Environmental regulation of virulence in Vibrio cholerae: integrating multi-omics, predictive models, and comparative insights across emerging Vibrio species

Abstract

Vibrio cholerae thrives at the interface between the aquatic environment and the human host dynamically through integration of environmental signals to highly coordinated virulence programs. This review examines how environmental sensing systems, regulatory networks, biofilm formation, and secretory systems have been integrated to maintain stability, transmission, and pathogenesis. By incorporating these advances in genomics, transcriptomics, proteomics, metabolomics, and predictive models, we demonstrate how multi-omics approaches have changed our understanding of condition-related virulence regulation at the system-level view. In addition, we extend this framework via comparative analysis among pathogenic Vibrio species and also reveal conserved regulatory architectures alongside species-specific adaptations that affect ecological fitness and pathological potential. Finally, discussing how multi-omics and machine learning integration can enable outbreak prediction, inform One Health approaches, and identify environmental regulating anti-virulence targets. This integrated insight positions environmental regulation as a central organizing agent of Vibrio pathogenicity and provides a roadmap for translating complex biological datasets to practical insights in the public health and treatment field.

Introduction

Vibrio species infections of humans, such as Vibrio cholerae, the causative agent of cholera, and other members of the Vibrionaceae family, such as V. parahaemolyticus and V. vulnificus, are on the rise globally. Climate changes in marine environments influence this trend [1]. V. cholerae is a gram-negative, rod-shaped, and mobile bacterium from the Vibrionaceae family and is taxonomically classified within the Gammaproteobacteria [2]. This waterborne bacterium has the capacity to survive in aquatic environments and cause severe diarrhea in humans [3]. Although more than 200 serotypes of V. cholerae have been identified, only the O1 and O139 serotypes are responsible for epidemic and pandemic cholera, with the O1 serotype having been divided into two biotypes, classic and El Tor, and the El Tor biotype being the dominant lineage in the current seventh pandemic [4–6]. Cholera remains an important global public health issue. According to the World Health Organization (WHO) global cholera situation update (17 August 2025), a total of 409,222 cholera/acute watery diarrhea (AWD) cases and 4738 deaths were reported across multiple WHO regions, especially where the availability of clean water is poor. Although the actual global burden of cholera is considered to be quite higher than reported cases due to underdiagnosis and limited surveillance capacity in endemic areas, the reported numbers still highlight the significant impact of the disease on public health. Environmental sensing enables V. cholerae to transition from aquatic reservoirs to human hosts, driving outbreaks in vulnerable populations of Vibrio [7]. Recent advancements in multi-omics technologies such as genomics, transcriptomics, and proteomics are clarifying molecular mechanisms that control these adaptations [8]. Additionally, comparative studies among other members of the Vibrionaceae family show both similar and distinct virulence strategies [9–11].

The environmental regulation of V. cholerae virulence relies on complex sensory systems. The membrane-associated regulatory protein couples ToxR/ToxS and TcpP/TcpH, perceives identical signals, such as pH and bile salts, and functions in conjunction with the activation of the transcription factor ToxT, which results in upregulation of cholera toxin (CT) and toxin-coregulated pilus (TCP). Both these agents are essential for intestine colonization [12, 13]. Bacteria also utilize quorum sensing (QS) and small regulatory RNAs (sRNAs) to control these responses with accuracy. Transcriptomics studies reveal sRNAs regulation of virulence gene expression under low-oxygen conditions, common in aquatic environments [14–16]. Biofilm formation is the foundation for V. cholerae persistence in water and host colonization [17–20]. Biofilms that are encapsulated with extracellular polymeric substances (EPS) enhance the stress resistance of bacteria and facilitate intestinal adhesion [18, 19]. Multi-omics analyses manifest the role of Vibrio polysaccharide (VPS) and rugosity and biofilm structure modulator (rbm) gene clusters in biofilm synthesis, and proteomics studies also indicate bile-induced matrix protein upregulation [21–23]. To complement these main virulence pathways, several other proteins participate in V. cholerae surveillance and host adaptation. Outer membrane protein U (OmpU) enhances the resistance to bile acids and also assists in maintaining membrane integrity during intestinal colonization [23]. Although secreted metalloprotease hemagglutinin/protease A (HapA) facilitates bacterial detachment in late stages of the infection and enhances its environmental dissemination [24]. Accessory toxins such as accessory cholera enterotoxin (ACE), zonula occludens toxin (Zot), and hemolysin A (HlyA), through adjustment of epithelium permeability and inflammatory response, in association with CT, exacerbate diarrhea [25]. Primary colonization is supported by flagellar motility genes like the flagellin A gene (flaA) that provide the possibility of initial contact, especially before TCP connection [26]. Regulatory proteins VpsT and VpsR activate biofilm-related gene clusters, although HapR, in high cellular density conditions, suppresses biofilm formation and regulates the transition between persistence and dispersion states. Finally, these agents integrate environmental sensing with host colonization strategies and reinforce the aquatic and pathogenic lifestyle of V. cholerae [27]. Environmental factors such as temperature and salinity can affect biofilm dynamics, with studies showing that warmer waters can increase biofilm formation and enhance cholera risks [28, 29]. Secretion systems, especially the type VI (T6SS) and type II (T2SS), are crucial in pathogenesis and transporting cytotoxins and effectors that disrupt host cells and competitors. Temperature regulates T6SS via QS and histone-like nucleoid structural proteins. Proteomic profiling identifies variable T6SS effectors that support interbacterial competition and host invasion. These systems serve as a bridge between environmental adaptation and virulence [30–32].

