Water Research xxx (xxxx) xxx

Contents lists available at ScienceDirect

Water Research
journal homepage: www.elsevier.com/locate/watres

Sargasso Sea Vibrio bacteria: Underexplored potential pathovars in a

perturbed habitat
Tracy J. Mincer a, b, 1, *, Ryan P. Bos a, Erik R. Zettler c, Shiye Zhao d, Alejandro A. Asbun c,
William D. Orsi e, Vincent S. Guzzetta f, Linda A. Amaral-Zettler c, g, h, 1, *
a
Harbor Branch Oceanographic Institute, Florida Atlantic University, Fort Pierce, FL, USA
b
Department of Biology, Wilkes Honors College, Florida Atlantic University, Jupiter, FL, USA
c
Department of Marine Microbiology and Biogeochemistry, NIOZ Royal Netherlands Institute for Sea Research, Den Burg, Texel, the Netherlands
d
Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushimacho, Yokosuka 237-0061, Japan
e
Department of Earth and Environmental Sciences, Paleontology and Geobiology,Ludwig-Maximilians-Universität München, 80333 Munich, Germany
f
Emory University, Medical College, Atlanta, GA, USA
g
Department of Freshwater and Marine Ecology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, the Netherlands
h
Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, MA, USA

A R T I C L E I N F O A B S T R A C T

Keywords: We fully sequenced the genomes of 16 Vibrio cultivars isolated from eel larvae, plastic marine debris (PMD), the
Vibrio pelagic brown macroalga Sargassum, and seawater samples collected from the Caribbean and Sargasso Seas of the
Pathogen North Atlantic Ocean. Annotation and mapping of these 16 bacterial genome sequences to a PMD-derived Vibrio
Plastic marine debris
metagenome-assembled genome created for this study showcased vertebrate pathogen genes closely-related to
Sargassum
Marine
cholera and non-cholera pathovars. Phenotype testing of cultivars confirmed rapid biofilm formation, hemolytic,
Oligotrophic and lipophospholytic activities, consistent with pathogenic potential. Our study illustrates that open ocean
Open ocean vibrios represent a heretofore undescribed group of microbes, some representing potential new species, pos­
sessing an amalgam of pathogenic and low nutrient acquisition genes, reflecting their pelagic habitat and the
substrates and hosts they colonize.

1. Introduction humans via seafood consumption, as well as morbidity and mortality via
wound infection (Baker-Austin et al., 2021; Daniels and Shafaie, 2000).
Gammaproteobacteria of the genus Vibrio inhabit freshwater, coastal Plastic marine debris (PMD), first described in surface waters of the
marine, open ocean, and abyssal habitats and can comprise a significant Sargasso Sea (Carpenter and Smith, 1972), has become a world-wide
fraction of plant and animal microbiomes residing in these myriad concern as it increasingly permeates habitats geographically distant
aquatic environments, fulfilling crucial roles in biogeochemical cycling from anthropogenic influence (Amaral-Zettler et al., 2020) and is known
and ecosystem health (Hasan et al., 2015; Daniels and Shafaie, 2000; to persist decades longer than natural substrates in the marine envi­
Michotey et al., 2020). Some vibrios are also known pathogens, such as ronment (Amaral-Zettler et al., 2020; Zettler et al., 2013). Previous
Vibrio cholerae, the causative agent of cholera pandemics, notorious for studies employing small-subunit rRNA gene amplicon surveys found
its historic human toll (Baker-Austin et al., 2021). Of growing public that Vibrio spp. are common members of PMD microbial biofilms (also
health concern, non-cholera Vibrio spp. are now recognized as the referred to collectively as the plastisphere) (Kirstein et al., 2019; Ober­
dominant cause of human mortality from the marine environment beckmann et al., 2015; Pedrotti et al., 2022; Zettler et al., 2013).
(Baker-Austin et al., 2021). Vibrio spp. such as V. alginolyticus, V. harveyi, Additionally, non-quantitative, cultivation-independent rRNA gene
V. parahaemolyticus, V. campbellii and V. vulnificus, among others, are amplicon surveys have implicated possible Vibrio pathovars consistently
potent pathovars impacting both aquaculture-reared and wild bivalve inhabiting the Great Atlantic Sargassum Belt (Michotey et al., 2020;
and finfish stocks, causing life-threatening foodborne illnesses in Theirlynck et al., 2023), as well as wild fish stocks (Senderovich et al.,

* Corresponding authors.
E-mail addresses: [email protected] (T.J. Mincer), [email protected] (L.A. Amaral-Zettler).
1
Co-corresponding authors.

https://doi.org/10.1016/j.watres.2023.120033
Received 5 January 2023; Received in revised form 25 April 2023; Accepted 1 May 2023
Available online 3 May 2023
0043-1354/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Please cite this article as: Tracy J. Mincer et al., Water Research, https://doi.org/10.1016/j.watres.2023.120033
T.J. Mincer et al. Water Research xxx (xxxx) xxx

