https://orcid.org/0000-0001-6509-9740
Figures
Abstract
Vibrio cholerae, a globally significant pathogen, causes both endemic and epidemic cholera and has also been associated with sporadic gastroenteritis and foodborne infections. Shrimp exports are a key source of revenue and employment in Bangladesh. However, Vibrio outbreaks pose serious public health and socio-economic risks through seafood consumption and the spread of antibiotic-resistant genes from aquaculture. The current study investigates the genomic and pathogenic features of the V. cholerae strain associated with seafood, with particular focus on the aquaculture environment of Bangladesh. Using Oxford Nanopore long-read sequencing technology, whole-genome sequencing (WGS) was performed on V. cholerae strain SU129B isolated from pacific white shrimp in Noakhali, Bangladesh, to explore its genetic diversity, antimicrobial resistance, and virulence determinants. The assembled genome exhibited high completeness (93.08%) and contiguity (N50 = 1), along with minimal contamination (1.33%). Average nucleotide identity (ANI) analysis showed 98.13% similarity with the reference V. cholerae strain RFB16, confirming species-level identification. Functional and pathway analyses indicated that the strain possesses a complex network of genes and metabolic systems that contribute to its survival in diverse environments. Analysis of the genome also revealed several biosynthetic gene clusters associated with the production of secondary metabolites, which contribute to osmotic tolerance, and metal acquisition. The genome also harbored multiple antibiotic resistance genes, which confer resistance via efflux systems, target modification, and membrane adaptation mechanisms. Pangenome analysis revealed 8,964 genes, including 2,085 core genes, 429 soft-core genes, 1,431 shell genes, and 5,019 cloud genes, demonstrating considerable genetic diversity and adaptability. Phylogenetic analysis showed a close evolutionary relationship between the shrimp and clinical strains, suggesting possible genome conservation across environmental and clinical isolates. The findings demonstrate that V. cholerae strain SU129B can evolve in aquaculture environments and may serve as a reservoir for virulence and multidrug resistance.
Citation: Uddin MS, Chamonara K, Masum MHU, Siddiqua A, Hossain I, Sultana S, et al. (2026) Genomic and phylogenomic exploration of Vibrio cholerae strain SU129B isolated from Penaeus vannamei: Resistance, virulence, and evolutionary dynamics in bangladeshi aquaculture. PLoS One 21(3): e0344787. https://doi.org/10.1371/journal.pone.0344787
Editor: Md. Asaduzzaman Shishir, DIU: Dhaka International University, BANGLADESH
Received: November 3, 2025; Accepted: February 25, 2026; Published: March 10, 2026
Copyright: © 2026 Uddin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All sequencing data generated in this study have been deposited in the NCBI BioProject database under accession number PRJNA1294324, and are publicly available through the Sequence Read Archive (SRA) (SRR34709123). The complete genome data have been submitted to NCBI GenBank with the accession JBQWMA000000000.1, while the assembly data have been deposited with the genome accession, GCA_052545435.1.
Funding: This research is funded by the Research cell, Noakhali Science and Technology University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
1. Introduction
Vibrio species are gram-negative, halophilic bacteria often found in aquatic habitats, including fish, mollusks, and crustaceans [1,2]. The species are the most often associated with human illnesses among the diverse bacterial pathogens present in seafood [3]. Vibrio cholerae, the species that causes cholera, may infect people through contact with fish, shellfish, or water [1]. Over 200 V. cholerae serogroups have been identified, yet only the pathogenic O1 and O139 serogroups are associated with cholera. However, V. cholerae from non-O1 and non-O139 serogroups can cause gastroenteritis and have been linked to rare foodborne outbreaks rather than widespread epidemics [4]. Cholera is primarily caused by the consumption of contaminated food or water, although it can also spread through direct contact between individuals [5]. The cholera-causing V. cholerae has both environmental and human phases in its life cycle [6]. Freshwater ecosystems may harbor the species as free-living entities, within biofilms composed of cells, or in association with plankton [7,8]. Unlike other hazardous non-cholera Vibrio species, V. cholerae is transmitted by the fecal-oral route and via person-to-person contact [6]. Upon entering the human host and traversing the small intestine, V. cholerae begins to express genes encoding virulence factors, such as cholera toxin and the toxin-coregulated pilus (Tcp). The structure of cholera toxin includes two subunits, CtxA and CtxB. The pentameric CtxB subunit binds to the ganglioside GM1, a sialylated glycosphingolipid, located on the plasma membranes of enterocytes [9]. The elements of bound cholera toxin