Vibrio parahaemolyticus: a review on the pathogenicity, antibiotic resistance, foodborne outbreaks, and detection methods

Vibrio parahaemolyticus is a Gram-negative bacterium that is a natural inhabitant of the marine habitat. V. parahaemolyticus is a human foodborne pathogen linked to the consumption of contaminated raw and undercooked seafood. V. parahaemolyticus pathogenicity has been linked to the presence of two virulence gene that is thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). The emergence of antibiotic resistant strain of V. parahaemolyticus is a menace to public health. V. parahaemolyticus is linked to several foodborne diseases in Asian countries including Japan, China and Taiwan and has been acknowledged as the major cause of human gastroenteritis in the United States. The emergence of pathogenic Vibrio species in shellfish in Malaysia requires persistent monitoring and public enlightenment on food safety. Several detection methods based on its virulence factors are used in detecting V. parahaemolyticus. This review will provide an insight on V. parahaemolyticus, its pathogenicity, antibiotic resistance, foodborne outbreaks and detection methods.


Introduction
Vibrio parahaemolyticus belongs to the Vibrionaceae family. They are Gram-negative, ubiquitous, halophilic facultative anaerobic bacteria found in marine, estuarine environments that are positive to the biochemical test catalase and oxidase. They can survive at a temperature between 5ºC and 43ºC and are grown optimally at 37ºC. The Vibrio genus consists of 142 species that are mainly found in the marine environment (Sawabe et al., 2013). V. parahaemolyticus is capable of causing infection in human. V. parahaemolyticus uses flagella to move freely in water or fixed to an animate object such as a shellfish (Godepotratz et al., 2011). V. parahaemolyticus possesses a dual flagella system which helps them adapt to the different environment. The polar flagella help with movement while the lateral flagella involved in biofilm formation (Broberg et al., 2011). V. parahaemolyticus is regarded as the most prevalent pathogen associated with seafood and many outbreaks have occurred worldwide as a result of ingestion of raw and undercooked seafood which resulted in the inflammation of the bowel. The availability of this pathogenic bacteria in the marine habitat should be a great concern to humans due to the consistent outbreak of the disease (Ceccarelli et al., 2013). V. parahaemolyticus causes infection by attaching itself to the fibronectin and phosphatidic acid on the host cell thereby unleashing different toxins into the cytoplasm of the host cell and this will lead to a lifethreatening illness (Gode-Potratz et al., 2011). V. parahaemolyticus was first discovered in the 1950s in Japan as a foodborne disease with a huge outbreak originating from the prevalent serotype O3: K6 from 1997to 2001(Hara-Kudo et al., 2012. Several studies have reported a high prevalence of V. parahaemolyticus in shellfish in Malaysia (Al-Othrubi et al., 2014;Sahilah et al., 2014;Tang et al., 2014;Malcolm et al., 2015). V. parahaemolyticus has been reported as the major causative agent of seafood associated gastroenteritis in several countries such as the United States and Asian countries (Scallan et al., 2011).

Pathogenicity of Vibrio parahaemolyticus
It is a known fact that a great number of V. parahaemolyticus strains detected from environmental samples are not pathogenic. A great number of the pathogenic V. parahaemolyticus are isolated from clinical samples. V. parahaemolyticus is capable of producing three hemolysins; thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH) and thermolabile hemolysin (TLH) encoded by the tdh, trh and tlh genes (Paranjype et al., 2012). The pathogenicity of V. parahaemolyticus is dependent on their ability to produce the virulent gene thermostable direct hemolysin (tdh) and thermostable direct hemolysin related hemolysin (trh) (Gutierrez West et al., 2013). The T3SS1 guarantee the survival of V. parahaemolyticus strain in its habitat. In addition, the T3SS1 are capable of causing damage to the infected host cell thus essential nutrients are released (Paranjpye et al., 2012). The thermolabile hemolysin (TLH) is another type of hemolysin encoded by the tlh gene and also causes the breakdown of red blood cells (Wang, Xiang, Feng et al., 2013). The tlh gene also causes the breakdown of human erythrocytes (Broberg et al., 2011). Aside V. parahaemolyticus possessing the virulence gene which increases its pathogenicity, the presence of the flagella for its movement also contributes to the pathogenicity because it is able to produce a capsule thereby helping the pathogen move freely and survive in its habitat and a human host (Broberg et al., 2011).