2010). Since 2011, rapidly expanding populations of these free-living 2.2. Strain isolation and phylogenetic prioritization and gene copy
brown macroalga Sargassum spp. in the Atlantic Ocean have been number analyses
observed, including frequent and unprecedented seaweed accumulation
events (Lapointe et al., 2021). This observation, in combination with During two oceanographic cruises in the Sargasso Sea in May of 2012
anomalous rising ocean temperatures, spotlights Vibrio spp. for future (C241) and 2013 (C247) a focus was placed on Vibrio bacteria cultivars
ocean concerns (Vezzulli et al., 2016). To the best of our knowledge, due to their presence on plastic debris and their underexplored nature in
little is known regarding the ecological relationship of vibrios with the open ocean. In all, 1188 total presumed Vibrio spp. cultures were
Sargassum spp. Additionally, genomic and metagenomic evidence has obtained and cryopreserved from our CaV culture isolations (500 cul­
been lacking as to whether vibrios colonizing PMD and Sargassum tivars from C241; 688 cultivars from C247, Fig. S1). We prioritized
indeed have pathogenic potential in humans (Wright et al., 2020). strains using growth morphology on CaV agar based upon sucrose
In this study, we describe the isolation and genomic characterization fermentation and pH indicator dye color ranging from pink to blue to
of Vibrio spp. from the Sargasso and Caribbean Seas of the North Atlantic colorless. Other prioritization parameters were: site obtained and sub­
Ocean from samples of eel leptocephali (larvae), PMD, Sargassum, and strates from which they were derived. We further prioritized our Vibrio
seawater. From a total of 1188 Vibrio spp. isolates collected from two cultivars employing heat shock protein 60 (hsp60) gene sequence
cruises in the North Atlantic Ocean, 16 cultivars were prioritized for full phylogenetic analysis (Preheim et al., 2011) of over 100 cultivars,
genome sequencing, annotation (Table 1) and comparative analyses yielding 4 major clades with closest affiliations with V. alginolyticus, V.
with a metagenome-assembled genome (MAG) specifically generated for campbellii, V. fortis, and V. parahaemolyticus by average nucleotide
this study, from a Sargasso Sea PMD sample where Vibrio spp. were identity (ANI) analysis in Fig. 1 in the main text and hsp60 phylogeny in
previously reported to be the dominant bacterial phylotype using small- Fig. S2.
subunit ribosomal gene sequencing (Zettler et al., 2013).
2.3. Bacterial adhesion assay
2. Methods
Vibrio cultures revived from cryopreserved stocks in YT broth (10 g
2.1. Collection of samples and isolation of Vibrio cultivars Bacto yeast extract, 5 g Bacto tryptone (Becton Dickinson, NJ, USA) in 1
L artificial seawater) (Mincer and Aicher, 2016) by overnight incubation
Samples of various substrates were collected using either a rectan­ with 100 RPM shaking at 23 ◦ C in culture tubes filled with 10 ml of YT
gular neuston net (1 m x 0.5 m, 335 µm mesh) towed at the surface, or a media were inoculated at a 1:1000 dilution and grown for 16–18 h. A
1-meter circular net (1-meter diameter, 335 µm mesh) at 10-m depth microbial attachment assay was adapted (O’Toole and Kolter, 1998), for
from the SSV Corwith Cramer as part of SEA Semester research cruises C- screening biofilm formation on plastic. Subsequent methods details can
241 in May of 2012 and C-247 in May of 2013 (Fig S1, Table 1). Sub­ be found in the Supplemental Methods section. Results are reported in
strates including Sargassum, leptocephalus eel larvae, and PMD were Table S1 and Fig. S3.
rinsed twice in 0.2 µm filtered seawater then streaked onto CHROMa­
gar™ Vibrio (CaV) agar plates (DRG, NJ, USA) and subsequently incu­
2.4. Microbial phenotype screening
bated at ambient temperature in the dark and examined every twelve
hours for colony growth. Individual colonies were isolated by trans­
Hemolysis assays were conducted using blood agar plates prepared
ferring to seawater tryptone plates (1 g Bacto tryptone, 20 gs BactoAgar
from defibrinated sheep’s blood (Fisher Scientific). Results were scored
(Becton Dickinson, NJ, USA) per 1 L natural seawater) to assure that
as alpha hemolysis (clear green or brown tinting to the blood, indicating
single colonies were obtained. Cultivars were cryopreserved in a 1:9
the presence of methemoglobin), beta hemolysis (an entire clearing of
solution of sterile glycerol:seawater tryptone broth at − 80 ◦ C.
the blood cells in the medium indicating full blood cell lysis), or gamma
hemolysis (no noticeable change in the blood agar medium) as

Table 1
Data detailing the substrate sources, location and clade phylogeny of the isolates and MAG.
Strain Source Substrate Lat Lon Cruise # (year) Geographic Area Clade
D417 Leptocephali 24.9519, − 64.8520 C247–012–2-MN (2013) Sargasso Sea V. alginolyticus

D449 Leptocephali 20.6510, − 64.6680 C247–006-NT (2013) Sargasso Sea V. fortis

B172 Polyethylene 18.6720, − 64.5850 C241–002-NT (2012) Tropical Atlantic V. parahaemolyticus

B181 Polyethylene 18.6720, − 64.5850 C241–002-NT (2012) Tropical Atlantic V. parahaemolyticus

B513 Polyethylene 27.3120, − 63.5600 C241–017-NT (2012) Sargasso Sea V. alginolyticus

B516 Polyethylene 28.0870, − 63.4820 C241–019-NT (2012) Sargasso Sea V. alginolyticus

