ABSTRACT

   Vibrio alginolyticus is a naturally occurring marine bacterium,
   recognized as an emerging pathogen in humans and animals and the second
   most common cause of vibriosis in the U.S. However, information
   regarding the physiology and growth of this species in the environment
   is limited. Here we evaluated the effects of temperature, salinity, and
   iron condition on the growth response across unique V. alginolyticus
   strains. A combination of growth kinetics and gas chromatography-mass
   spectrometry-based metabolomics studies was used to evaluate the
   optimal and tolerable ranges of growth and to characterize the
   metabolic effects of iron supplementation. All V. alginolyticus strains
   tested demonstrated broad temperature and salinity tolerance, resulting
   in growth at all measured temperatures (24°C–40°C) and salinities
   between 1% and 6% (wt/vol) NaCl with optimal growth between 30°C–36°C
   and 2%–4% NaCl. Environmental strains showed no growth limitation at
   iron concentrations ranging from 0.5- to 20.0-µM ferric Fe but
   demonstrated reduced growth at 0.2 µM. Likewise, the number of
   significantly upregulated metabolites in V. alginolyticus cultures
   grown in iron-replete (4-µM) media was greater than that in
   iron-deficient (~0 µM) media but varied with prior growth conditions.
   Detected compounds were associated with key metabolic pathways, namely,
   amino acid, carbohydrate, lipid, and nucleotide metabolism, suggesting
   that introduced iron facilitated broad activation of V. alginolyticus
   metabolism and helped to promote growth responses. Combined, these
   results demonstrate that V. alginolyticus strains are capable of rapid
   growth under a broad range of favorable temperature and salinity
   levels, which can be affected by the presence of iron.

IMPORTANCE

   Transmission of V. alginolyticus occurs opportunistically through
   direct seawater exposure and is a function of its abundance in the
   environment. Like other Vibrio spp., V. alginolyticus are considered
   conditionally rare taxa in marine waters, with populations capable of
   forming large, short-lived blooms under specific environmental
   conditions, which remain poorly defined. Prior research has established
   the importance of temperature and salinity as the major determinants of
   Vibrio geographical and temporal range. However, bloom formation can be
   strongly influenced by other factors that may be more episodic and
   localized, such as changes in iron availability. Here we confirm the
   broad temperature and salinity tolerance of V. alginolyticus and
   demonstrate the importance of iron supplementation as a key factor for
   growth in the absence of thermal or osmotic stress. The results of this
   research highlight the importance of episodic iron input as a crucial
   metric to consider for the assessment of V. alginolyticus risk.

   KEYWORDS: Vibrio alginolyticus, iron, physiology, metabolomics, growth
   kinetics, tolerance

INTRODUCTION

   Vibrio alginolyticus is a ubiquitous marine bacterium native to coastal
   and estuarine waters worldwide. As an opportunistic pathogen, this
   species is an important agent of both human and animal disease
   affecting a broad range of host species including marine fishes ([32]1,
   [33]2), crustaceans ([34]3, [35]4), mollusks ([36]5), echinoderms
   ([37]6), corals ([38]7, [39]8), marine mammals ([40]9), sea turtles
   ([41]10), and humans ([42]11, [43]12). Animal infections have been
   widely described in association with the aquaculture industry and range
   in severity with disease signs manifesting as mild epidermal lesions
   ([44]9) to systemic organ dysfunction and hemorrhage often leading to
   mass mortality ([45]1, [46]4). Reported human infections, which have
   doubled in the U.S. from 2009 to 2019 ([47]13), are strongly associated
   with recreational and/or occupational exposure to seawater and manifest
   primarily as opportunistic infections of the ears and pre-existing or
   sustained wounds ([48]11, [49]12). While often severe in aquaculture
   settings [i.e., causing a higher percentage of mortality in affected
   fish and crustaceans ([50]1, [51]4)], human infections are typically
   non-life threatening, presenting as self-limiting or readily treatable
   through the administration of antibiotics such as ciprofloxacin and
   tetracycline ([52]14, [53]15). However, infections in immunocompromised
   patients have been shown to progress to invasive conditions such as
   bacteremia and sepsis, greatly increasing the chance of mortality
   ([54]11, [55]16). Collectively, the burden of V. alginolyticus
   infections imposes a substantial economic and regulatory encumbrance to
   aquaculture and public health, with annual cost estimates in excess of
   one million dollars (USD) for the treatment of human infections in the
   United States ([56]17, [57]18) and a global estimated cost of three
   billion dollars (USD) for the treatment or culling of Vibrio
   aquaculture outbreaks (of which V. alginolyticus is a major
   contributor) ([58]19).