separate within the endoplasmic reticulum (ER) following endocytosis and subsequent retrograde transport. The release of the enzymatic CtxA subunit from the ER into the cytosol allows ARF6 to initiate allosteric processes through ADP ribosylation [10,11]. The activation of adenylyl cyclase follows the binding of the active CtxA subunit to ARF6, leading to the catalysis of ADP ribosylation on a G protein-coupled receptor. The cystic fibrosis transmembrane receptor (CFTR) undergoes phosphorylation by protein kinase A (PKA) when there are increased levels of cellular cAMP [11]. This leads to the movement of ions and water into the lumen of the small intestine, resulting in diarrhea [3]. Bangladesh ranks among the world’s largest fish producers, with an estimated harvest of approximately 5 million metric tons in 2023–24. The fisheries sector contributes 2.53% to the national Gross Domestic Product (GDP), 22.26% to agricultural GDP, and 0.90% to export revenue [12,13]. Despite growth, the fisheries GDP declined from 2.7% in 2019 to 2.41% in 2023 due to rising production costs, disease outbreaks, and environmental challenges [12]. Shrimp exports provide millions in revenue and provide employment for others; nonetheless, Vibrio outbreaks present significant socio-economic and health hazards [13]. This public health concern extends beyond the direct consumption of contaminated seafood, encompassing the horizontal dissemination of antibiotic resistance determinants to other clinically significant bacterial pathogens affecting humans [14,15]. The overuse or abuse of antibiotics in aquaculture leads to the development and spread of antibiotic resistance [16].
This study focuses on V. cholerae isolated from pacific white shrimp collected at a local fisheries market in Noakhali, Bangladesh. The pangenome was analyzed to elucidate the virulence traits and genetic diversity of V. cholerae. These findings will contribute to understanding the pathogenic mechanisms of this opportunistic organism and the molecular factors that facilitate V. cholerae's adaptation and survival in diverse environments.
2. Materials and methods
2.1 Ethical statement
The Ethics Approval Committee of Noakhali Science and Technology University (NSTUEC). reviewed and authorized the study protocol (Approval no. NSTU/SCI/EC/2025/451).
2.2 Sample collection, processing and antibiotic resistance profiling
The pacific white shrimp (Penaeus vannamei) (n = 103) samples were obtained from different fish markets in Noakhali district. The samples were collected aseptically and kept in a sterile plastic zipper container. Thereafter, the samples were conveyed on ice in a cooler box (kept at a temperature below 4°C) from the sampling site to the Department of Microbiology at Noakhali Science and Technology University (NSTU) and were processed shortly. The shrimp samples were homogenized and enriched in alkaline peptone water at 37°C. The enriched cultures were inoculated onto thiosulfate–citrate–bile salts–sucrose (TCBS) agar plates and incubated for 18–24 hours at 37 °C [17]. The Vibrio isolates were presumably identified based on their physical traits, such as size, shape, and color. Suspected Vibrio isolates were then identified using direct microscopy and biochemical tests, including oxidase, catalase, methyl red, Voges-Proskauer, and urease tests (Table S1) [18,19]. Final confirmation was obtained by polymerase chain reaction (PCR) targeting the genus-specific groEL gene. Amplification utilized the primer pair groVc1 (5′-GATCTTGACTGGCGGTGTTGTG-3′) and groVc2 (5′-GTCACCCACCAGAGAAGAGAGT-3′) under primer-specific PCR conditions [20]. Antibiotic resistance of the experimental isolate was assessed using standardized protocols from the Clinical and Laboratory Standards Institute (CLSI, M45) [21], European Committee on Antimicrobial Susceptibility Testing (EUCAST, v.16.0) [22], and Clinical and Laboratory Standards Institute (CLSI, M100) [23]. Fourteen antibiotics were tested to determine the isolate's susceptibility profile (Table S2). However, among the V. cholerae isolates, the experimental isolate was selected based on its antimicrobial resistance profile and subsequently subjected to whole-genome sequencing.
2.3 Whole genome sequencing, base calling and quality control
The total DNA from the isolates was extracted using the QIAamp® DNA Mini Kit (QIAGEN, Germany) and then quantified using a Qubit 4 fluorometer (Thermo Fisher Scientific, USA). Before DNA sequencing, a native barcoding kit (SQK.NBD.114.24) was used for library preparation, and sequencing was performed on the Oxford Nanopore MinION™ Mk1C platform with the FLO-MIN114 (R10.4.1) flow cell. All information was gathered using the MINKNOW application (1.11.5). To generate pass reads (FASTQ format) with an average quality score higher than 9, the MinIONTM Mk1C sequencing data (FAST5 files) were basecalled using the Guppy (v.6.3.2) in high accuracy mode. Trimming was done by Porechop (v.0.2.4) to remove the adaptor and barcode sequences [24]. Low-quality bases (q < 10) were removed using Nanofilt [25].
