Tools to Enumerate and Predict Distribution Patterns of Environmental Vibrio vulnificus and Vibrio parahaemolyticus

Vibrio vulnificus (Vv) and Vibrio parahaemolyticus (Vp) are water- and foodborne bacteria that can cause several distinct human diseases, collectively called vibriosis. The success of oyster aquaculture is negatively impacted by high Vibrio abundances. Myriad environmental factors affect the distribution of pathogenic Vibrio, including temperature, salinity, eutrophication, extreme weather events, and plankton loads, including harmful algal blooms. In this paper, we synthesize the current understanding of ecological drivers of Vv and Vp and provide a summary of various tools used to enumerate Vv and Vp in a variety of environments and environmental samples. We also highlight the limitations and benefits of each of the measurement tools and propose example alternative tools for more specific enumeration of pathogenic Vv and Vp. Improvement of molecular methods can tighten better predictive models that are potentially important for mitigation in more controlled environments such as aquaculture.


Introduction
Vibrio are Gram-negative, flagellar bacteria that typically live in marine to estuarine waters [1].Three major human pathogens in the genus include Vibrio vulnificus, V. parahaemolyticus, and V. cholerae, the first two of which are considered in this review.As facultative anaerobes, Vibrio metabolic demands can be met even in suboxic waters [2].Vibrio vulnificus (henceforth abbreviated Vv) can enter open cuts or wounds via exposure to contaminated seawater and may cause necrotizing fasciitis (infamously known as "flesh-eating disease").Systemic cases often necessitate surgery or peripheral amputations, and in rare cases, result in death [3,4].Vv can also cause gastrointestinal disease through the ingestion of raw or undercooked shellfish and is the leading cause of reported acute gastroenteritis and septicemia cases due to foodborne illness [5].Over 95% of all Vv cells associated with oysters are in the meat [6], and roughly 50% of Vv human infections are transmitted through food consumption [7].Similarly, V. parahaemolyticus (Vp) is transmitted through the consumption of raw or undercooked shellfish, especially oysters [8].Both Vv and Vp can exist in the water column in a planktonic state, but in response to certain molecular signaling events, can become sessile on surfaces such as shellfish, finfish, plankton, or suspended particulate material [9,10].Although antibiotic-resistant V. cholerae resulting from the use of aquaculture probiotics may confer resistance to non-cholera Vibrio species, as reviewed elsewhere [11], numbers of isolates from environmental samples that are identified as V. cholerae can be quite low [12].This review focuses on environmental and climate-related parameters known to be associated with higher incidences of Vv and Vp in several types of environmental samples.
Unfortunately, the global occurrence of outbreaks and rates of reported Vv and Vp cases may increase [13][14][15].Vibriosis cases are predicted to rise, in part, due to anthropogenic climate change and associated extreme meteorological events and downstream changes to the waterbodies where Vibrio naturally reside [16][17][18][19].Antibiotics usage in aquaculture areas may also lead to Vibrio populations with antibiotic tolerance ("persisters", please see below).Additional considerations are the specific genetic and phenotypic properties of strains, serovars, and "serovariants", which are extensive in both Vv and Vp species, as evident from just a single study of oyster-associated strains [20].An increase in variant numbers among strains, i.e., increased genetic diversity, should be considered, especially when enumerating with functional genes, such as those that confer fitness in aquaculture facilities, fitness for sessile living state, and/or higher pathogenicity to humans [21].Hence, it is particularly important to recognize the limitations and strengths of the various molecular tools for detection of Vibrio in environmental samples.

Objectives
The primary aims of this review are to (i) provide an overview of virulence genes used to call for and enumerate Vv and Vp in environmental samples, (ii) summarize current knowledge of environmental factors driving Vv and Vp success, and (iii) present examples of alternative molecular tools for ecological surveys and better predictions of Vv and Vp distribution changes with various environmental and climate-related perturbations.