Pathogen fixation to the host cell
The ability of the bacteria to fix itself into the host cell is a crucial step in which disease develops. V. parahaemolyticus is capable of binding to several host cells such as epithelial cell, fibroblasts and macrophages (Stones and Krachler, 2015) due to the presence of the multivalent adhesion molecule 7 (MAM7) found in many Gram-negative bacteria (Krachler et al., 2011). The MAM7 is made up of a water-repelling stretch of 44 amino acids at its N terminus (Letchumanan et al., 2014) MAM7 consists of two host surface receptor which is; host membrane phosphatidic acid lipids (PA) which helps the bacteria bind firmly to its host cell and the extracellular matrix protein fibronectin which acts as a co-receptor to enhance the entry of the pathogen to the host cell (Lim et al., 2014) MAM7 is made up of seven mammalian cell entry domain and all seven possess the PA binding (Lim et al., 2014). When MAM7 bind to fibronectin and PA, an obstruction can occur during the fixation of MAM7 to the host cell if one of the surface receptors is blocked. Furthermore, MAM7 is important for primary host binding once the disease occurs and for T3SS-mediated cell death in some cell types. This information on MAM7 gives an insight on the relationship between bacterial and host cell (Krachler et al., 2011). T3SS is important for the pathogenicity of different Gram-negative bacteria and mainly used by the bacteria for its growth and survival inside the host cell (Dean, 2011). T3SS uses a needle-like apparatus to transfer toxins and bacterial proteins also called effectors into the host cells thereby targeting and hijacking several eukaryotic signalling pathways. The release of these effectors from bacteria is caused as a result of contact between the bacteria and host (Dean, 2011).

Toxins produced by Vibrio parahaemolyticus
V. parahaemolyticus exhibit different virulence factors such as the production of toxins which increases their chances of causing gastrointestinal infection (Liu and Chen, 2015). V. parahaemolyticus are largely found in the marine and coastal habitat but not all strains are virulent (Velazquez-Roman et al., 2012). The thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh) are the main toxins found in V. parahaemolyticus. V. parahaemolyticus becomes virulent when they possess the tdh and trh gene which are responsible for hemolysis and cytotoxicity activity in the host cell (Broberg et al., 2011;Zheng et al., 2014). The strains collected from the environmental samples do not possess the tdh and trh gene. Thus, most environmental strains do not cause disease to humans and animals (Gutierrez West et al., 2013). However, studies have shown that even when the tdh and/or trh genes are absent, V. parahaemolyticus remains virulent and this shows there are other virulence factors attributed to the bacterium (Jones et al., 2012;Pazhani et al., 2014). Compared to the environmental samples, the clinical samples that cause gastroenteritis in human possess the tdh and trh gene which are able to break red blood cells on Wagatsuma blood agar. This hemolytic activity is called the Kanagawa phenomenon (KP) (Alipour et al., 2014). Out of all environmental samples, only 1 to 2% is said to be KP-positive while others are regarded as KP-negative (Alipour et al., 2014). TDH is a toxin that is capable of forming pores on the red blood cells and this allows for free movement of water and ions through the membranes which later leads to swelling of the cell and death in most cases (Raghunath, 2015). The TDH targets the epithelial and intestinal cell thereby causing severe diarrhoea (Shimohata and Takahashi, 2010). TRH is similar to TDH in terms of its hemolytic activity on blood cells (Ham and Orth, 2012) TRH can be destroyed when subjected to heat (Shimohata and Takahashi, 2010) even though it is being reported to be more pathogenic than TDH (Saito et al., 2015). In addition, TLH is another type of toxin that influences the pathogenicity of V. parahaemolyticus (Zhao et al., 2011) and it is often referred to as a species-specific marker. The TLH possess the phospholipase activity and are able to break down the human red blood cell (Broberg et al., 2011) Thus, TLH and TDH play a similar role in the pathogenicity of V. parahaemolyticus (Wang et al., 2015). V. parahaemolyticus is classified based on the somatic (O) and capsular (K) antigen which is formed in different environmental condition (Nair et al., 2007). Serovar is also used in detecting V. parahaemolyticus and its pathogenicity. Since V. parahaemolyticus is a multi-serovar bacterium that comprises 12 different O antigens and more than seventy different K antigens in its capsule . Out of this serovar, the serovar (O3:K6, O4:K68 and O1:K untypeable) are the most lethal and deadly to humans and are recognized as the main cause of the foodborne disease (Jones et al., 2012). The toxic strains of V. parahaemolyticus cause severe inflammation of the bowel and are usually spread through consuming raw or semi-cooked seafood (Zarei et al., 2012). In February 1996, a distinctive serotype O3:K6 of V. parahaemolyticus accompanied by a discrete genetic marker instantly occurred in Kolkata, India. Consecutively, a similar serotype isolated from India has been isolated from other foodborne disease outbreak in Southeast Asia, Europe, Africa and the United States (Velazquez-Roman et al., 2014). The prevalent O3:K6 strain was detected in Peru and then spread to Chile in 1998 with over 16,804 occurrences, then to the United States in 1998 with over 700 occurrences than to Brazil in 2001 with more than 18 occurrences and Mexico in 2004 with more than 1200 occurrences (Velazquez-Roman et al., 2014).

