IV. Results

II. REVIEW OF LITERATURE

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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

Review of literature

Page 5

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


Page 6

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


Page 7

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


Page 8

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


Page 9

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


Page 10

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


Page 13

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


Page 15

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


Page 17

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.


Page 18

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


Page 19

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


Page 20

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


Page 21

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


Page 22

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+.


Page 23

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


Page 24

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).


Page 25

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).


Page 26

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).


Page 27

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)


Page 28

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


Page 29

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


Page 30

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.


Page 31

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)


Page 32

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


Page 33

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


Page 34

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.


Page 35

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–


Page 36

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.


Page 37

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


Page 38

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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