Culture-Dependent and -Independent Method Considerations
Microbiologists often use culture-dependent methods.However, the limitation of first relying on growth in liquid or solid media before enumerating the bacteria may lead us to underestimate the numbers in the substrates of interest, such as fish tissue, sediment, and water column samples.The "viable but not culturable" (VBNC) state is a survival mechanism in which bacteria are alive, but do not grow in culture media.Entering the VBNC state often occurs in response to unfavorable conditions, such as, in the case of Vv, extreme salinity changes or large temperature drops [22]."Persister" cells are those that are possibly induced by the usage of antibiotics in aquaculture areas, as previously reviewed elsewhere [23] Briefly, persisters are similar to VBNC cells, in that both types are more stress-tolerant than vegetative cells, but persisters are in a less dormant state than VBNC cells; additionally, persister cells are generally antibiotic-tolerant.Some surveys of the isolates of aquaculture samples resulted in significant number of isolates with multiple Antibiotic Resistance Genes (ARGs), especially from aquaculture waters where antibiotics usage was low [24].A few ARG marker genes are provided in Table 1, and a more comprehensive list is available in [24].For further review on Vv VBNC and persisters, please see [23], and for a proteomics study of resuscitation mechanisms of Vp from the VBNC state, see [25].An extensive review of Vibrio VBNC and persister states in survival and epidemiological implications was recently published elsewhere [26].While lower temperatures may lead to underestimation of environmental or mammalian-host-associated Vv [27,28], the opposite may be true for Vp in controlled culture conditions where salinity, not temperature, is the important factor affecting the VBNC state [29].In slightly different culture conditions, increased temperatures can somewhat reverse the VBNC issue [30].Additionally, physiological responses of oysters to hypoxia may influence the accumulation of Vibrio in the meat [31].Other environmental stressors on the host organism, such as salinity, temperature, and metal concentrations may also influence Vibrio loads in the oyster [6,32].
A variety of methods have been reported for enumeration and screening for Vibrio in water, sediment and aquaculture samples.These include those based on solely culture-independent ("molecular") tools, where DNA or RNA is extracted directly from the substrate and evaluated using deep sequencing of a marker gene ("amplicon" libraries), qPCR, RT-qPCR, metagenomics, or metatranscriptomics.Since the culture-dependent step is skipped, the bacterial VBNC state does not affect detection.In waterbodies annually experiencing wide temperature ranges, hence possibly inducing the VBNC state of Vibrio, considerations should be made when enumerating during cold months.For example, in the Chesapeake Bay, Vv could comprise ~10% to 50% of culturable bacterial populations in oys-ters [33], easily enumerated via direct-plating/colony hybridization (DP/CH; i.e., a method that is inherently culture-based).However, in the same estuary, Vibrio numbers declined with temperature, thereby evading detection with these standard culture methods [34].
Currently, most studies rely on a combination of enrichment followed by molecular verification.An example is MPN (most probable number)-qPCR (MPN-qPCR) or MPN-PCR, where the substrate is diluted in liquid culture, counts are based on the MPN method, and then turbid cultures are subjected to either non-quantitative PCR or quantitative PCR (qPCR).The non-quantitative (PCR) or quantitative (qPCR) step is designed to verify presence, or to obtain gene abundances from the species or strains of interest.MPN-PCR and MPN-qPCR have long been used for pathogen screening of environmental or food samples [35][36][37].Other methods based on culture-dependent, followed by culture-independent tools include DP/CH and DP-colony PCR, both relying on the success of the Vibrio to first grow on a solid culture medium.All such methods, liquid-and solid-culture-based (DP/CH, DP/CHfollowed by PCR confirmation, DP followed by colony PCR, MPN-PCR, and MPN-qPCR) rely on the "cultivability" of the species of interest.Thus, rather than using only conventional microbiological methods for detection and quantification of bacterial communities, it can be more valuable to use molecular methods that evaluate DNA or RNA for confirming the presence/absence or providing quantities of the strains of interest.Care must be taken to interpret the findings of any type of study (culture-dependent, or -independent) when modeling predictions of increased pathogenic Vv or Vp with any environmental or ecological factor [38].A newer type of quantitative PCR (digital droplet PCR, ddPCR) has recently been used to quantify pathogenic Vibrio species in the environment [39].Benefits of pathogenic screening bypassing the culture step, and performing qPCR or RT-qPCR directly on DNA or RNA extracted from the substrate, has been extensively reviewed elsewhere [40][41][42].

Marker Genes
Sequencing of conserved taxonomic marker genes, such as the 16S rRNA gene, 23S rRNA gene, and rpoA, can help in the taxonomic identification of various Vibrio isolates, regardless of species, strain, or pathogenicity status [43,44].However, due to the highly conserved sequences of these phylogenetic markers, PCR of these genes are usually not suitable for screening for species or specific strains, and thus cannot be used to reliably differentiate between pathogenic and non-pathogenic strains.Genus-level molecular tools were used in earlier studies of environmental Vibrio [44][45][46], some of which are included in this review for describing the early findings of environmental parameters driving Vibrio success."Functional" genes are now more often used as molecular markers to enumerate and/or score samples as positive or negative for Vibrio.Both Vv and Vp possess specialized genes that contribute to pathogen virulence by promoting phenotypes such as increased epithelial (intestinal) cell adhesion or erythrocyte (red blood cell) lysis (i.e., hemolysis).Some enteropathic bacteria are capable of host invasion through mucus-binding proteins, including those encoded by gbpA of V. cholerae [47] and V. vulnificus [48].An early phenotypic screening tool for pathogenic Vibrio was the modified Elek test method [49].A positive result, called the Kanagawa phenomenon, is characterized by the halo formation around colonies grown on Wagatsuma agar, signifying the colony contains a Vp strain actively expressing the ß-hemolysin.The Kanagawa phenomenon test must be validated with culture-independent methods, since pathogenic strains from human patients, fish and water that score tdh-positive do not always present the Kanagawa phenotype [50].
The Vv-specific virulence gene, vvhA, encodes an extracellular cytolysin (VVH) which, if present, often confers higher likelihood of pathogenesis, possibly by helping the Vv invade host intestines, thereby allowing for its entry into the bloodstream [51].Published vvhA PCR primer pairs (Table 1) are routinely used to detect Vv in DNA from seawater, sediments, fish or shellfish tissues.Recently, vvhA has been used to enumerate total pathogenic Vv, while the virulence-correlated gene (vcg) is now considered as a stronger indicator of virulence in Vv.Both environmental (vcgE) and clinical (vcgC) genes are strongly associated with distinct types of the hemolysin gene, vvhA, among clinical and environmental isolates [52,53].
Table 1.Noteworthy Vibrio marker genes.Common marker genes that are used to identify and enumerate Vibrio vulnificus (Vv) and V. parahaemolyticus (Vp) in environmental samples are listed here.Although most studies rely on PCR to detect species-specific genes, selected genes from recent publications relying on transcriptomics, genomics or metagenomics are also presented.