2.4 Genome assembly, quantification and strain identification
The de novo assembly was performed using Flye (v.2.9.4) [26]. This tool assembles long sequencing reads from platforms like as PacBio and Oxford Nanopore, using a repetition graph approach to address the elevated error rates associated with long reads [26]. Polishing of the genome was done by Racon (v.1.5.0) [27]. The Type (Strain) Genome Server (TYGS) was employed to evaluate the species. The TYGS is an easily accessible and effective web server designed for prokaryotic taxonomy based on genomic data. It generates phylogenetic trees at the genome scale and provides advanced estimates for species and subspecies boundaries using automatically identified closest type genome sequences [28]. The potential contamination within the genome and its completeness were evaluated using ConEst16S (https://www.ezbiocloud.net/tools/contest16s) [29], CheckM (v.1.2.4) [30], and BUSCO (v.6.0.0) (https://busco.ezlab.org/) tools [31]. The genome statistics were calculated by QUAST (v.5.2.0) [32], while the average nucleotide identity (ANI) was assessed using the FastANI tool [33].
2.5 Genome annotation, and mapping
The annotation of the assembled genome was performed by Prokaryotic genome annotation pipeline (Prokka) [34], Rapid annotations using subsystem technology toolkit (RASTk) [35], and Prokaryotic genome annotation pipeline (PGAP) tools [36]. Prokka is a widely used rapid annotation tool for prokaryotic genomes, using ab initio gene prediction and homology-based annotation approaches within a modular and hierarchical framework [34]. Additionally, the RASTtk is a high-throughput pipeline developed for annotating prokaryotic genomes, utilizing subsystem-based functional annotation along with automated gene prediction algorithms [35]. However, the PGAP, the official genome annotation system of the National Center for Biotechnology Information (NCBI), offers standardized, automated, and evidence-based annotation of bacterial and archaeal genomes [36]. Finally, the CIRCOS plot of the annotated genomes was constructed using the Proksee server [37].
2.6 Subsystem and metabolic pathway analysis
Understanding subsystems and metabolic pathways is essential for the analysis of bacterial genome data and for comparing gene activities with cellular physiology [38,39]. The subsystems and functional metabolic pathways of the strain were predicted by Patric server [40] and RAST [35]. Biosynthetic gene clusters (BGCs) are groups of adjacent genes in bacterial genomes that produce secondary metabolites, which are small bioactive molecules important for various ecological and physiological functions, such as antibiotics, pigments, toxins, siderophores, and signaling compounds [41]. In the assembled genomes, biosynthetic gene clusters were identified using the antiSMASH (v.7.0) tool [42].
2.7 Antimicrobial resistance gene, mobile genetic elements, and virulence gene analysis
Antimicrobial resistance genes were assessed using the CARD (v.6.03) tool [43]. Mobile genetic elements (MGEs) are DNA segments capable of translocating within or across genomes, promoting genetic diversity and horizontal gene transfer (HGT) in bacteria [44]. Subsequently, the MGEs were explored through mobileOG-db (v.1.1.3) [45]. The virulence profile of a bacterium encompasses the whole array of genes, factors, and molecular pathways that facilitate host infection, immune evasion, tissue damage, and assure survival and transmission [46]. The virulence genes were predicted using Victors [47].
2.8 Pangenome analysis and pathogenicity profiling
A comprehensive pangenome and comparative genomic analysis was conducted to elucidate the genetic diversity of the target strain. The objective was to identify specific variations in the presence or absence of genes or gene families among strains from diverse geographical regions and sources. Thirty-one complete genome sequences of V. cholerae strains, obtained from both human and non-human origins across multiple countries, were sourced from the Bacterial and Viral Bioinformatics Resource Center (BV-BRC). The analyses utilized these 31 V. cholerae genomes, including the genome sequenced in this study, and were performed using Roary (v. 3.13.0) [48]. This tool can rapidly build large-scale pan genomes, identifying the core and accessory genes [48].
