh1 and tdh2 varied in seven out
of 165 residues and the products of tdh1 and tdh2 genes were immunologically indistinguishable
(Nishibuchi and Kaper, 1990). Some strains of V. parahaemolyticus produced weak haemolysis
on Wagatsuma agar (KP intermediate strains) and reason for the weak haemolysis was due to
presence of a single chromosomal copy of tdh gene.
The TDH phenotypes of tdh gene-positive but showing weak Kanagawa phenomenon
(KP+W) or Kanagawa phenomenon negative (KP-) strains were distinctly different from those of
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Kanagawa positive (KP+) strains. The KP- tdh carrying strains have usually one chromosomal
copy of tdh gene, termed as tdh3. In one exceptional KP- strain, an additional tdh gene was
associated with plasmid and this gene was designated as tdh4 (Nishibuchi and Kaper, 1990; Yoh
et al., 1991). The tdh5 gene was cloned from a KP- strain that also carried trh gene and produced
TDH at a very low level (Baba et al., 1991). The V. parahaemolyticus strain carrying tdh3 and
tdh4 gene copies did not produce detectable amounts of tdh-specific RNA transcript (Nishibuchi
and Kaper, 1990). The five variants of tdh gene had >96.7 % sequence identity and encoded
haemolysins with similar biological activities (Baba et al., 1991; Yoh et al., 1991; Nishibuchi
and Kaper, 1995).
The sizes of all the tdh coding sequences examined were identical containing 567 bp
(Nishibuchi and Kaper, 1995). The tdh2 gene was mostly responsible for TDH production
(Nishibuchi et al., 1991). Gene tdh2 contributed to >90 % of the KP while tdh1 was accounted
for 0.5-9.4 % of total TDH under various culture condition (Nishibuchi and Kaper, 1990; 1995).
This difference in the level of expression among the five variants of gene is directly related to the
difference in the promoter strength at position -24 and -34. Okuda and Nishibuchi (1998)
demonstrated that the representative tdh genes, (other than tdh2 genes) i.e., tdh1, tdh3 tdh4, and
tdh5 genes could achieve KP positive level of TDH production by point mutation in the promoter
region at position -34. Other variants of tdh gene are expressed at very low levels in V.
parahaemolyticus (Nishibuchi et al., 1991; Lin et al., 1993; Okuda and Nishibuchi, 1998).
ToxRS is widely distributed and highly homogeneous among Vibrio species, and it
mediates environmentally induced regulation of the virulence gene expression including tdh and
ctxAB expression in different Vibrio species (Lin et al., 1993; Reich and Schoolnik, 1994; Lee et
al., 2000; Crawford et al., 2003). Thus toxRS operon of V. parahaemolyticus plays an important
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role in the regulation of tdh2 gene. However, the strength of the tdh promoter plays a major role
in regulation of tdh expression than toxR gene (Lin et al., 1993; Okuda and Nishibuchi, 1998).
Honda et al. (1992) studied the haemolytic mechanism of TDH, a possible virulence
factor of V. parahaemolyticus, the study shown that TDH acts as a pore-forming toxin. The
effect of TDH is detoxified when it is subjected to the temperature at the range of 60 to 70°C but
is reactivated by additional heating above 80°C. This phenomenon, known as the Arrhenius
effect, is due to the fibrillogenicity of the TDH protein (Fukui et al., 2005). Purified TDH protein
having a molecular weight of 44 kDa is composed of two identical subunits of 22 kDa and
having isoelectric point of 4.9 (Miyamoto et al., 1980). Amino acid analysis of purified TDH
reveals that presence of higher amount of acidic amino acid residues accounted for nearly 43 %
of the total amino acid content and 11 % basic amino acids (Sakurai et al., 1973).
As a pore-forming toxin, TDH damages the erythrocyte membrane by making pores
estimated at 2 nm in diameter and also has the ability to lyse target eukaryotic cells by punching
holes in the plasma membrane (Honda et al., 1992). Evidence suggests that TDH causes
haemolysis by three sequential steps, binding to the erythrocyte membrane by the N-terminal
region, followed by formation of a transmembrane pore, and then disruption of the cell
membrane (Honda et al., 1992; Tang et al., 1995). TDH produced by the V. parahaemolyticus
has been implicated in the pathogenesis of diarrhoeal disease caused by this organism
(Nishibuchi et al., 1992). The activity of TDH in in vitro systems revealed that TDH is
responsible for intestinal fluid secretion as well as cytotoxicity in a variety of cell types
(Raimondi et al., 2000) and the TDH receptor is known to be GT1 ganglioside (Takeda et al.,
1976) and the activity of which enhances in the presence of Ca2+.