   Environmental factors that enhance or inhibit the growth of V.
   alginolyticus populations in the environment are critical to the
   estimation of exposure risk for this species. Prior studies have shown
   that temperature and salinity are the two leading environmental
   determinants of growth for most Vibrio species ([59]20, [60]21) and
   that increased temperature positively correlates with increased Vibrio
   abundance ([61]20, [62]22 [63]– [64]25). This correlation has been
   corroborated for V. alginolyticus specifically ([65]26, [66]27) and
   provides a mechanism for the strong seasonality of infections
   associated with warmer months ([67]12, [68]28). V. alginolyticus can
   tolerate temperatures ranging from 5°C to 42°C with faster growth
   typically occurring between 22°C and 37°C and optimal growth (fastest
   growth rate) at 35°C ± 2°C ([69]29 [70]– [71]33). Second to
   temperature, salinity is a critical factor for the establishment of
   Vibrio range with species-specific optimal growth occurring from 0 to
   35. V. alginolyticus has been shown to be tolerable of a wide salinity
   range from 0.5 to 60.0 with optimal growth occurring at 30–35 ([72]33).
   The expansive thermo- and halotolerance of V. alginolyticus suggests
   that this bacterium is well adapted to tropical/temperate waters and
   may only be limited within these systems by seasonal cooling, the
   presence of freshwater input, and/or atypical hypersaline environments.

   Temperature and salinity largely define the broad geographical range of
   V. alginolyticus. However, in warm coastal regions such as Florida and
   Hawaii, where V. alginolyticus infection reports are high ([73]13),
   other more episodic environmental determinants, such as nutrient
   availability, may play an important role in shaping the local and
   short-term Vibrio community structure ([74]34 [75]– [76]36). Iron is an
   essential cofactor for bacterial metabolism that is often limiting in
   marine waters ([77]37, [78]38). Prior research has established the
   specific importance of episodic iron input for the enrichment of Vibrio
   populations during Saharan dust deposition events ([79]35). During
   these events, aerosolized ferric (Fe^3+) and ferrous (Fe^2+) iron is
   transported from Northern Africa via the Atlantic trade winds and
   deposited into the oligotrophic waters of the Southeastern United
   States and the Gulf of Mexico ([80]35, [81]36, [82]39). Microbial
   community surveys have shown that these events trigger a substantial
   increase in the relative and absolute abundance of Vibrio in the
   microbial population, which can swell to 5–30× the background
   concentration for 24–72 h following the onset of deposition ([83]35,
   [84]36, [85]40). Termed “Vibrio blooms,” these events have the
   potential to increase the risk of exposure to opportunistic Vibrio
   pathogens, including V. alginolyticus, and are important but
   understudied factors to consider for risk characterization.

   In addition to facilitating population growth, iron acquisition is an
   important characteristic of virulence for V. alginolyticus ([86]41). V.
   alginolyticus has developed a sophisticated iron acquisition system
   designed to compete for and scavenge iron from the ambient environment.
   The two major factors that comprise this system are siderophores and
   the TonB energy transduction system ([87]41 [88]– [89]43). Siderophores
   are small molecular weight compounds that have a high affinity to
   chelate ferric iron. These compounds are secreted extracellularly where
   they bind ambient iron and are recognized by outer membrane proteins
   ([90]42, [91]44, [92]45). Ferrisiderophore complexes are internalized
   via TonB, a transmembrane protein system that facilitates transfer of
   energy from the inner cell membrane to the outer cell membrane,
   enabling active transport ([93]41, [94]42). While these systems enhance
   the competitiveness of V. alginolyticus in environmental settings, they
   also contribute to its establishment during infection by outcompeting
   host iron sequestration mechanisms or directly scavenging iron from
   heme in blood cells, thus increasing the iron pool available to
   infecting cells ([95]41, [96]43). Increased iron availability is known
   to increase bacterial replication ([97]46) and promote biofilm
   formation ([98]47) in Vibrio spp., which can contribute to the onset
   and severity of infection. Despite this importance, the relationship
   between iron concentrations in seawater and V. alginolyticus growth and
   metabolism is poorly understood, and there is a substantial need for
   baseline characterization.

