International Journal of Health and Pharmacy Unveiling the Toxin Genes of Vibrio cholerae in Seafood: A Collaboration of Phenotypic and Genotypic Approaches Ni Luh Budi Artaningsih1.
I Putu Bayu Mayura2*.
Ni Nyoman Sri Budayanti3
1,2,3
Program Studi Magister Ilmu Biomedik.
Fakultas Kedokteran Universitas Udayana.
Indonesia Corresponding author:
Email: bayu_mayura@unud.
Abstract.
Vibrio cholerae is a pathogenic bacterium that causes cholera, a severe diarrheal disease transmitted through contaminated food or water, particularly seafood such as fish, shrimp, and shellfish.
This review aims to provide a comprehensive overview of studies that have identified and characterized toxin genes of V.
cholerae in seafood using phenotypic and genotypic approaches.
The literature review was conducted using online databases including PubMed.
ScienceDirect.
Scopus.
Web of Science, and Google Scholar with keywords AuVibrio choleraeAy.
Auvirulence geneAy.
Aucholera toxinAy.
Auphenotypic-genotypic detectionAy.
Aucholera infectionAy.
Selected articles were published between 2015 and 2025 in English or Indonesian and discussed the identification of V.
cholerae toxin genes from marine products.
The findings revealed that V.
cholerae isolates from seafood commonly harbor virulence genes such as ctxA, hlyA, ompW, tcpA, zot, ace, and rtx, which play crucial roles in colonization, toxin production, and pathogenicity.
Phenotypic detection using TCBS agar and hemolysis tests serves as an initial screening, whereas genotypic identification through PCR and sequencing offers higher sensitivity and specificity.
Combining both methods enhances detection accuracy and risk assessment.
Therefore, monitoring virulence genes of V.
cholerae in seafood is essential for strengthening food safety surveillance and preventing cholera outbreaks in coastal and tourism areas.
Keywords: Vibrio cholerae, virulence gene, cholera toxin, phenotypic-genotypic detection and cholera infection.
INTRODUCTION
Cholera is an acute diarrheal infection that can be life-threatening if not properly treated.
It is estimated that there are 1.
3 to 4.
0 million cases of cholera worldwide each year, resulting in 21,000 to 143,000 deaths.
The primary cause of cholera infection is the consumption of food or water contaminated with the bacterium Vibrio cholerae (WHO, 2.
Vibrio cholerae is a Gram-negative bacterium belonging to the family Vibrionaceae, with a curved-rod shape and a length of approximately 2Ae4 AAm (Fernandez et al.
(Yoon and Waters, 2.
(Jawetz and AdelbergAos, 2.
Cholera is closely associated with poor sanitation, as well as direct contact with or ingestion of contaminated water and/or food .
uch as water used for drinking, cooking, bathing, and crop irrigatio.
(Jawetz and AdelbergAos, 2.
Seafood, including fish, shellfish, and shrimp, has been identified as one of the major sources of food-borne disease transmission, including cholera (Mumpuni and Hasibuan, 2.
(Brauge et al.
, 2.
There are more than 200 serogroups of Vibrio cholerae based on the O antigen, but only serogroups O1 and O139 are responsible for epidemic and pandemic cholera (Jawetz and AdelbergAos, 2.
The virulence of Vibrio cholerae is closely related to its ability to produce cholera toxin (CT), the principal factor responsible for the diseaseAos pathogenesis (Herrera and J.
Satchell, 2.
The major toxin produced by V.
cholerae is cholera toxin .
, while additional accessory toxins include toxin-coregulated pilus (TCP), zonula occludens toxin .
, accessory cholera enterotoxin .
, hemolysin A .
lyA), and several other supporting virulence genes (Montero et al.
, 2.
Several studies have demonstrated the presence of V.
cholerae toxin genes in marine-derived samples, such as the detection of the ctxA gene in Tenualosa ilisha fish sold in traditional markets in Bangladesh (Hossain et al.
, 2.
Another study conducted at Kedonganan Fish Market.
Bali, reported the detection of cholerae carrying the hlyA gene in shrimp and shellfish samples (Sukrama et al.
, 2.
Moreover, the ompW gene, which serves as a molecular marker for V.
cholerae identification, was also detected in shrimp and shellfish from the same market (Praja et al.
, 2.
Detection and characterization of V.
cholerae and its toxin genes in seafood samples are generally performed using a phenotypic approach with Thiosulfatehttps://ijhp.