Species
Encodes 1 Name of protein encoded by gene of interest. 2Published PCR primer pair name, name of primer used to call for gene of interest using molecular methods (N/A: PCR was not used, gene sequence groups identified via deep-sequencing or genomics/metagenomics tools). 3Reference: study in which PCR primer pair was first developed and tested or employed to enumerate Vv or Vp in environmental samples. 4IAC = Internal Amplification Control. 5 ARG = Antibiotic Resistance Gene.For ARGs, a more comprehensive list of PCR targets for screening of aquaculture samples is provided by [24].
The pilus has a major role in Vibrio pathogenesis.In pathogenic Vv strains, pilF and pilA encode major pilus structural subunits.Those, and potential regulator genes like gacA that may control pil expression, are involved in biofilm formation, oyster colonization, and adherence to host gut epithelial cells [66][67][68]92].Common PCR primer pairs for enumerating the pilA and pilF genes show promise for seafood pathogen screening [69][70][71].The expression of the toxic lipoprotein, plp, causes necrotic cell death of epithelial cells and lyses human erythrocytes of membranes.For example, Vp persisting in Pacific oyster tissues rely on types I and IV pili, as well as polar and lateral flagellar systems [93].Not only has this study helped us to better understand the pilus mechanism at the Vp-oyster interface, but also provided a gateway for more in-depth studies, for evaluating potential effects of relevant environmental parameters, including joint salinity-pH condition.Expression of the aforementioned genes is directly involved in pathogenicity.However, important regulatory genes responsible for the expression of virulence genes are also used to assess potential pathogen loads (Table 1, regulatory gene examples).These include transcriptional activator HlyU [85], periplasmic regulator RseB [86], heme and hemoglobin receptor hupA [74], and quorum-sensing master regulator SmcR [76].Another example is BrpR, a regulatory protein upstream of several genes known to be involved in Vv biofilm formation and rugose colony formation [72], indicative of the potential for pathogenicity (see Table 1, brpL, brpG).
Similarly, there are several virulence-associated genes in Vp.Generally, the hemolysin genes of major interest for screening environmental samples are tdh, tlh, and trh.Like VVH, Vp hemolysins increase the permeability of host cell membranes, act as toxic porins and could result in cell lysis [94].The genes encoding the thermostable-direct hemolysin (tdh) and the thermostable-direct hemolysin-related hemolysin (trh) are the most commonly used Vp virulence gene markers (Table 1).The gene encoding the thermolabile hemolysin (tlh) is typically a species-specific marker that is used to call for and enumerate all Vp, regardless of pathogenicity.Not all strains of Vp are pathogenic, and samples that score tdhor trhpositive (or both) are generally only those that are scored as "hemolysin-producing" [59].Interestingly, synteny analyses of Vp hemolysin and nearby genes indicate gene order may confer differences in pathogenicity of strains isolated from human wounds or feces, water samples, and shellfish [95].
The ToxR protein, first described in V. cholerae, is a regulatory protein strongly associated with the upregulation of the gene encoding tdh in Vp [62] or vvhA in Vv [63].The toxR gene can be used to identify pathogenic Vp and Vv in environmental studies [96,97], and a homolog is present in V. alginolyticus, another important related pathogen [64].The Vp metalloprotease, encoded by vpm, promotes pathogen invasion via degradation of fish host tissue, especially type I collagen [65]; the vpm gene is another example of an alternative PCR target used to screen environmental samples [97].Table 1 presents the selected PCR primer pairs for these and other example marker genes, along with additional genes described in other molecular-based studies using tools other than PCR.Described below are many of the environmental parameters thought to drive Vibrio success, inferred from studies using a combination of culture-dependent and -independent techniques.

Predictors, Published Patterns, and Correlated Ecological Factors
Several water quality parameters may have strong influence on pathogenic Vibrio dynamics, partly due to their known optimal ranges.Temperature and salinity are often reported as drivers of Vibrio populations, regardless of geographical location of the waterbody.A variety of parameters are correlated with abundance and distribution in various regions and waterbody types and can be generally grouped into three categories: i) hydrological and/or meteorological factors, ii) abiotic factors that may be indicative of biological activity, and iii) purely biological factors.Additional considerations should be made when evaluating aquaculture studies, such as the now-known strains of Vp that are resistant to food pressure treatments [98,99].An overview of environmental parameters potentially driving Vibrio success are provided here and in Table 2.    4 Gene(s) detected with probes or amplified via PCR or qPCR.Primer pair or probe name and reference provided. 5Reported pH conditions between 7.0 and 9.0. 6Factors directly associated with tidal influence, including tidal range, tidal coefficient, or salinity stratification. 7External physical factors related to resuspension of the water column, including wind direction, wind speeds, and storm events. 8Nutrient concentrations.TDN: total dissolved nitrogen.TDP: total dissolved phosphorus.DIN: dissolved inorganic nitrogen.Si: silicate.DON: dissolved organic nitrogen.TP: total phosphorus.TN: total nitrogen.TKN: total Kjeldahl nitrogen. 9Fecal indicator bacteria (fecal enterococci, total coliforms). 10Other microorganisms including cyanobacterial harmful algal blooms (HABs), eukaryotic phytoplankton HABs, zooplankton. 11Species include Cylindrospermopsis raciborskii, Anabaena spp. 12 Heterocapsa rotundata. 13Heterosigma akashiwo, Gyrodinium instriatum. 14following extreme storm event. 15Akashiwo sanguinea. 16Change in seagrass density.Strong, positive correlations between water temperature and Vv distribution and/or abundance have been demonstrated in numerous studies, such as [46,55,110,114,120,123,140] among others.Using a combination of DP-CH and MPN-qPCR with primer pairs previously described [61], Vibrio concentrations were enumerated in surface waters, sediments, and oysters in coastal waters of two states in the subtropical northern Gulf of Mexico region [100].Several environmental parameters, especially sea surface temperature (SST), contribute to Vibrio success in one or more of these substrates, along with chlorophyll a (chl a) and turbidity in the water column.Although many of the earlier studies finding increased Vibrio with higher sea surface temperatures were focused on subtropical or tropical locales, this strong positive relationship is also seen in additional, more temperate regions.For example, in North Carolina Atlantic east coast estuaries, DP/CH also confirmed strong relationships between temperature and Vv and Vp, but additional abiotic and biotic factors were also important considerations [12].In the North Sea, where water temperatures were low and salinities high, infection occurrence was lower [141].Warm waters were associated with high concentrations of Vv and Vp in surface waters of subtropical northern Gulf of Mexico but also in more temperate waters in the Pacific Northwest in the Chesapeake Bay [101].
There is little "correlation" of Vibrio with SST when short-term, within-season, evaluations are conducted in both culture-dependent [128] and culture-independent [134,142] types of studies.This is especially evident from studies where the entire sampling period occurs in the warm summer months [134] or from single-year evaluations where temperature is a strong factor [143].Long-term, there are positive relationships between increased temperature with Vibrio in the zooplankton and phytoplankton seawater fraction [122], in the water column [112], and with Vp associated with shellfish [106].However, in some cases, there may be a non-significant relationship of long-term effects of SST with Vv and Vp abundances in the water column [18].Evaluation of epidemiological data and concomitant increases in sea surface temperatures [144][145][146] and other studies, reviewed by [147], are especially important for forming more confidence in ecological and environmental predictor models.
Increases in water temperature could also decrease water quality and induce favorable conditions for pathogen growth, such as salinity changes during droughts.For example, insubtropical surface waters in the northern Gulf of Mexico near Galveston, TX, USA contained the highest abundances of culturable Vv in waters with salinities of 7-16% and water temperatures between 30°C and 37°C [103,148].Temperature was a driving factor in waters near Honolulu, HI, where Vv was low during the summer dry season, when waters were warm, and importantly, more saline [104].In certain cases, droughts can promote distribution and abundance of pathogenic bacteria.However, some droughts do not seem to have much of an effect.For example, abundance of Vv in North Carolina estuaries was in fact reduced during a drought season compared to an above-average freshwater inflow event [149].Similarly, a longer-term study (10 years) in the Neuse River Estuary showed that culturable Vibrio numbers were not necessarily associated with the three most commonly reported factors for predicting estuarine Vibrio abundance: salinity, temperature, and dissolved oxygen [150].In a subtropical estuary, Charlotte Harbor of southwest Florida, there were no significant relationships between the presence of Vv and pH, salinity, turbidity, dissolved nutrients, or estuarine bacteria, an outlier from other studies [151].These conflicting findings suggest that increased temperature coupled with a specific salinity range may provide more favorable Vibrio growth conditions.

