Cholera is an acute diarrheal disease with 2.9 million cases and 95,000 deaths estimated to occur each year in at least 47 countries across the world (Ali et al., 2015). The majority of severe cases occur in children under 5 years old. Cholera is often described as a disease of inequity, disproportionately affecting the poorest populations of a country or community (GTFCC, 2017). This longstanding disease has been thoroughly studied in a wide range of research fields from basic science to therapeutics. Mechanisms for prevention, intervention, and possible elimination of cholera have been clearly described and continue to be investigated but lack practical implementation in many vulnerable populations. The etiological agent of cholera is the gram-negative bacterium Vibrio cholerae, specifically those strains belonging to serogroups O1 and O139. Strains belonging to other serogroups may cause less severe non-cholera diarrhea or no disease symptoms at all and are collectively referred to as non-O1/non-O139 strains. V. cholerae is highly motile in aquatic environments, using a single, polar flagellum to propel itself (Echazarreta and Klose, 2019). This bacterium is also readily found in biofilms that form on hard surfaces, i.e., rocks or pipes, as well as in association with shellfish and vertebrate fish (Baine et al., 1974; Silva and Benitez, 2016). Humans can become infected with V. cholerae O1/O139 by consuming contaminated food or water, granting the bacteria access to the small intestine. Here, the bacteria aggregate using the toxin co-regulated pilus (TCP) and other colonization factors to colonize the intestine in a non-invasive manner (Almagro-Moreno et al., 2015; Silva and Benitez, 2016). Once established in the small intestine, the pathogen induces production of cholera toxin (CT), which results in an ion imbalance in the host intestine, leading to the rapid loss of fluids and electrolytes and potentially deadly dehydration via profuse, watery diarrhea (Thiagarajah and Verkman, 2005). Bacteria are disseminated from the intestine with the diarrhea, known as “rice water stool,” of an infected patient and exhibit hypervirulence for a limited time period (Butler et al., 2006). In many cases, cholera-containing fecal matter then contaminates a shared drinking water source, enabling infection with V. cholerae O1/O139 to spread rapidly through an entire community in the form of localized outbreaks (GTFCC, 2017). This massive fluid loss can lead to hypovolemia and can be up to 50% lethal if untreated (Sack et al., 2004). Fortunately, fatality rates drop significantly, to just 1%–2%, with standard treatment using an oral rehydration solution (ORS) (Desjeux et al., 1997). Prompt implementation of ORS therapy counteracts the fluid and electrolyte loss caused by V. cholerae infection and keeps the patient alive while the body naturally clears the infection over a period of several days.

Over the past 200 years, seven pandemics of cholera have been recorded, though instances of cholera-like illness have been described for millennia (Blake, 1994). The long-established nature of this disease has enabled evolutionary differentiation of the pathogen into thousands of strains ranging from environmental strains to those capable of causing endemic and pandemic cholera.

Cholera is estimated to cost $2 billion each year in global healthcare costs and loss of productivity (GTFCC, 2017). As global society becomes increasingly interconnected, cholera is perpetuated by human travel and transmission to naïve populations, lack of adequate infrastructure or disruption of existing infrastructure due to poverty, war, or natural disaster, and shifting weather patterns resulting from climate change. Fortunately, an abundance of research continues to emerge to better describe this pathogen and the disease it causes. This review aims to describe recent advancements in cornerstone cholera research related to genetic evolution of V. cholerae strains, molecular mechanisms of pathogenesis, improvements in animal models, pathogen interactions with the host microbiome and immune response, and disease epidemiology, presentation, and mitigation initiatives.

