Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e50983, enero-diciembre 2023 (Publicado Ago. 04, 2023)

Assessment of Vibrio populations in a transect of Rhizophora mangle

in Punta Galeta, Panamá: culture-dependent analyses reveal

biotechnological applications

Joel Sánchez-Gallego1,2; https://orcid.org/0009-0007-7473-4313

Librada Atencio3; https://orcid.org/0000-0003-3668-5577

Jacinto Pérez1; https://orcid.org/0000-0001-5048-614X

Omar Dupuy1; https://orcid.org/0000-0002-3880- 339X

Edgardo Díaz-Ferguson2; https://orcid.org/0000-0002-2314-5021

Filipa Godoy-Vitorino4*; https://orcid.org/0000-0003-1880-0498

1. Facultad de Ciencias de la Salud-William Gorgas, Universidad Latina de Panamá, Panamá; joeljsanchezg@gmail.com,

omarielsag@yahoo.com, jacintoperez@med.ulatina.edu.pa

2. Estación Científica Coiba (COIBA-AIP), Clayton, Ciudad del Saber, Panamá; ediaz@coiba.org.pa

3. Centro de Biodiversidad y Descubrimiento de Drogas, Instituto de Investigaciones Científicas y Servicios de Alta

Tecnología (INDICASAT AIP), Panamá; latencio@indicasat.org.pa

4. Department of Microbiology and Medical Zoology, Microbiome Laboratory, University of Puerto Rico, School of

Medicine, San Juan, Puerto Rico, USA; filipa.godoy@upr.edu (*Correspondence)

Received 17-II-2023. Corrected 22-III-2023. Accepted 25-VII-2023.

ABSTRACT

Introduction: Rhizophora mangle is considered an ecological niche for microorganisms with potentially novel

and complex degrading enzymes.

Objective: To characterize Vibrio populations using culture-dependent methods, using samples collected from

sediments and water along a red mangrove transect composed of three sites.

Methods: Strains were characterized according to their distribution, capacity to degrade of organic matter and

other environmental parameters. Additionally the sequence diversity was assessed using 16S rRNA sequencing.

Results: Bacterial densities were strongly associated with temperature and salinity. A total of 87 good-quality

sequences representing the isolates from the three sites, were binned into eight OTUs (Operational taxonomic

units). Taxonomic assignment indicated that the dominant members were Vibrionaceae. Beta diversity analyses

showed that bacterial communities clustered by sample source rather than spatial distribution, and that alpha

diversity was found to be higher in water than in sediment. Three percent of the strains from water samples could

degrade carboxyl-methyl cellulose with the smallest enzymatic indexes compared to 4 % of the strains from sedi-

ment samples that showed the highest enzymatic indexes. Two strains identified as Vibrio agarivorans degraded

cellulose and agarose, producing the highest enzymatic indexes.

Conclusions: We found higher bacterial densities and diversity in the bacterial communities of the water

samples compared to the sediment, with different OTUs including those similar to Ferrimonas, Providencia,

or Shewanella which were not isolated in the sediment. Vibrio OTUs were shown to degrade cellulose in both

sample types. The results of this study highlight the importance of red mangroves as Vibrio habitats and as res-

ervoirs of potential enzyme sources with biotechnological applications.

Key words: mangrove bacteria; cellulose; agarose; genetic diversity; biodegradation.

https://doi.org/10.15517/rev.biol.trop..v71i1.50983

BIOTECNOLOGÍA

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e50983, enero-diciembre 2023 (Publicado Ago. 04, 2023)

INTRODUCTION

Mangrove forests are important keystone

of coastal ecosystems and are located along

tropical and subtropical areas, covering up to

75 % of the world’s coastline. They are produc-

tive ecosystems with a unique combination

of land and sea, which harbor numerous spe-

cies of plants and animals (Zhu et al., 2022).

Among mangrove plant species, Rhizophora

mangle (red mangrove) typically grows in the

intertidal regions of the sheltered tropical and

subtropical coasts (Ellison et al., 2015). The

species R. mangle is native to the Atlantic coast

from Florida to Southern Brazil, and in West-

ern Africa, from Senegal to Angola (Ellison et

al., 2015), and typically dominates the zone

proximal to open water. Such environmental

patterns are also observed in Punta Galeta,

Panama, where red mangroves stand 10 m from

the water limit and extend up to 180 m from

the coast’s edge (Sousa & Mitchell, 1999).

It grows from a shrub to a small tree size, as

high as 1-8 m in the Caribbean, and can be

reproductively mature at < 1 m (Ellison et al.,

2015). This species has adapted their anatomy

and developed physiological strategies to sur-

vive to the constant changes, such as salinity,

nutrients, temperature, and frequent tropical

storms, of their environment (DeYoe et al.,

2020). Amongst the structures developed by R.

mangle to grow and handle harsh conditions,

include thickened leaves with secretory glands,

flexible stems, and extensive network of roots

or rhizophores (DeYoe et al., 2020).