D404 Polyethylene 20.6510, − 64.6680 C247–006-NT (2013) Sargasso Sea V. fortis

D409 Polyethylene 20.0688, − 64.6000 C247–008-NT (2013) Sargasso Sea V. alginolyticus

D420 Polyethylene 23.6686, − 64.5186 C247–010-NT (2013) Sargasso Sea V. fortis

D421 Polyethylene 23.6686, − 64.5186 C247–010-NT (2013) Sargasso Sea V. alginolyticus

D431 Polyethylene 26.6344, − 64.6833 C247–014-MN (2013) Sargasso Sea V. alginolyticus

B511 Sargassum 24.8120, − 64.5700 C241–013-NT (2012) Sargasso Sea V. alginolyticus

D401 Sargassum 20.6510, − 64.6680 C247–006-NT (2013) Sargasso Sea V. parahaemolyticus

D406 Sargassum 20.0688, − 64.6000 C247–008-NT (2013) Sargasso Sea V. alginolyticus

D415 Sargassum 24.9519, − 64.8520 C247–012–2-MN (2013) Sargasso Sea V. alginolyticus

D173 Seawater 18.2174, − 64.7180 C247-SS-001 (2013) Carribean Sea V. parahaemolyticus

MAG Polypropylene 31.628, − 41.415 C-230–01 (2010) Sargasso Sea V. parahaemolyticus

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T.J. Mincer et al. Water Research xxx (xxxx) xxx

Fig. 1. Vibrio pangenome analysis of isolates analyzed in this study. The circular phylogram was created using Anvi’o 7.0 analyses of 16 environmental Vibrio
isolates. The colors indicate the respective marine substrate from which each cultivar was isolated: eel leptocephalus (orange), plastic marine debris (red), Sargassum
(green), seawater (blue), and type strains (black). All environmental cultivars were tested for biofilm formation, bioluminescence, hemolysis (see Section 2.4 for
description), and phospholipase activity (see Supplemental Information). Results are color-coded in the legend. Average nucleotide identity (ANI) ranges between
100% (dark red) and 70% (pink) are also indicated, with the cladogram at the top right indicating ANI clustering. SCGs indicated by the outer circle denotes single
copy genes. Strain names indicated at top continue through the circular dendrogram to the ANI and clade diagram.

previously described (Brown, 1919). 2.6. Metagenomic sequencing and recruitment analyses
Extracellular phospholipase activity was screened on LB agar con­
taining additional salt (LB 25 g, Milli-Q water 1 L, BactoAgar 15 g, NaCl DNA extraction, library preparation, quality control and sequencing
7 g, CaCl2, 0.11 g) supplemented with 1% (v/v) egg yolk emulsion of our metagenomic DNA library followed the same protocol as for the
(Fisher Scientific, CA, USA) using a modified method (Kurioka and Liu, genomic sequencing but employed the DNA extraction protocol of Zet­
1967). Escherichia coli MG1655 incubated in two separate conditions, tler et al., 2013. An entire lane of sequencing on an Ilumina HiSeq 1000
23 ◦ C or 37 ◦ C, was used as a negative control. Subsequent methods was dedicated to this sample, yielding approximately 243,882,048 raw
details can be found in the Supplemental Methods section. reads (121,941,024 paired-end reads).

2.5. Genomic sequencing and analyses

2.7. Pangenomic analysis

Vibrio spp. genomic DNAs were extracted using a modification of a

The Vibrio spp. pangenome was created using the freely available
previously published method (Zettler et al., 2013). See Supplemental
analysis and visualization platform Anvi’o (version 7 dev) (Eren et al.,
Methods section for details.
2015) with modification of the pangenomic workflow (Delmont and
We assembled Vibrio spp. reads into contigs using CLC genomics
Eren, 2018). Open reading frames were identified using Prodigal
workbench v.7 (http://www.clcbio.com). The average coverage for
(V2.6.3) (Hyatt et al., 2010), and contig databases for all genomes were
each genome was between 40X and 117X. Genomes were not fully
produced using the anvi-gen-contigs-database command. Hierarchical
closed, and thus represent draft genomes. However, completeness of
clustering of prodigal-designated genes was prepared using BLASTp, and
draft genome sequencing was assessed by single copy gene (SCG) rep­
putative homologous gene clusters were subsequently grouped using the
resentation, and all genomes were found to contain the full suite of
Markov Cluster Algorithm (Hyatt et al., 2010) (minbit 0.5; inflation 2)
universally conserved SCGs using BUSCO indicating that genomic
and aligned with MUSCLE (V3.8.1551) using default parameters
coverage was near complete for each isolate.
(Edgar, 2004). The average nucleotide identity of genomes was
SCGs were identified using Benchmarking sets of Universal Single-
computed using the program pyANI (V0.2.10; ANIb) with a range of
Copy Orthologs (BUSCO) (Simão et al., 2015) with the vibrionale­
70–100% similarity, and these data were used to construct a phyloge­
s_odb10 database as a reference. The percentage of SCGs from each
netic tree (distance: Euclidean; linkage: Ward) displayed in Fig. 1.
isolate genome that were BUSCO notated as complete (C), duplicated
Contig databases were annotated using COG20 categories, functions,
(D), fragmented (F), and missing (M) are displayed in Fig. S4.
and pathways (Galperin et al., 2021), as well as KOfams (Aramaki et al.,
2020) and Pfams (Mistry et al., 2021). Additional query files containing
the major and minor Vibrio virulence factors were obtained from the