Salinity
Increases in salinity due to drought may actually decrease the levels of Vv associated with oysters, even when both culture-dependent and culture-independent methods of Vibrio measurement are employed [152].Hindsight analyses indicate that Vv are more abundant in "wet" vs. "dry" years in the temperate Chesapeake Bay system [153].In certain areas, such as subtropical estuaries, salinity can be a stronger determinant for Vibrio success than temperature.Vibrio are considered to be mesohalophiles (3-16 PSU).Thus, ecological surveys commonly report a negative relationship with salinity, especially at sites proximal to freshwater input [100,118,127,128].Additional considerations should also include prevalence of the bacteria in surface waters as compared to bottom waters, where surface and bottom Vv may be differentially affected by temperature in the long term [154], a phenomenon likely present in any type of stratified estuarine environment.Some studies suggest that numbers increase proportionally with salinity.For example, a study near the Mississippi Gulf Coast reported a significant relationship between salinity (and turbidity) with Vp in surface waters and oysters [129].Vibrio exposure can increase from exposure to low-salinity standing water caused by hurricane-induced flooding [155], but prior wind (see Section 4.1.4and storm surge may also contribute to this problem.Systems affected by abundant freshwater influence, such as Indian tropical monsoonal estuaries, may also experience high Vv and Vp abundances, but in addition to salinity, other correlative factors such as eutrophication (see Section 4.2), reduced flushing leading to hypoxia, and wind direction reversal, are important [156].
Neither salinity nor SST alone is a strong predictor of pathogenic Vv and Vp, suggesting the need to consider other environmental factors influencing their distribution.Many studies of environmental samples consider the specific relationship between temperature and salinity to be the most primary consideration in Vibrio ecology.Warm waters with moderate-to-high salinity are typical for high abundances of Vibrio measured in both temperate and warmer locales [55,110].The first study to detect and quantify co-occurring Vibrio species in West African coastal waters also confirmed that SST and salinity were driving environmental factors of Vibrio densities, and Vv was highest at the end of the wet season [124].However, wind events (highest in this region's dry season) were not considered.In myriad examinations among a variety of geographic regions and types of waterbodies, the one environmental parameter that is least predictive is salinity, where Vibrio abundances can be positively or negatively correlated with it, or where bimodal or non-linear models best fit the data (see "Relationship" column in Table 2).

Alkalinity
Fluctuations in pH levels may also affect Vibrio ecology, particularly when preceded by heavy rainfall [157,158].In Dauphin Island Bay, AL, USA, the optimal water pH for Vv and Vp were found to be ~7.0-9.0 [127].In more temperate South Carolina, USA, Vp abundances were negatively correlated with pH, where HABs were prevalent in coastal stormwater detention ponds [115].In a group of subtropical estuaries, pH was found to be correlated positively with Vv, but negatively with Vp [128].Evidently, variation in driving factors can be highly region-specific and potentially seasonally driven, but tidal influence or freshwater inputs on fluctuating pH may also be the driving factor in Vibrio abundances.Tidal influence is an especially important predictive factor in estuaries, particularly shallow ones or those subjected to stratification [110,128,132].

Wind
Importantly, besides SST, other considerations such as wind speed and air temperature should be included in ecological models.In a limited geographical area (tropical Florida, USA) there were relationships between coastal clinical cases and multiple meteorological factors [159].One study elucidated the ecology and culturable Vv and Vp abundances in three substrates (surface waters, bottom sediments, and invertebrate biofilms) within the Pensacola Bay System, FL.High concentrations of Vv and Vp in surface waters may have been due to high wind speeds during the days leading up to sampling, which caused resuspension bacteria that would have otherwise been present within the sediments [128].Additionally, wind-induced surface agitation may promote oxygenation, favoring Vibrio species whose oxygen preferences align with surface water conditions.For example, in the Chesapeake Bay, differential abundances of Vv and Vp were seen before and after an extreme storm event (Hurricane Irene), possibly because of increased turbidity from vertical mixing.Abundances in the water column, sediment, or both, were significantly correlated with several environmental parameters, notably secchi depth, total suspended solids, and tidal height [132].Wind direction is also an important consideration, where the shape of the estuary dictates mixing depending on the vector [131], or where wind direction reversal, such as before or after significant storm events [156], can influence the water column Vv and/or Vp abundances.