The Vibrio cholerae genome is approximately 4.0 MB in size and organized into two, distinct circular chromosomes (Trucksis et al., 1998; Heidelberg et al., 2000). Nearly 75% of the genome is contained on chromosome 1 with the remainder on chromosome 2, though a few isolates have been identified that contain a single, fused chromosome and are termed Natural Single Chromosome Vibrio (Chapman et al., 2015; Johnson et al., 2015; Yamamoto et al., 2018; Sozhamannan and Waldminghaus, 2020). While strain-specific differences exist, the entire V. cholerae genome generally contains between 3,600 and 3,900 coding sequences (Heidelberg et al., 2000; Thompson et al., 2011). Chromosome 1 serves as the core genome, encoding most of the essential housekeeping genes and conserved virulence genes. In contrast, chromosome 2 encodes many hypothetical proteins and open reading frames that appear to have been obtained from external sources through horizontal gene transfer events (Heidelberg et al., 2000). While many chromosome 2 genes serve redundant or unknown functions, at least a dozen essential genes have been identified on this chromosome and encode ribosomal proteins L35 and L20 as well as an NAD synthetase and ParA family protein involved in chromosome partitioning (Cameron et al., 2008; Hui et al., 2010; Chao et al., 2013; Kamp et al., 2013). Strains of V. cholerae are classified into serogroups according to the unique structure of the O-antigen associated with the strain’s outer membrane lipopolysaccharide (LPS) molecule. In a recent study, Murase et al. (2022) explored the genetic relatedness of all 210 reported serogroups and identified critical distinctions in structural biosynthesis gene clusters on both chromosomes. While only the O1 and O139 serogroups have been known to cause pandemic cholera, members of the remaining serogroups have had significant impacts as environmental strains. While not typically life-threatening, non-O1/non-O139 serogroups can cause sporadic cases of non-cholera diarrhea, sometimes closely resembling cholera, and some have been shown to act as evolutionary intermediaries in virulence gene acquisition via homologous recombination and horizontal gene transfer (Li et al., 2014, 2019a).

In the environment, V. cholerae is often associated with chitinous surfaces, such as those found on mollusks and other shellfish, and with phytoplankton (Baine et al., 1974; Tamplin et al., 1990; Hounmanou et al., 2019). Vertebrate fish have more recently been proposed as potential V. cholerae reservoirs, as both environmental and toxigenic strains have been isolated from numerous fish species, including tilapia (Oreochromis niloticus) (Senderovich et al., 2010; Halpern and Izhaki, 2017). Zebrafish (Danio rerio) have been shown to be naturally susceptible to infection and colonization by V. cholerae and have been developed as a V. cholerae model (Runft et al., 2014; Mitchell et al., 2017; Mitchell and Withey, 2018; Nag et al., 2018b, 2020). Aquatic reservoirs harboring both environmental and pandemic V. cholerae strains provide rich conditions for genetic evolution, and many non-O1/non-O139 have been found to contain partial pathogenicity islands and fully intact virulence genes, including the Vibrio seventh pandemic islands 1 and 2 (VSP-1, VSP-2), toxin co-regulated pilin (tcpA), hemolysin A (hlyA), and the Type 6 secretion system (T6SS) (Li et al., 2014, 2019a; Yan et al., 2022; Santoriello et al., 2023).

A mixed-transmission dynamic model of V. cholerae developed by Mavian et al. (2020) made use of spatiotemporal V. cholerae distribution following the single-source introduction in Haiti to model the establishment of aquatic reservoirs and the potential for evolutionary gene transfer events. These reservoirs are particularly relevant in cholera-endemic countries where seasonal cholera blooms result in multiple, periodic outbreaks with strains of varying virulence. Ongoing susceptibility to infection despite previous exposure to V. cholerae has been associated with serotype switching—a phenomenon in which V. cholerae O1 serogroup strains can express alternative surface antigens to present as either Ogawa, Inaba, or very rarely Hikojima serotypes—and is likely enabled by gene transfer under selective conditions in environmental reservoirs during off-peak cholera seasons (Baddam et al., 2020; Ramamurthy et al., 2020). Non-O1/non-O139 strains that primarily exist in the environment may use the same virulence genes in a different manner. For example, the recently identified T6SS gene cluster known as Aux3 has been shown to readily excise from the genome and recombine in a new location. However, this activity is typically only observed in environmental strains, while pandemic strains have integrated the feature into the chromosome (Santoriello et al., 2020).