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2. 11. 2. Thermostable direct haemolysin-related haemolysin (TRH)
Till 1987, it was believed that the production of TDH is the major virulence property in
strains responsible for the cause of gastroenteritis. The outbreak of gastroenteritis in the Republic
of Maldives showed that apart from the TDH, Kanagawa negative strains also involved in the
pathogenicity of V. parahaemolyticus. The investigation revealed that some clinical strains did
not produce TDH, but produced a haemolysin very similar to it designated as TRH (Honda et al.,
1987a). The gene which encodes for the production of TRH is represented by the trh1 and trh2
subgroups. Molecular genetic analysis of the trh encoding TRH revealed that the trh gene was 68
% homologous to the tdh gene (Nishibuchi et al., 1989). The trh2 gene encoded a polypeptide
composed of 189 amino acid residues which differed from the products of the trh1and tdh2
genes by 30 and 69 residues respectively and found to be partially identical with trh1 and tdh
gene products (Kishishita et al., 1992). The study also revealed that the expression level of trh1
to be higher than that of trh2 genes. Unlike tdh genes, trh genes are transcribed at low levels
(Okuda and Nishibuchi, 1998).The haemolysin produced by the Kanagawa negative V.
parahaemolyticus (TRH) is composed of two subunits of 23 kDa with an isoelectric value of 4.6.
TRH displayed biological properties similar to TDH with respect to enterotoxicity and
cardiotoxicity. TRH also showed marked difference in the sensitivity of various erythrocytes
compared to TDH (Honda and Miwatani, 1988). Like TDH, TRH also showed fluid
accumulation in the rabbit ileal loop test, molecular structure and antigenecity, but distinct from
TDH in amino acid sequence and heat stability (Nishibuchi et al., 1989; Honda et al., 1990).
2. 12. Urease (ure) gene
Urease enzyme present in several bacteria, plants, fungi and algae catalyzes the
hydrolysis of urea to yield ammonia and carbamate (Mobley and Hausinger, 1989, 1995; Lee et
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al., 1992). Urease is a high molecular weight multimeric nickel- containing enzyme and the
tightly bound nickel in urease appears to participate in catalysis. The genes encoding urease
enzyme have been cloned and characterized from various species (Mobley et al., 1995). The
amino acid sequences for all ureases of plant and bacterial origin are closely related (Mobley et
al., 1995). Urease gene, ure has been found to play a significant role in pathogenesis in many
bacteria implicating it as a virulence factor.
Like other vibrios, V. parahaemolyticus does not generally produce urease and thus
considered as negative for Urease activity. However, several studies have reported urease-
positive V. parahaemolyticus was isolated from clinical sources, and environmental samples,
implicated in human gastroenteritis. Interestingly, the urease producing phenotype of V.
parahaemolyticus was characterized by the possession of the trh gene. The urease production
also served as a tool to differentiate many members of enterobacteriaceae, vibrios and
Aeromonas spp. Isolation of the urease positive strains of V. parahaemolyticus from a clinical
sample was first reported in 1979 (Huq et al., 1979). Subsequently, isolation of urease positive
strains from cases of gastroenteritis was reported from various parts of the world (Huq et al.,
1979; Lam and Yeo, 1980; Oberhofer and Podgore, 1982; Nolan et al., 1984; Abbott et al., 1989;
Kaysner et al., 1990; Cai and Ni, 1996; Okuda et al., 1997). Urease production does not seem to
be essential for pathogenicity as most of the isolates with negative urease activity were
associated with virulence (Suthienkul et al., 1995). However, association of ure gene with either
trh and/or tdh genes (Suthienkul et al., 1995; Obata et al., 1996; Okuda et al., 1997) made urease
production a reasonably good marker for pathogenic V. parahaemolyticus carrying trh+ gene
(Suthienkul et al., 1995).