Turbidity
In coastal environments, an increased rate of fluvial input often increases turbidity, possibly driving Vibrio success [12,100,129].Mixing and resuspension of bottom sediments transports sediment-dwelling Vibrio upward into surface waters, increasing their overall distribution and consequently reintroducing them to the water column.Not only does particle association allow Vibrio to evade grazing, particles are sources of nutrients and organic carbon [160,161].In a recent study of coastal Alabama, USA, 30-50% of total Vibrio was associated with particles sized 5 µm or larger in a freshwater-driven system [131].In a nearby northern Gulf of Mexico coastal region, light attenuation was, not unexpectedly, positively correlated with total suspended solids, but neither surface nor sedimentary Vibrio abundances were correlated with turbidity or light attenuation [128].

Abiotic Factors Chlorophyll and Nutrients
Chlorophyll a (chl a) and nutrient concentrations are considered as abiotic factors, but they are indicative of biological activity within the ecosystem.Both biotic and abiotic factors generate selective pressures in ecosystems, which facilitate the emergence of pathogenic traits in the community [162].In a northern temperate estuary, Vp associated with oysters were positively correlated with water column chl a [163], and the relationships of sedimentary and water column Vibrio with chl a were significant in several other regions [46,110,147].Two regions near the subtropical Gulf of Mexico coast showed positive correlations of Vv and Vp with chl a in surface waters and sediments [100,128].Eutrophication often promotes algal blooms.As blooms die and decompose, the dissolved and particulate organic matter released become fodder for heterotrophic bacteria such as Vv and Vp, which are efficient scavengers that often outcompete other non-pathogenic heterotrophs.
There is a clear connection between elevated nutrients and concentrations of Vv and Vp.In nutrient-rich detention ponds of South Carolina, USA [115], concentrations of a variety of nutrients-dissolved organic nitrogen, dissolved inorganic nitrogen, total dissolved phosphorus, and silicate-were important, where almost all correlated positively with both Vv and Vp.Similarly, in the Pensacola Bay System, surface water Vp positively correlated with total dissolved inorganic nitrogen and total Kjeldahl nitrogen [128].Another study showed that Vp was moderately correlated with total dissolved phosphorus and total dissolved nitrogen in Rehoboth Bay, Delaware, USA [133].Conversely, a strong inverse relationship was observed with ammonia for both Vv and Vp in North Carolina estuaries [12].However, a predictive modelling survey in this region indicated that high dissolved organic carbon alone may not have a direct impact but can likely promote more rapid Vibrio growth when temperature and salinity ranges are viable [164].

Biological Factors 4.3.1. Other Bacterial Pathogens
A positive correlation of Vibrio with total bacteria and/or coliforms is not unexpected, especially when Vibrio abundances are enumerated with culture-based methods [165].Relationships between surface water Vv and fecal enterococci [124] was consistent with other earlier, culture-based studies [12,166].Co-occurrences of Vp and other pathogenic bacteria were seen in a northern estuary with fecal contamination, concomitant with positive correlations with particulate matter and zooplankton abundances [167], again suggesting multivariate considerations must be made in predictive modeling.Indeed, in North Carolina estuaries, correlations of Vv, Vp, or both, were seen with several chemical, physical, and biological parameters, including salinity, turbidity, dissolved oxygen, concentrations of inorganic nutrients, and loads of other human pathogens such as E. coli and total coliform bacteria [12].