From 1817 until the 1960s, pandemic cholera was caused by the V. cholerae O1 Classical biotype, characterized by the presence of a distinctive CT, TCP, and Vibrio pathogenicity island (VPI). Cholera in the current, ongoing seventh pandemic has been caused by a new O1 biotype, El Tor. El Tor biotype emerged as the primary causative agent of pandemic cholera beginning in 1961 and is defined by its resistance to polymyxin B, production of hemolysin A, and presence of two unique pathogenicity islands, VSP-1 and VSP-2, all of which Classical biotype lacks (Chart, 2012). Several diagnostic methods have been developed to distinguish between Classical and El Tor biotypes in recent years. PCR-based genotypic assays typically screen for specific sequence variations in virulence genes including tcpA, ctxA, ctxB, and toxR, while another genome-based method targets unique small RNA genes (Crumfield et al., 2018; Greig et al., 2018; Ahmed et al., 2019). Biotype can be distinguished phenotypically by evaluating antibiotic and phage susceptibility, capability for hemolysis and proteolysis, and variations in metabolism of citrate and glucose (Crumfield et al., 2018; Lee et al., 2020). One simple diagnostic measure for distinguishing between Classical and El Tor biotype strains in clinical settings has been the susceptibility of Classical strains to polymyxin B while El Tor strains have demonstrated resistance to this antibiotic, though this may not always be a reliable metric as El Tor strains continue to evolve (Crumfield et al., 2018).

When V. cholerae was first introduced to Haiti, it had devastating effects on the population which, initially, was largely attributed to the naivety of the previously unexposed region. While this certainly played a role in the rapid transmission and severe disease observed during this outbreak, Haitian variants of El Tor have been identified as more virulent than their southeastern Asian predecessors inducing elevated levels of inflammation and damage to the intestinal mucosa (Ghosh et al., 2019). These variants have also demonstrated greater production of CT, increased motility, and enhanced colonization dynamics in both human disease and animal host models (Satchell et al., 2016; Ghosh et al., 2019). Interestingly, recent epidemics in India and West Bengal have been caused by El Tor strains that more closely resemble Haitian variant strains in their genetic profile but also exhibit polymyxin B-sensitivity (Samanta et al., 2018; Shaw et al., 2022). These polymyxin B-sensitive strains have also exhibited hypervirulent traits compared to El Tor strains previously isolated in the same regions of southeast Asia (Samanta et al., 2018). These Classical-like features of more recently evolved El Tor isolates will likely require the development of new measures for phenotypic identification of biotype, particularly in resource-limited settings. Additionally, five clinical isolates from Kolkata, India revealed the inability to replicate the cholera toxin phage (CTXΦ) for the secretion of infectious particles by some El Tor variants, a feature that was commonly observed in El Tor strains between the 1970s and early 2010s (Ochi et al., 2021).

With the rapid development and decreasing cost of genome sequencing and analysis in the early 2000s, extensive genomic comparison analyses have described the evolution of the El Tor biotype and its evolutionary successors in detail. Whole-genome studies have revealed the similarities and differences in key virulence factors, including the CTXΦ, and have used these data to characterize the evolution and spread of El Tor variants into three waves (Kim et al., 2014). Hu et al. (2016) detailed key genomic events between the early 1900s and the 1960s that enabled El Tor’s maturation from a relatively benign form of V. cholerae to the virulent pathogen credited with causing the ongoing 7th pandemic of cholera. These events included the acquisition of an El Tor-specific tcpA gene that enabled human colonization, pathogenicity islands VSP-1 and VSP-2, and an El Tor-specific CTXΦ. Since this initial characterization of the lineage leading to the 7th pandemic by El Tor, others have explored genetic variation within El Tor strains to assess virulence trait acquisition. Large genomic fragments carrying genes for antimicrobial resistance, including the integrative and conjugative element (ICE) known as the SXT element, have been acquired by some El Tor strains isolated from the natural environment, and afford resistance to tetracycline, streptomycin, and even chloramphenicol (Ahmed et al., 2005; Sarkar et al., 2019).

An analysis of over 300 V. cholerae O1 strains revealed El Tor strains typically contained more virulence-related genes than Classical strains, as well as the presence of many redundant genes across the El Tor genome (Li et al., 2019b). While V. cholerae readily takes up DNA from the environment, defense mechanisms have also been developed to prevent the acquisition of unwanted or detrimental features. Both small, multicopy- and large, low copy number plasmids can be degraded by defense mechanisms identified as DdmABC and DdmDE should they prove to be detrimental to overall fitness of the V. cholerae host cell (Jaskólska et al., 2022). Phage defense in El Tor strains has been attributed in part to activity of genes on the VSP-1 and VSP-2 pathogenicity islands, including an antiviral cytidine deaminase which disrupts normal availability of nucleotides to deprive infecting phage of necessary components to replicate (Hsueh et al., 2022; O’Hara et al., 2022).