3. Results
3.1 Multidrug resistance profile of V. cholerae strain SU129B
Among the V. cholerae isolates (n = 17), SU129B was selected as the experimental isolate for further analysis due to its multidrug resistance. The isolate demonstrated resistance to erythromycin, ceftazidime, chloramphenicol, meropenem, azithromycin, and cefotaxime. It exhibited an intermediate pattern to nitrofurantoin and amikacin. The isolate remained susceptible to gentamicin, ciprofloxacin, nalidixic acid, ampicillin, imipenem, and levofloxacin (Table S1). Its resistance to at least one agent in three or more antimicrobial categories resulted in its classification as a multidrug-resistant strain based on established resistance profiles (Fig. 1A).
(A) The phenotypic antimicrobial resistance profile of the strain SU129B followed by CLSI guideline, (B) The ANI profile of the strain SU129B with the reference genome strain RFB16, (C) The functional annotation of the genome of strain SU129B utilizing PGAP, RASTk, and Prokka.
3.2 Genome quality and functional profile
The genome of V. cholerae strain SU129B was sequenced and analyzed to evaluate its structural and functional attributes. A total of 819,028 reads were obtained, yielding a sequencing depth of 247.4x. The assembled genome size was approximately 4.05 Mbp, consistent with the reference V. cholerae genome (strain RFB16), and had a GC content of 47.63%. The assembly quality metrics indicated that the genome was exact and complete. The genome exhibited coarse and fine consistencies of 99.7% and 96.7%, respectively. The completeness was 93.08%, while the contamination level was negligible at 1.33%. The assembly included just three contigs, with a contig N50 value of 2,888,403 bp and an L50 of 1, signifying a highly contiguous and well-structured genome assembly (Table 1). Taxonomic validation by ANI showed 98.13% similarity with reference V. cholerae isolates (RFB 16), confirming precise species-level classification (Fig. 1B). Subsequently, isolate SU129B was designated as “Vibrio cholerae strain SU129B.” The complete genome sequence of the strain has been submitted to GenBank at NCBI, assigned GenBank, and assembly accession numbers JBQWMA000000000.1, and GCA_052545435.1, respectively (Table 1).
Functional annotation revealed 2,730 protein-coding genes with assigned functions and 1,074 genes with no known functions, collectively accounting for 93.84% of all anticipated properties. Notably, 28.23% of the identified proteins were classified as hypothetical, suggesting the presence of uncharacterized or novel genes warranting further investigation. Moreover, 96.58% of the characteristics were associated with local protein families, suggesting significant genetic conservation and functional consistency within the species (Table 1). The identified genes were annotated into various gene categories, including coding sequences (CDS), ribosomal RNA (rRNA), and transfer RNA (tRNA). The Prokka annotation revealed 3,742 genes, whereas the RASTk server and the PGAP pipeline reported 3,707 and 3,938 genes, respectively.
Prokka identified a total of 3,606 CDSs, whereas RASTk predicted 3,804 CDSs, and PGAP identified 3,568 CDSs. However, each tool identified 104 tRNA genes. In the genome, 31 rRNA genes were identified by Prokka and PGAP, while 30 were identified by RASTk (Fig 1C, Fig 2).
3.3 Subsystem and metabolic profile
Analysis of the superclass revealed that the strain SU129B possessed genes related to metabolism, protein processing, stress response and defense, respiration, membrane transport, cellular processes, regulation, and cell signaling (Fig 3A). The analysis of the subclass showed that the strain contains significant genes associated with biotin biosynthesis, DNA uptake, RNA processing, DNA repair, and the metabolism of lysine, threonine, methionine, cysteine, proline, and pyrimidines (Fig 3B, S1 Data). Subsystem analysis identified several essential functional modules with varying gene abundances. The lipopolysaccharide transport (Lpt) system, TAM transport system, peptidyl-prolyl cis-trans isomerases, ribosomal large subunit, RNA processing and degradation system, type IV pilus system, chorismate synthesis, and glycine cleavage system were predominant (Fig 3C, S1 Data).
(A) The superclass, (B) Subclass and (C) subsystem analysis of the strain SU129B.