Multi-omics integration with machine learning (ML) significantly improves the V. cholerae dynamics prediction. Environmental factors such as sea surface temperature (SST), chlorophyll a concentration, and salinity have been used as disease outbreak prediction models, especially in the coastal regions of South Asia. For example, ML models trained on SST and phytoplankton bloom satellite images forecast seasonal cholera cases in the coastline of India and Bangladesh [33, 34]. The strategies supplement early warning systems, especially with climate change contributing to the rise in the risk of Vibrio species development [35, 36]. Comparative analyses between Vibrionaceae family members show different mechanisms of virulence [9]. V. parahaemolyticus uses T3SS for effector transmission, while V. vulnificus relies on hemolysins [37, 38]. Multi-omics studies reveal QS and species-specific responses to any osmotic stressors, and bioinformatics analyses also show divergent regulatory motifs. These findings enlighten interventional strategies [39–41]. Future perspectives are predictive models, the One Health framework, and novel therapeutics. Real-time environmental and genomic data enhance surveillance and monitoring in cholera-prone areas [42]. Aquaculture is one of the ecological factors that is addressed by One Health approaches [43]. Bacteriophage treatments and clustered regularly interspaced short palindromic repeats (CRISPR) antimicrobials are also manifesting potential in combating V. cholerae multidrug-resistant biofilms [44–46]. Rather than acting separately, environmental persistence, virulence regulation, and host colonization in V. cholerae act as a component of one unified adaptive strategy regulated by complex signal transduction and regulatory pathways [25, 47]. Even though many environmental V. cholerae strains have preserved their environmental-sensing and stress response regulatory systems, most of them have remained non-pathogenic due to their inability to acquire, maintain, or express virulence determinants, such as CTXφ and VPI-1. Successful pandemic lineages appear to combine environmental persistence capacity with regulatory architectures capable of efficiently integrating virulence activation under host-associated conditions [48, 49]. In this context, integrated multi-omics approaches and predictive modeling frameworks provide prominent insights to understand how environmental adaptation, regulatory switching, and pathogenic potential form cholera emergence and transmission dynamics.

Collectively, these results enrich our knowledge of how environmental signals trigger virulence in V. cholerae. Bringing molecular, ecological, and computational perspectives together, this review intends to enhance outbreak prediction and novel therapies development in the face of emerging climate-related challenges.

Environmental regulation of virulence in V. cholerae

To thrive in both aquatic and intestinal environments, V. cholerae utilizes a multi-layered sensory network that translates environmental triggers to genetic and physiologic responses. This network acts as a molecular interface between a bacterium and its surrounding environment and regulates processes such as virulence, motility, and biofilm formation through external and internal signal integration. Based on mechanistic insights, sensing systems are divided into three main categories, including two-component systems (TCS), QS, and intracellular second-messenger pathways [50–53]. TCS is considered one of the most fundamental sensory modules in V. cholerae. They typically consist of a membrane-bound protein histidine kinase that detects environmental changes and a cytoplasmic regulator that controls gene expression downstream [54]. For example, ToxR–ToxS and TcpP–TcpH systems, in response to signals such as bile salts, osmolality, pH, and temperature that represent intestinal milieu, activate and inculcate ToxT expression synergistically [55–59]. ToxT, as a main regulator protein, stimulates virulence gene expression, including ctxAB and TcpA [60]. Recent studies show that ToxR undergoes certain structural changes in response to bile salts that enhance its ability to bind to promoters and activate transcription. As a result, a direct link between environmental sensors and pathogenic expression is established [58, 61].

The second layer of the network is the QS. QS is considered an advanced intercellular link that coordinates population-wide behavior based on cell density [62, 63]. V. cholerae utilizes multi-autoinducers such as cholera autoinducer 1 (CAI-1) and autoinducer-2 (AI-2); these molecules finally affect the phosphorylation cascade, including LuxU and LuxO [64, 65]. In low cell density, phosphorylated LuxO increases the small regulatory RNA (Qrr sRNAs) expression that inhibits the translation of HapR, a global transcription factor. This inhibition activates virulence and motility gene expression; this regulation is reversed at high cell density [62, 66–68]. Recent studies show that QS isn’t just an on/off switch but also is continuous and remittent dynamic [63]. Regulatory feedbacks between sRNAs and Hfq protein control the timing and severity of virulence responses precisely [63, 67].

The third layer is intracellular second messengers, especially Cyclic di-guanosine monophosphate (c-di-GMP), that control the switch between motile and sessile lifestyles [69]. Elevated c-di-GMP levels activate transcriptional regulators such as VpsR and VpsT which increase exopolysaccharide synthesis and biofilm formation, although they inhibit cell motility. In contrast, c-di-GMP reduction increases motility and facilitates host colonization [70–73]. The balance of c-di-GMP has been maintained by diguanylate cyclase (DGC) and phosphodiesterase (PDE), which are influenced by metabolic and environmental signals [69]. Multi-omics studies show that spatial distribution of c-di-GMP can guide collective behaviors and biofilm patterns in the microbial communities [74–76]. In addition to these classic pathways, V. cholerae uses chemosensory arrays and mechanosensory channels to detect physical forces, nutrient gradients, and chemical compounds [77, 78]. Some host-derived biliary lipids can modulate the TcpP/TcpH–ToxR axis and also change the efficacy of ToxT activation and virulence expression [12, 79].

Beyond signal conception, V. cholerae uses complex regulatory networks to translate these signals to coordinated behavioral and transcriptional outputs [80]. In the center of these networks, the ToxR–TcpP–ToxT regulatory cascades serve as a central hub and link environmental inputs to virulence factor expression [81]. QS and c-di-GMP signaling act as subsidiary regulatory layers and modulate these cascades based on cell density and environmental states [25, 51]. Global regulators such as RpoS, carbon storage regulator A (CsrA), and ferric uptake regulator (Fur) also incorporate metabolic and stress signals in this hierarchy. CsrA connects carbon metabolism with pathogenicity factor expression through regulating ToxT transcription. However, RpoS participates in stress adaptation during transition from aquatic to intestinal environments [67, 82, 83]. This integration confirms that virulence programs are activated just in optimum physiological and environmental conditions [80]. These multi-layered interactions are amplified by temporal feedback loops. QS regulates c-di-GMP levels; c-di-GMP in turn influences the access of transcription factors to promoters, and sRNAs provide quick, reversible post-transcriptional control [52, 67]. This architecture enables V. cholerae to organize responses that are both sensitive to subtle environmental changes and resistant to external noises [84].

The output layer of this system is gene adaptation that converts the integrated signals to complex phenotypic states. Recent advances in RNA sequencing (RNA-seq), single-cell transcriptomics, and multi-omics have revealed that V. cholerae represents highly dynamic and heterogenous patterns of gene expression [63, 85, 86]. Environmental induction of ToxR/TcpP increases ToxT and downstream virulence gene transcription drastically [84]. Simultaneously, QS co-regulated sRNAs change the transcriptome through the inhibition or stabilization of main messenger RNAs (mRNAs). These shifts change not only virulence pathways but also metabolic and stress-response genes [67]. Emerging approaches that combine multi-omics, structural biology, and machine learning are going to reveal new and previously unknown regulatory nodes that control V. cholerae adaptation [87, 88]. Predictive network models enable the correlation between environmental signals with virulence potential and outbreak dynamics [89, 90]. Targeting key parts in sensory-regulatory functions such as ToxR and LuxO, or c-di-GMP modulating enzymes, represents promising approaches for developing anti-virulence treatments [58, 91, 92]. In general, understanding these interconnected sensory and regulatory pathways not only provides a system-level view of V. cholerae biology but also provides the basis for designing interventional strategies in the prevention and control of cholera disease [50, 62, 67].