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T.J. Mincer et al. Water Research xxx (xxxx) xxx

Pathosystems Resource Integration Center (PATRIC) virulence factor 2.10. Assessing MAG completeness
database (Gillespie et al., 2011) and queried using Anvi’o, against all
genomes using BLASTn (perc_identity 70; wordsize 18; evalue 1e-10) Mapped metagenomic reads to the different isolates were first der­
(Eren et al., 2015). Absence, or presence, as well as copy number of eplicated with a 100% identity using Vsearch v2.8.0 (Rognes et al.,
genes was verified by analyzing the aligned genome region (length) 2016) and mapped against the MEGAHIT assembly. In order to increase
relative to the full query gene length (qlen), with a combination of the confidence of the mapping, resultant .sam files were filtered with a
BLASTn, TBLASTx (evalue 1e-10), exported amino acid sequences of minimum required quality of 30. Raw reads belonging to the mapped
target genes stored in Anvi’o, and COG20 functions, KOfams, and Pfams contigs and not present in our already selected contigs were also
(all Vibrio spp. isolate annotation data included in Data S1 Table 1). included in our new set of raw reads.
Anvi-get-enriched-functions-per-pan-group was used to calculate Due to the complex nature of the metagenomic sample, the assembly
functional enrichment scores for annotation sources for each substrate of metagenome assembled genomes MAGs was challenging, requiring a
category (Shaiber et al., 2020); eel leptocephali; plastic; Sargassum; custom workflow (Fig. S5 and Data S1 Table 5). However, we were able
seawater), and these scores were programmatically matched with to validate the completeness of a MAG belonging to the Vibrio clade in
unique identifiers of exported gene detection and coverage values for the subset of raw reads according to the following approach. First, we
mapped metagenome reads (Supplemental Tables). Functions with a ran BUSCO v4.0.6 (Simão et al., 2015) to the assembled genome of Vibrio
q-value less than 0.05 were considered enriched. Strains D404 and D449 B172 (Vibrio sp. isolated from a piece of plastic which was determined
displayed an ANIb within the cutoff to warrant a new species, as dis­ closest to the MAG of interest) finding 1444 complete single-copy
cussed in the Main Text, 86% in Anvi’o and 89% with GTDB-Tk, against BUSCO genes out from 1445 from the vibrionales_odb10 BUSCO line­
the nearest fully-sequenced type strain V. fortis Dalian14 (Chaumeil age. Next, using bwa mem, the selection of raw reads was mapped
et al., 2020; Ke et al., 2017). against the same reference genome, and subsequently using BEDTools
v2.26.0 (Quinlan and Hall, 2010) with the coordinates of the identified
SCGs by BUSCO and the mapping results, the coverage for each
2.8. Mutation analysis
single-copy genes was computed, finding 1400 single-copy genes.
Further results can be found in Data S1 Tables 5 and 6.
Nucleotide sequences from gene clusters of interest were exported
from Anvi’o and subsequently aligned with each clade’s type strain
3. Results
using the local pair parameterization in MAFFT (v7.475) (Falda et al.,
2012). The aligned sequences were imported into MEGA X for mutation
3.1. Potential new species of Vibrio from the North Atlantic Ocean
analysis (Stecher et al., 2020) and the pairwise comparisons of the
number of non-synonymous (dN) and synonymous (dS) mutations were
Environmental Vibrio spp. cultivars segregated into 3 major clades by
calculated using the Nei-Gojobori method under the assumption that
hsp60 phylogeny (Fig. S2) and Average Nucleotide Identity using BLAST
homologous recombination occurred at uniform rates (Nei and Gojo­
(ANIb) with type strains V. alginolyticus, V. campbellii (known as a
bori, 1986). Full-length gene alignments with a ratio of dN/dS <1 were
member of the “Vibrio Harveyi core” clade (Ke et al., 2017)), and V. fortis
categorized as having experienced purifying selection, whereas genes
(Fig. 1). Strains D404, and D449 displayed an ANIb of 86% in Anvi’o and
with dN/dS >1 were classified as having underwent positive selection
89% with GTDB-Tk, against the nearest fully-sequenced type strain
(Data S1 Table 2). Amino acid sequences were also exported for studying
V. fortis Dalian14, suggesting they represent a new species within the
the syntenic region of the MSHA operon across isolates and type strains
Vibrio genus.
using the same parameters and tools as described above (Data S1
Table 3).
3.2. Pathogenic physiology and genes present in isolates and MAG