Analysis of functional annotation and KEGG pathways revealed a diverse range of metabolic pathways, demonstrating the organism's considerable metabolic flexibility and ecological adaptability. These pathways include essential processes such as carbon fixation, energy metabolism, biosynthesis of vital cofactors, production of secondary metabolites, and degradation of xenobiotics. Various biosynthetic pathways for cofactors and vitamins have been identified, including those involved in pantothenate and CoA biosynthesis, biotin metabolism, nicotinate and nicotinamide metabolism, and ubiquinone and other terpenoid-quinone biosynthesis. A significant number of pathways for the biosynthesis of secondary metabolites were identified, encompassing terpenoid backbone biosynthesis, diterpenoid biosynthesis, anthocyanin biosynthesis, betalain biosynthesis, and tetracycline and streptomycin biosynthesis. The existence of polyketide sugar unit biosynthesis and the production of nonribosomal peptides, including siderophore groups, suggests a promising capacity for generating bioactive compounds. Significantly, multiple pathways linked to xenobiotic degradation were identified, comprising the degradation of tetrachloroethene, toluene, xylene, styrene, bisphenol A, naphthalene, anthracene, and 1,4-dichlorobenzene, along with benzoate degradation via hydroxylation and the metabolism of xenobiotics by cytochrome P450 (S2 Data). The genome of V. cholerae strain SU129B contained numerous significant biosynthetic gene clusters, including six predominant biosynthetic gene clusters, as well as additional transport and regulatory genes. The main biosynthetic gene clusters were non-ribosomal peptide (NRP)-metallophore, non-ribosomal peptide synthetase (NRPS), terpene precursor, isocyanide, betalactone, and ectoine (Fig 4).
3.4 Antimicrobial resistance genes, and mobile genetic elements analysis
The antimicrobial resistance gene profile of the V. cholerae strain SU129B revealed a spectrum of resistance-related factors across several antibiotic classes. A unique genomic cluster including almF, almG, almE, and varG homolog was discovered in the 0.9–1.0 Mbp region. A gene identified as Escherichia coli parE was also found at this locus. In the downstream region, between 1.8 Mbp and 2.6 Mbp, additional gene loci were found, including the CRP regulator, the vanT gene, and rsmA, all of which are associated with antibiotic resistance or transcriptional regulation. Additionally, the QnrVC4 gene was found at approximately 3.8 Mbp (Fig 5). The genome also showed several MGEs involved in replication, recombination and repair, phage integration and excision, horizontal transfer, and stability/defense mechanisms. The antimicrobial resistance gene CRP resides near the mobile genetic elements rnr and dam_1 (between 90 kbp and 125 kbp). The vanT gene is part of a vanG antimicrobial resistance cluster approximately 660–700 kbp. This locus is closely associated with various MGEs, including replication-related elements (rep) and transfer-associated modules (priB, dnaB) (Fig 5). Within the genomic region ranging from 860–920 kbp, MGEs are the most prevalent, surrounding the antimicrobial gene rsmA. The close association of transfer-related genes (ygaD) and recombination factors (recA, mutS). The genomic region between 2800 and 2840 kbp encompasses a clinical region that includes the parE gene. The location of this resistance gene within a cluster abundant in MGEs (recJ, xerD, parC, cca) (Fig 5).
3.5 Virulence associated gene analysis
More than 100 virulence-associated genes were identified in the genome of their V. cholerae strain SU129B. The virulence-associated genes identified can be categorized into functional groups based on their biological roles. A substantial number of these genes play roles in regulation and signal transduction, including toxR, crp, rpoS, rpoE, ompR, arcA, relA, hfq, sspA, cyaA, and fur. Additionally, a significant category consists of genes associated with toxin synthesis and secretion, including rtxA, tolC, tolB, irgA, and VC1716. Other genes, such as aceF, pykF, deoC, sucA, pta, zwf, frdC, atpA, galK, galM, ilvD, purF, purK, carA, carB, guaB, glyA-1, glmM, fabG, and acpS, are associated with metabolic processes and energy production (S3 Data). In addition, the strain can withstand oxidative and environmental stress in the host by utilizing DNA repair and stress response genes, including recO, ruvB, trxA, trxB, clpB, gshB, and mukB, which contribute to genomic stability. Moreover, the remaining genes related to protein synthesis and translation, including tufA, rpmJ, rpmF, prfC, and VC1715. Finally, other putative or uncharacterized genes (e.g., VC0727, VC1639, VCA0583, VCA0910, VC2660, VCA0600) are located within genomic regions often associated with virulence clusters (S3 Data).
3.6 Comparative genome analysis and genome-wide pathogenicity profiling
Comprehensive pangenomic and comparative genomic analyses were conducted using 31 whole-genome sequences of V. cholerae SU129B isolates from diverse nations and origins, including the isolate under study. The pangenome study revealed a core genome of 2085 genes, including 429 soft-core genes, 1431 shell genes, and 5019 cloud genes. The present study analyzed a pangenome comprising 8964 genes (Fig 6A). The pan-genome-wide analysis revealed that V. cholerae strains from non-human sources showed considerable genomic and phylogenetic similarity to clinical isolates from human disease. The V. cholerae strain SU129B, obtained from Bangladesh, had a significant phylogenetic relationship with the clinical isolate V. cholerae strain BC1071 from Germany. Furthermore, the V. cholerae strain M18C 4, obtained from a migratory bird in China, and the strain Vc306, a fish isolate, were classified within the same clade, indicating a significant degree of phylogenomic relatedness (Fig 6B, 6C).