Biofilm formation and persistence of V. cholerae

Biofilm in V. cholerae is not only a defensive structure but also an evolutionary strategy that allows the bacteria to transition between aquatic and intestinal environments. Recently, the research perspectives of biofilm have exceeded the descriptive and genetic level and moved toward understanding a complex network of adaptation, persistence, and transmission. The network in which environmental signals, host-imposed pressure, regulatory pathways, and metabolic programs have been integrated to generate diverse physiologic conditions [93–96].

In aquatic environments, V. cholerae faces diverse stressors that trigger the transition to a biofilm lifestyle. Attaching to biological and non-biological surfaces such as chitinous skeletons of crustaceans, phytoplankton, zooplankton, and also microplastics whose presence increasingly stimulates early biofilm formation [97–99]. Surface sensing is mainly carried out through mannose-sensitive hemagglutinin (MHSA) pili and N-acetylglucosamine-binding protein A (GbpA) adhesions, which trigger downstream regulatory cascades. Once attached, V. cholerae enters a preserved habitat where exopolysaccharide production, matrix protein secretion, and changes in cell physiology induce long-term environmental permanence [93, 100–102]. Biofilms also act as a reservoir for cells that enter a conditionally viable environmental cells (CVEC) or viable but non-culturable (VBNC) state, which provides an additional layer of stability under extreme conditions. One of the key findings of recent studies is that biofilm-related cells can persist for a long period of time, even when planktonic cells die. With the return of appropriate conditions, CVEC cells or low-activity biofilm cells can reactivate and contaminate the environment, contributing to the seasonal cholera outbreaks [96, 98, 103]. In a human host, biofilm-derived cells show enhanced tolerance to bile, acid, and oxidative stress, and often exhibit increased colonization potential [104]. Host-derived elements such as mucins, bile salts, and nutrient changes, activate dispersal responses and liberate active motile cells from mature biofilms, which then colonize the gut more effectively [105, 106]. Multi-omics analyses have also shown that the gene expression pattern of biofilm-derived cells is significantly different from standard planktonic cultures, supporting the idea that environmental biofilms prime V. cholerae for infection [86, 107].

Biofilm construction in V. cholerae is organized by a multi-layered regulatory network. VPS which is synthesized by the vps gene clusters, is the pre-eminent part of this process. This polymer forms the biofilm skeleton, and matrix proteins such as rugosity and biofilm structure modulator A (RbmA), RbmB, RbmC, and biofilm-associated protein 1 (Bap1) make this structure stable and functional through enhancing structural incorruptibility, mediating cell-cell interactions, and shaping the biofilm architecture [25, 93, 108–110]. Transcription regulation is mainly controlled by two key global regulators, including VpsT and VpsR, both of whom respond to changes in the intracellular second messenger c-di-GMP. An increase in c-di-GMP levels activates these regulators, then shifting cells from motile toward biofilm formation via turning on vps genes and turning off flagellar genes [47, 94, 111]. Recent studies indicate that c-di-GMP levels are regulated by a large and variable set of DGC and PDE that each of them responds to specific environmental inputs. This modulator-regulating architecture allows V. cholerae to control biofilm formation precisely [47, 94]. Phenotypic diversity also contributes to biofilm strength. The rugose variant, which is characterized by an increase in vps production and folded colony morphology, repeatedly has been seen under stress conditions and also is associated with increased tolerance to antimicrobial agents [112, 113]. Conversely, non-matrix-producing subpopulations maintain their motility and are poised for dispersal, revealing the inherent heterogeneity within the biofilm. One of the emerging findings in recent studies is injury-derived biofilm induction, in which one subpopulation of cell lysis releases signals that rapidly trigger the remaining cells to increase biofilm formation. This collective response to the stress provides a mechanism for rapid rearrangement of the microbial community under inappropriate conditions [86, 95, 114].

The recent integration of genomics, transcriptomics, proteomics, metabolomics, and single-cell approaches is considerably expanding our understanding of V. cholerae biofilm mechanisms [86, 115]. Omics-based studies manifest novel regulatory agents, including small RNAs, metabolite-responsive regulators, and post-transcriptional changes that influence matrix protein stability and signaling enzymes [67, 115, 116]. Multi-omics analyses show that biofilm formation requires a synchronized metabolic change. Key changes such as alteration of central carbon metabolism, redox balance, and lipid biosynthesis indicate that forming a biofilm is energy-intensive and needs a targeted allocation of resources [87, 115]. Proteomic studies have also shown that matrix-forming agents, nutrient transporters, and stress-responsive proteins are expressed differently in each spatial region of biofilm. Single-cell transcriptomics and spatial metabolomics reveal that V. cholerae biofilms are not uniform structures. Rather, the presence of nutrient gradients, oxygen diffusion limitations, and matrix density create different microenvironments that lead to specific and different behavior of each subpopulation cell. These findings support the idea that biofilms are emergent multicellular structures with distinct functional regions [86, 117, 118]. The shift from environmental persistence to host-mediated virulence in V. cholerae is associated with a series of physiological and regulatory modifications affecting both environmental persistence and infection capability. Some of the differences between these two adaptations are illustrated in Table 1. Collectively, multi-omics datasets provide a system-level insight into how V. cholerae incorporates environmental signals, regulatory circuits, and metabolic demands to maintain biofilm communities.

Table 1.