   Here we investigate growth characteristics of V. alginolyticus in
   response to a range of temperature, salinity, and iron concentrations
   to better understand how these factors can influence population
   responses across tested strains and to provide context for rapid growth
   that supports local bloom formation. Additionally, the metabolic
   response of iron stimulation was further evaluated in a recently
   isolated environmental strain using gas chromatography-mass
   spectrometry (GC-MS)-based metabolomic profiling to better understand
   the specific biochemical response elicited by this bacterium in
   relation to iron supplementation and deprivation. Together, these
   findings can be used to better predict the environmental conditions
   favorable to the proliferation of this bacterium and can be used to
   mitigate infection risk for humans and in aquaculture settings.

RESULTS

   The results of growth kinetics experiments demonstrated the optimal and
   tolerable limits of temperature, salinity, and iron concentration for
   three unique strains of V. alginolyticus. Growth curves for all tested
   strains were constructed from OD[600] measures to determine the
   duration of lag phase and the doubling time, which represented the time
   required to adapt and the productivity of the strain under the given
   environmental conditions, respectively. Optimal range was defined as
   the conditions where all three strains demonstrated the fastest
   strain-level doubling time, whereas the tolerable limit was defined as
   the conditions where no growth inhibition was observed. Tested strains
   included two unique environmental isolates, JW16-551 and JW16-580,
   originally collected in 2016 from water near Looe Key Reef, off the
   coast of the Florida Keys (USA), during a Saharan dust deposition event
   ([99]26), and the V. alginolyticus type strain, American Type Culture
   Collection (ATCC) 17749, originally isolated in 1961 from spoiled fish
   in Japan ([100]48).

Temperature effects on growth

   Optimal V. alginolyticus growth occurred between 30°C and 36°C for all
   strains when grown at a 3% NaCl concentration in non-iron-limiting
   media [lysogeny broth (LB), with an estimated iron content of 17 µM
   ([101]49)] ([102]Fig. 1; Fig. S1; Table S4). The fastest doubling time
   was observed at 32°C (81.6 min), 36°C (71.3 min), and 30°C (96.4 min)
   for strains JW16-551, JW16-580, and ATCC 17749, respectively. The
   shortest lag phase duration was observed at 40°C (2.0 h), 40°C (2.2 h),
   and 36°C (2.7 h) for strains JW16-551, JW16-580, and ATCC 17749,
   respectively. Within the tested temperature range, all three V.
   alginolyticus strains showed similar patterns of doubling time and lag
   phase duration up to 36°C. At temperatures ≥36°C doubling time diverged
   with strain JW16-580, showing a relatively unchanged rate, a
   progressively longer doubling time for JW16-551, and a substantial
   increase in doubling time for strain ATCC 17749. A similar divergence
   was noted for lag phase duration at temperatures of ≥38°C, where time
   in lag phase continued to shorten for strains JW16-551 and JW16-580 but
   increased for strain ATCC 17749 at elevated temperatures. Growth was
   not inhibited within the tested temperature range (24°C–40°C); however,
   longer doubling times and lag phase durations were observed at
   temperatures ≤26°C and ≥38°C for all strains.

Fig 1.

   [103]Fig 1
   [104]Open in a new tab

   Growth response of V. alginolyticus at varying temperatures. Optimal
   growth range indicated by dashed vertical lines. All cultures grown in
   lysogeny broth plus salt (LBS) 3% (wt/vol) NaCl under non-limiting iron
   conditions. Linerange values represent the standard error of reported
   metrics. (A) V. alginolyticus doubling time from 24°C to 40°C. (B) V.
   alginolyticus lag phase duration from 24°C to 40°C. N = 12 for each V.
   alginolyticus strain.