The roots can create a variety of micro-

habitats suitable for colonization by a range of

microorganisms (Gomes et al., 2010; Gomes

et al., 2014; Hamzah et al., 2018; Zhang et

al., 2017). Culture independent studies con-

ducted on the roots of R. apiculata from China

RESUMEN

Caracterización de poblaciones de Vibrio en un transecto de Rhizophora mangle en Punta Galeta,

Panamá: análisis dependientes del cultivo revelan aplicaciones biotecnológicas

Introducción: Rhizophora mangle se considera un nicho para microorganismos con enzimas degradantes poten-

cialmente novedosas y complejas.

Objetivo: Caracterizar poblaciones de Vibrio con métodos dependientes de cultivo, provenientes de muestras de

sedimentos y de agua recolectadas a lo largo de un transecto de R. mangle compuesto por tres sitios.

Métodos: Las cepas se caracterizaron según su distribución, diversidad, degradación de materia orgánica y

parámetros ambientales.

Resultados: Las densidades bacterianas estuvieron fuertemente asociadas con la temperatura y la salinidad.

Un total de 87 secuencias de buena calidad que representan los aislamientos de los tres sitios se agruparon en

8 OTUs (Unidad taxonómica operativa). La asignación taxonómica indicó que los miembros dominantes eran

Vibrionaceae. Los análisis de diversidad beta mostraron que las comunidades bacterianas se agruparon por fuente

de la muestra en lugar de distribución espacial, y se encontró que la diversidad alfa era mayor en el agua que en

los sedimentos. El 3 % de las cepas de muestras de agua fueron capaces de degradar carboxi-metilcelulosa con

índices enzimáticos más bajos en comparación con el 4 % de las cepas de muestras de sedimentos que mostra-

ron los índices enzimáticos más altos. Dos cepas identificadas como Vibrio agarivorans degradaron celulosa y

agarosa, produciendo los índices enzimáticos más altos.

Conclusiones: Encontramos mayor densidad bacteriana y diversidad en comunidades bacterianas de muestras de

agua que en las de sedimento, con diferentes OTUs, incluyendo aquellos similares a Ferrimonas, Providencia,

o Shewanella, que no fueron aislados en el sedimento. OTUs de Vibrio degradaron celulosa en ambos tipos de

muestras. Los resultados del estudio resaltan la importancia de mangle rojo como hábitat de Vibrio y reservorio

de fuentes potenciales de enzimas con aplicaciones biotecnológicas.

Palabras clave: bacteria de manglar; celulosa; agarosa; diversidad genética; biodegradación.

Nomenclature: SMT1: Supplementary material Table 1; SMF1: Supplementary material Figure 1.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e50983, enero-diciembre 2023 (Publicado Ago. 04, 2023)

showed that Proteobacteria and Bacteroidetes

were the dominant phyla (Zhang et al., 2017).

Similar findings were reported in the roots of

R. mangle from Brazil, where Proteobacteria

and Bacteroidetes also dominated (Gomes et

al., 2010). Both studies have in common that

within the Proteobacteria, Gammaproteobac-

teria, and Deltaproteobacteria were the most

prevalent and abundant bacterial classes across

samples This might suggest the Rhizophora’s

roots harbor specific bacterial groups, regard-

less of the geography and the different species

of Rhizophora.

On the other hand, culture-dependent stud-

ies conducted in R. mangle from Brazil showed

low diversity among two different geographical

locations with Bacillus being the most common

genus, with highest endoglucanase activity (Sá

et al., 2014). Other studies have characterized

the phylosphere and sediments of R. mangle,

and confirmed low bacterial density (Dias et

al., 2012). Unlike the phyllosphere, sediments

of R. mangle were more diverse being domi-

nated by Alphaproteobacteria and Firmicutes

and with the highest endo- and exoglucanase

activity (Soares Júnior et al., 2014). The man-

grove’s sediments were reported to harbor

bacteria from orders Vibrionales, Actinomyce-

tales, and Bacillales with promising enzymatic

activities (amylases, proteases, esterases, and

lipases) for industrial applications (Dias et

al., 2009) and antibiotics (Thatoi et al., 2013).

Strains belonging to Vibrionales have shown a

variety of enzymatic activities, including amy-

lases, proteases (Dias et al., 2009), lipases, and

cellullases (Bibi et al., 2017). Most revisions

suggest increased Vibrio densities on waters

surrounding coastal ecosystems during the

summer, due to temperature increase (John-

son, 2015; Takemura et al., 2014). However,

exceptions were found in a study with Brazil-

ian mangrove sediments, where Vibrio strains

were more abundant during the winter (Dias et

al., 2009). Currently there are only few reports

exclusively on Vibrio spp. isolated from man-

groves (Rameshkumar & Nair, 2009). Studying

culturable Vibrio in these marginal forests is

essential, as its diversity and composition will

help us shed a light on their ecological role.

This study aimed to 1) examine spatial

distribution of culturable bacteria in water and

sediments along a transect of red mangroves,

2) evaluate the relationship between bacterial

densities and their environmental parameters,

and 3) explore the cellulolytic potential of

the bacterial isolates. We used both selec-

tive culture-dependent DNA sequence methods

applied to the isolates, to assess the bacterial

abundance and diversity along three sampling

points across a transect in Galeta Point man-

grove forest. Galeta lies next to an urban area

and industrial encroachment, due the proximity

of Panama Canal and Colon city.