Comparison of physiological and regulatory features of environmental persistence vs. host virulence in vibrio cholerae

Feature	Environmental persistence state	Host-associated virulent state
Dominant lifestyle	Biofilm-associated/sessile	Motile/planktonic
Intracellular c-di-GMP	High	Low
Main regulatory pathways	HapR, VpsR, VpsT	ToxR, TcpP, ToxT
Primary biological function	Environmental survival and persistence	Host colonization and toxin production
Virulence gene expression	Minimal or absent	High (CTX, TCP, colonization-associated factors)
Phenotypic characteristics	Rugose phenotype, VPS production, stress tolerance, VBNC/CVEC states	TCP expression, cholera toxin production, intestinal colonization
Ecological niche	Aquatic environments	Human intestinal tract
Motility	Reduced	Increased
Biofilm formation	Enhanced	Reduced/dispersed
Epidemiological importance	Long-term environmental reservoir	Disease transmission and outbreak potential
Environmental/host cues	Nutrient limitation, osmotic stress, surface attachment	Bile salts, bicarbonate, host-derived signals

Mechanistic integration in V. cholerae

QS regulation in V. cholerae

To our knowledge, most bacterial quorum-sensing systems provoke biofilm formation and virulence expression, which are induced by autoinducers at high cell density. Unlike several pathogens (most notably Pseudomonas aeruginosa), V. cholerae QS functions to repress virulence gene expression and biofilm formation in response to accumulated QS signals [119]. This regulation is thought to have a self-limiting nature and adaptation [120].

V. cholerae uses QS autoinducers to switch between biofilm (sessile communities) and planktonic cells and to control virulence factor expression. Two QS circuits occur in V. cholerae. The major system consists of CAI-1 and its receptor, cholerae QS sensor (CqsS), while a second system consists of autoinducer 2 (AI-2) and its receptor, LuxPQ [121, 122]. The main QS autoinducer is CAI-1, which is synthesized by the enzymatic activity of CqsA. CqsA conjugates in vivo S-adenosyl-L-methionine (SAM) and decanoyl-coenzyme to produce a signaling molecule, 3-aminotridec-2-en-4-one (Ea-CAI-1). Ea-CAI-1 is subsequently converted to CAI-1 via an intermediate molecule, tridecane-3,4-dione (DK-CAI-1) [123]. CAI-1 accumulates to higher levels within biofilm communities than in the planktonic state, resulting in the expression of QS regulator HapR [124]. Furthermore, there are two QS regulators, LuxO and the master regulator HapR, which are responsible for cellular mechanisms including motility, protease, biofilm formation, and virulence expression [125]. QS circuits are displayed in Fig. 1.

Fig. 1.

Regulation of QS in different conditions, depending on the density of the bacterial population. In high cell density, elevated levels of CAI-1 and AI-2 are sensed by CqsS and LuxPQ, resulting in dephosphorylation of LuxO and subsequent activation of HapR. HapR downregulates both virulence and biofilm-related genes. The opposite effect can be detected in low cell density conditions. Created with BioRender.com

High Cell Density (HCD) regulation

The QS system negatively regulates biofilm formation through the regulators AphA and HapR [122]. At high cell density, accumulation of the autoinducer CAI-1 inactivates and dephosphorylates LuxO and terminates the transcription of sRNAs, releasing the repression on HapR [126]. HapR binds a specific site on the aphA promoter (positions − 85 to − 58), blocking its transcription. Activated HapR then negatively regulates aphA, represses vps genes, and reduces intracellular c-di-GMP levels, collectively inhibiting VpsT-dependent biofilm formation (see Fig. 1) [127]. The resulting low AphA level inhibits the virulence gene cascade by blocking TcpP and ToxR-regulon expression, lowering virulence factors under HCD conditions [127, 128]. Moreover, at HCD, expression of the hap gene encoding the Hap protease is induced via the expression of HapA, facilitating the detachment of the cellular aggregation.

Low Cell Density (LCD) regulation

In contrast, initially at LCD, the quorum-sensing receptors are unliganded, resulting in the phosphorylation of LuxO. Phospho-LuxO activates the transcription of regulatory RNAs (qrr sRNAs), which in response repress HapR and conversely activate the expression of aphA. High aphA levels increase the vps genes transcription and the biofilm activator VpsT, facilitating biofilm formation. Therefore, LuxO negatively regulates the expression of the HapR protein (Fig. 1). Accordingly, reduced HapR levels repress the expression of the virulence regulator TcpP; however, at this state, it promotes the activation of virulence factors within the ToxR regulon [121, 129]. These factors enable the V. cholerae to colonize in the small intestine and produce biofilm formation initially.

c-di-GMP signaling & biofilm control

In V. cholerae, QS regulates the intracellular concentration of the second messenger cyclic diguanylate (c-di-GMP), thereby controlling the transition between planktonic and biofilm life [130, 131]. Accumulation of the second messenger c-di-GMP, synthesized by multiple diguanylate cyclases (CdgA, CdgH, CdgL, CdgK, CdgM), triggers the activation and dimerization of VpsR and VpsT to induce biofilm formation (see Fig. 2) [131]. The expression of the vps and rbm clusters is positively regulated by VpsR and VpsT. This results in the induction of the genes within the vps and rbm clusters that encode the polysaccharide and protein components of the biofilm extracellular matrix [132]. In parallel, c-di-GMP binds to FlrA, inhibiting its activity and repressing flagellar gene expression, thereby decreasing the motility of V. cholerae [72]. Moreover, five proteins containing PilZ domains may have some unclear role in biofilm formation, as their deletion resulted in reduced motility, diminished biofilm formation, and colonization [133]. In contrast to its role in biofilm formation, high levels of c-di-GMP might repress virulence gene expression [134, 135].

Fig. 2.

V. cholerae regulates biofilm formation via a c-di-GMP-dependent manner. c-di-GMP active 2 gene clusters, contributing to biofilm formation. Furthermore, c-di-GMP suppresses Flagel and toxin synthesis, making V. cholerae less virulent (potentially dormant phase). Created with BioRender.com

During infection, V. cholerae constantly adjusts its intracellular c-di-GMP concentration to coordinate transitions between motile and sessile states based on intestinal-environmental factors. At the early colonization stage, decreased c-di-GMP level through activation of PDE and repression of DGC promotes optimal expression of motility and virulence genes [136]. Later in infection, recombination-based in vivo expression technology (RIVET) revealed activation of GGDEF-domain DGCs, indicating that the bacterium raises its c-di-GMP intracellular concentration before leaving the host to promote biofilm aggregation for environmental survival [135]. This surprisingly shows that V. cholerae may occupy transitional states between planktonic and biofilm forms within the intestine, influenced by location-specific factors [137, 138].

Collectively, these results accentuate how quorum sensing, c-di-GMP signaling, and virulence-associated regulatory networks integrate the transition between environmental persistence and host-associated colonization in V. cholerae.

Virulence gene regulation

V. cholerae contains a virulence regulon of more than 20 genes involved in colonization, toxin production, and adaptation. The virulence regulon is regulated by a cascade of transcriptional regulators that includes ToxR, TcpP, and ToxT.