International Journal of Health and Pharmacy Citrate-Bile Salts-Sucrose (TCBS) agar to observe colony morphology, and a genotypic approach through molecular assays such as Polymerase Chain Reaction (PCR) (Jawetz and AdelbergAos, 2.
Given the high seafood consumption in Indonesia, early detection and characterization of virulence genes in various marine products are essential, considering the potential risk of cholera transmission through contaminated seafood, the limited local data on pathogenic V.
cholerae strains, and the need to strengthen food safety and disease prevention efforts in coastal and tourism areas.
Based on these considerations, this article aims to provide a comprehensive, evidence-based review of existing studies on the toxin genes of Vibrio cholerae detected in commonly consumed seafood.
II.
METHODS
This review employed a literature-based approach focusing on the toxin genes detected in seafood through both phenotypic and genotypic methods.
Data were collected from scientific journals discussing the discovery of toxin genes from Vibrio cholerae in various marine products such as fish, squid, shrimp, shellfish, and others.
Scientific articles were collected from major electronic databases, including PubMed.
Scopus.
Web of Science, and Google Scholar.
The literature search was performed using combinations of the following keywords like Vibrio cholerae, virulence gene, cholera toxin, phenotypic-genotypic detection, cholera infection.
Selected articles were published between 2015 and 2025 in English or Indonesian and discussed the identification of V.
cholerae toxin genes from marine products.
This review not only highlights the development of phenotypic detection methodsAisuch as the use of selective TCBS agar and hemolysis testsAibut also evaluates the effectiveness of genotypic approaches including PCR and sequencing in identifying major toxin genes such as ctxA, hlyA, and ompW.
The analysis was carried out by examining previous studies that provided data on pathogenesis, types of virulence genes, and detection techniques applied in Vibrio cholerae research.
This methodological approach ensures that all presented findings are based on documented scientific evidence and reflect the most recent advances in molecular detection of Vibrio cholerae II.
RESULTS AND DISCUSSIONS
Pathogenesis Transmission of Vibrio cholerae primarily occurs through the fecalAeoral route, either via contaminated water or food, or through mechanical vectors such as houseflies (Musca domestic.
, which can carry the bacteria on their legs and mouthparts (Sebrina et al.
, 2.
Once inside the host.
cholerae must overcome the acidic barrier of the stomach before reaching the small intestine.
Pathogenic strains possess acid tolerance mechanisms that allow them to survive and remain infectious under low-pH conditions (Guli.
In the intestinal lumen, the bacteria encounter multiple layers of host defenses, including the commensal microbiota, epithelial integrity, rapid epithelial turnover, and protective mucus layer (Guntina.
Agung and Kusuma, 2.
However.
cholerae is able to overcome these barriers through adhesion mechanisms mediated by toxin-coregulated pilus (TCP), fimbriae, adhesin proteins, and outer membrane proteins such as ompW, which bind to GM1 ganglioside glycolipid receptors on the intestinal mucosal membrane (Sebrina et al.
, 2.
Following colonization.
cholerae produces its major virulence factor, cholera toxin (CT), encoded by the ctxAB gene.
The toxin consists of two subunits: subunit B, which binds to GM1 receptors, and subunit A, which activates adenylate cyclase, leading to an increase in intracellular cyclic AMP .
AMP) levels (Guli, 2.
(Siregar et al.
, 2.
Elevated cAMP inhibits sodiumAechloride absorption and enhances chloride and bicarbonate secretion, resulting in massive fluid loss into the intestinal lumen.
Additionally, increased prostaglandin levels further contribute to excessive fluid secretion (Sebrina et al.
, 2.
This condition causes profuse watery diarrhea, which may exceed one liter per hour, often accompanied by vomiting, rapid dehydration, and circulatory shock.
https://ijhp.
International Journal of Health and Pharmacy Virulence Genes In its pathogenesis.
Vibrio cholerae employs a series of mechanisms that enable it to survive, colonize, and infect the human gastrointestinal tract.
These processes are complex and are largely regulated by the expression of virulence genes that control colonization, toxin production, and immune evasion.
The major virulence genes of V.
cholerae and their functions are summarized below.
Table 1.
Virulence Genes of Vibrio cholerae Virulence Factor Function/Role Refrence Major Factor Virulence The principal toxin responsible for severe, life-threatening diarrhea (Pant.
Das .
It is encoded within the filamentous phage genome and Bhadra, known as CTX, which carries the ctxA and ctxB genes.