MATERIALS AND METHODS

Study area and site characteristics: The

study was conducted in the mangrove forest

of Punta Galeta, Panama, near Punta Galeta-

Smithsonian Marine Station (9°24’ 29.861’’

N & 79°51’39.6’’ W). A transect of 180 m in

length was drawn, which started at the coast

and ended up where the red mangrove mixed

with the white mangrove. The 180 m transect

was divided into three areas. Each sampling

site was separated from the other by 80 -90

m in length. GPS coordinates were registered

using a GPS eTrex 10 (Garmin, Kansas, US)

(Table 1, SMT1).

Field sampling collection and environ-

mental measurements: The collection was

held on February 9 and 10, 2016 between 9

am and 11 am. The environmental temperature

during sampling was 28.6 º C ± 1.19. A total of

60 samples were analyzed at the three transect

sites (30 sediment and 30 water samples, 10

per sampling site). Samples of each source

were collected aseptically and placed in sterile

bags (sediment) at a depth of 5 cm and in 50 ml

falcon tubes (water). Samples were transported

at 4 °C to the laboratory, where they were

processed. Environmental parameters (pH,

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e50983, enero-diciembre 2023 (Publicado Ago. 04, 2023)

temperature, and salinity) measurements were

made in triplicate, in situ, from ten sediment

and ten water samples using Ohaus pens ST10

(pH) and Ohaus ST20s (Salinity and tempera-

ture) (Ohaus, New Jersey, USA).

Isolation and bacterial enumeration:

Ten water samples (0.1 ml) and sediment (0.1

g) from each site were serially diluted (101-103)

with 0.9 ml of NaCl 0.85 %, and plated (0.1

ml) onto Thiosulfate-Citrate-Bile Salts-Sucrose

(TCBS) agar (Himedia, Mumbai, India) for

total Vibrio enumeration. The culture plates

were incubated at 30 °C for 48 h. Because

samples were directly plated to this selective

medium, with no enrichment steps, the Col-

ony-Forming Units (CFUs) reported here are

likely under-estimates of actual bacterial levels

(Blackwell & Oliver, 2008). All yellow and

green colonies were counted as presumptive

vibrio isolates and reported as CFUs per ml of

water and CFU per g of sediment.

Screening in Congo red and Lugol agar

overlay method: A total of 360 strains were

stored at -80 °C in a cryoprotectant solution

of Trypticase Soy Broth (TSB) at 2 % NaCl

supplemented with 15 % glycerol. Strains were

grown at 30 °C on Trypticase Soy Agar (TSA)

(Himedia, Mumbai, India) with 2 % NaCl for

24 h. Agar plugs were cut and plated into Zobel

marine agar 2216 (Himedia, Mumbai, India)

medium containing 1 % Carboxyl methylcellu-

lose (CMC) (Sigma, Saint Louis, USA). Plates

were incubated at 30 °C for 48 h. Strains with

cellulolytic activity were detected by the for-

mation of clear zones with orange edges around

colonies through the Congo red overlay method

(Teather & Wood, 1982). A 10 mL aliquot of

Congo red dye (2.5 g/l) was then added to each

plate. After 30 min, the solution was discarded,

and the cultures were washed with 10 mL of 1

M NaCl. Cellulase production was indicated

indirectly by areas of hydrolysis. The halos

were measured for subsequent calculation of

the enzymatic index (EI) using the expres-

sion: IE = diameter of hydrolysis zone/ colony

diameter (Florencio et al., 2012). The strains

that showed CMC hydrolysis were tested for

their capacity to degrade agarose. Strains were

grown at 30 °C on TSA with 2 % NaCl for 24

h. Agar plugs were cut and placed into plates

onto solid Zobel marine agar 2216 medium.

Colonies that formed pits or clearing zones

on agar plates were dyed with Lugol’s iodine

solution (1.0 g iodine crystals and 2.0 g KI)

and dissolved in 300 mL distilled water (Hu et

al., 2009).

Genomic DNA extractions: Due to phe-

notypic similarities among isolates, a total of

90 strains were randomly selected for genomic

DNA extractions according to a previously

described method (Blanco-Abad et al., 2009),

Table 1

Correlations of environmental parameters in water samples versus bacterial densities transformed to a logarithmic scale

Sites Environmental Parameters N Rho P

1 (0 m) Salinity 10 -0.535 0.11

pH 10 -0.078 0.83

Temperature 10 -0.013 0.97

2 (80 m) Salinity 10 0.042 0.91

pH 10 0.177 0.62

Temperature 10 0.729 0.01

3 (180 m) Salinity 10 0.727 0.01

pH 10 -0.674 0.03

Temperature 10 0.358 0.31

N = number of samples; Rho = Spearman´s rank correlation; P = probabilistic value. P ≤ 0.05 is considered significant

P-values are in bold.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e50983, enero-diciembre 2023 (Publicado Ago. 04, 2023)

from an initial 1 ml of each pure culture. Cul-

tures were grown overnight in Luria Bertani

broth supplemented with 2 % NaCl at room

temperature, then centrifuged at 10 000 rpm

for 2 min. The supernatant was discarded, and

the pellet re-suspended in 500 µl of 5 % Che-

lex-100 (BioRad, California, USA). These sus-

pensions were vortexed for a few seconds and

incubated at 56 ºC for 20 min, boiled at 100 °C

for 10 min and then placed on ice for 2 min and

centrifuged at 13 000 rpm for 5 min The super-

natant containing the DNA was transferred to a

new tube and stored at -20 °C.