Emergence of Vibrio parahaemolyticus pandemic clone
The incidence of V. parahaemolyticus occurs irregularly showing no link between distinct serovar of V. parahaemolyticus and gastroenteritis (Hernandez-Diaz et al., 2015). In molecular epidemiological studies, a distinct serovar O3:K6 was isolated from clinical samples (Velazquez-Roman et al., 2012) and this has been a global menace in Africa (Ansaruzzaman et al., 2008), Asia (Li et al., 2016), Europe (Martinez-Urtaza et al., 2005) and the United States (Velazquez-Roman et al., 2014). The swift spread of the O3:K6 serovar has made V. parahaemolyticus an important foodborne pathogen to the general public worldwide (Ceccarelli et al., 2013). In 2007, several serovars of V. parahaemolyticus has been discovered with similar genotypes and molecular profile as the O3:K6 and were termed 'serovariants', in which O4:K68, O1:K25 and O1:KUT are the most common serovar (Nair et al., 2007). V. parahaemolyticus genetic diversity was made easy due to the influence of a multilocus sequence typing (MLST) scheme (González-Escalona et al., 2008). The O3:K6 serovar was able to spread worldwide due to the presence of the open reading frame 8 (orf8) through phage f237 infection thereby increasing its potency level because the orf8 protein product influences the ability to be able to bind to the intestine of the host cell (Ceccarelli et al., 2013).