2.9. MEGAHIT approach for identifying Vibrio reads from the All 16 Vibrio spp. isolates were tested for substrate adhesion to the
metagenome polymers PVC, PP, PS and glass, as well as the ability to oxidize or lyse
red blood cells (alpha or beta hemolysis, respectively) and hydrolyse
We used trimmomatic v. 0.36 (Bolger et al., 2014) to quality trim and phospholipids. Interestingly, cell density appeared to introduce a factor
remove adapters from 121,941,024 raw reads, leaving 99.89% of the of variability in substrate adhesion in our assays for strain D449, sug­
reads remaining. We used Kraken v. 1.0 (Wood and Salzberg, 2014) to gesting a quorum sensing mechanism could be influencing this pheno­
assess the taxonomic composition of the trimmed reads and then type (Fig. S3). Hemolysis and phospholipase assays were positive for 14
assembled the reads using MEGAHIT v.1.1.3 (Li et al., 2015) with a and seven of the 16 isolates, respectively (Fig. 1). The MAG mapped to
k-min set to 21, k-max to 141 and a k-setp of 12. We used Quast v4.6.3 to about 48% of the B172 genome, the isolate that was most closely related
evaluate the resulting assembly quality. The assembly coverage was by ANI (Data S1 Table 6).
performed by mapping-back the reads to the assembly using bwa-mem Components of the accessory colonization factor (ACF) gene cluster,
v. 0.7.17-r1188. The resultant .sam file was sorted and converted to a under control of the ToxR regulon in V. cholerae (Peterson and Meka­
.bam file using SAMtools v. 1.8 (Li et al., 2015). We obtained the depth lanos, 1988), were partially present in our isolates and the MAG, Fig. 2.
of coverage using the jgi_summarize_bam_contig_depths script available The acfA gene, present in 12 of 16 isolates and the MAG, is required for
with Metabat2 (Kang et al., 2015). Metagenome-assembled genomes full virulence and intestinal colonization in V. cholerae isolates in com­
(MAGs) where computed using BinSanity v.0.2.6.4 (Graham et al., bination with the toxin co-regulated pilus gene cluster and the acfB, C, D
2017) with the BinSanity-wf command and a minimum contig length of accessory genes (Everiss et al., 1994), present to varying degrees in our
500 bp resulting in four Vibrio bins of low completeness according to isolates and more consistently in the MAG, which was missing only acfB
CheckM v. 1.0.11 (Parks et al., 2015) results (9.83, 6.54, 4.39, and 0). (Fig. 2). The ilpA gene, found in all isolates and the MAG, encodes a li­
Given that previous analyses showed a significant number of Vibrio poprotein known in V. vulnificus to enable full adhesion to human in­
reads, unbinned contigs were further identified in the assembly and testinal epithelial cells (K. J. Lee et al., 2010). Our MAG and all isolates
coding sequences (CDS) detected with Prokka v1.14-dev (Seemann, except D404, D420, D449 possessed the multivalent adhesin molecule 7
2014). Protein sequences were then annotated using diamond (MAM7), a trans-membrane protein that is essential for lethality in
v0.9.22.123 (Buchfink et al., 2014) against the NCBI NR database mouse assays conducted with V. parahaemolyticus (Lim et al., 2014;
downloaded on April 2019. Subsequent methods details can be found in Stones and Krachler, 2015). Genes encoding the mannose-sensitive
the Supplemental Methods section. haemagglutinin Type IV pili (MSHA) were well-represented in all

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T.J. Mincer et al. Water Research xxx (xxxx) xxx

Fig. 2. Hallmark pathogenic genes present in our isolates and the PMD metagenome assembled genome (MAG) within the functional categories of adherence,
chemotaxis and motility, toxins, regulatory elements, nutrition resistance factor (NTR) related genes, also known as persister genes, and the RS1 element of the
cholera toxin phage. Heatmap colors indicate gene copy numbers from a range of 0 to 15.