The expression of virulence genes is associated with the ToxR signal cascade, along with the QS system. QS regulates the production of virulence factors via the AphA–HapR regulatory system, depending on the density level. This transcriptional cascade is initiated with TcpPH. AphA, which is a member of a new and relatively uncharacterized regulatory family, displays high homology to PadR, a regulator of phenolic acid decarboxylases [139]. aphA and aphB enhance the transcription of the transmembrane regulators TcpP and TcpH [140, 141]. Apha activates TcpPH expression by allowing the binding of the LysR-type regulator, aphB, to the promoter [142]. TcpPH, in coordination with transmembrane regulators ToxR/S, activates the expression of the soluble AraC-family regulator ToxT [143, 144]. ToxT is in coordination with the ctxA and tcpA promoters, enabling the production of CT and TCP [145].

The two virulence factors, accompanied by vps genes that are expressed by V. cholerae O1 and O139, CT, is responsible for the severe rice-watery diarrhea typical of this disease, and TCP is required for intestinal colonization. The genes encoding CT subunits (ctxA and ctxB) form an operon within the prophage form of the filamentous phage CTXF [146]. In contrast, the genes associated with the TCP establish a cluster known as the Vibrio pathogenicity island (VPI) or TCP island, in which tcpA encodes the major pilus subunit. Another virulence factor is the sheathed polar flagellum driven by sodium motive force (SMF) [147], which is expressed by a hierarchical regulatory cascade that involves the alternative RNA polymerase subunits σ54 and σ28 and the σ54-dependent transcriptional activators FlrA and FlrC [148]. A recent study claims that its participation in virulence gene expression with the mutant flagellum enhances the transcription of ctxA and tcpA [149].

Moreover, histone-like nucleoid structuring protein (H-NS) is a redundant protein that functions as a transcriptional silencer, repressing horizontally acquired virulence genes (ctxAB, tcpA) and biofilm-matrix genes (vps, rbm) [150, 151]. Fluctuations in the intracellular c-di-GMP pool mediate the antagonism of H-NS repression via sequential activation of VpsR and VpsT [152].

Host-derived signals, particularly intestinal bile and bicarbonate, regulate the V. cholerae cascades. Intestinal bile is a combination of bile acids, cholesterol, and unsaturated fatty acids and is subject to numerous chemical transformations. Bile mixtures stimulate RNA polymerase subunits σE and σS envelope-stress responses and elevate intracellular c-di-GMP, thereby enhancing VpsR and VpsT-mediated biofilm activation, while unsaturated fatty acids repress premature transcription of ctxAB and tcpA through inhibition of ToxT [153, 154]. On the contrary, bicarbonate, concentrated near the intestinal villi, restores ToxT activity and favors expression of major virulence factors [155, 156].

The dual environmental condition of V. cholerae introduces stress-responsive σ-factor circuits (σS/RpoS and σE/RpoE) as upstream integrators linking external signals to the QS and c-di-GMP regulatory system [157, 158].

Microbiome-based protection against cholera (Metagenomics)

Shotgun metagenomics of the gut microbiome allowed high-resolution profiling of microbial genes, species, and strains associated with susceptibility to V. cholerae infection. A Random Forest model was used by Levade et al. to identify predictive features based on their relative weighting in the RF model at the species/genus/strain level, comparing bacterial abundances in infected and uninfected contacts [159]. Species including Eubacterium rectale, Campylobacter hominis, Ruminococcus gnavus, Bacteroides vulgatus, Veillonella parvula, and members of the Prevotella and Eubacterium genera were most abundant in uninfected individuals. Conversely, in contacts who developed V. cholerae infection, species of the genera Bifidobacterium, Actinomyces, and Collinsella were primarily associated with asymptomatic cases, while Clostridium ventriculi, Streptococcus parasanguinis, members of Veillonella, and Shigella were profuse in symptomatic patients [159].

The gut microbiome (GM) can defend against enteropathogenic infections, including V. cholerae. After exposure to V. cholerae, the exuberance of Blautia obeum increased, modulating V. cholerae pathogenicity via inhibiting QS in a mouse model [160].

Short-chain fatty acid (SCFA) production is primarily associated with protection against infection. E. rectale, R. gnavus, and B. vulgatus produce SCFAs from mucin glycans [161, 162]. B. vulgatus increases SCFA abundance in the gut, which inhibits V. cholerae colonization in mice [163]. These compounds induce immune cell maturation particularly via histone deacetylase inhibition and alterations in gene expression [164, 165]. SCFAs have been shown to suppress CT-induced chloride secretion in the mouse gut, limiting water and sodium loss, and they can also enhance CT-specific antibody responses [166–168].

In contacts who developed asymptomatic V. cholerae infections, Bifidobacterium species showed an increased level. Bifidobacteria are major producers of acetate and lactate to protect the gut against enteric infection and suppress V. cholerae biofilm formation, respectively [169, 170]. Bifidobacterium bifidum and Bifidobacterium adolescentis change bile acids to reduce the activity of V. cholerae type VI secretion systems [171].

Iron competition may provide protection against V. cholerae, as several gene families involved in iron transport, iron regulation, and riboflavin metabolism were enriched in protected individuals [172]. Increased microbial competition for iron can reduce its availability in the gut, and both riboflavin and iron are necessary for V. cholerae colonization and virulence. This iron limitation may consequently impair the growth of the bacteria [173, 174].

Instead of acting independently, lactate, SCFAs, and iron competition form an integrated metabolic insight that collectively influences oxidative stress, nutrient availability, and virulence regulation in V. cholerae. This convergence shapes a mechanical framework that explains why microbiomes enriched in protective metabolic functions are associated with asymptomatic infection, although their absence correlates with symptomatic disease outcomes (see Fig. 3) [175].

Fig. 3.

Microbiota-derived metabolites and iron competition form intestinal redox state, pH, and V. cholerae infection. The gut microbiome produces SCFAs and L-lactate that maintain luminal pH and host redox balance. During infection, V. cholerae inhibits the gut microbiome and alters metabolite availability via iron competition. Oxidative and acidic stresses induce V. cholerae antioxidant and pH homeostasis responses, which cause CT release and intestinal inflammation. Created with BioRender.com

Genomics of non-O1/O139 V. cholerae

A comprehensive proteome and secretome study of non-O1/O139 V. cholerae with different genotypes and phenotypes isolated from aquatic animals identified common extracellular and intracellular proteins, including virulence and resistance-associated proteins. Regarding genomics, none of the non-O1/O139 V. cholerae isolates carried the major toxin genes ctxAB, tcpA, ace, or zot. However, high prevalence of virulence-associated genes was found, including rtxABCD, hlyA, tlh, and hapA [176].