Antibiotic resistance of Vibrio parahaemolyticus
Antibiotics resistance has now been seen as a global menace to the public health and food industry (FAO, 2016). The continuous use and abuse of antibiotics by humans, in the marine industry, agriculture and livestock have led to the emergence and spread of resistant strains. Also, aside from the antibiotic resistance strains, the multidrug-resistant bacterial strain is another menace because the bacterial becomes resistant to several antibiotics that would have been able to kill or inhibit its growth. The multidrug-resistant bacterial could be a result of chromosomal DNA mutations, enzymatic inactivation, transformation as well as conjugation (Van Hoek et al., 2011). In the marine industry, antibiotics are used indiscriminately in fish farming to enhance the growth of aquatic animals and also reduce the risk of bacterial infection most especially the Vibrio species. Since Vibrio species are mainly inhabitants of the marine environment capable of causing infections in aquatic fish (Sudha et al., 2014). Hence, the continuous use of this antibiotics has led to an increase in the antibiotic resistant strains of Vibrio species (Letchumanan et al., 2015). In fish farming, tetracyclines, erythromycin, sulphonamides, oxytetracyclines, chlortetracycline and amoxicillin are used in some part of Asian including Malaysia, Myanmar and Philippines whereas nitrofurans, chloramphenicol and dimetridazole were banned in most countries (Weese et al., 2015). V. parahaemolyticus infection has been linked to oysters, clams, cockles, mussels, crabs and shrimps (Malcolm et al., 2015). The Malaysian communities are seen as a huge consumer of seafood with an increase of above 40kg/capita/ year since 1970 thereby generating a means of income to the fish farmers (Witus and Van, 2016). An increase in the aquaculture industry as a result of the demand for fish has led to a weakened immune system thereby exposing the fish to bacterial infections. Continuous use of antibiotics will increase the resistant strains of Vibrio species (Lee and Raghunath, 2018). Also, these resistant strains can be transferred from human through the consumption of contaminated foods and can also be transferred to other bacteria thereby causing harm to the public (Kim et al., 2013). In recent studies, Carbapenems was reported as the last resort for treating Gram-negative and Gram-positive infections and infections caused by multidrug-resistant bacteria. However, there is an increased occurrence of carbapenem-resistant bacteria (Martin et al., 2018). Carbapenem-resistant Vibrio spp. has been detected from environmental seafood samples (Gu et al., 2014). The consistent increase in antibiotic resistant and multidrug-resistant strain of V. parahaemolyticus is an important public health issue (Xie et al., 2017;Syamimi-Hanim and Tang, 2019 A foodborne disease outbreak is a continuous occurrence of the same disease emerging as a result of consumption of the same food. In the United States, about 97% of the fish and shellfish consumed are imported and this percentage has increased over time. Between 1996Between -2014 outbreaks were associated with foods imported to the country which led to 10,685 illness, 1,017 hospitalizations and 19 death (USDA, 2016). Also, the Asian countries are most susceptible to the V. parahaemolyticus epidemic because the temperature and climate in this region support the development of this bacteria. Recently, the ministry of health has reported a foodborne outbreak in which about 3% of the disease was linked to V. parahaemolyticus (Bilung et al., 2005). Foodborne outbreaks caused by V. parahaemolyticus mainly occur in places with high consumption of seafood. In the United States, (Iwamoto et al., 2010) reported an outbreak of the disease via the consumption of undercooked seafood. The infection can occur after consuming food that has been contaminated and the disease gives symptoms such as frequently passing out watery faeces and vomiting (Yeung and Boor, 2004). V. parahaemolyticus associated with consumption of raw or improperly cooked seafood commonly occur in summer (Khan et al., 2002). According to the Korean Ministry of food and drug safety, consumption of contaminated seafood has been one of the leading causes of food poisoning. In Korea, V. parahaemolyticus outbreak occurs mainly in August and September with just a few cases in June, July and October but in 2004, there was an outbreak of the disease in April and May. There has been an increase in V. parahaemolyticus in Canada since 2009 from 0.3/100000 in 2005 to 1.4/100000 in 2014. The reason for the increase remains unknown but it could also be as a result of environmental temperature or a deliberate consumption of raw shellfish. Also, it has been reported in several states in Venezuela an outbreak of V. parahaemolyticus leaving 36% of the people infected hospitalized but no death was recorded (CDC, 2018). Thus, following the detection of V. parahaemolyticus, more than 25% of the foodborne outbreak caused by the consumption of contaminated seafood has been linked to this bacterium (Alam et al., 2002).