isolates and the MAG, Fig. 2. In addition to MSHA type IV pili, Type II, B181 and D173, sharing near-identical amino acid similarity to
III, and VI secretion system pathways were nearly complete for the MAG V. campbellii (Data S1 Table 1).
and all isolates that possessed genetic components associated with those
pathways (Fig. 2 and Data S1 Table 7). All strains were capable of 4. Discussion
reproducible biofilm formation in our assays, on either multiple plastic
polymer or glass substrates except for strains B516 and D449 (Table S2), 4.1. Vibrio ecology and pathology
emphasizing the importance of an attached lifestyle for these isolates.
Nutritional resistance (NTR) genes such as the full hutWXZ operon The ecology and pathology of vibrios are well-studied in coastal re­
with multiple copies of the hutZ gene were present in all cultivars, but gions with many cultivars physiologically characterized and fully-
absent in the MAG (Fig. 2). HutZ, shown to be a crucial protein in sequenced (1738 were represented in the IMG database at the time of
V. cholerae for obtaining heme-bound iron once red blood cells are lysed analysis, adding confidence to our annotations) (Markowitz et al.,
(Wyckoff et al., 2004) and an important pathogenic determinant in the 2012). With 147 species and 4 subspecies described, 24 Vibrio spp. are
fish pathogen Edwardsiella piscicida (Shi et al., 2019) among other mi­ recognized as plant or animal pathogens (Grimes, 2020). Open ocean
crobial fish pathovars (Lemos and Balado, 2020), was present in mul­ habitats present an extreme scenario, oligotrophic conditions force ‘feast
tiple copies in all Vibrio cultivars, but absent in the MAG. Bile salt or famine’ situations where key nutrients such as phosphorous are
hydrolase (BSH), an important gut colonization factor in vertebrate limiting, particularly in the Sargasso Sea (K. J. Lee et al., 2010). In the
pathovars, was present in isolates B172, D173, B181, D401, and MAG. case of vibrios in these habitats it appears that pathogenic attachment,
Other NTR genes polyphosphate kinase (ppk) and the SOS response potential toxin production and hydrolytic enzymes targeted towards
regulated cell division inhibitor sulA were present in nearly all isolates animal and plant tissues, likely provide an advantage.
and the MAG (Fig. 2). Repeats in toxin (RTX) which were hallmarks in Attachment is a key factor in pathogenesis and secretion-based
the outbreaks of El Tor cholera epidemics, were present in all isolates attachment factors are well-represented in the Vibrio spp. isolates and
and the MAG (Lin et al., 1999) as were toxR and toxS genes (crucial MAG (Fig. 2 and Data S1 Table 7). Variability is common in secretion
members of the ToxR regulon, involved in expressing cholera entero­ systems and function does not necessarily rely on a full complement of
toxin (CT) and TCP in epidemic V. cholerae strains (Bina et al., 2003) all known genes in a secretion system pathway (Izoré et al., 2011). The
(Waldor and Mekalanos, 1996). Only tcpE was found in D404 and D449 MSHA pilus is one of several biofilm and adhesion mechanisms in
(isolated from PMD and Leptocephalus, respectively) whereas the other V. cholerae, which in concert with the toxin coregulated pilus TCP, is
11 TCP pilus assembly genes were absent (Data S1 Table 1). Multiple full responsible in the formation of multilayered, complex biofilms on chitin
copies of various hemolysin genes were detected in all of our isolates and (Yildiz and Visick, 2009). The MSHA system is also important in adhe­
many presented this phenotype in our assay (Fig. 1, Fig. 2 and Supple­ sion to diatom produced chitin, aiding persistence in the water column
mental Tables). Interestingly, isolates D401 and D406 were found to (Frischkorn et al., 2013) and has also been implicated in early coloni­
possess the rs1A, B, and R genes (Fig. 2), known to be part of a hori­ zation and adherence to plastic polymers (Bos et al., 2023). Multiple
zontally transferred lysogenic phage, RS1Φ using the same receptor as copies of the mshA genes are known to enhance adhesion, biofilm for­
the CTXΦ phage (Lin et al., 1999). However, no presence of cholera mation, DNA uptake, twitching motility, and virulence (Ellison et al.,
toxin was observed in any of our isolates or MAG. Additionally, the gene 2018). Of note, isolate B172 has seven copies of mshA, which is high
encoding the Zonula occludens toxin, zot, required for the weakening of compared to a calculated average of 2.1 (n = 1738) for other vibrios
tight junctions in the vertebrate intestinal epithelial lining (Mauritzen from our analysis (Fig. 2; Table S3; Data S1 Tables 1 and 2). An analysis
et al., 2020) was present in a majority of our isolates and the MAG of synonymous versus non-synonymous nucleotide substitutions showed
(Fig. 1, Fig. 2; Data S1 Table 7). Analysis of dN/dS also showed that the that specific mshA alleles were under positive selection with dN/dS ra­
zot genes appear to be under strong positive selection ranging from 3.1 – tios as high as 2.8, suggestive of strong positive selection (Data S1
1.5 (Data S1 Table 3). Many of the isolates contained phosphate/­ Table 3). Biofilm formation in many Vibrio spp. is commonly under
phosphonate ABC transport systems and strikingly isolates D404 and flagellar influence (Yildiz and Visick, 2009), with motility being an
D449 possessed a near-complete phosphonate utilization pathway as important feature of the copiotrophic lifestyle of Vibrio spp. (Hunt et al.,
characterized in E. coli (phnCDEFGHIJKLMNOP) missing only phnF 2008) and pathovars (Daniels and Shafaie, 2000). Complementing
(Data S1 Table1). However, a recent study showed through cloning motility, chemotaxis and lateral and polar (swarming) flagellar gene sets
studies in E. coli that phnGHIJKLM is only necessary for full phosphonate were well-represented in all isolates and the MAG (Fig. 2).
utilization (Jochimsen et al., 2011), suggesting isolates D404 and D449 Additionally, synteny of the MSHA operon varied as two funda­
possess a functioning carbon-phosphorous lyase pathway. Another mental patterns (independent of strain phylogeny), and high diversity of
interesting adaptation was the presence of proteorhodopsin in isolates amino acid compositions of mshA at the amino acid level (Figs. S6 and

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T.J. Mincer et al. Water Research xxx (xxxx) xxx