The natural habitat of V. cholerae in biofilms is often composed of richly diverse communities of Vibrio species and other aquatic bacteria. Some of the species present in these biofilms are capable of natural competence and can release significant amounts of DNA (>100 μg/ml) into the environment. Biofilms readily form on chitinous surfaces, a biopolymer that has been shown to induce natural competence in some V. cholerae strains (Baur et al., 1996; Meibom et al., 2005). Additionally, V. cholerae killing of non-kin bacterial competitors mediated by the type six secretion system (T6SS) enables the uptake of large DNA fragments (>150 Kbp) following lysis of the target cell (Joshi et al., 2017; Matthey et al., 2019). Exposure to high concentrations of free DNA in these chitin-rich conditions enables the rapid genomic diversification of V. cholerae which is modeled in the evolution of El Tor strains.

In the aquatic environment, V. cholerae does not produce the human-specific virulence factors that are required to cause cholera. After ingestion by a human, the bacteria sense signals in the gut that initiate a complex cascade of transcription factors that ultimately induce production of the major virulence factors, CT and TCP, together with a collection of other accessory virulence factors (Matson et al., 2007). At the top of the cascade are transcription factors AphA and AphB (Kovacikova and Skorupski, 1999; Skorupski and Taylor, 1999; Kovacikova and Skorupski, 2001; Kovacikova et al., 2004). AphA is translated at low cell density, and its genomic targets were recently described using CHiP-Seq (Haycocks et al., 2019). AphB senses low oxygen and low pH and becomes active as V. cholerae passes through the stomach and into the upper small intestine (Kovacikova et al., 2010). AphA and AphB work together to activate transcription of the next level of the cascade, which is composed of the TcpPH and ToxRS pairs of integral membrane proteins. While ToxRS is thought to be constitutively produced, TcpPH production requires the activity of AphA and AphB. When produced, TcpPH senses the bile salt taurocholate (Yang et al., 2013; Xue et al., 2016), whereas ToxRS senses other bile salts in the intestinal lumen (Midgett et al., 2017; Bina et al., 2021). TcpP and ToxR then bind directly to the promoter of the master virulence regulator, ToxT, and activate its production (Krukonis et al., 2000; Krukonis and DiRita, 2003; Goss et al., 2010; Fan et al., 2014; Haas et al., 2015). ToxT binds directly to the promoters upstream of ctxAB and tcpA and activates their transcription. However, ToxT activity is repressed by unsaturated fatty acid components of bile to prevent virulence factor production in the lumen of the small intestine (Chatterjee et al., 2007; Koestler and Waters, 2014; Plecha and Withey, 2015). As motile V. cholerae enters the mucus layer, the large fatty acids cannot penetrate, and ToxT becomes activated by the presence of bicarbonate, which is secreted by epithelial cells (Abuaita and Withey, 2009). Unsaturated fatty acids and bicarbonate have opposing roles in affecting the affinity of ToxT for its DNA binding sites (Withey and DiRita, 2006; Thomson and Withey, 2014; Plecha and Withey, 2015; Thomson et al., 2015). Thus, CT and TCP production only occurs when V. cholerae has reached the ideal location for colonization, within the intestinal mucus layer and close to the epithelial surface in crypts, where CT can enter cells and play its toxic role, resulting in voluminous watery diarrhea (Millet et al., 2014).

Vibrio cholerae has two distinct phases in its lifecycle: the highly motile, free-swimming state, and the sessile, virulent state. Motility is important in an aqueous environment, while attachment and biofilm formation is necessary for colonization in the human small intestine or on the surfaces of fish, plankton, and other chitinous material (Tamplin et al., 1990; Donlan and Costerton, 2002; Senderovich et al., 2010; Hathroubi et al., 2017). Responding to environmental signaling is crucial for V. cholerae survival, and robust methods of inverse regulation over virulence factors and motility are required.