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2. 13. Type three secretion system (T3SS)
The term Type III Secretion System (T3SS) was coined in 1993. These system composed
of complex protein secretion system to deliver bacterial effector proteins into host cells that then
modulate host cellular functions (Galan and Collmer, 1999). These bacterial devices are present
in both plant and animal pathogenic bacteria and are evolutionarily related to the fagellar
apparatus. The T3SS in many Gram negative organisms such as Salmonella, Yersinia,
enteropathogenic Escherichia coli (EPEC), Pseudomonas aeruginosa, Bordetella spp., Ralstonia
spp. and Chlamydia contribute to pathogenesis of the disease (Chevance and Hughes, 2008,
Ghosh, 2004, Gijsegem et al., 2000). The most researched T3SSs are from species of Shigella
(causes bacillary dysentery), Salmonella (typhoid fever), Escherichia coli (food poisoning),
Vibrio (gastroenteritis and diarrhea), Burkholderia (glanders), Yersinia (plague), Chlamydia
(sexually transmitted disease), Pseudomonas (infects humans, animals and plants) and the plant
pathogens Erwinia, Ralstonia, Rhizobium, and Xanthomonas
The T3SS is composed of approximately 30 different proteins, making it one of the most
complex secretion systems in bacteria (Fig 2. 3). Its structure shows many similarities with
bacterial flagella and some of the proteins of T3SS also share amino acid sequence similarity to
flagellar proteins. To be precisly speaking, T3SS is used both for secreting toxins or proteins
related to infection and flagellar components. Although T3SSs are substantially conserved, the
effector molecules they deliver are unique for each bacterial species. The core of the T3SS
apparatus is often referred to as the needle complex. The needle complex is composed of two
distinct portions: (i) a needle structure (ii) a cylindrical basal body. The needle structure projects
from the outer membrane of bacteria and is used in injecting effectors into host cells. The basal
body spans the outer and inner membranes of the bacterium including the periplasmic region and
functions as a channel to transport effector proteins (Ghosh,2004, Ogino et al., 2006).
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2. 13. 1. T3SS in V. parahaemolyticus
Through the complete genome sequencing, two chromosomes have been identified, a
large chromosome I and a small chromosome II. The comparison of V. parahaemolyticus
genome with that of V cholerae indicates that their chromosome I does not differ much in size
(3·3 Mb of V.parahaemolyticus versus 3·0 Mb of V.cholerae) but chromosome II of V.
parahaemolyticus is much larger than that of V cholerae being 1·9 and 1·1 Mb, respectively.
This size difference could have arisen during evolution through acquisition of genes, gene
duplication or by horizontal transfer as in V. parahaemolyticus or through frequent gene decay or
deletion as in V. cholerae. The sequencing also revealed the presence of gene encoding type III
secretion systems (T3SS). It consists of two non-identical T3SS encoded within a pathogenicity
island (Makino et al., 2003). Both T3SSs are located in pathogenicity islands, one on the larger
chromosome I (T3SS-1) and the other on the smaller chromosome II (T3SS-2).
T3SS1 is known to be present in V. parahaemolyticus isolated from clinical and
environmental sources regardless of their pathogenicity and has a G+C content similar to the rest
of the genome indicating that this region is ancestral to the species. T3SS-2 is located
predominantly in the chromosome II of highly virulent strains of V. parahaemolyticus recovered
after 1995, whereas most clinical isolates recovered before 1995 do not encode T3SS-2
indicating that the region is not essential for virulence (Makino et al., 2003, Hurley et al., 2006).
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Fig. 2. 3. Diagrammatic representation of type secretion system in Gram negative bacteria.
(A) T3SS injectisome with the two rings spanning the membranes and the needle
protruding outside the bacterium. (B) Mechanism of T3SS injectisome during infection.
The translocators form a pore into the target cell membrane, and the effectors are
translocated into the cytosol of the target cell. (Source: Troisfontaines and Cornelis, 2005)
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The pathogenicity island of V. parahaemolyticus located on chromosome I encode a
T3SS that is highly homologous to the T3SS found in Yersinia spp. The locus is in the reverse
order of the locus of Yersinia spp. and there is a set of twelve open reading frames (ORFs) that
are predicted to encode hypothetical proteins (Fig.2. 4).
Fig. 2. 4. Organization of V. parahaemolyticus T3SS1 cluster located on chromosome 1
The pathogenicity island located on chromosome II exhibits many of the characteristics
of a “classic” pathogenicity island. The island has insertion elements located at both ends and the
G+C content is lower (39.8%) than the rest of the genome (45.4%) suggesting the acquisition of
these genes through a recent lateral transfer (Terai et al., 1991; Schmidt and Hensel et al., 2004).
This island encodes the tdh genes and the toxRS genes that are known to be involved in
virulence. In addition this, the island also contains the necessary structural genes for a second
T3SS. The list of identified virulence determinants of V. parahaemolyticus are detailed in Table
2. 2. There are also other genes located in the island that are candidates for virulence factors
including VPA1321, a homologue of the E.coli protein cytotoxic necrotizing factor-1. The T3SS-
2 has been demonstrated in rabbit ileal loop model to be involved in cytotoxicity and
enterotoxicity. This data along with the presence of this system in all clinical isolates implies that
it is required for virulence in humans. The gene product of T3SS-1 has been found to induce
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cytotoxicity in cell culture but does not appear to affect enterotoxicity in a rabbit ileal loop
model (Park et al., 2004). In 2009, a second type of T3SS-2 was discovered in trh+ V.