(A) The pangenome composition of the strains comprising the core, shell, cloud and soft genes, (B) The phylogenetic analysis of the Vibrio strains from different sources and origins, (C) The heatmap depicts the conserved genes among the Vibrio isolates.
4. Discussion
The current study evaluates the genomic and pathogenicity traits of V. cholerae strain linked to seafood, specifically in Bangladesh's aquaculture environment. This species is the most clinically relevant, causing both endemic and epidemic cholera globally [3]. Pathogenic serogroups O1 and O139 are typically associated with cholera outbreaks; however, non-O1/non-O139 strains can also cause occasional gastroenteritis and foodborne illnesses [4]. The ecological adaptability and public health relevance of V. cholerae are further highlighted by its dual existence as a human pathogen and a free-living aquatic organism [7,8]. Genomic surveillance of V. cholerae isolates from aquaculture sources is essential in Bangladesh to identify virulence factors, monitor the dissemination of resistance, and assess zoonotic potential. In this context, WGS was utilized to characterize V. cholerae isolated from white Pacific shrimp collected in Noakhali, Bangladesh. The study aimed to elucidate the strain's genetic diversity, and antibiotic resistance profile through comparative and pan-genome analyses. The V. cholerae strain SU129B demonstrated extensive multidrug resistance, exhibiting complete resistance to erythromycin, ceftazidime, chloramphenicol, meropenem, azithromycin, and cefotaxime. Resistance to at least one agent from three or more antimicrobial classes categorizes SU129B as multidrug-resistant, underscoring its potential clinical and environmental significance as a reservoir of antibiotic resistance determinants. The WGS analysis produced 819,028 reads with a depth of 247.4 × , yielding an assembled genome of approximately 4.05 Mbp and a GC content of 47.63%. These values closely align with non-O1/non-O139 V. cholerae reference genomes (strain RFB16), confirming that the isolate belongs to the non-agglutinating (NAG) lineage rather than the epidemic O1/O139 groups. The assembly also exhibited high contiguity, consisting of three contigs, with an N50 of 2,888,403 bp and an L50 of 1.
The genome-wide analysis of V. cholerae strain SU129B revealed the presence of genes associated with protein processing, membrane transport, and cellular signaling, suggesting that V. cholerae strain SU129B possesses robust mechanisms for host colonization and immune evasion. The lipopolysaccharide transport system and the TAM transport system are especially important for outer membrane biogenesis and the secretion of virulence-associated proteins. Lipopolysaccharide (LPS) is critical in initiating host inflammatory responses [49], whereas the TAM complex promotes the assembly of surface-exposed virulence factors, thereby increasing bacterial adherence and persistence [50]. The type IV pilus system, identified through subsystem analysis, is a significant contributor to virulence. Type IV pili are essential for motility, biofilm formation, and attachment to host cells, all of which are critical for effective intestinal colonization during infection [51]. Similarly, peptidyl-prolyl cis-trans isomerases facilitate protein folding and adaptation to stress, allowing the bacterium to maintain functionality under oxidative and osmotic stress imposed by the host [52]. The metabolic pathways revealed by KEGG analysis highlight the organism's ability to adapt to its environment and its strength in facing pathogenic challenges. The identification of various pathways for xenobiotic degradation underscores the strain's capacity to thrive in contaminated or chemically stressed environments, including aquaculture systems that contain antibiotic and chemical residues [53]. This resilience could support the ongoing presence and spread of harmful, antibiotic-resistant strains within aquatic ecosystems.