Animal models for cholera research can be divided into two categories: mammalian models and non-mammalian models. These models, their uses, and their advantages and disadvantages are summarized in Table 1. The most commonly used mammalian model for enteric diseases generally is the specific-pathogen free (SPF) adult mouse (Mus musculus), but these hosts can be resistant to intestinal colonization by some strains of orally inoculated V. cholerae, due in part to the presence of gut microbiota (Sit et al., 2019). The streptomycin-treated adult mouse is sometimes used as a model for V. cholerae colonization. However, in this model V. cholerae colonizes the colon but not the small intestine (SI), where V. cholerae colonizes during human infection. This model is TCP-independent, and there are no detectable disease symptoms (Nygren et al., 2009; Wang et al., 2018). Pretreatment with clindamycin or ketamine to disrupt intestinal microbiota and motility can permit oral V. cholerae infection in the adult mouse (Olivier et al., 2009; You et al., 2019). Clindamycin is used as an antibiotic, like streptomycin, to clear the gut microbiota, and ketamine is a dissociative anesthetic that can be used to slow down the bowel movement and give V. cholerae a better chance to colonize. The most widely used mammalian model for V. cholerae colonization is the 3- to 5-day-old infant mouse, where oral challenge of V. cholerae can initiate TCP-dependent colonization of the SI (not the colon) and CT-dependent fluid accumulation within 16 h (Angelichio et al., 1999; Ritchie and Waldor, 2009). Advanced microscopy of the infant mouse small intestinal tissue has revealed the localization patterns of V. cholerae in the intestine control virulence gene regulation during infection (Millet et al., 2014; Gallego-Hernandez et al., 2020). Different competition studies in the infant mouse model have recently emphasized the contributions of fatty acid and carbon metabolism, cell wall maintenance, and V. cholerae LPS modifications during intestinal colonization (Hayes et al., 2017; Wang et al., 2018; Fleurie et al., 2019; Yang et al., 2020; Sit et al., 2021).

Two adult rabbit surgical models have been widely used for decades to test the colonization and fluid secretion resulting from V. cholerae infection. The removable intestinal tie adult rabbit diarrhea (RITARD) model is used for studying the toxin-mediated diarrheal disease caused by V. cholerae (Spira et al., 1981; Sinha et al., 2015). The ileal loop model is a widely used surgical model to study the fluid accumulation following by V. cholerae colonization by surgically creating sealed loops in the small intestine of the adult rabbit (Burrows and Musteikis, 1966; Mondal et al., 2014; Withey et al., 2015). These adult rabbit surgical models have largely been replaced by the infant rabbit (Oryctolagus cuniculus) model of cholera, which exhibits rapid CT-dependent lethal diarrheal illness, along with TCP-dependent SI colonization (Ritchie et al., 2010). High numbers of V. cholerae can be readily collected from the infant rabbit diarrheal fluid, which is quite similar to human rice water stool in chemical composition (Ritchie et al., 2010). Infant rabbit studies have revealed the genetic landscape of colonization factors in V. cholerae through transposon-insertion sequencing (Tn-Seq) screens; however, these screens can be largely limited by bottleneck effects in infant mice (Fu et al., 2013; Kamp et al., 2013; Pritchard et al., 2014; Hubbard et al., 2018). V. cholerae RNA-seq, metabolomic, and proteomic datasets, as well as insights into V. cholerae population dynamics during infection, have been successfully studied in the infant rabbit model (Mandlik et al., 2011; Abel et al., 2015; Zoued et al., 2021). Recently, a transcriptomic study in infant rabbits revealed novel roles for CT in the shaping of the pathogen’s nutritional microenvironment (Rivera-Chavez and Mekalanos, 2019).

There are several non-mammalian species for studying V. cholerae as well, such as fruit flies (Drosophila melanogaster), nematodes (Caenorhabditis elegans), wax moths (Galleria mellonella), and zebrafish (Danio rerio). The intestinal anatomy and immune system of Drosophila is similar to mammals, enabling the use of flies to investigate pathogenicity, quorum sensing, and host response to V. cholerae infection (Hang et al., 2014; Vanhove et al., 2017; Kamareddine et al., 2018; Davoodi and Foley, 2019). The introduction of CT can induce death in flies although the infection and host-killing by V. cholerae in these models is neither CT-dependent nor TCP-dependent (Blow et al., 2005). Even with these limitations, this model is useful as it offers reproducibility, low cost, and easy genetic manipulation compared to mammalian hosts. Cholera research in C. elegans is important for studying the known and presumed accessory V. cholerae virulence factors and toxins (Vaitkevicius et al., 2006; Logan et al., 2018). The ability of V. cholerae to kill G. mellonella larvae and form biofilms in these hosts imitates the pathogenic potential with mammalian model organisms (Nuidate et al., 2016; Bokhari et al., 2017).