PCR amplification and sequencing of

16S rRNA genes: PCR reactions were per-

formed according to the method by Frank et

al. (2008). The bacterial 16S rRNA gene was

amplified from the genomic DNA, using univer-

sal eubacterial 16S rRNA primers comprised by

27F (5’-AGAGTTTGATCMTGGCTCAG-3’)

and 1492R (5’- TACGGYTACCTTGTTAC-

GACTT-3’) (Frank et al., 2008). PCR reactions

were performed in a 50 µl reaction mixture

containing 3 µL of DNA as the template, each

primer at a concentration of 0.4 µM and the

mixture of deoxynucleotides triphosphate at

a concentration of 0.8 mM, as well as 1 U of

FastStartDNA polymerase (Roche Diagnostics

GmbH, Mannheim, Germany) and buffer to 1X

(Roche Diagnostics GmbH, Mannheim, Ger-

many). PCR amplifications were performed in

a 2720 thermal cycler (Applied Biosystems,

California, USA) with an initial denaturation

for 5 min at 95 °C, 30 cycles consisting of

10 cycles of denaturation at 95 °C for 45 sec,

annealing at 58 °C for 45 sec. (decreasing by

0.6 °C per cycle) and extension step of 72 °C

for 1.2 min; and 20 cycles of denaturation at 95

°C for 45 sec., annealing at 54 °C for 45 sec.

and extension step of 72 °C for 1.2 min plus a

final elongation step of 72 °C for 7 min. The

PCR product was confirmed by gel (1.6 %)

electrophoresis and visualized under UV light

after staining the gel with ethidium bromide

(0.5 µg/ml). PCR amplicons were purified and

sent for custom Sanger sequencing to Macro-

gen Inc (Seoul, South Korea).

Data analyses: Bidirectional full-length

sequences were visually inspected using

Geneious R11 (Biomatters Ltd, Auckland, New

Zealand) software. A total of 87 isolates passed

QC via analyses with Quantitative Insights into

Microbial Ecology (QIIME) 1.91(Caporaso et

al. 2010). Sequences were inspected for chime-

ras with the USEARCH61 hierarchical cluster-

ing method (Edgar et al., 2011). Sequences

were aligned using the SINA aligner (Pruesse

et al., 2012). Sequences were binned into OTUs

in QIIME using Mothur (Schloss et al., 2009).

Taxonomy: The taxonomy assignment of

the representative OTUs was carried out using

the RDP database (Wang et al., 2007) with

a confidence threshold of 97 %. An OTU

table was created using make_otu_table.py

using Biological Observation Matrix (BIOM)

(McDonald et al., 2012) with the taxonomic

classifications. Taxa summaries were built by

modifying the QIIME L6 taxonomy table with

the R package reshape2 (Wickham, 2007).

Pie charts of water and sediment samples

were made with the OTU table using Micro-

biomeAnalyst (Dhariwal et al., 2017). Tax-

onomy bar plots with dendrogram and metadata

annotations were built using RStudio (https://

cran.r-project.org) with the ggplot2 package

(Wickham, 2016). We constructed a dendro-

gram from the results of hierarchical clustering

analyses, using the hclust function (Müllner,

2013). The average-linkage algorithm in the

hclust model was applied to cluster the samples

based on their source. Clustering data was

extracted with the ggdendro package (De Vries

& Ripley, 2016). The distance matrix was

computed using Bray-Curtis distance using the

Vegan package (Oksanen et al., 2020). Finally,

dendrogram, annotations, and histograms were

arranged into a grid using the cowplot library

(Wilke, 2017). Megablast analyses was per-

formed to confirm closest match to the NCBI

database to some isolates.

Alpha Diversity: Alpha-diversity analy-

ses was done with option –min_rare_depth

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(the lower limit of rarefaction depths). Chao1

abundance-based estimator (Chao, 1987) was

calculated for the water and sediment samples

with the script for alpha diversity and boxplots

were prepared using the ggplot2 library.

Beta Diversity: Microbial community

structure variations between water and sedi-

ment samples were performed with beta diver-

sity analyses via Principal Coordinate Analyses

(PCoA) on a Bray-Curtis (BC) distance matrix

using the Ordinate function from R Phyloseq

package (McMurdie & Holmes, 2013). For

this, the OTU table and sample metadata were

imported in R Studio and merged into one

phyloseq object. The OTU counts were trans-

formed into relative abundance matrix previ-

ously stored as an OTU table-class. Ordination

point colors were assigned according to each

sample type variable and customized using aes-

thetic functions from ggplot2 package.