Salinity effects on growth

   Optimal V. alginolyticus growth occurred between 2% and 4% (wt/vol)
   NaCl concentrations when cultures were incubated at 30°C in
   non-iron-limiting media (LB) ([105]Fig. 2; Fig. S2; Table S4). Fastest
   doubling time was observed at NaCl concentrations of 2% (90.6 min), 3%
   (91.5 min), and 4% (141.6 min) for strains JW16-551, JW16-580, and ATCC
   17749, respectively. The shortest lag phase duration was observed at
   NaCl concentrations of 2% (2.8 h), 3% (2.5 h), and 3% (3.0 h) for
   strains JW16-551, JW16-580, and ATCC 17749, respectively. At a NaCl
   concentration of 1%, doubling time slowed for all tested strains. Lag
   phase duration remained relatively stable at 1% for both environmental
   strains but was notably longer for ATCC 17749. Complete inhibition (no
   growth) was observed in salt-free trials (0% NaCl) for all strains. At
   increased salinities (5%–8% NaCl), a progressive slowing in doubling
   time and lengthening of lag phase duration were noted for all strains.
   Substantial inhibition of growth occurred at NaCl concentrations of
   ≥7%, which prevented accurate calculation of bacterial doubling time,
   although sufficient growth was observed to allow determination of the
   lag phase duration at these concentrations.

Fig 2.

   [106]Fig 2
   [107]Open in a new tab

   Growth response of V. alginolyticus at varying NaCl concentrations.
   Optimal growth range indicated by dashed vertical lines. All cultures
   grown in non-iron-limiting LBS broth amended to the NaCl concentration
   designated by the experimental condition and incubated at 30°C.
   Linerange values represent the standard error of reported metrics. (A)
   V. alginolyticus doubling time from 1% to 8% (wt/vol) NaCl. (B) V.
   alginolyticus lag phase duration from 1% to 8% (wt/vol) NaCl.
   Substantial inhibition of all strains was observed at salt
   concentrations of ≥7%, preventing accurate calculation of doubling
   time. However, minor increases in optical density were detected; thus,
   lag time duration measures were collected for these concentrations. N =
   12 for each V. alginolyticus strain.

Iron effects on growth

   Environmental V. alginolyticus strains were amenable to growth at all
   measured iron concentrations (0.2–20.0 µM as provided in FeCl[3]) when
   incubated at 30°C with a 3% (wt/vol) NaCl concentration in defined
   minimal media (termed VibFeL). However, strain ATCC 17749 was
   substantially inhibited by the minimal media regardless of iron
   concentration. This inhibition prevented accurate calculation of
   doubling time and lag phase duration for most experimental trials with
   this strain, although detection of minimal growth at iron
   concentrations of ≥3 µM enabled determination of lag phase duration
   from 3 to 20 µM and doubling time at 20 µM ([108]Fig. 3; Fig. S3; Table
   S4). Of the two environmental strains, the fastest doubling time was
   observed at 20 µM (69.1 min) and 10 µM (52.0 min), and the shortest lag
   phase duration was observed at 10 µM (4.7 and 5.4 h) for strains
   JW16-551 and JW16-580, respectively. Both environmental strains
   demonstrated similar patterns of doubling time response throughout the
   experiment with faster rates observed between 0.5- and 20.0-µM iron
   concentrations and markedly slowed rates at 0.2 µM. This was also
   observed for lag phase response where increasing iron facilitated a
   progressively shorter lag phase duration for both environmental
   strains, peaking at 10–20 µM. No growth inhibition was observed at an
   iron concentration of 20 µM; therefore, no upper optimal or tolerable
   limit could be determined.

Fig 3.

   [109]Fig 3
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   Growth response of V. alginolyticus at varying iron concentrations.
   Optimal growth occurred at all values ≥0.5 µM (indicated by dashed
   vertical line) with no discernable upper limit. All cultures grown in
   VibFeL broth at 3% NaCl (wt/vol) and incubated at 30°C. Linerange
   values represent the standard error of reported metrics. (A) V.
   alginolyticus doubling time from 0.2- to 20.0-µM iron. (B) V.
   alginolyticus lag phase duration from 0.2- to 20.0-µM iron. Growth of
   strain ATCC 17749 was substantially inhibited at all tested
   concentrations of iron; thus, accurate calculation of the growth rate
   was not possible for this strain except for the 20-µM concentration.
   Minor increases in optical density were observed at iron concentrations
   of ≥3 µM, allowing for calculation of lag phase duration from 3 to 30
   µM. N = 12 for each V. alginolyticus strain.