Cyanobacterial and Eukaryotic Algal Blooms
The two different potentially pathogenic Vibrio species may respond differently to blooms following extreme storm events.In the Neuse River Estuary, North Carolina, USA, after an extreme storm event (Hurricane Matthew), several types of HABs were observed in surface waters, including cyanobacterial types and eukaryotic dinoflagellates, both measured using deep sequencing of the hsp60 gene [136].Although Vibrio abundances were estimated via relative abundances in deep sequencing, significant correlations with the typical "environmental factors" were seen, but differing relationships with HABs, depending on the type of HAB and the Vibrio species, were also observed.Correlations with a cyanobacterial bloom with Vp and Vv were positive and negative, respectively, but both Vibrio species were positively correlated with dinoflagellate blooms.Therefore, after extreme storm events, water quality, including various photosynthetic pigments such as chl a (as a proxy for potential HABs) should therefore be monitored closely when assessing aquaculture risks.These risks were also assessed in coastal Mediterranean, in a fish aquaculture area, where several water quality markers were correlated with multiple Vibrio species, based on 16S rRNA profiles and culturable bacteria counts [168].Interestingly, but not surprisingly, bacterial species often associated with fish lesions and/or mortality were associated with relatively high levels of the human pathogens in the Vv and Vp species markers found in the deep sequencing amplicon data.
Species within the copiotrophic Vibrio genus are often associated with surfaces, which could include association with harmful algal bloom (HAB)-forming cyanobacteria, eukaryotic phytoplankton and zooplankton.Indeed, a combination of higher SST and chitinous plankton abundance are associated with higher Vv [143], and distinct pathogenic types are associated with distinct groups of chitinous eukaryotic plankton and follow particular patterns with respect to environmental conditions such as salinity and temperature [39].Capabilities of extracellular polysaccharide matrix formation for Vibrio and their resulting increased fitness were previously reviewed elsewhere [160,169].Archived plankton samples may be screened with molecular methods to further understand long-term SST increases and prediction of Vibrio dynamics.Using genus-specific PCR primers, Vibrio associated with plankton were positively correlated with SST, in addition to other climate indices, namely, the Northern Hemisphere Temperature and the Atlantic Multidecadal Oscillation [122].
Co-occurrences of high relative abundances of Vibrio and HAB species are also of particular interest.In temperate coastal and inland waters of Delaware, USA, higher abundances of Vibrio are concomitant with various eukaryotic species, often associated with blooms, as assessed with both size fractionation and in whole water analyses [44,133].The early Delaware study relied solely on phylogenetic markers to identify relative abundances of Vibrio species of interest, but the 2022 Rosales study in the same geographic region used more specific genetic markers that are most likely to reflect the more pathogenic strains of Vv and Vp.Additionally, in subtropical estuaries of the Gulf of Mexico coast (Alabama USA), the findings are similar to those of temperate Delaware [131].Size fractionation prior to DNA extraction and analysis using qPCR with the most reliable marker genes indicate that Vv and Vp are associated with blooms of Akashiwo sanguinea and Heterocapsa spp.Even more notably, and supporting other findings [136], the effect of wind prior to the HAB bloom influences the strength of the responding Vibrio increased abundances.
In both northern and southern temperate regions, as well as subtropical waters, abundances are positively correlated with higher relative amounts of markers of algal or cyanobacterial biomass, including but not limited to photosynthetic pigments, microscopy counts, HAB marker genes, or relative HAB abundances based on phylogenetic marker gene datasets [131,137].Geographic considerations, including the maximum or persistence of SST must also be included in predictive modeling.In a Prorocentrum bloom of temperate coastal surface seawater (East China Sea), based on Vibrio-specific genes in a metagenomics dataset, as well as 16S rRNA relative abundances in amplicon deep sequencing data, there was a strong (but not statistically significant) association between Vibrio genera gene abundances and HAB algal biomass [135].In contrast to findings of coastal South Carolina, USA, tidal creeks and nearby retention waterbodies, where increases in either or both Vv and Vp were associated with harmful cyanobacterial blooms and harmful eukaryotic algal blooms [115], there were no such associations in a partner study in Puget Sound of Washington state, USA, a more temperate environment [170].Notably, correlations of Vp with HABs are more pronounced with higher SST.In the Puget Sound, there were clear blooms of three harmful species during the study period (Pseudo-nitzschia, Alexandrium and Dinophysis), but these events did not coincide with increases in Vp [170].Also in this study, the only water column nutrient significantly associated with Vp was silicate, unlike the findings of many other studies suggesting strong correlations between Vv and Vp abundances with nitrogen-and phosphorous-containing nutrients (see Section 4.2).
The known association of Vv and Vp in the "particle-associated" fraction (often meaning associated with eukaryotic plankton) also emphasizes the need for size fractionation of water column samples prior to screening for the bacteria.This type of size fractionation is rare, but is becoming more common in environmental studies such as [131], resulting in insightful conclusions about the Vibrio ecology considerations when planning and modeling to improve aquaculture safety.Indeed, the composition of the plankton community may dictate the relative success of Vv and Vp [131] or bacteria in the Vibrio genus [143].Based on a study using only culture-independent methods, relative abundances of planktonic Vibrio are found to be higher in the FL fraction as compared to PA (>3 micron), but the qPCR primers used were only able to enumerate Vibrio at the genus level [142].

Seagrass Density
Coastal areas with high water movement can facilitate Vibrio transport and distribution.As recently examined with culture-dependent tools [128], it has long been known that Vibrio are enriched in sediments, where their abundances can exceed water counts by a log or more, and temporal fluctuations of Vv and Vp abundances in sediments are pronounced and tied closely to SST [171].Wind intensity, changes in direction, and changes in wind patterns may mix sedimentary Vibrio into the water column, increasing chances of human encounters or bringing more into contact with oyster and other shellfish.Fortunately, it is possible that these effects may be mitigated by submerged aquatic vegetation that stabilize sediments.In temperate eelgrass (Zostera marina) meadows, all Vibrio and Vv abundances were reduced, based on culture-dependent measurements [138].
Similarly, a negative correlation of Vibrio plus other pathogens was seen with other types of seagrasses in tropical coasts of Indonesia based on deep sequencing of the 16S rRNA gene [139].Unfortunately, inferring the relationship of grass density with strain-and species-specific Vibrio abundances is not possible with this method, and to date, the number of seagrass density studies is limited.Additionally, it has recently been suggested that surfaces of macroalgae or seagrasses could harbor higher Vibrio densities than in environs absent of macrophytes [172].The promise of decreased pathogen loads in waterbodies with high-density seagrass beds, however, is likely due to the sediment stabilization by healthy seagrasses.

Other Anthropogenic Factors
In addition to all the environmental factors described above, introduction of microplastics and heavy metals into waterbodies near aquaculture facilities may also increase abundances.For example, Vp enumerated with trh qPCR was positively correlated with heavy metals in water samples, but the impact of metals was also seen for general bacteria, E. coli, Pseudomonas aeruginosa, among other groups [134].Additionally, growth and biofilm formation by both pathogenic and non-pathogenic types of Vv are positively correlated with increasing iron concentrations [173].Introduction of heavy metals, antibiotics, or both, may affect the rate or mechanism ofARG transfer on transposable elements [174].There are several recent general reviews on antibiotic resistance genes in Vp and Vv, including [81,175,176].
Extracellular polysaccharide matrix formation by Vv and Vp leads to fitness on surfaces, both sessile and planktonic [160,169].Therefore, it is not surprising that microplastics loads in the water column are a risk factor for enhanced Vibrio abundances.Vibrio bacteria are also enriched in the plastisphere.A survey from the North and Baltic Seas reported that Vibrio-attached microplastics tend to be more brittle, perhaps due to degradation by high winds and storm events [177].These degraded microplastics may increase surface area and create greater attachment sites for these particle-associated microbes.More general molecular tools for enumerating bacterial families, including Vibrioaceae, were previously used in the context of fluorescence in situ hybridization (FISH) to follow succession of colonizers on plastic surfaces; Vibrio are important in early biofilm colonization [178].
In northern estuarine waters, Vibrio have different responses to various artificially introduced substrates, where wood or plastics result in the expression of different groups of genes, resulting in different proteomes of the different substrates' bacteria [179].Colonizing sargassum and microplastics in subtropical waters of the Caribbean and Sargasso Seas all apparently rely on the expression of the pilus-related MSHA operon, including the mshA gene.The MSHA is necessary for adhesion and biofilm formation, regardless of substrate [180].As extensively reviewed [181], sessile Vibrio abundances on these artificial surfaces are similar to those in naturally occurring substrates, both in fixed environments as well as on planktonic surfaces such as floating particles or larger plankton.