Conventional method
The most probable number (MPN) is one of the common methods used to detect and enumerate V. parahaemolyticus in food samples, but the method is time-consuming and tedious. In order to improve sensitivity and detection, alkaline peptone water serves as an enrichment broth for V. parahaemolyticus because it has a pH level (8.5-9) which supports the growth of V. parahaemolyticus and a high sodium chloride content (NaCl) thereby inhibiting the growth of other bacteria (DePaola and Kaysner, 2004). Other selective broths used in place of APW are salt polymyxin broth (SPB), alternative protein source (APS) broth, salt colistin broth, glucose salt teepol broth and bile salt sodium taurocholate broth (Bisha et al., 2012). TCBS is the most widely used selective agar for detection of V. parahaemolyticus and more preferred because of its sucrose/bromothymol blue diagnostic system which separates sucrose positive Vibrios such as V. cholerae from other forms of Vibrio. V. cholerae will produce a yellow colony on TCBS agar (Mrityunjoy et al., 2013) while V. parahaemolyticus will produce a green or bluegreen colony on TCBS agar (Bisha et al., 2012). With several studies on the detection of V. parahaemolyticus from seafood, scientists have not been able to differentiate between the colonies of V. parahaemolyticus from other Vibrio colonies on TCBS agar since TCBS is a general selective media for all Vibrio spp. (Bisha et al., 2012). Therefore, a new enrichment and selective agar solely for the identification of V. parahaemolyticus in seafood was discovered by Hara-Kudo et al (2001). The samples were enriched in SPB and streaked on chromogenic CHROMagar. The CHROMagar is made of a colourimetric substrate for β-galactosidase and produced mainly for the identification of ortho-nitrophenyl βgalactosidase-positive V. parahaemolyticus from another identical Vibrio spp. (Bisha et al., 2012). V. parahaemolyticus produce a mauve colour on CHROMagar which makes it easier to differentiate from other Vibrio species. Therefore, several scientists have concluded that CHROMagar gives more sensitive than TCBS agar in detecting V. parahaemolyticus (Su and Liu, 2007). In addition, V. parahaemolyticus can also be detected via colony hybridization. This process involves counting the bacteria on agar plates and confirming the bacteria counted via DNA hybridization. In most cases, every bacterium has its own probes based on variable regions of the 16S rRNA (Thompson et al., 2004). Several studies have shown the counting of bacteria using the radioactive DNA probes or non-radioactive DNA probes in colony hybridization (Deepanjali et al., 2005). Thus, colony hybridization is mainly used to ascertain the identity and amount of pathogenic V. parahaemolyticus present in seafood (Suffredini et al., 2014). MINI REVIEW pathogenic gene that influences the production of a distinct hemolytic ring on blood cell agar plates in high salt concentration with D-mannitol as carbon source and this process is called the Kanagawa phenomenon (KP) (Nishibuchi and Kaper, 1995). In past studies, KP was the benchmark for the identification of pathogenic and non-pathogenic V. parahaemolyticus strains (Ono et al., 2006) but this method is not realistic, time-wasting and a lot of samples are used . The Wagatsuma agar consists of human or rabbit blood with NaCl, mannitol, crystal violet and K 2 HPO 4 . This agar is mainly used to differentiate between the tdh and non-tdh strains. Since V. parahaemolyticus strains containing the tdh gene will break down the Wagatsuma agar (Alipour et al., 2014). Therefore, the development of a more sensitive and rapid method for identification and detection is important for the general well-being of the public.