Statistical analysis: Bacterial counts were

log transformed and the distributions of the

counts were determined using Shapiro-Wilkson

test (P < 0.05). Spearman correlations (Rho)

were calculated to evaluate the relationships

between the transformed bacterial densities

on a logarithmic scale (CFU per mL of water

and CFU per g of sediment) and environ-

mental parameters. The significance of the

correlation was declared when P ≤ 0.05. All

the univariate analyses were carried out in R

studio and plots were generated using ggplot2.

Significant differences of alpha diversity were

calculated using a non-parametric two-sample

t-test using 999 Monte-Carlo permutations

using the QIIME script compare_alpha_diver-

sity.py using the collated alpha diversity file

resulting from the alpha rarefaction analyses.

The comparison was done between water and

sediments samples, created via the input cat-

egory passed via “-c” on the mapping file.

Permutational Multivariate Analysis of Vari-

ance (PERMANOVA) was the significance

test applied to the beta diversity analyses using

the vegan package. This PERMANOVA test is

determined through permutations and provides

strength and statistical significance on sample

groupings using a Bray-Curtis distance matrix

as the primary input.

RESULTS

Measurements of environmental param-

eters by site: Measurements of environmental

parameters indicated an increase in salt as

measurements were made from the coast to the

mangrove forest (varied from 30.85 to 33.68

ppt) (SMT1). Regarding the pH of the water

samples, they indicated that the most alkaline

point is site 2 (6.99) with a very low pH varia-

tion between sites 2 and 3. Water temperature

measurements did not vary drastically along

the transect. Measurements of environmental

parameters in the sediment were fairly constant

throughout the transect (SMT2). Site 1 was the

coldest because is always flooded. No large

variations were observed in the temperature

measurements between sites 2 and 3.

Spatial distribution of bacterial densi-

ties: All colonies were counted, and bacterial

populations were expressed as Log CFUg-1 of

fresh sediment and Log CFU mL-1. The high-

est bacterial density was found in the sediment

samples from site 1 (1.53 Log CFU g-1). The

lowest bacterial density was given in water

samples from site 1 (0.32 Log CFU g-1). Fur-

thermore, for sediment samples, site 2 varied

very little in bacterial density compared with

site 3 that showed a density of sediment sam-

ples. A comparison of bacterial load was made

between CFUs from bacteria isolated from

water and sediment samples (Fig. 1).

Relationship between environmental

parameters and Log CFU ml-1 or g-1: The

relationship between the bacterial densities and

the temperature of the water samples indicated

a positive correlation (P ≤ 0.05) (Table 1) for

site 2 (Rho = 0.729, P = 0.01). In site 3, we

found a correlation between the bacterial densi-

ties and the salinity of the water samples (Rho

= 0.727, P = 0.01. In contrast with positive

correlations, we found a negative correlation

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e50983, enero-diciembre 2023 (Publicado Ago. 04, 2023)

between pH and Log CFU ml-1 of bacterial

densities in site 3 (Rho = -0.674, P = 0.03)

(Table 1).

Bacterial isolation: For each site and type

of sample (water and sediment), 60 strains

were isolated randomly resulting in a total of

360 strains, comprising 180 strains for water

samples and 180 strains for sediment samples.

All strains showed growth on TCBS, 2 % NaCl

TSA, and marine agar 2 216 under the same

culture conditions.

Genetic diversity of bacterial strains:

A total of 87 good-quality sequences from

phenotypically distinct isolates, 42 sequenc-

es obtained from sediment strains and 45

obtained from water samples were analyzed

using QIIME 1.91. These sequences were

binned into 4 OTUs (sediment) and 6 OTUs

(water) respectively. Beta diversity analyses

shown in the PCoA diagram (Fig. 2A), indi-

cate a slight separation of samples according

to sample source (sediment and water). The

sediment strains of sites 2 and 3 were grouped

close to each other sharing less dissimilarities,

compared to OTUs from water strains (Fig.

2A), as also shown by Bray-Curtis dissimilar-

ity analyses separated the water sample from

site 1 (RW1) from the rest of water samples

(RW2 and RW3) (Fig. 2A). The PCoA showed

that the sum of both axes explained almost all

(99.6 %) the variation. The sample source, from

either water or sediment, is a selectively strong

force for the bacterial community structure,

rather than each of the sampling sites. Box-

plots representing the Chao1 index between

sample types showed a higher diversity in the

water samples (Chao1 = 4.5) compared to sedi-

ment samples (Chao1 = 2.87), with no differ-

ences in diversity between type samples (P =

0.274) (Fig. 2B). Although the community is

represented by a small number of individuals

(rarefaction only by 10 sequences). There was

a total of 8 clusters (OTUs) from the combined

87 initial sequences (Fig. 3, SMT3). From the

87 selected strains for genetic characteriza-

tion, the recovery and selectively of TCBS for

Vibrionaceae members was higher in sediments

samples (Fig. 3A), obtaining 98 % (N = 41)

Fig. 1. Distribution of bacterial densities. CFU g-1 (Sediment) or CFU ml-1(Water) across samples and sites along transects.

8Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e50983, enero-diciembre 2023 (Publicado Ago. 04, 2023)

strains belonging to Vibrionaceae and only 2

% (N = 4) of strains were identified as Proteus.

Overall, TCBS showed a high selectivity (88.5

%) by inhibiting the growth of heterotrophic

bacteria. The pie charts of water taxa (Fig. 3B)

indicated that we could recovered 81 % (N =

36) Vibrionanceae members, and 20 % (N = 9)

were identified as non-Vibrionaceae members,

including members of Proteobacteria phylum:

Ferrimonas, Providencia, and Shewanella. We

determined which genera changed between

two sources and the three sites depicting the

relative abundance through bar plots (Fig. 4).

The PERMANOVA test showed no differences

between the genera present in water and sedi-

ments samples (P = 0.104). Notably, the first

site from the water samples within the highest

genera diversity, harboring different taxa Vib-

rionaceae family. The taxa diversity decreased

as it entered mainland for both sample types

(water or sediment). The taxonomic resolution

of 16S rRNA identified Vibrio genera in water

Fig. 2. Alpha and Beta diversity analyses on the 87 isolates. A. Beta diversity comparisons by PCoA. Ordinations of Bray-

Curtis dissimilarity between the bacterial strains from two different sources. The color of the spheres indicates the source

of the samples. B. Chao1 alpha diversity indexes of strains according to the sampling source. Blue dots indicate the means

of the chao1 metric.

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samples exclusively. The dendrogram based on

dissimilarities indicated that sediments strains

shared less dissimilarities than water strains.

Screening of cellulose and agar degra-

dation of strains: The overlay method with

Congo red was used to evaluate, semi-quan-

titatively, the ability of the strains to degrade

cellulose. All strains (N = 360) were screened

for cellulose degradation with 1 % CMC on

the marine agar 2 216 in triplicate. This test

was based on the observation of growth and

measurement of the hydrolysis halo that is used

for the calculation of the enzyme index (EI).

The halos produced by the cellulose hydrolysis

were directly related to the action region of

the cellulolytic enzymes, since the dye only

remained bound to regions where there likely

is β-1, 4-D-glucanohydrolase bonds (Florencio

et al., 2012). The absence of color or pale halo

around the colonies corresponds to the area of

CMC degradation. Strains from sediment that

Fig. 3. Pie charts depicting the sample composition among sediment and water samples. A. Relative abundance of the taxa

in sediment samples. B. Relative abundance of taxa in water samples. The numbers before each bacterial taxa represent the

8 OTU clusters.

10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e50983, enero-diciembre 2023 (Publicado Ago. 04, 2023)

showed CMC hydrolysis on marine agar (4.4

%) were respectively (Sediment site 1: S1-24,

S1-31, S1-35 and Sediment site 2: S2-7, S2-8,

S2- 9, S2-12, S2-37). Strains isolated from

water samples that showed hydrolysis of CMC

on marine agar were lower than those of sedi-

ment (3.3 %) (W1-5, W1-8, W2-10; W2-30,

W3-55, W3-56). The diameter of the halo zone

was useful for the selection of strains that can

efficiently degrade polysaccharides such as

cellulose, xylan and amylose. In addition, the

enzymatic index can be used as a simple and

rapid methodology to select strains that may

have potential to produce enzymes (Florencio et

al., 2012). The results of EI (Table 2) obtained

for the strains after 2 days of incubation at 30

°C represent the average of measurements for

3 experiments performed independently under

the same conditions. The largest enzymatic

indexes for cellulose degradation were found

in the strains from sediment samples, S1-31

and S2-37 (Table 2). Similar results could be

seen in agar degradation with only two strains

having agarolytic activity, S1-31 (EI = 4.3)

and S2-37 (EI = 3.5). A megablast analyses

indicated that the closest match for the strains

S1-31 and S2-37 is Vibrio agarivorans.

DISCUSSION

The bacterial densities of cultured bac-

teria along a red mangrove transect in Pan-

amá showed a pattern defined according to

the sample origin (sediment and water). We

observed that in water samples the bacte-

rial densities increased as it entered mainland;

the inverse happened in sediment samples.

However, the abundance remained steady in

sites 2 and 3 for both types of samples. This

invariability in Vibrionaceae densities was like

Fig. 4. Taxa summary at the genus levq12el of all bacterial strains. Average-linked clustering (Bray-Curtis) in sediment and

water bacterial taxa according to pH, source, and salinity data. Samples derived from sediment are indicated as RS and those

from water are indicated as RW.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e50983, enero-diciembre 2023 (Publicado Ago. 04, 2023)

a distribution pattern found in sediments of

three different sites from Mexico populated

by black mangroves (Gonzalez-Acosta et al.,

2006). Although our results share a common

stability pattern, the abundance of culturable

Vibrio spp. in sediments of Mexico was higher

(7.4 log CFU g–1) (Gonzalez-Acosta et al.,

2006) compared with our bacterial densities

in sediments (0.82 Log CFU g-1). A study on

Brazilian mangrove sediment bacteria (Dias et

al., 2009), showed no recovery of Vibrio spp.

from mangroves sediment at a depth of (0-10

cm). Conversely, we could isolate Vibrionaceae

members from our sediment samples at a depth

of 5 cm. Regarding the water samples, the

densities of Vibrio spp. in estuaries from some

investigations during the dry season were simi-

lar to our strain densities in water (Pfeffer et

al., 2003; Turner et al., 2009; Wetz et al., 2008).