All Factors
Historically, several basic water quality parameters such as temperature and salinity were thought to have strong influence on pathogenic Vibrio dynamics, partly due to their known optimal ranges.As described in detail in Section 4.1 through Section 4.3, myriad examples exist demonstrating a combination of factors, both biotic and abiotic, that correlate very well with increased Vv and Vp abundances.Wind-mixing of sediments, seagrass bed densities, and other climate-related factors can influence both Vibrio distribution, as well as the distribution of other planktonic groups such as zooplankton and phytoplankton, now known to be the biotic predictors of Vibrio blooms.Importantly, it is now clear that multivariate analyses are needed to predict Vv and Vp abundances' correlations with meterological, biological and abiotic environmental factors [39,182], since oceanographic and hydrographic perturbations affect all trophic levels, including those to which Vibrio may be attached.In addition, interpretation of correlative relationships should also consider the enumerating tools used, which are overviewed in Section 5 (below).

Future Outlooks, Advancements, and New Molecular Tools
Ecological or environmental parameter predictions, to date, are based on various enumeration methods, each of which has its own set of limitations.Limitations of enumeration methods could include sensitivity, PCR primer bias, or underestimations due to "cultivability" of the environmental strains of interest.Common to many types of naturally occurring bacteria, the "Viable but Not-Culturable" (VBNC) state can confound models.Bacteria in the VBNC state may cause us to miss those "overwintering" Vibrio from sediments, or Vibrio associated with oysters, when using culture-based methods [32,183].The in situ water temperature also dictates culture growth rates, in particular environmental Vv from cooler samples [184].Although the VBNC state, metabolic survival and pathogenesis responses of these bacteria have been extensively reviewed elsewhere [26,[185][186][187], most current environmental monitoring types of studies still do not consider the impact of inclusion of culture-based methods on Vibrio abundance underestimates.Modeling based on data from most studies using partially culture-based methods described in Table 2, such as liquid culture-based (MPN-) or solid medium (DP-) culturing are at risk of underestimating Vv and Vp that are possibly pathogenic but resist growth on our current culture media.
Abundances gleaned from metagenomics studies of samples associated with enteric pathogens can be as reliable as, or at least consistent with, culture-based methods.For screening environmental samples, tying strain taxonomy with important functional genes such as individual ARGs or multi-drug resistance transporters is possible with metagenomics [188].Other more specialized types of molecular-only types of screening tools are available, such as LAMP-PCR-based assays for Vp, can be used, but these methods are limiting due to cost, extensive pretreatment needs, or other problems [189].Care should be taken with interpretations of studies using tools enumerating Vibrio at the genus level, or even at the species level without considering relative species abundances of pathogenic and non-pathogenic species [18].
Sensitivity of current assays, both those that include a culture-dependent step, as well as those that bypass enrichments, is generally not a problem.For example, methods in DP studies or others that use an enrichment step prior to PCR/qPCR, all have very low detection limits, ranging from ~<1 [21] to 5-15 colony forming unit (CFU) per mL of seawater [54,128].From oysters, DP methods can detect as low as 5-10 CFU per gram of oyster homogenate sample [121,190].Multi-step assays that rely on liquid culture-based enrichment steps first, prior to PCR or qPCR, are even more sensitive, since the enrichment allows for growth prior to detection.Ranges of sensitivity for these types of studies are quite variable.For example, in a variety of substrates from both subtropical and temperate locales, the lower detection limit of either Vv or Vp in all substrates tested was 1 to >80,000 CFU per unit of substrate tested, specifically 1-250 CFU/mL (water), 10-25,000 CFU/g (oyster), and 100 to 83,333 CFU/g (sediment) [101].
PCR primer bias could also lead to underestimation of true Vibrio abundances.For example, most of the recent studies employing PCR-based verification use PCR primers designed and published over a decade ago ("Gene" column in Table 2).One exception is a recent study that developed newer functional gene assays, including for one for sodB and related genes, which may assist Vv in transitioning from the environment into a virulent state associated with its host [74].PCR primer biases may result from direct PCR (or methods that use a culture-based step first followed by PCR or qPCR), since primer pairs (Table 1) may not match well to all representative strains' sequences.PCR primer bias does not create an issue with sensitivity, rather the community composition of pathogenic strains of Vibrio may not be accurately represented with use of outdated primer sequences.To remedy this, continual development of qPCR primer pairs should be carried out, refining them to be inclusive of more recently submitted sequences from known pathogenic strains.
However, there is hope; examples of more efficient direct analyses of DNA are becoming more prevalent, such as direct qPCR [191], and metagenomics [192], where both of these types of methods by-pass the culture-dependent step completely.Sensitivity of qPCR-only studies is, like with methods containing a culture step, very good.For example, Vp detection is as low as 1 to 10 copies per mL of seawater with trh primers [134]; with these and other primers for Vp and for other Vibrio species, a similar range is detectable with the very sensitive qPCR method [137].Detection of Vv with direct qPCR is also very sensitive; cell equivalents in DNA enumerated with vvhA qPCR is as low as 15 CFU per mg of homogenized fish tissue [54].PCR primer bias can be overcome with the use of PCR-independent detection of functional genes in genome segments of the entire microbial consortia.For instance, in metagenomics, sensitivity may be a problem, but the benefits gleaned (new sequences of functional genes obtained without a PCR step) may lead to improved PCR and qPCR primer pairs [192].