parahaemolyticus strain TH3996 (Okada et al., 2009) which showed significant homology to the
T3SS-2 of KP positive V. parahaemolyticus RIMD2210633. After the report of novel type III
secretion system in trh-positive V. parahaemolyticus, it has been classified into two distinct
phylogroups, T3SS2α (tdh+ve RIMD2210633) and T3SS2β (trh+ TH3996). Genetic organization
of T3SS2α and T3SS2β of V. parahaemoluyticus is depicted in Fig. 2. 5. T3SS2 are also found
in other human pathogenic vibrios such as V. cholerae non-O1/non-O139 strains and V. mimicus.
These findings demonstrate that these two distinct types are distributed not only within a species
but also beyond the species level (Henke and Bassler, 2004; Okada et al., 2010).
Fig. 2. 5. Genetic organization of T3SS2α and T3SS2β of V. parahaemoluyticus located on
the chromosome 2. Red arrows designate type III-related genes, blue arrows designate
genes encoding putative regulatory and putative effector proteins, gray arrows indicate
genes encoding hypothetical proteins (Source: Okada et al., 2009)
The distribution of T3SS2α and T3SS2β is not limited to within a species but goes
beyond the species level. Recently the presence of T3SS2β in V. mimicus has been reported
(Okada et al., 2010). The presence of these secretion system in microorganisms beyond the
species level suggests that the possession of such secretion systems may confer some common
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beneficial effect(s) on the organisms (Fig. 2. 6). Although the nature of such benefit(s) is as yet
unknown.
Fig. 2. 6. Schematic depiction of the hypothetical evolutionary acquisition of a T3SS related
gene cluster in V. parahaemolyticus, V. cholerae and V. mimicus (Source Okada et al., 2009)
2. 13. 1. 1 . Effector proteins of V. parahaemolyticus T3SS2
A number of bacterial pathogens utilize toxins and T3SSs to subvert host signaling
system as a strategy to promote their survival and replication during infection. The pathogenic
strains of V. parahaemolyticus have acquired an additional T3SS in the second chromosome later
during evolution to ensure successful infection in the human host. Various effectors secreted by
T3SS2 target many host factors including MKKs, small Rho GTPases, and F-actin to manipulate
critical signaling pathways and actin cytoskeleton organization. The overall mechanism of
effector proteins of T3SS1 and T3SS2 in inducing infection is detailed in Fig. 2. 7 and Fig. 2. 8.
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VopA/P (Vibrio outer protein A/P) is a homolog of Yersinia effector protein YopJ which
known to inhibits mitogen-activated protein kinase (MAPK) and NFκB signaling pathway (Orth
et al., 1999; Mukherjee et al., 2006; Mukherjee et al., 2007). These effectors belongs to the
acettyltrnaferase group which targets kinase pathway. The VopA/P only inhibits MAPK
pathway and not NFκB signaling (Trosky et al., 2004). Yersenia is primarily an extracellular
bacteria and its effector protein YopJ target multiple pathways and block the host innate immune
response.
Fig. 2. 7. The mechanism of action of effector proteins from T3SS1 of V. parahaemolyticus.
The effectors are translocated into host cells and activates MAPK pathways, rapid
autophagy. disassembly of the actin cytoskeleton and cell rounding, membrane blebbing
and contributing to cell lysis (Source: Zhang and Orth, 2013)
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Fig. 2. 8. Mechanism of action of effector proteins from T3SS2 of V. parahaemolyticus.
T3SS2 injects at least five known effectors into eukaryotic cells which results in actin
rearrangement, bacterial invasion, inhibition of MAPK signaling pathway, formation of
actin stress fibers (Source: Zhang and Orth, 2013)
It also promotes cell death by blocking the NFκB survival pathway. The mechanisms
underlying the MAPK inhibition by YopJ and VopA/P are also different. YopJ acetylates the
critical serine and threonine residues located on the activation loop of MAPK kinases and IKKβ
blocking the phosphorylation sites that are necessary for the activation by upstream kinases.
VopA/P also acetylates the same serine and threonine residues of MKKs thereby preventing
kinase activation in a similar manner. Interestingly, VopA/P modifies an additional lysine also
present in the catalytic loop that is essential for binding to the γ-phosphate of ATP (Trosky et al.,
2007).