Proteomics and secretome analysis

Under virulence-inducing (ToxT-activated) conditions, outer membrane vesicle (OMV) proteomics identified 90 proteins, involving TcpA, TcpC, and CtxB, showing the physiological state of the bacteria in vivo. Integration of OMV proteomics with functional genomic datasets (RNA-seq, Tn-seq) demonstrated that several OMV-associated proteins are essential for growth or gastrointestinal colonization, including virulence factors such as TCP.

DegP is one of the essential OMC proteins, and its protease and chaperone mechanisms may be used as an antibacterial drug target, as it is found in both Gram-negative and Gram-positive organisms [177, 178]. DegP has an underlying role in guiding OMPs through the periplasm [179]. DegP induces nine proteins with abundances, including RmbA, RmbC, and Bap1, that serve as the major components of biofilm formation [180]. Reportedly, Bap1 adheres to the biofilm on the internal surface of the intestine, while RmbC and Bap1 encapsulate cell clusters [181]. HAP protein is associated with biofilm formation, and it’s shown to be dependent on DegP in OMV [182]. As a result, the shortage of DegP significantly causes a colonization defect, as it is reported for Streptococcus mutans and Porphyromonas gingivalis [183, 184].

The lack of DegP significantly causes a colonization defect, as it is shown in the DegP mutant EC956. It is unclear why the characterized DegP mutant suppresses host colonization in infant rabbits, whereas the lack of DegP-dependent proteins did not result in colonization defects. The colonization defect of the DegP mutant EC956 might be correlated with the Bap1 expression. The mutant rbmA gene leads to colonization defects, while the double-mutant rbmC bap1 did not result in similar defects [21]. Additionally, Zhu et al. showed that a mutation in the VPS gene (VC0920) did not considerably affect the colonization in the infant mouse model [185].

Comparative insights across pathogenic vibrio species

Environmental factors exhibit a great effect on the V. cholera. Seasonal fluctuation in temperature can induce phytoplankton growth. Phytoplankton and V. cholerae are in a symbiotic relationship; hence, the promotion of phytoplankton growth leads to an increase in the bacteria population. The optimum temperature for this bacterium is 37 °C, but it can survive in a range of 16°C to 42°C. It has been demonstrated that a temperature rise can increase the Vibrio growth index in aquatic environments [186]. In this context, sunlight can affect phytoplankton, indirectly influencing V. cholerae. It has been proposed that warm weather, accompanied by stasis of the flow, results in overgrowth of Vibrio [187]. Water depth is another factor. In the Bakerganj area, the depth of wells has a negative correlation with Vibrio infection. One possible explanation may be that shallow water causes faster warming (accompanied by a higher concentration of bacteria in a volume unit) and provides better conditions for the bacteria and infection [188]. Vibrio species are halophilic and have an optimum point for salt concentration. Alteration in salinity can accelerate V. cholerae growth as well as upregulate the toxin.

Simplified model for environmental regulation of V. cholerae toxicogenesis

Cholera has 2 main virulence factors, including CT and TCP. CT binds to the GM1 receptor, resulting in retrograde endocytosis and G protein–coupled receptors (GPCRs) activation. In the following, adenylate cyclase (AC) becomes activated and up-regulates Cystic fibrosis transmembrane conductance regulator (CFTR) [189]. TCP has a flexible structure, contributing to colonization in the intestine [190]. The main regulator of these factors is ToxT, influenced by several signaling pathways and determines the expression of CT and TCP. In a simplified model, two major sensors, including ToxR–ToxS and TcpP–TcpH, sense environmental signaling. In the following, they control ToxT activity, which in turn regulates expression of virulence genes. In the following, we discussed this regulatory system in more detail.

ToxT regulation

ToxT has 4 alleles, including AY, AF, SY, and SF. All these alleles are similar in coding sequences, but their promoter regulation is specific to each serotype. For instance, in Eltor serotypes such as N16961 and IB5230, AF and AY alleles are active, and the two remaining alleles are silent. The reverse pattern is seen in serotype IB5230. This shows that the regulation of ToxT expression is determined by regulatory interactions specific to each genetic background [60]. Opposite to El-Tor, all 4 alleles in O395 can stimulate expression of virulence genes. One possible explanation is the concept of the “self-amplifying loop”. As mentioned previously, ToxT expression is based on two major factors, including ToxR and TCP. The ToxR–TcpP system becomes active, resulting in the expression of the ToxT protein. ToxR binds to the ToxT promoter region located between − 100 and − 69 upstream of the transcriptional start site. This molecule serves as a promolecule, enhancing TCP binding to the region located on −51 to −32 [191]. ToxR-TcpP-induced production of ToxT is just at a basic level. In the following, the ToxT protein promotes its expression by itself (self-amplifying loop) [192, 193]. This causes overproduction of ToxT, which can upregulate virulence genes [60]. An overview of the ToxR regulatory system of V. cholerae is illustrated in Fig. 4.

Fig. 4.

ToxR regulatory system of V. cholerae and effect of environmental factors on virulence genes expression. ToxR and TcpP are sensors contributing to the shifting of V. cholerae between the virulent and dormant phenotypes. Created with BioRender.com

ToxR and TcpP as sensors for environmental factors

As previously said, ToxR and TcpP serve as environmental sensors. These molecules are both membrane-localized transcription factors that sense environmental signals and regulate gene expression machinery in V. cholerae. In that case, ToxT and TcpP bind to their cognate proteins, including ToxS and TcpH, respectively. An alkaline condition and nutritional deficiency trigger a process called regulated intramembrane proteolysis (RIP) in ToxR. ToxR undergoes proteolysis via RseP protease, causing the downregulation of the bacteria’s main virulent genes, initiating the dormant phase. This phase is also known as VBNC, exhibiting the bacterial state in the aquatic environment [194]. ToxS can protect ToxR from rapid degradation, slowing down the rate of bacterial inactivation [195]. TcpP and ToxT also undergo RIP in adverse environmental conditions. The same protective function has been detected in TcpH [196]. In contrast to the previous process, appropriate conditions upregulate bacterial virulence genes.