Vibrio cholerae persists in the aquatic environment between outbreaks where they fight with different predators and environmental stressors. Some models were developed to study the environmental survival of V. cholerae. The mannose-sensitive hemagglutinin (MSHA) type IV pilus is crucial for attachment and initiation of colonization of V. cholerae in the pharynx of the worm, C. elegans, which could be linked to a fitness advantage of V. cholerae upon contact with bacterium-grazing nematodes (List et al., 2018). The soft-shelled turtle has potential value as an animal model to study the colonization and the transmission of V. cholerae as V. cholerae can colonize both on the dorsal side surface and in the intestine of turtles. MSHA is necessary for body surface colonization whereas, toxin-coregulated pili (TCP) or N-acetylglucosamine-binding protein A (GbpA) play important roles for colonization in the intestine (Wang et al., 2017). The type VI secretion system (T6SS) of V. cholerae is capable of conferring virulence toward eukaryotic and prokaryotic hosts. VasX, a functional protein for T6SS confers the virulence of V. cholerae toward Dictyostelium discoideum, a species of soil-dwelling amoeba commonly known as slime mold (MacIntyre et al., 2010; Miyata et al., 2011). These models are also very important to demonstrate the lifestyle of aquatic organisms in the presence of the aquatic bacterium, V. cholerae.

Zebrafish (D. rerio) are an emerging non-mammalian model to investigate V. cholerae pathogenesis, including colonization, transmission, host response, and competition with intestinal microbiota. The zebrafish is a natural host for V. cholerae which means V. cholerae can colonize the zebrafish intestine without any modification to the host’s microbiota (Runft et al., 2014). V. cholerae infection can be initiated through a natural, oral route in zebrafish, which eventually leads to human cholera-like symptoms including liquid stool in excreted water (cloudy water) and secretion of mucin and protein in excreted water within 24 h of initial exposure (Runft et al., 2014; Mitchell et al., 2017; Nag et al., 2018b). Zebrafish also show promise to study innate and adaptive immune responses during V. cholerae infection (Farr et al., 2021, 2022). Studies in the zebrafish model have been informative regarding V. cholerae’s interaction with gut microbiota and the involvement of T6SS in this interaction (Logan et al., 2018; Breen et al., 2021b). However, as most non-mammalian studies remain to be validated in either human or small mammal cholera models, their applicability to our understanding of human cholera may be limited (Sit et al., 2022).

The human gut microbiome contains the majority of commensal bacteria in the body (Qin et al., 2010; Sender et al., 2016). In humans, the dominant phyla are Firmicutes and Bacteroidetes, though the composition of the gut microbiome can vary among individuals. Intrinsic factors, including host genetics, age, and sex, and extrinsic factors, such as diet and lifestyle, all affect the gut microbiome (Arumugam et al., 2011; Faith et al., 2011; Schmidt et al., 2018). The mucus lining of the gut is also an important component. The mucus layer acts as a natural barrier to protect the intestinal epithelium. It is primarily made of heavily-glycosylated proteins called mucins that are metabolized by the resident bacteria that reside in the intestinal mucus layer. Therefore, the mucus itself must be present to maintain a diverse microbiome (Kashyap et al., 2013; Li et al., 2015; Sicard et al., 2017).