VopC is a cytotoxic necrotizing factor which has been identified recently in V. parahaemolyticus
(Zhang et al., 2012). This work highlights the new evidence, demonstrating that V.
parahaemolyticus, which has been no longer thought to be an extracellular pathogen, invades the
host cell (Zhang et al., 2012). V. parahaemolyticus enter the host cells and remain intracellular
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by a process of that is mediated by a T3SS2 effector VopC. VopC shares sequence similarity to
cytotoxic necrotizing factor (CNF) toxins found in Yersinia spp., Bordetella spp., and pathogenic
E. coli. These toxins, once secreted into the host cell, target diverse eukaryotic factors to subvert
host cell systems for the benefit of the pathogen, such as facilitating the invasion of bacteria into
the host cell. The invasion is mediated by the enzymatic activity of VopC, a
deamidase/transglutaminase activity shared by other CNF toxins with conserved catalytic
residues. The deamidation of small Rho GTPases such as Rac and Cdc42 (on glumatine 61) by
VopC renders them constitutively active and promotes actin cytoskeleton rearrangement of the
infected cell so it can engulf the bacteria.
VopL contains three Wiskott-Aldrich homology 2 (WH2) domains that bind to actin monomers
and promote actin nucleation (Liverman et al., 2007). The bacterial cell shape and motility is
controlled by the highly dynamic process of assembly and disassembly of actin cytoskeleton.
Homeostasis is so crucial for cell survival, the actin cytoskeleton is one of the major targets of
many bacterial effectors and is manipulated by diverse mechanisms. VopL triggers an actin-
related phenotype during infection which is to induce massive stress fibers throughout the cell.
2. 14. Genome of V. parahaemolyticus
Genomic Islands (GI) are gene cassettes in the chromosomal region of
V.parahaemolyticus that used to be mobile and are now fixed. It can code for many functions
like symbiosis or pathogenesis and may help an organism's adaptation. Genomic islands have
unusual base composition compared with the core genome of the organism. Hurley et al., (2006)
identified seven genomic islands (Vp-PAI-1 to Vp-PAI-7) in the genome of V. parahaemolyticus
strain RIMD2210633, O3:K6 serotype isolated in Japan in 1996. Five GI regions were present
on chromosome 1 and two on chromosome 2. The GIs namely, Vp-PAI-1, Vp-PAI-2, Vp-PAI-3
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and Vp-PAI-4 are inserted adjacent to tRNA genes. The size of each region ranged from 10 kb to
81 kb and had an unusual base composition as compared to the core genome of V.
parahaemolyticus RIMD2210633. GIs of V. parahaemolyticus RIMD2210633 are flanked by
direct repeats and have lower G+C content (ranging from 38 % to 43%) than the overall genome
G+C content of 45%. These GIs may increase their fitness either for survival in the aquatic
environment or in their ability to infect humans. (Hurley et al., 2006; Boyd et al., 2008; Chao et
al., 2009, 2011). All GIs encoded an integrase gene with the exception of Vp-PAI-7, which
contained a number of transposase genes. Vp-PAI-7 is an 81 kb region that encodes both, a T3SS
and tdh gene and was previously identified as a potential pathogenicity island. The identified Vp-
PAI regions are known to encode putative virulence genes and therefore these regions may
represent potential pathogenicity islands. The study on genomic analysis of pandemic strain of
KP-positive V. parahaemolyticus (RIMD2210633) demonstrates that more than 86 % of the
RIMD2210633 genes are conserved and genes acquired through lateral gene transfer formed a
gene cluster. Among the genes that were variably present, 11 loci were found to be specifically
present in the pandemic strains. This suggests that difference between pandemic and
nonpandemic strains is not due to a simple genetic event (Izutsu et al., 2008). The gene cluster
from VPA1310 to VPA1396 was found to be exclusively conserved in KP-positive pathogenic
strains but not in KP-negative strains. This cluster is described as the “pathogenicity island Vp-
PAI” (Makino et al., 2003, Izutsu et al., 2008) and found to be unique and common to all KP-
positive strains suggesting a strong correlation with pathogenicity. The Vp-PAI is known to be
absent in the KP-negative strains. It is observed that tdh genes and other putative virulence
genes, including T3SS-2, are often located in the 80 kb region of Vp-PAI. From the literature, it
seems that not only the tdh gene but the whole region of Vp-PAI is required for the pathogenicity
of KP-positive V. parahaemolyticus strains.
