Vibrio alginolyticus growth kinetics and the metabolic effects of iron

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.

This manuscript evaluated the effects of temperature, salinity, and iron condition on the growth response in V. alginolyticus.Moreover, the metabolic effects of iron supplementation was investigated by GCMS.The authors found 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.Overall, this is a comprehensive work, which is worthy for publication.Some comments are as follows: 1.Line 35 and 327, what does the "Fe" indicated?ferriciron or ferrous iron or the total of iron?2.Line 133, "Scavenging of host-derived iron leads to increased bacterial replication resulting in V. alginolyticus persistence and increased severity of infection (Kustusch et al., 2011)".Is this view right?I did not find this opinion in the review article wrote by Kustusch et al., (2011).In addition, the low iron levels have been shown to induce the expression of a number of bacterial toxins and virulence factors (PMID: 16912433).And many researchers verified that iron promote biofilm formation in vivo and in vitro (PMID: 36383258;PMID: 35410114).So I think the author should reconsider the description here.3.Figure 1, please use the integrated name of strains instead of abbreviation.4.Line 218, SFE+ should be SFe+ 5.The conclusion can be condensed.
Reviewer #2 (Comments for the Author): Norfolk et al. perform a study on the Vibrio alginolyticus growth kinetics and the metabolic effects of iron.Authors used growth conditions, and metabolomics to analyze the effects of temperature, salinity, and iron on vibrio survival and growth.The study is interesting and merits publication, however, I have a number of remarks that require attention.1-Please confirm that OD derived counts match cell cytometry or CFU counts, or cell microscopy counts.The numbers should be similar.2-Please confirm by bioinformatics tools that the genome sequences of the studied strains reflect the temperature and salinity ranges obtained in the in vitro tests.The authors may use a bioinfo tool to verify congruence between their experiments and data retrieved directly from genome sequences, the so called in silico phenotypes.10.1186/1471-2164-13-S7-S3 , 10.1093/bioinformatics/btz059 3-why did you chose 4uM of Iron for the metabolomics?Also it is nuclear if how these vibrios would tolerate higher amounts of iron in the media.This is relevant in the face of possible iron inputs in the ocean and on coral reefs.Alginolyticus may appear in the water column in some occasions.https://peerj.com/articles/741/, 10.1126/science.1106028 .High levels of Iron might induce the growth of putative vibrios in the water 10.1016/j.scitotenv.2019.135914 , 10.1016/j.scitotenv.2018.11.112 which in turn might kill corals .Is 4 uM much in terms of possible marine systems vibrio may play a role?Ex bays, reefs.This concentrations of iron are common in such systems?4-the metabolomics part is preliminar?I had the impression the analyzes could provide the specific molecules and metabolites names induced by iron.Not only the general keeg groups.Also match metabolome data with genome data to elucidate gene function and in silico phenotyping.I do not think the so called unique metabolic pathways was clear enough and they require molecule ids.For instance, which terpenoids and polyketides were identified?
Minor remarks -Impossible to see a blue line (ATCC) in fig3A.
-as a study control, the authors could have used Vibrio parahaemolyticus which is a syster species of V. alginolyticus.The comparison would be useful for many reasons.
-please improve conclusions and avoid results now.

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Reviewer #1 (Comments for the Author):
This manuscript evaluated the effects of temperature, salinity, and iron condition on the growth response in V. alginolyticus.Moreover, the metabolic effects of iron supplementation was investigated by GCMS.The authors found 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.Overall, this is a comprehensive work, which is worthy for publication.Some comments are as follows: 1.Line 35 and 327, what does the "Fe" indicated?ferric iron or ferrous iron or the total of iron?Thank you for your comment.These levels are indicative of ferric iron in the media added as designated FeCl3 amendments.We have modified word use in the abstract as well as added additional information to the methods section to clarify these concentrations.
Lines: 481-486."Prior work indicates that LB provides ~17 µM Fe (Abdul-Tehrani et al., 1999).Iron effects were measured between 0.2 and 20 µM Fe(III) at 0.2, 0.5, 1, 3, 4, 10, and 20 µM added FeCl3.These levels were selected to represent a range of iron concentrations from environmentally relevant (0.2-4 µM) to highly elevated levels (10-20 µM).Iron concentrations above 20 µM were not evaluated due to the formation of precipitate which interfered with the accurate collection of optical density measures."2.Line 133, "Scavenging of host-derived iron leads to increased bacterial replication resulting in V. alginolyticus persistence and increased severity of infection (Kustusch et al., 2011)".Is this view right?I did not find this opinion in the review article wrote by Kustusch et al., (2011).In addition, the low iron levels have been shown to induce the expression of a number of bacterial toxins and virulence factors (PMID: 16912433).And many researchers verified that iron promote biofilm formation in vivo and in vitro (PMID: 36383258; PMID: 35410114).So I think the author should reconsider the description here.Thank you for your comment.We have amended the wording of this section of the introduction and added additional references citing the importance of iron for Vibrio spp.virulence.
Lines: 129-136."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 (Wang et al., 2008;Kustusch et al., 2011).Increased iron availability is known to increase bacterial replication (Wright et al., 1981) and promote biofilm formation (Çam & Brinkmeyer, 2019) in Vibrio spp.which can contribute to the onset and severity of infection."3.Figure 1, please use the integrated name of strains instead of abbreviation.Thank you for pointing out this inconsistency, we have replaced the legend names for figures 1-3 to match the full names in text.

4.Line 218, SFE+ should be SFe+
Thank you, we have corrected this typo.
5.The conclusion can be condensed.Thank you for your comment.We have condensed the language of the conclusion to cover the broad findings of this work.