Temperature and salinity have been the

most exhaustively studied environmental fac-

tors associated with Vibrio densities (Johnson,

2015; Takemura et al. 2014). Our results are

supported by other studies where positive cor-

relation with temperature (Janelidze et al.,

2011; Pfeffer et al., 2003; Wetz et al., 2008)

and salinity (Pfeffer et al., 2003; Turner et

al., 2009) were observed in estuarine waters,

using similar culture techniques. Our correla-

tion analysis suggested that the environmental

factors were the driven forces to shape the

culturable community in water samples, where

we found that alpha diversity was higher com-

pared to the sediment. Site 1 made the highest

contribution to the taxa abundance reported

in water samples. One possible explanation

about this was this site was the closest to the

sea and therefore was under the influence of

tidal hydrodynamics, which probably led to

the selection of different taxa. It is likely that

spatial distribution of Vibrionaceae was higher

in water samples of the first site due several

factors that go beyond the focus of this study,

such as: residence time, net growth rate, graz-

ing rates by protists, stress, nutrient availabil-

ity, and viral lysis rates (Mackey et al., 2017).

Overall, the variation of the bacterial communi-

ties in the mangrove ecosystem is characterized

by periodic flooding of the tides; and environ-

mental factors such as salinity, temperature

and nutrient availability are highly variable and

result in unique and specific features (Holguin

et al., 2006).

In this study, we observed that the sample

type (water/sediment) have a high impact in the

composition of Vibrionaceae taxa compared to

Table 2

Enzymatic indexes of cellulose degraders and their corresponding halo sizes.

Strains Closest match (Megablast) Mean Halo (mm) Mean Hydrolisis halo (mm) EI SD

S1-24 N.D. 9 2.67 0.30 0.58

S1-31 Vibrio agarivorans 9 43.33 2.41 1.53

S1-35 N.D. 9 3.67 0.41 0.58

S2-7 N.D. 9 4.67 0.52 0.58

S2-8 N.D. 9 5.67 0.63 0.58

S2-9 N.D. 9 7.67 0.85 0.58

S2-12 N.D. 9 8.67 0.96 0.58

S2-37 Vibrio agarivorans 9 33.67 2.59 1.53

W1-5 N.D. 9 2.00 0.22 0.00

W1-8 N.D. 9 1.67 0.19 0.58

W2-10 N.D. 9 3.67 0.41 0.58

W2-30 N.D. 9 2.67 0.30 0.58

W3-55 Vibrio alginolyticus 9 5.67 0.63 0.58

W3-56 N.D. 9 3.67 0.41 0.58

EI = enzymatic indexes. N.D. = Not determined; SD=Standard deviation.

12 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e50983, enero-diciembre 2023 (Publicado Ago. 04, 2023)

each sampled mangrove habitat. Conversely to

our findings, a study conducted in Brazilian

mangrove sediments, found that each site of a

transect select and influence specific the bac-

terial communities (Rocha et al., 2016). This

same study reported the prevalence of Vibrio

in their first collection site as well in the last

section of them transect (site 3). Although their

transect was longer than ours - 450 m distance-

(Rocha et al., 2016), it revealed similar Vibrio

levels to those reported here.

According to our genetic characterization

at the genus-level, Proteus was only found to

be present in one of the sediment samples - a

bacterium known to be an indicator of fecal

contamination (Drzewiecka, 2016). We also

report the presence of Shewanella, Providencia,

and Ferrimonas in water samples. Despite the

selectivity of TCBS for vibrio isolates, other

genera such as Staphylococcus, Flavobacte-

rium, Pseudoalteromonas, and Shewanella can

grow on TCBS as well, (Thompson et al., 2004)

as we found. Perhaps, these non-Vibrionaceae

members have a terrestrial origin as a result

of freshwater inputs. The identification using

16s rRNA gene showed a good resolution for

water strains at the genus level. This limitation

could be circumvented using different phyloge-

netic and evolutionary methodologies such as

Multi-Locus Sequence Typing (MLST), Mul-

tiple-Locus Variable-Number Tandem-Repeat

Analysis (MLVA), or Pulsed-Field Gel Electro-

phoresis (PFGE) which allows to distinguish

strains with little genetic variation (Lüdeke

et al., 2015). Factors that maintain genetics

and species diversity act in concert in marine

ecosystems (Robinson et al., 2010). In this

research, changes in species composition and

other levels of diversity (genetic and species

diversity) along the study gradient seems to

be both related to substrate water exposure.

The same pattern has been observed in other

benthic intertidal taxa where water exposure,

salinity, temperature, habitat complexity and

substrate spatial heterogeneity shape the com-

munity structure and diversity (Archambault &

Bourget, 1996).