Cholera
(CTX)
The ctxA gene increases intracellular cAMP levels in intestinal epithelial cells, resulting in ion and water secretion into the intestinal lumen and leading to profuse watery diarrhea.
The ctxB gene encodes the B subunit that binds to GM1 ganglioside (Hsiao and receptors, facilitating entry of subunit A into the host cell.
Zhu, 2.
Virulence Accessory Factors Facilitates bacterial aggregation and microcolony formation through (Montero et pilusAepilus interactions in the human intestine.
TCP expression is al.
, 2.
Toxin-coregulated usually coupled with CTX production and also acts as a receptor for pilus (TCP) CTX, contributing to the evolution of virulence in non-toxigenic cholerae strains through sequential acquisition of VPI followed by CTX.
Disrupts tight junctions between intestinal epithelial cells, (Lopezincreasing mucosal permeability and inducing diarrhea.
Zot also joven et al.
Zonula Occludens indicates pathogenic potential since it can be found in both CTX 2.
Toxin (Zo.
and its precursor pre-CTX, thus playing a role in genetic diversification and horizontal transfer of virulence factors among V.
cholerae strains.
Alters ion transport in the ileum, leading to mild diarrhea.
Ace can (Montero et Accessory Cholera trigger early intestinal secretion before CTX acts by stimulating al.
, 2.
Enterotoxin (Ac.
Ca-dependent ClA/HCOCEA symporters, resulting in extracellular Ca influx.
Encodes the El Tor hemolysin, a pore-forming toxin with (Diez et al.
hemolytic, cytotoxic, and enterotoxic activities.
It contributes to 2.
mucosal damage and intestinal fluid accumulation.
Expression is Hemolysin (HlyA) regulated by complex factors such as HlyU .
HapR, and Fur .
, and is influenced by environmental conditions including oxidative stress.
Exerts cytotoxic effects on host cells through actin (Chow.
Ng Repeat in Toxin depolymerization and cross-linking, causing cell morphological and Yuen, (RTX toxi.
changes, tissue necrosis, and intestinal inflammation .
Detection Methods Phenotypic Tests Phenotypic detection of Vibrio cholerae is performed through isolation and identification based on colony morphology, biochemical characteristics, and growth on selective media such as Thiosulfate Citrate Bile Salts Sucrose (TCBS) agar.
Colonies of V.
cholerae typically appear yellow on TCBS agar due to their ability to ferment sucrose (Guntina.
Agung and Kusuma, 2.
Prior to isolation, environmental samples are https://ijhp.
International Journal of Health and Pharmacy commonly subjected to an enrichment step using alkaline peptone water (APW) at pH 8.
6, which selectively promotes the growth of Vibrio species due to their tolerance to alkaline conditions and ability to proliferate rapidly under microaerophilic conditions.
Following enrichment, presumptive V.
cholerae colonies may also be isolated on additional selective and differential media such as Tellurite Taurocholate Gelatin Agar (TTGA), where colonies appear translucent with a characteristic dark center and surrounding halo resulting from gelatin hydrolysis, as well as on CHROMagarE Vibrio, which facilitates differentiation based on species-specific chromogenic reactions.
Presumptive isolates are subsequently purified on non-selective media and further characterized using basic biochemical tests, including oxidase activity, which is typically positive for V.
Serogroup determination for epidemiologically significant strains is conducted using slide agglutination assays with O1 and O139 antisera, allowing phenotypic differentiation of pandemic-associated strains from non-O1/non-O139 environmental isolates (Huq et al.
, 2.
Genotypic Tests Gene-based genotypic detection has become the primary method for identifying V.
cholerae due to its high sensitivity and specificity.
Several molecular approaches are commonly used:
Conventional PCR Conventional PCR remains the mainstay method for the initial detection of V.
cholerae because it is costeffective, has simple procedures, and can be performed in many laboratories.
Common species-specific markers include ompW and toxR, both known for their high sensitivity and specificity in detecting clinical and environmental strains.
To distinguish epidemic serogroups, the rfb O1 and rfb O139 markers are often combined with virulence genes such as ctxA or tcpA.
This multiplex approach allows for simultaneous identification of species, serogroup, and toxigenic status within a single amplification.
Such dual-target strategies are particularly valuable in environmental surveillance, minimizing false positives caused by noncholera Vibrio species frequently found in aquatic samples (Kachienga et al.
, 2.
Real-time PCR .
PCR) and Digital Droplet PCR .
dPCR) Recent advances in genetic detection technologies have introduced digital droplet PCR .
dPCR) as a complementary or comparative method to real-time quantitative PCR .