Increases in vibriosis associated with finfish aquaculture has been reviewed extensively elsewhere [193].A recent systematic review of foodborne-Vibrio evaluated temperature and other non-environmental parameters provides insights into predictions of Vibrio dis-tributions in finfish aquaculture [194].Although specific serovars can affect the health of aquaculture species such as eels and other teleosts [195], the scope of this review is limited to human pathogens among Vv and Vp, and thus, particular attention is paid to increasing Vibrio abundances in shellfish [190].Oysters, often eaten raw, are well-known to harbor high abundances of both Vv and Vp, in their liquor, on their shells, and/or in the meat.Seafood-borne infections are dominated by these two species as well as V. cholerae [64].Oyster-derived Vp outbreaks in northern US environments on both Atlantic and Pacific coasts and in the Gulf of Mexico region [196], as well as Vv outbreaks in a variety of locales [197] indicate the pervasive nature of these naturally occurring species.
Numerous earlier, and all subsequent, studies (too many to list, but many of which are listed with "Oyster" in the Substrate column of Table 2) emphasize the need for good screening methods to improve aquaculture efforts.Importantly, there may be differential influences in the changes in salinity, TSS, or even water depth on the loads of pathogenic Vv in oysters and clams, as well as an increased chance of finding pathogenic Vp in cooler, rather than warmer, waters [38].These studies have demonstrated how using genomics and molecular methods to call for and/or enumerate these genes can distinguish virulent strains of Vibrio in environmental samples.
Genes of interest for many studies focused on those in pathogenic Vv and Vp, including tdh, trh, and vvhA.A 1999 study successfully defined and developed a 5 nuclease probebased qPCR method for quantifying Vp in oyster tissue homogenate [59].When compared to positive detection in pure culture strains, trh, but not tdh, was detected in one strain, suggesting that assaying for both genes may be necessary to detect all hemolysin-producing strains.Some published primer pairs also exist for detection and enumeration of Vv with qPCR forgoing the use of probes [56].Such a design relies solely on SYBR Green I fluorescence in qPCR for the detection of amplicon production during the course of the PCR, a more cost-effective approach than probe-dependent assays.Additionally, careful PCR primer design may improve detection since TaqMan probes add a layer of specificity, reducing the ability to include degeneracy codes to better capture the full diversity of pathogenic strains' gene sequences [198].
Pathogenicity-associated target genes may appear to be present in species other than Vp.For example, trh, a target gene that was only reported to be present in Vp [199] was found in what was later identified as V. alginolyticus, a near-neighbor species, suggesting a positive PCR result could be associated with presence of pathogenic Vp [200].The lineage of a newly evolved pathogenic Vp strain expanded its distribution along the North American Atlantic coast.It was dubbed as a new "sequence type", number 631 (ST631) exhibiting a similar virulence gene profile to members of a related ST36 clade, a group of Vp known to contain both tdh and trh [201].Methods of detecting such highly adaptable pathogens need to be attuned often to accommodate the effects of a rapidly changing environment.
Increasing global temperatures due to climate change portends an increase in frequency and/or intensity of heavy rainstorms, which could promote Vibrio abundance.However, there are now better predictive tools such as machine learning modeling of hydrodynamic factors influencing particle movement [202], along with multivariate analyses to predict Vv and Vp abundances' correlations with meteorological, biological and abiotic environmental factors [39,182].There is promise for improved predictive models without sampling oyster tissues; this is especially true in aquaculture environments with low tidal range.Recently described in a study of water column and oyster abundances, the predictive patterns with fluctuations in environmental parameters may be determined by water column abundances alone [110], potentially allowing for time and cost-savings.Finally, the knowledge that healthy densities of submerged aquatic vegetative material can possibly mitigate some of these problems is a good start; further studies on seagrass beds and associated water column, sediment and shellfish-associated Vibrio are needed.These improved models and additional information on the potential for seagrass mitigation may assist local, regional, and other policy-makers in planning improvements for aquaculture facilities and human recreation.Methodological shifts in measurement approaches, away from those that depend on culturing as a first step, and towards evaluation of DNA or RNA extracted directly from the substrate will also allow for more rapid detection, accurate quantification, and a more comprehensive characterization of the types of bacteria present.

Table 2 .
Known environmental factors associated with changes in Vv and Vp abundances.Studies listed show strong positive or negative correlations of Vibrio species with factors listed (Env.Column).Method: tools used to enumerate Vv or Vp are indicated (if PCR and/or qPCR primers were used, the original reference for the primer sequences is provided).Quantification methods for studies examining correlating environmental factors included: MPN (most probable number), DP (direct plating), CH (colony hybridization), PCR (polymerase chain reaction) or qPCR (quantitative PCR), BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR), or simply "culture", indicating liquid culture methods.CHROMAgar™ is a specialized direct plating method and is listed separately from DP.Some studies used only metagenomics and/or deep sequencing of amplicon libraries of a single gene, where the genes were either 16S rRNA or other phylogenetic markers.Genes specific to Vv and/or Vp are listed in Table1.Positive, negative, or other non-linear relationships of environmental factors with either or both Vv and Vp species are indicated, where a check mark indicates the effected Vibrio species, and a check in parentheses indicates that species-level confirmation is not possible due to the limitations of the enumerating tool used.