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The molecular analysis of the worldwide clinical isolates of pandemic and non-pandemic
V. parahaemolyticus belonging to pre-1996 and post-1995 demonstrated the presence of a 24 kb
region named V. parahaemolyticus island-1 (Vp-PAI-1) in O3:K6 and related strains recovered
after 1995. Further analysis of the other regions of genomic islands showed that Vp-PAI-4, Vp-
PAI-5 and Vp-PAI-6 are also highly unique to the virulent clonal complexes of pandemic strains
recovered after 1995 (Hurley et al., 2006). GIs specific to the pandemic strains are Vp-PAI-1 (24
kb), Vp-PAI-4 (17 kb), Vp-PAI-5 (12 kb) and Vp-PAI-6 (27 kb) and among them, Vp-PAI-1 is
suggested to be one of the markers of pandemicity due to the presence of a virulence-associated
gene (Wang et al., 2006, Nishioka et al., 2008). Chao et al. (2009) determined the presence of
functional pandemic strain specific markers (tdh, toxRS new, GS-PCR, orf8, Vp-PAI-1 Vp-PAI-
5, Vp-PAI-7 including T3SS-2). They noted that all the pandemic strains harboured Vp-PAI-1
and Vp-PAI-5 except for one pandemic strain O4:K68 isolated from an outbreak of food
poisoning that lacked Vp-PAI-5 which was possibly due to the chromosomal rearrangement.
From the several studies it is apparent that the O3:K6 strains post-1995 isolated from
different countries had probably evolved from a common ancestor. The arbitrarily primed PCR,
ribotyping, and pulsed field gel electrophoresis (PFGE) on post-1995 O3:K6 strains showed that,
they were genetically similar but significantly different from the genetically variable pre-1995
O3:K6 strains. In addition, the newly emerged O3:K6 clone has diversified into various other
serotypes such as O1: KUT, O4:K68, O1:K25, O4:K12 etc. since its initial isolation. These
strains are postulated to be clonal derivatives of the O3:K6 serotype because they are genetically
similar based on group-specific (GS)-PCR for toxRS and ORF8 PCR for detection of the f237
filamentous phage and the epidemiological techniques of arbitrarily primed PCR, PFGE, and
ribotyping. DNA restriction patterns of O3:K6 strains isolated in Peru and Chile have shown
profiles closely related to strains from Asian countries obtained from the first epidemic in 1996–
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1997 (Gonzalez-Escalona et al., 2005; Martinez-Urtaza et al., 2008). Similar relationships were
obtained through MLST using seven housekeeping genes (Gonzalez-Escalona et al., 2008).
The study of Han et al. (2008) showed that the pandemic clone post-1996 (new O3:K6)
and its serovariants might have emerged or evolved from the old-O3:K6 clone, which was
promoted by stepwise acquisition of genomic islands, toxRS/new sequence and differentiation of
O/K antigen genes. The study also provides direct evidence for the concept that the post-1996
strains of pandemic serovariants evolved from the new O3:K6 (Han et al., 2008). They also
demonstrated that, the acquisition of toxRS/new sequence led to the phylogenesis of the
intermediate-O3:K6 clade from the old-O3:K6 clone, and that the post-1996 new O3:K6
stemmed from this intermediate clade after the acquisition of tdh, Vp-PAI-5 and other
unidentified genes. It is concluded that, intermediate-O3:K6 clade served as the phylogenetic
intermediate between new-O3:K6 and old-O3:K6 and the differentiation of O/K antigen genes
promoted the derivation of new-O3:K6 serovariants from new-O3:K6. Several published
literature highlight the point that new pandemic O3:K6 and its serovariants originated from the
non pathogenic environmental strain of O3:K6 by lateral transfer of virulence genes from other
vibrios (Nair et al., 2007; Boyd et al., 2008; Chao et al., 2011) and the acquisition of additional
serotypes may be a selected response to host immunological pressure. Chowdhury et al.( 2004)
surmised that the transition of major serovars occurred among the pandemic strains and change
in serovar was possibly related to the change in the environmental conditions.
Multilocus sequence typing (MLST) is a procedure to characterize isolates of bacterial
species using the DNA sequences of internal fragments of multiple housekeeping genes. It plays
a role in studying epidemiology of infectious diseases, generating the information necessary for
identifying, tracking, and intervening against disease outbreaks. (Maiden et al., 1998; Urwin et
al., 2003). MLST provided strong molecular evidence for the clonal origin of V.
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parahaemolyticus O3:K6 and revealed that isolates within such a clonal group may acquire
previously identified serotypes of V. parahaemolyticus. The MLST study also confirmed genetic
diversity among the V. parahaemolyticus strains that prevailed before O3:K6 and genetic
uniformity between O3:K6 and its serovariants in spite of their serotype diversity.