PCR Detection
Polymerase chain reaction (PCR) is a reliable and easy way for distinguishing specific pathogenic bacteria. Multiplex PCR targets the toxR, tlh, tdh, trh of V. parahaemolyticus from environmental and clinical samples (Paydar et al., 2013). The PCR technique is highly sensitive and specific when detecting virulent bacteria from clinical and environmental samples (Nelapati et al., 2012). The toxR gene stimulates the formation of tdh gene which is found in pathogenic or non-pathogenic V. parahaemolyticus isolates (Sujeewa et al., 2009). In some cases, the thermolabile hemolysin (tlh) in V. parahaemolyticus can be used to produce a multiplex PCR technique for the detection of pathogenic V. parahaemolyticus (Yi et al., 2014). Although, tlh is not recognised as a virulent gene in V. parahaemolyticus but it is a threshold of the bacteria (Su and Liu, 2007). The multiplex PCR assay is a recognized and most used method for distinguishing between V. parahaemolyticus, V. cholerae and V. alginolyticus and the real-time PCR can be used to process a large number of samples and it gives a consistent result in amplifying the targeted gene . In recent times, PCR has been used in different fields of study such as forensics, molecular biology, molecular evolution, sequencing, genome mapping and even in diagnosing hereditary diseases. PCR was first developed by Kary Mullis in 1983 and is now used worldwide due to its availability, flexibility and versatility. PCR is often used for the swift detection of V. parahaemolyticus and its virulence gene. The PCR enables for swift amplification of specific DNA fragments from several pools of DNA by detecting its accumulation in real-time by measuring the increase in a fluorescent signal. The technique works by amplifying DNA fragments by using the DNA polymerase and two primers and then the agarose gel electrophoresis is used to detect the amplified fragments. A serogroup-Ospecific PCR assay was used in the isolation and identification of V. parahaemolyticus from clinical and environmental samples (Chen et al., 2012). The multiplex real-time PCR with separate fluorescent probes was used in detecting pathogenic V. parahaemolyticus in different seafood (Robert-Pillot et al., 2010). A quantitative PCR technique with propidium monoazide has been used to amplify the viable V. parahaemolyticus cells in raw seafood . Therefore, detection of V. parahaemolyticus using PCR gives a rapid result which is very accurate, but the PCR cycler needs to be optimized frequently to get an accurate result (Letchumanan et al., 2014).
With different technology advancement, Loopmediated isothermal amplification (LAMP) has been developed for the detection of V. parahaemolyticus and often used in place of the PCR (Notomi et al., 2000). It is a technique for DNA amplification under isothermal condition using specific primers (Qi et al., 2012). The benefit of the LAMP-based assay to the PCR is that during LAMP, amplification of nucleic acid takes place at a single temperature, thus the thermal cycler is not needed. The LAMP is used to identify tdh-positive isolates of V. parahaemolyticus targeting six regions of the tdh gene and comparing the results to PCR for detection of tdh and reverse passive latex agglutination for tdh detection (Nemoto et al., 2009). A novel LAMP in its original place was used in identifying V. parahaemolyticus strains and it gave a more rapid detection with high accuracy and less time compared to the regular LAMP and other PCR techniques (Wang, Shi, Su et al., 2013). In a recent study, Zeng et al. (2014) developed a novel LAMP-based method that combines the LAMP assay with immunomagnetic separation to identify V. parahaemolyticus in raw oysters.
Nevertheless, LAMP is a cost-effective method to detect pathogenic bacteria at a single temperature without using a cycler like the PCR, but the methods of targeted separation and enrichments influence the application of LAMP. In addition, the rpoD and toxR gene of V. parahaemolyticus strains were identified using the LAMP assay and this method gave an accurate result of 3.7 and 459 CFU per test in pure culture (Nemoto et al., 2011).

Immunological detection
The immunological detection method is often used for the swift identification and measurement of foodborne pathogens present in seafood. This method is dependent on the monoclonal antibodies of the pathogen.  (Sakata et al., 2012). Although, monoclonal antibodies are not enough to detect all environmental and clinical V. parahaemolyticus strains because other bacteria present in the samples can interfere with the process thereby giving a false interpretation (Prompamorn et al., 2013). In recent times, the use of recombinant antibody fragments like the single-chain variable fragments (scFvs) is important for researching, identifying and treating diseases (Wang et al., 2014). Wang et al. (2012) concealed a virulent tlh V. parahaemolyticus strain in a high affinity scFv antibody. The concealed scFv-LA3 antibody is active against the tlh V. parahaemolyticus strain. The scFv-LA3 precisely identified the tlh produced by the V. parahaemolyticus (Wang et al., 2014). Therefore, this method can be used to detect the tlh V. parahaemolyticus strains in seafood .

Conclusion
V. parahaemolyticus is a bacterium naturally occurring in marine and estuarine habitat and a leading cause of seafood-borne diseases. The research about the health implications of V. parahaemolyticus will most likely continue in future as long as seafood remains the vehicle of transmission of this bacterium. V. parahaemolyticus possess several virulence factors that cause hemolytic activity, gastroenteritis and sometimes death. The pathogenicity of V. parahaemolyticus is dependent on its virulence factors. Therefore, it is essential to gather more knowledge on the different virulence factors and how to regulate them. The high occurrence of antibiotic resistant V. parahaemolyticus in the marine environment is due to the continuous exposure to conventionally used antibiotics. Therefore, it is important to implement the use of probiotics in order to reduce the use of antibiotics thereby reducing threats to public health. The consistent outbreak of V. parahaemolyticus shows that the existing preventive measures to control the infection have not been effective as the infection continues to occur annually. Thus, it is important to develop a coordinated measure to eradicate the occurrence of the infections. Many detection methods are used in isolating and identifying V. parahaemolyticus depending on its virulence factors. Nonetheless, the PCR method gives a rapid, reliable, and more efficient result.

Conflict of interest
The authors declare no conflict of interest.