In terms of microbiome models to study V. cholerae outside of human fecal samples, experimentation is limited. Mammalian animal models commonly used for V. cholerae infection are not ideal for observing microbiome dynamics after colonization. Infant and adult mice, or the adult rabbits used in ileal loop or RITARD models, lack a complex gut microbiome due to age, antibiotic use for colonization, or surgery, respectively (Sawasvirojwong et al., 2013; Matson, 2018). One option is a human or mouse fecal transplant into axenic or gnotobiotic mice to observe V. cholerae colonization in the presence of a “humanized” mouse microbiome (You et al., 2019; Alavi et al., 2020). Non-mammalian animal models that are colonized by V. cholerae present an advantage due to the fecundity and shorter development period. The Drosophila model possesses a simple gut microbiome of low diversity that can be easily manipulated (Wong et al., 2011). The zebrafish model has the advantage of being a natural V. cholerae host, and zebrafish are able to keep complex intestinal microbiomes largely intact throughout the entire period of V. cholerae exposure, infection, and clearance; the noninvasive method of infection is particularly relevant (Senderovich et al., 2010; Wong et al., 2011; Runft et al., 2014; Breen et al., 2021a). The affordability of high-throughput sequencing of the 16S ribosomal subunit has allowed for more in-depth microbiome studies within the past decade; therefore, the diversity and dynamics of gut microbiomes in relation to pathogens and general perturbation is of growing interest (Gibbons and Gilbert, 2015).

As previously mentioned, V. cholerae strains of the O1 serogroup have been subdivided into two biotypes: Classical and El Tor. Though rarely occurring now, Classical infections were characterized by robust action of the CT and severe diarrheal symptoms lasting about 3 days (Hu et al., 2016). Initial infections with the El Tor biotype were mild, often failing to result in any cholera symptoms, but these infections were persistent, with V. cholerae isolates found in the stool of infected individuals for 1–2 weeks. The binding subunit of CT encoded by early El Tor strains differs structurally by 2 amino acids compared to the Classical CT which is hypothesized to account for the disparity in disease severity exhibited by Classical and El Tor infections (Raychoudhuri et al., 2009; Baek et al., 2020). Acquisition of the Classical CTXΦ by El Tor in the early 2000s led to increased disease severity comparable to that caused by Classical strains and of prolonged duration as was characteristic of early El Tor strains (Na-Ubol et al., 2011; Hu et al., 2016). Previous exposure to V. cholerae causing symptomatic cholera provides protective immunity against subsequent exposure, though cross-protection is not generally achieved for other serotypes or in cases where initial exposure did not produce symptoms (Leung and Matrajt, 2021).

From genomics to therapeutic development, V. cholerae research continues to make great strides towards a more complete understanding of how this pathogen survives in the aquatic environment, interacts with hosts, and causes disease. Analyses of evolutionary genetics have identified critical events that must occur for V. cholerae to transition from an environmental marine microbe to the pandemic strains that plague countries around the world today. Novel potential reservoirs in which V. cholerae continues to acquire new genetic material and gain fitness in the environment and the host are being identified in vertebrate fish in addition to the well-established reservoirs of copepods, phytoplankton, and shellfish. Studies focusing on motility, chemotaxis, and biofilm formation provide key insights to how V. cholerae survives in the environment and navigates colonization of the human host. Animal models in mice and rabbits offer means to study CT production and virulence gene expression in mammals while the zebrafish model can be used to gain a well-rounded perspective of natural infection including interactions with other members of the normal microbiome. Both the direct action of the T6SS and indirect activity of rapid fluid loss have been shown to disrupt the composition of the normal intestinal microbiota and open a new avenue of exploration regarding interspecies dynamics during V. cholerae infection. Increased understanding of microbiome composition that affords resistance against infection with V. cholerae offers potential for use of probiotics to reduce risk of infection and limit disease severity. Disease presentation has shifted with the changing genetic composition of El Tor variants, which exhibit various antimicrobial resistance patterns and a range of symptom severity depending on the exact etiological agent. Advances in cholera disease modeling have enabled better prediction of outbreak scenarios and, in turn, faster implementation of prevention initiatives and response efforts. Taken together, these advancements offer a holistic approach to the study of a persistent pathogen that has plagued the world for centuries. Continued efforts to explore V. cholerae dynamics as a model for genomic evolution, bacterial pathogenesis, and its role as a natural member of the aquatic ecosystem are needed, both to advance knowledge in the basic sciences and to develop relevant, effective public health measures to protect the most vulnerable populations.

MW led the writing of the manuscript. DN and IC contributed to writing the manuscript. JW contributed to the writing, edited the manuscript, and provided funding (grant numbers R01AI127390 and R21AI171072). All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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