The sequence analysis of the amplified house keeping genes (gyrB, recA, dnaE and gnd) of
different serovars of V. parahaemolyticus showed that prepandemic strains were highly variable
and the pandemic O3:K6 isolates shared two alleles (Chowdhury et al., 2004b). The study on
molecular analysis of the emergence of pandemic V. parahaemolyticus, clustered the diverse
panel of 42 V. parahaemolyticus belonging to 10 different serotypes into two closely related but
distinct groups based on the MLST data (Boyd et al., 2008). The former contained highly
virulent isolates whereas the latter comprised mainly environmental isolates recovered in early
2000. González-Escalona et al. (2008) found that the O3:K6 pandemic clone was the clone
complex CC and confirmed the first reported pandemic spread of V. parahaemolyticus. CC
corresponded to the pandemic strains with four different sequence types, ST-3, ST-42, ST-27
and ST-51. ST-3 was defined as the ancestral type or founder of the clonal complex CC and
unequivocally established the clonal relationship of the pandemic complex. The common feature
between nonpathogenic and pandemic strains is the presence of Vp-PAI-3. Further it has been
shown that, the acquisition of Vp-PAI-3 was the first step to form pandemic strains. The ancestor
of O3:K6 pandemic clone is believed to be O3:K6, ST-3 environmental non-pathogenic strain
(Chao et al., 2011). Literature suggests that the origin of pandemic clones was not from tdh+
(embedded in Vp-PAI-7) or trh+ strains, and the Vp-PAI-7 was not the island obtained after Vp-
PAI-3 and Vp-PAI-2. As Vp-PAI-1, Vp-PAI-5 play an important role in forming pandemic
clone, Vp-PAI-7 was acquired after Vp-PAI-1 and Vp-PAI-5; previous studies showed that Vp-
PAI-4 may be the last genomic island acquired by pandemic clones (Chao et al., 2011). In
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conclusion, based on the environmental strain (O3:K6, ST-3), the pandemic O3:K6 clone was
formed around 1996 by lateral transfer of large fragments of genes to obtain systematic virulence
genes and genomic islands.
Table 2. 1. Chronology of appearance of Vibrio parahaemolyticus O3:K6 and its serovariants in different countries
Serotype Country (Yr of isolation) Reference(s) Serotype Country (Yr of isolation) Reference(s) O3:K6 Taiwan (1995)
India (1996) Chiou et al., 2000 Okuda et al., 1997, Chowdhury et al., 2000
O1:KUT India (1998) Chowdhury et al., 2000b
Vietnam (1997) Chowdhury et al., 2004 Bangladesh (1998, 2000) Bhuiyan et al., 2002, Matsumoto et al., 2000
Laos (1997) Matsumoto et al., 2000 O4:K12 Thailand (1998–1999) Laohaprertthisan et al., 2000 Indonesia (1997) Okuda et al., 1997 Vietnam (1998–1999) Chowdhury et al., 2004 Korea (1997–98) Matsumoto et al., 2000 Chile (2004) Gonza´lez-Escalona et al., 2005 Chile (1998 and 2004) Cabello et al., 2007, Gonza´lez-Escalona et al., 2005 United states (2006) Balter et al., 2006 Bangladesh (1996–2000) Bhuiyan et al., 2002 O1:K41 Thailand (1998–1999) Laohaprertthisan et al., 2000 Japan (1998) WHO, 1999 Vietnam (1998–1999) Chowdhury et al., 2004 Thailand (1999) Chowdhury et al., 2000,
Laohaprertthisan et al., 2000 O1:K56 Vietnam (1998–1999) Chowdhury et al., 2004
Russia (2001) Smolikova et al., 2001 O3:K75 Vietnam (1998–1999) Chowdhury et al., 2004 France (2004) Quilici et al., 2005 O4:K8 Vietnam (1998–1999) Chowdhury et al., 2004 Mozambique (2004) Ansaruzzaman et al., 2005 O4:KUT Vietnam (1998–1999) Chowdhury et al., 2004 Europe (2004) Martinez-Urtaza et al., 2005 O5:KUT Vietnam (1998–1999) Chowdhury et al., 2004 Peru (1996) Gil et al., 2007 India (2004) Nair et al., 2007 Italy (2007-2008) Ottaviani et al., 2008; 2010 O5:K17 India (2002) Nair et al., 2007 Northeast Brazil (2008) Leal et al., 2008 O5:K25 India (2002) Nair et al., 2007 China (2005-08) Chao et al., 2009 O1:K33 India (2002) Nair et al., 2007 Thailand (2001-2002, 2000 -03) Bhoopong et al., 2007; Serichantalergs et al., 2007 O2:K3 India (2002) Nair et al., 2007