GC-MS metabolomics

   Endo- and exometabolite profiles for V. alginolyticus strain JW16-551
   were compared across four different conditions related to the iron
   content of the initial culture used for inoculation (referred to as the
   starvation condition) and the experimental culture (referred to as the
   iron condition). These trials included (i) non-starved, iron replete
   (NSFe+), where cultures were initially grown under non-limiting iron
   conditions and inoculated into iron-replete experimental media (4-µM
   FeCl[3]); (ii) non-starved, iron deficient (NSFe-), where cultures were
   initially grown under non-limiting iron conditions and inoculated into
   iron-deficient experimental media (0-µM FeCl[3]); (iii) starved, iron
   replete (SFe+), where cultures were initially starved of iron for 5
   days in iron-deficient media then inoculated into iron-replete
   experimental media; and (iv) starved, iron deficient (SFe-), where
   cultures were initially starved of iron for 5 days in iron-deficient
   media then inoculated into iron-deficient experimental media ([111]Fig.
   4). Growth was substantially reduced in all trials using the
   iron-deficient experimental media regardless of prior starvation
   condition ([112]Fig. 5). At 18 h of growth, iron-replete cultures
   (NSFe+ and SFe+) reached a mean of 3.50 × 10^7 and 4.03 × 10^7 colony
   forming units (CFU)/mL, whereas, iron-deficient cultures (NSFe- and
   SFe-) grew to a mean of 2.07 × 10^6 and 6.53 × 10^5 CFU/mL for
   non-starved and starved cultures, respectively. This equates to a
   15.9-fold (P value = 0.07) and 60.7-fold (P value = 0.06) increase in
   culturable V. alginolyticus under iron-replete conditions for
   non-starved and starved cultures, respectively. Furthermore,
   pre-starved cultures responded more rapidly when transferred to
   iron-replete media compared to non-starved cultures. Pre-starved
   cultures showed a mean of 2.78 × 10^7 CFU/mL at 11 h of growth in
   iron-replete media, whereas non-starved cultures only reached 5.03 ×
   10^6 CFU/mL at the same time point, representing a 4.5-fold increase (P
   value = 0.003) based on starvation ([113]Fig. 5).

Fig 4.

   [114]Fig 4
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   Sample preparation scheme for iron metabolomics experiments. Starvation
   condition [non-starved (NS) or starved (S)] represents the iron content
   of the initial inoculum culture where NS cultures were grown in
   non-limiting LBS 3% broth for 18 h at 30°C before inoculation, and S
   cultures were grown in iron-deficient VibFeL (0-µM FeCl[3]) for 5 days
   before inoculation. Iron condition (Fe+ or Fe−) represents the iron
   content of the experimental culture where iron-replete (Fe+) cultures
   were grown in VibFeL broth amended with 4-µM FeCl[3] and iron deficient
   (Fe−) were grown in non-amended VibFeL broth (0-µM FeCl[3]). All
   cultures were inoculated with V. alginolyticus strain JW16-551. All
   experimental VibFeL broth cultures were amended to 3% (wt/vol) NaCl
   concentration and incubated aerobically for 18 h at 30°C under 100 rpm
   of shaking agitation.

Fig 5.

   [116]Fig 5
   [117]Open in a new tab

   V. alginolyticus growth response (CFU/mL) of iron metabolomic samples.
   Starvation conditions (NS or S) represent the iron content of the
   initial inoculation culture, and iron conditions (Fe+ or Fe−) represent
   the iron content of the experimental culture. NSFe+ represents
   non-starved iron-replete cultures; NSFe− represents non-starved
   iron-deficient cultures; SFe+ represents starved iron-replete cultures;
   and SFe− represents starved iron-deficient cultures. Cultures measured
   at 0, 4, 11, and 18 h prior to collection for GC-MS analysis.