In the nutrient-rich state (such as the host intestine), ToxR becomes activated, binding to the promoter area of ompU and ompT. OMP refers to outer membrane proteins, which are porins for nutrients and molecules (amino acids, etc.) delivered through the bacterial membrane. ToxR increases the expression of ompU while downregulating ompT. It looks like ToxR is in a balance between “host survival mode” (with ompU) and “environmental persistence” (with ompT) [197]. Consistent with that, it has been manifested that ompU shows downregulation in the middle of the logistic phase of bacterial growth, while ompT is suppressed in this phase [197]. In addition to nutrient-rich conditions, an acidic environment (such as in the stomach) also activates ToxR, upregulating ompU expression, which is capable of protecting the bacteria from organic acids [198].

Other factors regulate the virulence of V. cholerae

There are some other factors affecting virulence genes. Unsaturated fatty acid suppresses CT and TCP in a ToxT-dependent manner. They inhibit ToxT to DNA and are also capable of suppressing ToxT expression [199]. In addition to fatty acids, bile salts are another organic compound affecting V. cholerae. Bile salts (such as taurocholate) may be an inducer of the ToxR regulon, which is important for virulence gene expression [12].

Temperature also has a significant effect on V. cholerae as well as other Vibrio species [200]. The optimal temperature for ToxT expression is 30 °C. However, some of the ToxT alleles can increase production of CT and TCP under laboratory conditions at 37 °C. In that case, ToxT is not the only regulator of virulence genes [13].

Although many environmental sensing and virulence-associated regulatory mechanisms are conserved across the genus Vibrio, distinct species have evolved lineage-specific adaptations that shape their ecological fitness, host interactions, and their pathogenic potential. Comparative analysis of these regulatory architectures can therefore help distinguish conserved virulence strategies from lineage-specific mechanisms associated with environmental persistence and disease emergence [27].

V. parahaemolyticus

Virulence factors of V. parahaemolyticus

V. parahaemolyticus pathogenicity is extremely dependent on two main factors: bacterial-derived toxins and the secretion systems, including T3SS and T6SS. The bacteria penetrate the host cell membrane with T3SS members, injecting the toxin directly into the cell. The major toxins of V. parahaemolyticus are thermostable direct hemolysin (TDH) and/or TDH-related hemolysin (TRH). The T3SS comprises two systems, including T3SS-1 and T3SS-2, which exhibit cytotoxic and enterotoxic effects, respectively [201, 202]. T3SS2, TDH, and some open reading frames (ORFs) create the VPI. ToxR analog proteins, including vrtA and vrtB, regulate the expression of the Vp-PAI gene [203].

Regulation of the T3SS system

Based on the important role of T3SS in V. parahaemolyticus pathogenesis, this system has been detected under a highly accurate regulatory system. There are 3 genes located at the terminal part of the T3SS-1 cluster genes, encompassing VP1698, VP1699, and VP1701. These genes are involved in environmental regulation of the T3SS-1 system. In adverse conditions (low nutrition, elevated NaCl concentration), VP1698 binds to VP1699, impairing its normal function to express T3SS-1. In trophic conditions, VP1701 binds to VP1698, preventing the formation of the VP1698-VP1699 complex. Hence, VP1699 is free to positively regulate T3SS-1 expression. In addition to the VP system (analog of the Exs system in Pseudomonas aeruginosa), another gene called H-NS downregulates VP1699, resulting in suppression of T3SS-1. This shows that T3SS-1 expression is regulated by two main systems [204].

Environmental factors and V. parahaemolyticus

Temperature

Environmental factors regulate the machinery of V. parahaemolyticus. In a study conducted by Urmersbach et al. expression of most virulence genes (including ToxR, TDH, T6SS-1, etc.) did not exhibit a significant difference in the response to temperature stress. Despite that, TS33-1 and some other pathogenic genes respond to cold or heat shock via up-/downregulation. On the opposite, structural genes are highly affected by temperature alternation [205]. Elevation of seawater temperature alters the V. parahaemolyticus pathogenicity significantly. Expression of genes involved in adhesion and biofilm formation (such as GlcNAc-binding protein A and mannose-sensitive hemagglutinin) increases in response to elevated temperature. The finding proposed that high temperature of the host body may facilitate bacterial adhesion to enterocytes and other cells [206].

Salinity and pH

V. parahaemolyticus is found in a wide range of salinity in different aquatic waters, as well as cholera. All Vibrionaceae family members carry the gene known as CosR, which regulates compatible solutes. These are compounds produced by bacteria in a hyperosmolar environment, which prevent bacterial shrinkage and death [207]. In a higher NaCl concentration, a hyperosmolar environment drains intrabacterial fluid, resulting in shrinkage. Additionally, this drainage can attenuate cytoplasmic pH. To reverse this effect, Vp upregulates a lysine decarboxylase (encoded by the cadA gene), converting lysine to cadaverine. This results in pH elevation and also increases intracellular osmolarity [202]. V. parahaemolyticus treated in 1% NaCl was extremely more toxic for the human host compared to the one in the 3% environment [208].

The tolerance of V. parahaemolyticus to acid exhibits how the bacteria transfer in the stomach and protect themselves from gastric acid. It has been revealed that V. parahaemolyticus, which adapts to the acidic environment of the stomach can exhibit more virulence, depending on upregulation of TDH and yscF (one of the major genes in the T3SS system) [209]. An acidic gastric environment results in upregulation of rpsJ and cspA, which are associated with stress proteins. In the following, stress proteins can conduct bacterial processes in a way to survive in acid. Among these alterations, V. parahaemolyticus exhibits downregulation of unnecessary pathways and also inhibits oxidative phosphorylation. In that case, the bacteria upregulate certain genes to initiate nitrate respiration, producing NH₃ to neutralize the gastric HCl [209].

Other factors

Bile acids also regulate V. parahaemolyticus. They bind a heterodimer structural member called VtrA/VtrC. This receptor transmits a signal to VtrB, resulting in transcription and activation of this gene. The product stimulates expression of the T3SS-2 gene cluster [210, 211]. This phenomenon is similar to V. cholerae, in that bile acids can activate the ToxR sensor (see above). Secondary bile acid can activate the T6SS-1 system and contribute to toxin transferring and bacterial competition (with other species) [212].