PCR).
One example of an advanced qPCR application is a quadruplex assay targeting hlyA, rfb O1, rfb O139, and ctxA genes simultaneously to determine species identity, serogroup, and toxigenic potential of V.
This assay demonstrates a limit of detection of approximately two colonies per reaction and 100% specificity for V.
cholerae compared to non-Vibrio species (Yang et al.
, 2.
Sequencing In addition to whole genome sequencing (WGS), amplicon sequencingAiusing either the Sanger method or targeted next-generation sequencing .
NGS)Airemains an essential strategy for specific genetic characterization of Vibrio cholerae (Chaguza et al.
, 2.
The targeted sequencing approach serves as a bridge between PCR-based molecular screening and comprehensive WGS analysis.
During outbreak investigations, a stepwise sequencing strategy can be implemented.
Initially, a PCR panel is used for rapid detection of key genetic markers, followed by amplicon sequencing of the ctxB and wbeT genes for preliminary strain stratificationAisuch as identifying ctxB7 alleles or determining Ogawa and Inaba Subsequently, a subset of representative isolates is selected for WGS.
This final stage enables detailed analyses, including determination of single nucleotide polymorphism (SNP) thresholds, mapping of antimicrobial resistance genes such as SXT.
R391, gyrA, and parC, and phylogenetic tracing across geographical regions.
Such a tiered framework ensures rapid response capabilities without compromising the resolution and precision required for public health decision-making (Chaguza et al.
, 2.
(Lassalle et al.
A Study On The Detection Of Vibrio Cholerae In Seafood Various studies have reported the presence of Vibrio cholerae in seafood products using diverse phenotypic and genotypic approaches.
A study on the transmission and toxigenic potential of V.
cholerae in Hilsha fish (Tenualosa ilish.
in Bangladesh demonstrated that the majority of samples were contaminated with V.
cholerae, with 81% of samples testing positive for the ompW gene and 20% harboring the ctxA gene.
https://ijhp.
International Journal of Health and Pharmacy A total of 158 isolates were identified, comprising serogroups O1 and non-O1/non-O139, highlighting the role of fish as a potential reservoir and transmission vehicle for toxigenic V.
cholerae (Hossain et al.
, 2.
Meanwhile, a study conducted in Shanghai involving 12 species of aquatic products revealed that although major virulence genes such as ctxAB, tcpA, ace, and zot were not detected, other virulence-associated genes, including hapA, hlyA, and rtxCABD, were frequently identified.
This study also reported a high level of antibiotic resistance, with more than half of the isolates exhibiting multidrug resistance patterns (Lassalle et , 2.
Another study carried out at the Kedonganan Fish Market in Bali highlighted the pheno-genotypic profiles of V.
cholerae isolated from shrimp and shellfish, where several isolates displayed hemolytic activity and carried the hlyA gene.
however, some isolates were genotypically positive but did not phenotypically express hemolysis.
These findings were further supported by subsequent research at the same location, which detected the ompW gene in a high proportion of shrimp samples and at a lower frequency in shellfish, with no detection of the ctxA gene.
This indicates that V.
cholerae contamination in local seafood products is predominantly associated with non-toxigenic strains.
Overall, these findings underscore the variability in the distribution of V.
cholerae, virulence profiles, and potential public health risks, which are influenced by the type of seafood commodity, geographical location, and detection methods employed(Praja et al.
(Sukrama et al.
, 2.
IV.
CONCLUSION
Vibrio cholerae is a pathogenic bacterium responsible for human cholera, typically transmitted through contaminated seafood such as fish and squid, especially when consumed raw or undercooked.
Detection of virulence genes such as ctxA, hlyA, ompW, tcpA, zot, ace, and rtx is crucial for understanding its colonization, toxicity, and pathogenicity.
Identification of V.
cholerae can be initiated through phenotypic screening using TCBS agar, but confirmation via PCR-based genotypic methods is essential due to their superior sensitivity and specificity.
The combination of phenotypic and genotypic approaches provides a more accurate assessment of infection risk.
Therefore, fish and squid may serve as important reservoirs of V.
cholerae, including virulent strains, making molecular detection of toxin genes vital for diagnosis, food safety surveillance, and prevention of seafood-associated cholera outbreaks.
ACKNOWLEDGMENT
The author declares that there is no acknowledgment.
VII.
COMPETING INTERESTS
The author declares that there is no competing interest.
REFERENCES