In general, there are several studies assess-

ing and predicting the dynamics of Vibrio

as these are impacted by recreation, used in

animal aquaculture and, therefore, represent

a public health issue with consequences for

humans and marine animals (Jacobs et al.,

2014; Johnson, 2015; Lüdeke et al., 2015;

Takemura et al., 2014; Turner et al., 2009; Wetz

et al., 2008). However, a few of these exploit

the biotechnological capabilities of Vibrio to

degraded natural biomass, despite the diverse

physiological properties and ubiquity in marine

environments. We showed that in our transect,

the strains that produced the largest hydroly-

sis halo on CMC media (S1-31, S2-37) and

caused a depression in the agar, were positive

for hydrolysis of agar using Lugol. Interest-

ingly, few Vibrio spp. have been reported to

possess multi-enzymatic machinery (Dias et

al., 2009, Liu et al., 2016), as those explained

here from red mangrove sediments. To the

best our knowledge, our study reports for first

time the presence of V. agarivorans in tropical

mangroves. The first and only report of V. a g a -

rivorans comes from Mediterranean seawater,

with which our two strains matched (Macián et

al., 2001). Further physiological characteriza-

tions are needed as well as quantifications of

the hydrolysis with the test of reducing sugars

with DNS (Di-nitro salicylic acid) for these

strains. This could shed a light to how these

two Vibrio strains adapted to the environmental

challenges present in the red mangrove forest

of Punta Galeta, being able to degrade 2 differ-

ent substrates.

Vibrio is a natural inhabitant along the

three sites of our mangrove transect and is

well known for its high frequency of gene

exchange (especially some clades), leading to

a very rapid evolution and genomic plastic-

ity (Tagliavia et al., 2019). In this sense, we

consider that sediment strains might adapted to

different characteristics in this habitat leading

to expression of higher enzymatic capabilities

than their counterpart water strains. Vibrio

species are Gram-negative, curved, rod-shaped

bacteria belonging to the class Gammaproteo-

bacteria and are still regarded by most marine

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e50983, enero-diciembre 2023 (Publicado Ago. 04, 2023)

microbiologists as the dominant culturable

bacteria in the ocean (Pruzzo, et al., 2005).

This feature made Vibrio a model to screening

enzymes with biotechnological applications

because working with the living organisms

allows to overcome some obstacles, such as

heterologous expression of proteins encoded

by environmental DNA in a surrogate host

(Tagliavia et al., 2019), generated while per-

forming functional metagenomics to screen for

microbial enzymes.

Spatial analysis like this, joined with

molecular genotyping, could provide important

information to identify microbial hotspots as

well as deserts in mangrove transects (Gonza-

lez et al., 2012). Additionally, correlations with

environmental parameters help develop new

hypothesis about dispersal limitation (Gonzalez

et al., 2012). Although sometimes it is difficult

to assign these physiological features to spe-

cies (Tagliavia et al., 2019). There is much

interest on the biotechnological capabilities

of mangrove microbes in which the variable

environmental conditions may impact micro-

bial structure and the enzymatic properties

of the communities.

This study reports the diversity of Vibrio

along a red mangrove transect in Panama,

indicating a moderate Vibrio load during the

time lapse of sampling and along a 180 m

transect. The correlations between the Vibrio

spp. load and temperature, indicate that this

parameter is a strong environmental driver for

the detection of Vibrio. Genetic analyses based

on the 16S rRNA gene indicate that there is

a higher diversity of Vibrio in water samples

compared to sediment, although Vibrio strains

from the sediment have cellulolytic activity. In

summary, our report highlights the isolation of

cellulose-degrading Vibrio from the red man-

grove niche, and indicates its potential appli-

cability in various industrial processes such as

biofuel production.

Ethical statement: the authors declare

that they all agree with this publication and

made significant contributions; that there is

no conflict of interest of any kind; and that we

followed all pertinent ethical and legal proce-

dures and requirements. All financial sources

are fully and clearly stated in the acknowledge-

ments section. A signed document has been

filed in the journal archives.

ACKNOWLEDGMENTS

We gratefully acknowledge the Minister

of Environment (MIambiente) for granting

permission to Joel Sanchez and Jacinto Perez

to make the collection. We thank Stanley

Heckadon and his scientific crew (Abilio, Ilia,

Gabriel, and Jairo) of The Smithsonian Tropi-

cal Research Institute- Galeta Marine station in

Colon, for their support and help. We also thank

Luis Mejia for his initial comments and sugges-

tions at the beginning of this project. This work

was partially funded by the National Secretary

of Science and Technology of Panama (SENA-

CYT, grant number APY-GC-2014-046) and a

fellowship granted to Joel Sanchez from Colon

Container Terminal-The Smithsonian Tropical

Research Institute (STRI). Special thanks to

Gilmary Ortiz for her input and contribution

during data analysis of the 16S rRNA for the

study. We appreciate the support of the PR-

INBRE P20GM103475 and the Inter-American

University of Puerto Rico.

See supplementary material

a40v71n1-SM1

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