Genome analysis of V. cholerae strain SU129B identified several conserved biosynthetic gene clusters involved in the production of secondary metabolites, such as ectoine, NRP-metallophore, terpene precursors, isocyanides, and β-lactone biosynthetic pathways. These clusters, recognized for their adaptability to environmental conditions, are considered to influence the pathogenicity and ecological viability of V. cholerae strain SU129B either directly or indirectly [54,55]. Ectoine production, mediated by the ectABC operon, is critical for osmoregulation and stabilizes cellular structures and proteins under hyperosmotic conditions. The biosynthesis of ectoine is vital for the survival of Vibrio species during saline and osmotic stress, thereby supporting persistence within host tissues [56]. This adaptation may increase the virulence of strain SU129B by maintaining bacterial viability under adverse conditions. In contrast, the NRPS-type biosynthetic loci in V. cholerae are primarily known for synthesizing vibriobactin, a catecholate siderophore that enables iron acquisition under limiting conditions. Vibriobactin acts as a chelator, sequestering iron from the environment and facilitating its uptake via specific transporters. Siderophore synthesis may improve the strain's survival in iron-deficient environments and promote colonization and competition with other microorganisms [57]. The terpene-related gene clusters identified in several bacteria encode enzymes that generate terpene precursors and subsequent terpene cyclases that transform these precursors into structurally diverse terpenoids. Microbial terpenoids serve multiple functions, including small-molecule signaling, membrane regulation, protection against oxidative stress, interspecific chemical defense, and direct modulation of host reactions. Thus, this gene cluster may facilitate the virulence of the strain SU129B [58,59]. Additionally, isocyanide-containing natural products are produced by isocyanide synthases (isnA/isnB-type genes) and exhibit a wide range of bioactivities, including bacterial digestion and metal chelation. Thus, in SU129B, these byproducts may function as electrophilic compounds that link metals or interact with proteins, making the species more competitive in its environment [60]. β-lactone biosynthetic gene clusters encode enzymes that produce metabolites containing β-lactones. This varied bioactive class inhibits proteolytic enzymes and regulates microbial relationships [61,62]. These clusters may thus indirectly modulate host-pathogen interactions or interbacterial competition in V. cholerae strain SU129B. The genome of V. cholerae strain SU129B also contained several genes conferring antibiotic resistance, including almF, almG, almE, varG, parE, and QnrVC4. The genes regulate the bacterial cell envelope and stress response systems. The alm gene cluster (almF, almG, and almE) is associated with lipid A modification, altering the LPS structure of the bacterial outer membrane. These modifications may affect membrane stability, susceptibility to host-derived antimicrobial peptides, and overall bacterial survival under harsh environmental circumstances [63,64].
The CRP is identified as a component of RND efflux systems, which provide resistance to multiple forms of antibiotics, including macrolides, fluoroquinolones, penams, and tetracyclines [65,66]. This gene resides near the mobile genetic elements rnr and dam_1, suggesting a possible genomic link between resistance factors and mobility-associated regions [67]. The presence of vanT genes within the vanG cluster indicates a potential for resistance through modifications to cell wall targets [68]. This gene confers vancomycin resistance and is closely associated with various MGEs, rep, priB, and dnaB, suggesting horizontal gene transfer may have introduced the cluster [67]. Additional multidrug resistance mechanisms were identified, including rsmA, a gene linked to the RND efflux pump that confers resistance to fluoroquinolones, diaminopyrimidines, and phenicols. The close association between MGEs and this gene, including ygaD, recA, and mutS, suggests a potential recombination hotspot [67]. These findings emphasize the strains’ ability to withstand antimicrobial agents, underscoring their clinical significance and the challenges in managing Vibrio infections [69]. The strain SU129B was shown to harbor a parE gene variant identical to that in Escherichia coli, associated with resistance to fluoroquinolone drugs [70]. A D476N point mutation in the parE protein confers resistance by inducing structural changes in the DNA gyrase subunit, the primary target of fluoroquinolones. This mutation may reduce the antibiotic's binding affinity, enabling bacterial survival in the presence of fluoroquinolones. This mechanism exemplifies the modification of antibiotic targets, and the identification of the mutant parE gene confirms that the strain exhibits a fluoroquinolone-resistant genotype [71]. The presence of this resistance gene within a cluster rich in MGEs, including recJ, xerD, parC, and cca, indicates a high potential for mobility through recombination or plasmid-mediated transfer [72]. The QnrVC4 gene confers resistance to fluoroquinolones by encoding a pentapeptide repeat protein. This protein protects DNA gyrase and topoisomerase IV from the inhibitory impacts of antibiotics, thereby maintaining the natural DNA replication process and reducing the bactericidal effect of the drug [73].