Reviewer #2 (Comments for the Author):
Norfolk et al. perform a study on the Vibrio alginolyticus growth kinetics and the metabolic effects of iron.Authors used growth conditions, and metabolomics to analyze the effects of temperature, salinity, and iron on vibrio survival and growth.The study is interesting and merits publication, however, I have a number of remarks that require attention.
1-Please confirm that OD derived counts match cell cytometry or CFU counts, or cell microscopy counts.The numbers should be similar.Optical density measurements were employed in this study to evaluate the growth kinetics of V. alginolyticus strains under the designated temperature, salinity, and iron conditions.Culturebased CFU counts were only used to quantify cellular growth for the metabolomics portion of the experiment prior to processing with gas chromatography mass spectrometry; thus, these values were not compared as they originate from different samples.
2-Please confirm by bioinformatics tools that the genome sequences of the studied strains reflect the temperature and salinity ranges obtained in the in vitro tests.The authors may use a bioinfo tool to verify congruence between their experiments and data retrieved directly from genome sequences, the so called in silico phenotypes.10.1186/1471-2164-13-S7-S3 , 10.1093/bioinformatics/btz059 Thank you for your comment.At current, only strain ATCC 17749 has a fully available genome for comparison using these methods.Furthermore, the intention of this research was to demonstrate the importance of temperature, salinity, and iron content together as determinants for V. alginolyticus growth.While the methods of in silico phenotyping are an interesting avenue to pursue -to the best of our knowledge, these methods have not been validated for salinity and iron tolerance in prokaryotes and thus are beyond the scope of this research.

3-why did you chose 4uM of Iron for the metabolomics?
The use of 4 µM iron for metabolomics experiments was selected to create an iron concentration consistent with levels observed on the upper end of normal for coastal systems.We have added additional context to the methods section to clarify this.Lines: 538-541."4 µM was designated as iron replete based on growth experiments for JW16-551 and prior work by Westrich et al. (2016).This level is also consistent with iron concentrations found in non-oligotrophic coastal waters (Zhu et al., 2018), offering an environmentally relevant concentration that would be sufficient for growth."Also it is unclear if how these vibrios would tolerate higher amounts of iron in the media.This is relevant in the face of possible iron inputs in the ocean and on coral reefs.Alginolyticus may appear in the water column in some occasions.https://peerj.com/articles/741/, 10.1126/science.1106028 .As stated in the results at lines 206-207, no growth limitation was observed for strains JW16-551 and JW16-580 at higher levels of iron suggesting that V. alginolyticus is able to tolerate iron concentrations up to 20µM iron.
Is 4 uM much in terms of possible marine systems vibrio may play a role?Ex bays, reefs.This concentrations of iron are common in such systems?Thank you for your comment.As stated above, an iron concentration of 4 µM is at the upper end of normal for most coastal systems.However, the type of system can substantially impact the typical iron concentration.Offshore oligotrophic systems such as coral reefs are typically highly iron deficient (nanomolar level) whereas estuarine systems are less limited (micromolar level) due to riverine input.We have added additional context to the discussion and methods section to clarify our iron selections and provide environmental and methodological context.Lines 340-343."This finding is particularly important for oligotrophic systems such as coral reefs which typically possess lower iron pools (Entsch et al., 1983;Kelly et al., 2011) compared to estuarine systems (Boyle & Edmond, 1977;Sunda, 2012) and may be more prone to the formation of iron-induced Vibrio spp.blooms." Lines 481-486.Noted above under reviewer one comment #1.4-the metabolomics part is preliminar?I had the impression the analyzes could provide the specific molecules and metabolites names induced by iron.Not only the general keeg groups.Also match metabolome data with genome data to elucidate gene function and in silico phenotyping.I do not think the so called unique metabolic pathways was clear enough and they require molecule ids.For instance, which terpenoids and polyketides were identified?Thank you for your comment.The specific molecules identified from the metabolomics portion of this research were identified and reported (both names and HMDB numbers) for each experimental condition in tables S1 and S2.Additionally, the use of KEGG metabolic pathways as the major result endpoint was selected based on previously published studies using untargeted metabolomics to assess metabolic function (https://doi.org/10.1128/spectrum.02067-22;https://doi.org/10.1128/Spectrum.00625-21;https://doi.org/10.1128/aac.02109-15).Furthermore, many metabolites identified in this study have the potential to function in multiple metabolic pathways thus, reporting based on pathway enrichment considers all possible pathways that may be represented by the specific chemical species identified.
Minor remarks -Impossible to see a blue line (ATCC) in fig3A.The blue line representing ATCC strain 17749 is shown in figure 3A under the 20µM iron treatment only.This is due to the fact that the iron limiting media utilized in this experiment severely limited the growth of this strain at all iron concentrations -with 20µM as the only concentration with sufficient growth to quantify doubling time.As such, the blue line only appears as a single linerange value under this treatment representing the standard error of growth for this strain.An explanation of this limitation can be found in the in the figure captions at lines 872-876.
-as a study control, the authors could have used Vibrio parahaemolyticus which is a syster species of V. alginolyticus.The comparison would be useful for many reasons.We appreciate that tests on additional Vibrio species would be informative, especially the closely related V. parahaemolyticus, but the intention of our study was to evaluate the abiotic tolerance parameters and importance of iron to V. alginolyticus, as this species is highly understudied compared to other non-Cholera vibrios.
-please improve conclusions and avoid results now.Addressed above in comment #5 from reviewer one.Re: Spectrum02680-23R1 (Vibrio alginolyticus growth kinetics and the metabolic effects of iron.)Dear Dr. William Anderson Norfolk, Dear Dr. Erin K. Lipp, your revised manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication.You will be notified when your proofs are ready to be viewed.
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