O4:K68 India (1998) Chowdhury et al., 2000a OUT:KUT India (2003–2004) Nair et al., 2007 Thailand (1999) Chowdhury et al., 2000b O3:KUT India (2003–2004) Nair et al., 2007 Bangladesh (1998 and 2000) Bhuiyan et al., 2002 O3:K5 India (2004) Nair et al., 2007 Vietnam (1998) Chowdhury et al., 2004 O4:K4 India (2004) Nair et al., 2007 Mozambique (2004) Ansaruzzaman et al., 2005 O4:K10 India (2004) Nair et al., 2007
O1:K25 India (1998, 2002) Nair et al., 2007, Chowdhury et al., 2000b, Matsumoto et al., 2000
O6:K18 Taiwan (2005) Nair et al., 2007
Thailand (1999, 2001-2002 ) Serichantalergs et al., 2007, Laohaprertthisan et al., 2000
O1:K26 China (2005-2008) Chao et al., 2009
Vietnam (1998–1999) Chowdhury et al., 2004 O4:K68 China (2005-2008) Chao et al., 2010 Bangladesh (1999–2000) Bhuiyan et al., 2002 O1:K36, China (2005-2008) Chao et al., 2010 China (2005-2008) Chao et al., 2009 O3:K25 China (2005-2008) Chao et al., 2010
O1:KUT India (1998) Chowdhury et al., 2000b O3:K68 China (2005-2008) Chao et al., 2010 Bangladesh (1998 and 2000) Bhuiyan et al., 2002, Matsumoto et al., 2000 O3 : K46 Thailand (2001-2002) Serichantalergs et al., 2007 Italy (2008) Ottaviani et al., 2010 China (2005-2008) Chao et al., 2009 Thailand (2001-2002) Serichantalergs et al., 2007
Table 2. 2. List of virulence factors present in V. parahaemolyticus
Name Domain Activity and Function References
Toxins and adhesin TDH Thermostable direct hemolysin It is an pore forming toxin cytotoxicity and enterotoxicity Nishibuchi and Kaper, 1995 TRH TDH-related hemolysin It is an pore forming toxin cytotoxicity and enterotoxicity Nishibuchi and Kaper, 1995 MAM7 mce domain Binds to fibronectin and phospholipid phosphatic acid
and helps in attachment to host cell Krachler et al., 2011
T3SS1 Effectors VopQ Unknown Unknown ---- VopS Fic domain AMPylates Rho family GTPases and Disrupts actin cytoskeleton ---- VPA0450 Inositol polyphosphate 5-phosphatase Disrupts plasma membrane integrity by hydrolyzing PI(4,5)P2 to PI4P
----
T3SS2 Effectors VopA/P Acetyltransferase Suppress immune response Trosky et al., 2004 VopC Cytotoxic necritizing factor Bacterial invasion Zhang et al., 2012 VopL WH2 domain Induce actin stress fiber Liverman et al., 2007 VopV Unknown Cytotoxicity and enterotoxicity ---- VopT ADP-ribosyltransferase Unknown ----
reChapter 2 Review.pdfChapter 2 Review.pdf2. 2. Characteristics of V. parahaemolyticus2. 3. Geographical distribution of V. parahaemolyticus2. 4. Distribution of V. parahaemolyticus in marine environments2. 5. Prevalence of V. parahaemolyticus in fish and shellfish2. 6. Association with planktonic organisms2. 8. Epidemiology2. 9. Pandemic clone of V. parahaemolyticus2. 10. Virulence properties2. 12. Urease (ure) gene2. 13. 1. T3SS in V. parahaemolyticus2. 14. Genome of V. parahaemolyticusGenomic Islands (GI) are gene cassettes in the chromosomal region of V.parahaemolyticus that used to be mobile and are now fixed. It can code for many functions like symbiosis or pathogenesis and may help an organism's adaptation. Genomic islands have...The molecular analysis of the worldwide clinical isolates of pandemic and non-pandemic V. parahaemolyticus belonging to pre-1996 and post-1995 demonstrated the presence of a 24 kb region named V. parahaemolyticus island-1 (Vp-PAI-1) in O3:K6 and rela...From the several studies it is apparent that the O3:K6 strains post-1995 isolated from different countries had probably evolved from a common ancestor. The arbitrarily primed PCR, ribotyping, and pulsed field gel electrophoresis (PFGE) on post-1995 O3...The study of Han et al. (2008) showed that the pandemic clone post-1996 (new O3:K6) and its serovariants might have emerged or evolved from the old-O3:K6 clone, which was promoted by stepwise acquisition of genomic islands, toxRS/new sequence and diff...Multilocus sequence typing (MLST) is a procedure to characterize isolates of bacterial species using the DNA sequences of internal fragments of multiple housekeeping genes. It plays a role in studying epidemiology of infectious diseases, generating th...
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