The genome of strain SU129B was expected to have many virulence-associated genes (toxR, crp, rpoS, rpoE, ompR, arcA, relA, hfq, sspA, cyaA, and fur), which facilitate the production of virulence proteins, stress response pathways, and quorum-sensing systems, allowing bacteria to adapt to fluctuating environmental and host circumstances [74–80]. Apart from other virulence-associated genes, rtxA encodes the RTX toxin, a crucial virulence factor [81]. Meanwhile, tolC and tolB are components of secretion systems that facilitate the release of toxins and antimicrobial substances [82]. However, many virulence-associated genes remain classified as hypothetical or uncharacterized. The presence of these genes suggests the potential for uncharacterized functional components within the genome. Hypothetical proteins are identified from open reading frames (ORFs) whose translated products are undefined and lacking functional annotation [83]. Comprehensive analysis of these proteins in V. cholerae may reveal novel biological functions and facilitate the identification of new diagnostic biomarkers or therapeutic targets for infection management. Future research should prioritize the functional characterization of these genes and their products through advanced computational approaches, including homology modeling, protein–protein interaction analysis, and machine learning-based prediction tools [84,85]. Special attention should be paid to hypothetical proteins that harbor sequence motifs or conserved domains linked to pathogenicity, antibiotic resistance, or biofilm formation. This research found that the V. cholerae pan-genome is expanding and shows significant genetic diversity among strains. Novel genetic elements arise through horizontal gene transfer, mutations, and environmental changes.
The study identified a core genome of 2,085 genes, representing the conserved genomic framework shared by V. cholerae strains. Comparative pangenomic and phylogenomic analyses showed that isolates from non-human sources have notable genomic and evolutionary similarities to clinical strains linked to human illness. The V. cholerae strain SU129B from Bangladesh is closely related phylogenetically to the clinical strain BC1071 from Germany. These findings suggest conservation of genomic features across geographically diverse strains, indicating shared evolutionary origins or horizontal gene transfer [86]. The genomic characteristics of V. cholerae strain SU129B reinforce a One Health framework, particularly regarding aquaculture and environmental surveillance in South Asia, including Bangladesh. The detection of virulence-associated genes, stress-response mechanisms, and multidrug resistance determinants in this non-O1/non-O139 environmental isolate emphasizes the role of aquatic reservoirs, particularly aquaculture systems, as significant sources of clinically relevant traits. The identification of xenobiotic degradation pathways and resistance genes associated with mobile genetic elements indicates selective pressure resulting from antibiotic and chemical usage in regional aquaculture. The phylogenomic similarity between SU129B and geographically distant clinical strains demonstrates the transboundary dynamics of V. cholerae evolution and highlights the necessity for integrated genomic surveillance across environmental, animal, and human health sectors. These results advocate for the integration of whole-genome sequencing into routine aquaculture and water-monitoring initiatives to enhance antimicrobial resistance monitoring and cholera preparedness in endemic areas.
5. Conclusion
This research provides detailed genomic insights into the Vibrio cholerae strain SU129B, isolated from white pacific white shrimp in Bangladesh's aquaculture setting. Comprehensive genomic and pan-genomic analyses revealed a remarkably flexible, multidrug-resistant strain that harbors a variety of biosynthetic gene clusters, virulence regulators, and resistance factors. Moreover, the existence of essential regulatory genes underscores the intricate genetic regulation of virulence, stress response, and metabolic adaptation. The multidrug resistance profile suggests that V. cholerae strain SU129B could serve as an environmental reservoir for the spread of antimicrobial resistance. A comparative phylogenomic analysis has uncovered a significant genetic link between the Bangladeshi strain SU129B and the clinical strain BC1071 from Germany, indicating a possible global genetic convergence among both environmental and pathogenic V. cholerae lineages. These findings highlight the ongoing genomic evolution and adaptability of V. cholerae in aquaculture systems, emphasizing the need for continuous genomic monitoring to mitigate zoonotic transmission and antimicrobial resistance. Future research should focus on characterizing unannotated and hypothetical genes to identify new mechanisms of virulence, resistance, and environmental survival.
Supporting information
S1 Table. The biochemical tests performed for the presumptive identification of the V. cholerae strain SU129B.
https://doi.org/10.1371/journal.pone.0344787.s001
(DOCX)
S2 Table. The phenotypic antibiotic resistance profile of the V. cholerae strain SU129B.
https://doi.org/10.1371/journal.pone.0344787.s002
(DOCX)
S1 Data. Subsystem analysis of the V. cholerae strain SU129B.
https://doi.org/10.1371/journal.pone.0344787.s003
(XLSX)
S2 Data. Metabolic pathway analysis the V. cholerae strain SU129B.
https://doi.org/10.1371/journal.pone.0344787.s004
(XLSX)
S3 Data. Virulence associated gene analysis of the V. cholerae strain SU129B.
https://doi.org/10.1371/journal.pone.0344787.s005
(XLSX)
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