V. vulnificus

V. vulnificus has specific virulent genes, including V. vulnificus hemolysin (VVH), multifunctional autoprocessing repeats-in-toxin (MARTX), siderophores, secretion systems, flagella proteins, etc. These genes are under an extremely regulated system, as well as other Vibrio species [213]. VVH (product of the vvhA gene) is a pore-forming toxin, stimulating cell apoptosis and lysis. These cytotoxic effects have been observed in various types of human cells, particularly endothelial cells and erythrocytes. The master regulator in V. vulnificus is HlyU, which induces expression of vvhA and rtxA1 (MARTX), which is an analog of RTX in V. cholerae. It has been manifested that HlyU is responsible for the pathogenic phenotype of the bacteria [214].

Environmental factors and V. vulnificus regulation

Iron

Iron has a significant effect on V. vulnificus. VVH hemolyzes erythrocytes to provide the iron demand of the bacteria. V. vulnificus expresses an iron-dependent regulator called Fur. Fur can bind to iron, forming a Fur-iron complex, which can bind to Fur box sequences and regulate expression of target genes. Fur can modulate VVH expression via two main arms: suppression of the VVH operon and upregulation of the VVH protease, including VvpE and VvpM [215].

Temperature and salinity

Temperature and salinity can also regulate V. vulnificus. In fact, temperature may be the most important factor in V. vulnificus emergence [216]. In fish, temperature is the main regulator of V. vulnificus pathogenesis. A temperature above 19°C has been documented as proper for V. vulnificus. It has been reported that V. vulnificus cannot tolerate cold temperatures, opposite to other Vibrio spp. capable of living in sub-zero conditions [217]. Temperature is also involved in V. vulnificus pathogenicity in the human body. It seems that temperature and iron amount control bacterial pathogenesis in a step-by-step manner. High temperature prepares bacteria for the initiation of infection, upregulating protease activity and mortality. In the following, appropriate iron concentration in the host body stimulates V. vulnificus to hemolysin secretion and other adverse effects [218]. Salinity also affects V. vulnificus. Although V. vulnificus can tolerate a wide range of salinity, lower salt concentration is favorable for bacterial proliferation. After 120 min, V. vulnificus loses cellularity in 5 M NaCl [216].

Other factors

Interestingly, it has been manifested that spills have a significant effect on V. vulnificus population and gene expression. Spills can elevate V. vulnificus density in the culture environment. Additionally, wastewater effluent induces the bacteria in the c-di-GMP-dependent pathway, resulting in biofilm formation [219, 220].

Host factors can affect V. vulnificus pathogenicity. Cholesterol can inactivate VVH via oligomerization. Additionally, calcium may inhibit V. vulnificus-induced hemolysis [221].

Conclusion

Environmental regulation provides a framework in which V. cholerae and other similar species coordinate their survival, transmission, and virulence across different ecological niches. Sensing systems, regulatory networks, biofilm dynamics, and secretion pathways work together as an adaptive circuit that responds to signals from the environment and the host, instead of acting independently. Multi-omics technologies play a key role in revealing regulatory complexity and enabling a system-level view of virulence programs that are highly context dependent. The convergence of multi-omics integration, adaptive genomics, and machine learning is making an important transition from descriptive models to predictive and mechanistic understanding of Vibrio pathogenicity. Comparative analysis between different Vibrio species not only highlights common regulatory principles but also reveals evolutionary divergences that shape emergence and outbreak potential. Putting these insights into One Health frameworks provides powerful opportunities for environmental surveillance, climate-based risk prediction, and early identification of outbreaks. Finally, targeting environmentally regulated virulence mechanisms represents a promising approach for anti-virulence interventions, which not only reduces selective pressure but also disrupted disease transmission. Advancing this integrated model is essential for anticipating and containing the global threat caused by Vibrio-associated diseases in a changing environment.

Abbreviations

AWD

Acute watery diarrhea

WHO

World Health Organization

ACE

Accessory cholera enterotoxin

CT

Cholera toxin

EPS

Extracellular polymeric substances

flaA

Flagellin A gene

HapA

Hemagglutinin/protease A

HlyA

Hemolysin A

ML

Machine learning

OmpU

Outer membrane protein U

QS

Quorum sensing

rbm

Rugosity and biofilm structure modulator

sRNAs

Small regulatory RNAs

RNA-seq

RNA sequencing

mRNAs

Messenger RNAs

SST

Sea surface temperature

T6SS

Type VI secretion system

TCP

Toxin-coregulated pilus

CRISPR

Clustered regularly interspaced short palindromic repeats

TCS

Two-component systems

VPS

Vibrio polysaccharide

Zot

Zonula occludens toxin

GPCRs

G protein-coupled receptors

AC

Adenylate cyclase

CFTR

Cystic fibrosis transmembrane conductance regulator

VBNC

Viable but non-culturable

RIP

Regulated intramembrane proteolysis

TDH

Thermostable direct hemolysin

TRH

TDH-related hemolysin

ORF

Open reading frame

VVH

V. Vulnificus hemolysin

MARTX

Multifunctional autoprocessing repeats-in-toxin

CAI-1

Cholera autoinducer-1

AI-2

Autoinducer-2

HCD

High cell density

LCD

Low cell density

CqsS

Cholerae quorum sensing sensor

SAM

S-adenosyl-L-methionine

Ea-CAI-1

3-aminotridec-2-en-4-one

c-di-GMP

Cyclic di-guanosine monophosphate

DGC

Diguanylate cyclase

PDE

Phosphodiesterase

VPI

Vibrio pathogenicity island

SMF

Sodium motive force

H-NS

Histone-like nucleoid structuring protein

RIVET

recombination-based in vivo expression technology

SCFA

Short-chain fatty acid

GM

Gut microbiome

OMV

Outer membrane vesicle

Fur

Ferric uptake regulator

CsrA

Carbon storage regulator A

CVEC

Conditionally viable environmental cells

Bap1

Biofilm-associated protein 1

GbpA

N-acetylglucosamine-binding protein A

MHSA

Mannose-sensitive hemagglutinin

RbmA

Rugosity and biofilm structure modulator A

Author contributions

Conceptualization: S.S.; writing – original draft: S.S.T.; visualization: Sh.Y.; writing—review and editing: A.R. and A.Kh.D.; supervision: O.P. All authors who contributed to the article have approved the final version of the manuscript before submission.

Funding

None.

Data availability

No datasets were generated or analyzed during the current study.

Declarations

Ethics approval and consent to participate

The ethical committee of Semnan University of Medical Sciences approved this study with the number: IR.SEMUMS.REC.1405.022.

Competing interests

The authors declare no competing interests.