Endometabolites

   Cell pellets were extracted to evaluate the endometabolomic response
   under differing iron and starvation conditions. Principal component
   analysis (PCA) shows distinct grouping of the cultures by exposure
   condition ([118]Fig. 6). Fluxes in the endogenous metabolome of
   iron-replete cultures show similar patterns of clustering and
   confidence interval overlap regardless of starvation condition, whereas
   iron-deficient samples show starvation-dependent groupings with minor
   overlap in principal component space. Comparison of iron conditions
   (NSFe+ vs NSFe−, and SFE+ vs SFe-) showed increased metabolic activity
   following transfer to iron-replete media with 49 and 47 significantly
   elevated metabolites identified in iron-replete trials for non-starved
   and starved cultures, respectively, compared to 20 elevated metabolites
   identified from iron-deficient trials (both starvation conditions)
   ([119]Table 1). Pathway analysis of metabolites from replete cultures
   (NSFe+ and SFe+) were found to be associated with 25 and 30 unique
   metabolic pathways (≥2 constituents detected) for non-starved and
   starved cultures, respectively. Alanine, aspartate, and glutamate
   metabolism was the most strongly represented pathway (the pathway with
   the greatest proportion of associated metabolites detected) under both
   starvation conditions ([120]Fig. 7). Conversely, metabolites from
   iron-deficient experimental conditions corresponded to only six and
   four pathways for non-starved and starved cultures, respectively.
   Aminoacyl-tRNA biosynthesis was the most strongly represented pathway
   regardless of prior starvation condition (SFe− and NSFe−). No unique
   metabolic pathways were detected in iron-deficient samples that were
   absent in iron-replete samples ([121]Table 1; [122]Fig. 7; Table S1;
   Fig. S4, S8, and S9).

Fig 6.

   [123]Fig 6
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   Principal component analysis of spectral features identified in strain
   JW16-551 GC-qToF/MS-based metabolomics; (A) polar endometabolites, (B)
   non-polar endometabolites, and (C) exometabolites. Shaded regions
   represent a 95% confidence interval of the sample group. N = 3 for each
   sample type.

TABLE 1.

   Summary of upregulated metabolites and associated metabolic pathways
   identified for iron and starvation comparisons
   Sample comparison[125] ^a ^,[126] b Iron condition Starvation condition
   Metabolite type Number of upregulated metabolites identified Number of
   associated metabolic pathways
   NSFe+/NSFe− Replete Non-starved Endometabolites 49 25
   NSFe−/NSFe+ Deficient Non-starved Endometabolites 20 6
   SFe+/SFe− Replete Starved Endometabolites 47 30
   SFe−/SFe+ Deficient Starved Endometabolites 20 4
   NSFe+/SFe+ Replete Non-starved Endometabolites 14 1
   SFe+/NSFe+ Replete Starved Endometabolites 30 19
   NSFe−/SFe− Deficient Non-starved Endometabolites 12 2
   SFe−/NSFe− Deficient Starved Endometabolites 19 3
   NSFe+/NSFe− Replete Non-starved Exometabolites 19 14
   NSFe−/NSFe+ Deficient Non-starved Exometabolites 15 6
   SFe+/SFe− Replete Starved Exometabolites 30 14
   SFe−/SFe+ Deficient Starved Exometabolites 10 6
   NSFe+/SFe+ Replete Non-starved Exometabolites 9 3
   SFe+/NSFe+ Replete Starved Exometabolites 11 9
   NSFe−/SFe− Deficient Non-starved Exometabolites 23 10
   SFe−/NSFe− Deficient Starved Exometabolites 9 6
   [127]Open in a new tab
   ^^a

   Sample comparison indicates the two metabolite profiles that were
   compared where elevated metabolite and pathway totals correspond to the
   sample in the numerator. Starvation conditions (NS and S) indicate the
   iron conditions of the initial inoculum culture, whereas iron
   conditions (Fe+ or Fe−) indicate the iron conditions of the
   experimental culture.
   ^^b

   Non-starved iron replete (NSFe+), non-starved iron deficient (NSFe−),
   starved iron replete (SFe+), and starved iron deficient (SFe−).

Fig 7.

   [128]Fig 7
   [129]Open in a new tab

   Metabolic pathways associated with significantly altered
   endometabolites detected in V. alginolyticus cultures under iron
   supplementation and iron starvation conditions. The left y-axis lists
   all associated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways;
   the right y-axis illustrates the broad category of each KEGG pathway;
   the fill color represents the normalized number of pathway hits found
   for the metabolites detected; and the x-axis shows the experimental
   comparison. From left to right, columns 1–4 illustrate iron comparisons
   and columns 5–8 represent starvation comparisons. TCA, tricarboxylic
   acid cycle. CoA, coenzyme A.