S7, respectively). This mshA gene diversity could be an adaptation to the 4.3. Open ocean vibrios as opportunistic ‘hunters’
ecological pressures of vibrios in the open ocean and the many sub­
strates that these isolates encounter, such as animal and plant tissues, in In the vast open ocean, Sargassum represents an ancient and unique
addition to plastic debris. Altogether, the adhesion and toxin genes ecosystem for marine fauna and many of their larval and juvenile forms,
present in the isolates and MAG display a genetic tool kit typical of including uniquely adapted species such as the Sargassum pipefish and
V. cholerae and V. parahaemolyticus host colonization and biofilm for­ seahorse (Coston-Clements et al., 1991). Predators have specifically
mation genes, with multiple degeneracy and copy number, a theme adapted to these macroalgal oases of life in the nutrient depleted sea as
similar to other Vibrio spp. pathovars (Yildiz and Visick, 2009). Posi­ well, notably, the endemic Sargassum fish (Histrio histrio) an aggressive
tively selected pathogenic genes such as MSHA and other Type IV pilus piscivore exquisitely adapted to the Sargassum canopy via mimicry
mechanisms may also provide an advantage in competing on surfaces (Rogers et al., 2010). Similar adaptations taking advantage of the
such as attachment to plastic and adhesion in the Sargassum phyco­ abundant sea life in these seaweed patches appear to have taken place at
sphere. More research is needed to understand the selective pressures on the microbial level. Members of the Vibrio Harveyi clade: V. campbellii,
the MSHA operon and the role of mshA specifically in surface coloni­ V. owensii and V. rotiferianus which includes relatives of our isolates, are
zation in the wild. All of our isolates also possessed alginate lyase, some recognized animal pathogens (Ke et al., 2017), while V. fortis has been
possessing multiple alleles (Data S1 Table 1), which could provide linked to crustacean and seahorse disease (Wang et al., 2016). With the
enhanced carbon utilization in the Sargassum phycosphere. Isolates increase of Sargassum and PMD in the Sargasso Sea and their growing
B181 and D173 also possessed proteorhodopsin, nearly identical to the co-occurrence in other parts of the open ocean such as the Great Atlantic
V. campbellii allele, previously determined to function as a proton pump Sargassum Belt in the North Equatorial Recirculation Region, open ocean
and provide protection against starvation under oligotrophic conditions Vibrio spp. colonizing these substrates are expanding as well. Sargassum
(Wang et al., 2012). Scavenging limited phosphorous through the beaching events are now so commonplace that efforts to upcycle this
carbon-phosphorous lyase pathway is an important adaptation in seaweed biomass in the form of livestock feed and fertilizer are under­
oligotrophic environments (Lee et al., 2017) and two of the Vibrio spp. way (Milledge and Harvey, 2016).
isolates (D404 and D449) appear to have a complete pathway necessary
for acquisition of phosphorous from phosphonate compounds (Joc­ 5. Conclusions
himsen et al., 2011). Interestingly, these are also the two strains in our
study that were the most divergent by ANI, possibly representing a new The MAG represents the first Vibrio spp. assembled from plastic
species within the Vibrio genus. debris, with its nearest relative to isolate B172 and possesses many
pathogenic genes. Overall, given the complement of toxin, adherence,
4.2. Potential for vertebrate host infection and phenotype expression hydrolysis, and nutritional resistance gene sets in the open ocean Vibrio
spp. isolates and MAG and additional positive functional screens for
Hallmark pathogenic gene sets suggest that some of our Vibrio cul­ hemolysis and phospholipase phenotypes, it appears that some Vibrio
tivars have the potential to infect vertebrate hosts, such as acfA, ilpA, spp. in this environment have an ‘omnivorous’ lifestyle targeting both
bile salt hydrolase, zot, MSHA, ToxR and ToxS, with all these gene sets plant and animal hosts in combination with an ability to persist in
mirrored in the Vibrio MAG from PMD (Fig. 2, Data S1 Tables 5 and 6). It oligotrophic conditions. With increased human-Sargassum-PMD in­
is interesting that this combination of pathogenic gene sets, particularly teractions, associated microbial flora of these substrates could harbor
acfA, ilpA and MAM7 all appear to be separate key pathogenic de­ potent opportunistic pathogens. In particular, caution should also be
terminants in V. cholerae, V. vulnificus and V. parahaemolyticus, respec­ exercised regarding the harvest and processing of Sargassum biomass
tively. The fact that the open ocean Vibrio spp. isolates and MAG possess until the risks are explored more thoroughly.
these genes illustrates their unique mosaic nature and selective envi­
ronmental pressures, compared to other well-characterized Vibrio spp. Funding
pathovars. The presence of the zot gene in these Vibrio spp. isolates and
our MAG, and evidence for its strong positive selection in the cultivars, is National Science Foundation grant OCE-1,155,671 (T.J.M.), Na­
intriguing and could have implications for the surrounding oligotrophic tional Science Foundation Emerging Frontiers in Research and Innova­
waters if it is causing vertebrate hosts to have a leaky gut and release tion Grand subaward number 432343 (T.J.M.), FAU World Class Faculty
nutrients to the environment which, in turn, could stimulate the growth and Scholar Program (T.J.M.), National Science Foundation grant OCE-
of Sargassum and other surrounding organisms. The presence of the 1,155,571 (L.A.A-Z.), National Science Foundation grant OCE-
RS1Φ is intriguing as this is typical of only V. cholerae strains (Lin et al., 1,155,379 (E.R.Z.), NSF TUES grant to E.R.Z. and L.A.A.-Z. (DUE-
1999). Such similarities among our isolates and V. cholerae suggest some 1,043,468).
shared ancestry or gene transfer events between these clades. Addi­
tionally, the isolates all shared the presence of microbial collagenase, Data and materials availability
similar to other known Vibrio spp. pathovars (Data S1 Table 1). Collagen
is a crucial component in animal tissue matrices, amounting to 25% of All BioProject, MAG, BioSample and SRA numbers and GenBank
mammalian proteins, for example (Grimes, 2020). accession numbers can be found in the Supplemental section Data S1
Interestingly, cell density appeared to introduce a factor of vari­ Tables.
ability in substrate adhesion in our assays, particularly for strain D449,
suggesting a quorum sensing mechanism could be influencing this CRediT authorship contribution statement
phenotype (Fig. S3). This mechanism has been found to be important in
biofilm formation and virulence gene expression in V. cholerae where Tracy J. Mincer: Conceptualization, Investigation, Methodology,
elevated cell density was shown to govern a committed point of no re­ Visualization, Funding acquisition, Project administration, Supervision,
turn, very similar to the biofilm formation phenomenon illustrated in Writing – original draft, Writing – review & editing. Ryan P. Bos:
Fig. S3 (Hammer and Bassler, 2004) Hemolysis and phospholipase as­ Investigation, Methodology, Visualization, Writing – review & editing.
says were positive for 14 and seven of the 16 isolates, respectively (Fig. 1 Erik R. Zettler: Funding acquisition, Project administration, Writing –
and Table S2). Hemolysis and phospholipase phenotypes, known hall­ review & editing. Shiye Zhao: Methodology. Alejandro A. Asbun:
marks of pathogenic vibrios, were mapped onto the clades of Methodology, Visualization, Writing – review & editing. William D.
V. alginolyticus, V. campbellii, V. fortis and V. parahaemolyticus, according Orsi: Visualization, Writing – review & editing. Vincent S. Guzzetta:
to their phylogenetic placement (Fig. 1). Methodology, Writing – review & editing. Linda A. Amaral-Zettler:

6
T.J. Mincer et al. Water Research xxx (xxxx) xxx

Conceptualization, Investigation, Methodology, Visualization, Funding Eren, A.M., Esen, O.C., Quince, C., Vineis, J.H., Morrison, H.G., Sogin, M.L., Delmont, T.
O., 2015. Anvi’o: an advanced analysis and visualization platformfor ’omics data.
acquisition, Project administration, Supervision, Writing – review &
PeerJ. https://doi.org/10.7717/peerj.1319.
editing. Everiss, K.D., Hughes, K.J., Kovach, M.E., Peterson, K.M., 1994. The Vibrio cholerae acfB
colonization determinant encodes an inner membrane protein that is related to a
family of signal-transducing proteins. Infect. Immun. https://doi.org/10.1128/
iai.62.8.3289-3298.1994.
Declaration of Competing Interest Falda, M., Toppo, S., Pescarolo, A., Lavezzo, E., Di Camillo, B., Facchinetti, A., Cilia, E.,
Velasco, R., Fontana, P., 2012. Argot2: a large scale function prediction tool relying
All authors declare that they have no competing interests. on semantic similarity of weighted Gene Ontology terms. BMC Bioinf. https://doi.
org/10.1186/1471-2105-13-S4-S14.
Frischkorn, K.R., Stojanovski, A., Paranjpye, R., 2013. Vibrio parahaemolyticus type IV pili
Data availability mediate interactions with diatom-derived chitin and point to an unexplored
mechanism of environmental persistence. Environ. Microbiol. 15 (5) https://doi.
org/10.1111/1462-2920.12093.
Data will be made available on request.
Galperin, M.Y., Wolf, Y.I., Makarova, K.S., Alvarez, R.V., Landsman, D., Koonin, E.V.,
2021. COG database update: focus on microbial diversity, model organisms, and
widespread pathogens. Nucleic. Acids. Res. 49 (D1) https://doi.org/10.1093/nar/
gkaa1018.
Acknowledgments Gillespie, Joseph J., Wattam, Alice R., Cammer, Stephen A., Gabbard, Joseph L.,
Shukla, Maulik P., Dalay, Oral, Driscoll, Timothy, Hix, Deborah, Mane, Shrinivasrao
We acknowledge R/V Corwith Cramer C-230 cruise Chief Scientist P., Mao, Chunhong, Nordberg, Eric K., Scott, Mark, Schulman, Julie R., Snyder, Eric
E., Sullivan, Daniel E., Wang, Chunxia, Warren, Andrew, Williams, Kelly P.,
Giora Proskurowski and participant Emelia Deforce for assistance col­ Xue, Tian, Yoo, Hyun Seung, Zhang, Chengdong, Zhang, Yan, Will, Rebecca,
lecting samples under the NFWF-NOAA Marine Debris Program Award Kenyon, Ronald W., Sobral, Bruno W., 2011. PATRIC: the comprehensive bacterial
No. 2009-0062-002. We further acknowledge SEA students and crew for bioinformatics resource with a focus on human pathogenic species. Infect. Immun.
79 (11), 4286–4298. https://doi.org/10.1128/IAI.00207-11.
their assistance collecting and processing samples aboard R/V Corwith Graham, E.D., Heidelberg, J.F., Tully, B.J., 2017. Binsanity: unsupervised clustering of
Cramer. We thank Greg Boyd, Beth Slikas, Donald W. Melvin, Ben Ong, environmental microbial assemblies using coverage and affinity propagation. PeerJ
Lia N. Corbett, and Leslie Murphy for technical assistance. Graphic ab­ 2017 (3). https://doi.org/10.7717/peerj.3035.
Grimes, D.J., 2020. The Vibrios: scavengers, Symbionts, and Pathogens from the Sea.
stract was created using BioRender, agreement number VD25CAOFX1.
Microb. Ecol. https://doi.org/10.1007/s00248-020-01524-7.
Hammer, B.K., Bassler, B.L., 2004. Quorum sensing controls biofilm formation in Vibrio
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Hasan, N.A., Grim, C.J., Lipp, E.K., Rivera, I.N.G., Chun, J., Haley, B.J., Taviani, E.,
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the online version, at doi:10.1016/j.watres.2023.120033. C., Han, C.S., Eisen, J.A., Huq, A., Colwell, R.R., 2015. Deep-sea hydrothermal vent
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