   Comparison of prior growth condition demonstrated that iron starved
   cultures responded more robustly when transferred to iron-replete media
   compared to non-starved cultures. Previously starved cultures
   transferred to iron-replete media (SFe+) showed significant
   upregulation of 30 unique metabolites corresponding to 19 different
   metabolic pathways with alanine, aspartate, and glutamate metabolism
   and beta-alanine metabolism as the most represented pathways.
   Comparatively, previously non-starved cultures transferred to
   iron-replete media (NSFe+) showed upregulation of 14 metabolites
   corresponding to only one biochemical pathway, glycerophospholipid
   metabolism. Transfer to iron-deficient media demonstrated little
   difference in the total number of significantly upregulated metabolites
   and associated pathways with 19 and 12 metabolites corresponding to 3
   and 2 metabolic pathways for starved (SFe−) and non-starved (NSFe−)
   trials, respectively ([130]Table 1; [131]Fig. 7; Table S1; Fig. S5,
   S10, and S11).

Exometabolites

   Exometabolomic assessments compared the extracellular metabolomic
   profiles of V. alginolyticus spent media in response to prior
   starvation and iron growth conditions. Results were consistent with
   those observed from endometabolite analyses, showing similar PCA
   patterns with respect to iron and prior starvation comparisons. PCA of
   the detected exometabolites showed distinct clustering by sample type
   where cultures transferred to iron-replete conditions showed similar
   patterns of grouping (NSFe+ and SFe+) and iron-deficient cultures
   separated markedly by prior starvation condition (NSFe− and SFe−)
   ([132]Fig. 6). Comparison of iron condition suggested an increase in
   metabolic response in conjunction with transfer to iron-replete media
   with significant upregulation of 19 and 30 metabolites for non-starved
   (NSFe+) and starved (SFe+) cultures, respectively ([133]Table 1;
   [134]Fig. 8). These compounds were mapped to fluxes in 14 total
   metabolic pathways for both starvation conditions (NSFe+ and SFe+) with
   alanine, aspartate, and glutamate metabolism representing the most
   represented pathway. Cultures transferred to iron-deficient media
   showed significant upregulation of 15 and 10 metabolites associated
   with six metabolic pathways each with glyoxylate and decarboxylate
   metabolism and C5-branched dibasic acid metabolism as the most
   represented for non-starved (NSFe−) and starved (SFe−) cultures,
   respectively. Of the detected pathways, five (amino sugar and
   nucleotide sugar metabolism, C5-branched dibasic acid metabolism,
   galactose metabolism, gluconeogenesis/glycolysis, and methane
   metabolism) were only identified in iron-deficient cultures ([135]Table
   1; [136]Fig. 8; Table S2; Fig. S6, S8, and S9).

Fig 8.

   [137]Fig 8
   [138]Open in a new tab

   Metabolic pathways associated with significantly upregulated
   exometabolites detected in V. alginolyticus cultures under iron
   supplementation and iron starvation conditions. The left y-axis lists
   all associated KEGG pathways; the right y-axis illustrates the broad
   category of each KEGG pathway; the fill color represents the normalized
   number of pathway hits found in the metabolites detected; and the
   x-axis shows the experimental comparison. From left to right, columns
   1–4 illustrate iron comparisons and columns 5–8 represent starvation
   comparisons.

   Comparison of prior starvation condition was also consistent with
   endometabolite results with increased metabolic activity in previously
   starved cultures when transferred to iron-replete conditions. Starved
   iron replete (SFe+) cultures showed significantly elevated levels of 11
   metabolites corresponding to 9 metabolic pathways with alanine,
   aspartate, and glutamate metabolism identified as the most represented
   pathway. Comparatively, previously non-starved cultures transferred to
   iron-replete conditions (NSFe+) showed significant elevation of nine
   metabolites corresponding to three pathways, alanine, aspartate, and
   glutamate metabolism, glutathione metabolism, and
   aminoacyl-tRNA-biosynthesis. Conversely, the number of metabolites and
   associated metabolic pathways were higher in previously non-starved
   cultures transferred to iron-deficient media (NSFe−) than in starved
   iron-deficient cultures (SFe−) with significant upregulation of 9 and
   23 metabolites corresponding to 6 and 10 metabolic pathways for starved
   and non-starved cultures, respectively ([139]Table 1; [140]Fig. 8;
   Table S2; Fig. S7, S10, and S11).

DISCUSSION

   As a naturally occurring pathogen, exposure risk for V. alginolyticus
   is strongly associated with its population abundance in the
   environment. Prior research has documented the importance of
   temperature and salinity as the two major factors influencing Vibrio
   populations, broadly, with temperature, in particular, playing an
   important role in controlling the geographical range across many Vibrio
   species [e.g., see references (20, 21)]. However, in tropical and