Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Diversity of Vibrio spp in Karstic Coastal Marshes in the Yucatan Peninsula

  • Icela Ortiz-Carrillo ,

    Contributed equally to this work with: Icela Ortiz-Carrillo, Neyi Eloísa Estrella-Gómez, Marcela Zamudio-Maya, Rafael Rojas-Herrera

    Affiliation Facultad de Ingeniería Química, Universidad Autónoma de Yucatán, Periférico Norte km 33.5, Tablaje catastral 13615, Colonia Chuburná de Hidalgo Inn, C.P., 97203, Mérida, Yucatán, México

  • Neyi Eloísa Estrella-Gómez ,

    Contributed equally to this work with: Icela Ortiz-Carrillo, Neyi Eloísa Estrella-Gómez, Marcela Zamudio-Maya, Rafael Rojas-Herrera

    Affiliation Facultad de Ingeniería Química, Universidad Autónoma de Yucatán, Periférico Norte km 33.5, Tablaje catastral 13615, Colonia Chuburná de Hidalgo Inn, C.P., 97203, Mérida, Yucatán, México

  • Marcela Zamudio-Maya ,

    Contributed equally to this work with: Icela Ortiz-Carrillo, Neyi Eloísa Estrella-Gómez, Marcela Zamudio-Maya, Rafael Rojas-Herrera

    Affiliation Facultad de Ingeniería Química, Universidad Autónoma de Yucatán, Periférico Norte km 33.5, Tablaje catastral 13615, Colonia Chuburná de Hidalgo Inn, C.P., 97203, Mérida, Yucatán, México

  • Rafael Rojas-Herrera

    Contributed equally to this work with: Icela Ortiz-Carrillo, Neyi Eloísa Estrella-Gómez, Marcela Zamudio-Maya, Rafael Rojas-Herrera

    rafael.rojas@correo.uady.mx

    Affiliation Facultad de Ingeniería Química, Universidad Autónoma de Yucatán, Periférico Norte km 33.5, Tablaje catastral 13615, Colonia Chuburná de Hidalgo Inn, C.P., 97203, Mérida, Yucatán, México

Diversity of Vibrio spp in Karstic Coastal Marshes in the Yucatan Peninsula

  • Icela Ortiz-Carrillo, 
  • Neyi Eloísa Estrella-Gómez, 
  • Marcela Zamudio-Maya, 
  • Rafael Rojas-Herrera
PLOS
x

Abstract

Coastal bodies of water formed by the combination of seawater, underground rivers and rainwater comprise the systems with the greatest solar energy flow and biomass production on the planet. These characteristics make them reservoirs for a large number species, mainly microorganisms. Bacteria of the genus Vibrio are natural inhabitants of these environments and their presence is determined by variations in the nutrient, temperature and salinity cycles generated by the seasonal hydrologic behavior of these lagoon systems. This study determined the diversity of the genus Vibrio in 4 coastal bodies of water on the Yucatan Peninsula (Celestun Lagoon, Chelem Lagoon, Rosada Lagoon and Sabancuy Estuary). Using the molecular technique of 454 pyrosequencing, DNA extracted from water samples was analyzed and 32,807 reads were obtained belonging to over 20 culturable species of the genus Vibrio and related genera. OTU (operational taxonomic unit) richness and Chao2 and Shannon Weaver diversity indices were obtained with the database from this technique. Physicochemical and environmental parameters were determined and correlated with Vibrio diversity measured in OTUs.

Introduction

The genus Vibrio is the most diverse and abundant group of marine bacteria with 74 described species, and its taxonomy is under constant review due to the incorporation of genotypic and molecular analyses that show this genus to be highly heterogeneous [1,2]. The species of clinical importance are V. cholerae, V. parahaemolyticus, V. vulnificus, V. alginolyticus, V. fluvialis, V. mimicus, V. hollisae, V. damsela, V. furnissii, V. cincinnatiensis, V. harveyi and V. metschnikovii. There are also species of ecological and probiotic importance, such as V. fischeri, V. splendidus, V. halioticoli, V. mediterranei and V. rotiferanius [2,3].

Saline environments comprise the natural habitat of vibrios [4] and although their salt requirements fluctuate, the majority develop well between 2 and 2.5% salt. They can be found in the water column, sediments, zooplankton, plankton, algae and the gastrointestinal tract of many marine organisms [58]. Another special characteristic of this genus is the ability of its species to live under conditions of low salt and nutrient concentrations, by reducing their size to 0.2 μm (microvibrios) [7,9].

The members of this genus are highly specialized because they have the capacity to perform specific metabolic activities with respect to the use of carbon depending on the depth at which they are found. For example, Vibrio profundum presents optimal growth at 2,000 atmospheres and produces a large quantity of fatty acids that help it to maintain flow through the cell membrane at high pressures and low temperatures [9,10]. These bacteria are capable of degrading labile polymers such as starch, casein and agar [11] and they also produce a wide variety of enzymes, of which quinolase and chitinase are of particular significance because they provide the ability to degrade chitin and use it as a source of carbon and nitrogen [1214].

Environmental gradients (temperature, salinity and nutrients) and biological factors influence the distribution and dynamics of Vibrio populations [5,12,1517]. The species of genus Vibrio possess the versatility to develop in different environments and temperatures, however the optimum development for most species has been reported above 17°C. Some species are capable of “hibernating” in sediments or associating with marine fauna, which allows them to store a large quantity of proteins and develop biofilms, ensuring that they respond efficiently to the constant changes in ecosystems [2,18,19].

Coastal bodies of water are systems that are subjected to significant variability in their determining ecological factors due to a wide range of influences, including hydrologic, climatic and anthropogenic factors. On the Yucatan Peninsula these bodies of water are karstic in nature, which makes them rich in carbonates and other salts. Furthermore, water residency is not constant and therefore results in significant changes in salinity levels[20]. These special characteristics of the region influence the development, prevalence and distribution of the different organisms that inhabit these lagoons.

In these lagoons riverine tourism and fishing activities are widely practiced thus making an important issue to know which epidemiological and ecological important microbial species inhabit these systems. For example, octopus, which has become a highly important exportation product for Mexico, are caught in riverine areas under the influence of coastal lagoons, while other shellfish species like shrimp and oysters are directly extracted from lagoons being both octopus and shellfish intended for human consumption frequently raw or undercooked. As stated above, several Vibrio species are clinically, ecologically as well as biotechnologically important and, as far as we are aware, there are no reports on the taxonomic structure of these microorganisms that dwell in the coastal lagoons of the Yucatan Peninsula. Therefore, the aim of this work was to study the taxonomic structure and diversity of the genus Vibrio in 4 coastal lagoons by enrichment in selective medium strategy and subsequent massive 16S rRNA gene sequencing.

Materials and Methods

Study sites

Samples of surface water were taken from 4 coastal lagoons on the Yucatan Peninsula in Mexico: Celestun (20° 45' N—90°22' W), Chelem (21°15' N—89°45' W), Rosada Lagoon (21°19' N—89°19' W), and Sabancuy Estuary (18°58' N—91°12' W). The sampling was performed from August to October of 2011 (Chelem 08/24; Laguna Rosada 09/06; Celestún 09/28; Sabancuy 10/25). The sampling was random without replacement and 10 samples of surface water were collected along a transect parallel to the coastal axis. Samples were deposited in sterile plastic bottles and conserved in refrigeration at 4°C. All samples were processed within 24 hours after sampling.

According to the Mexican laws and regulations no permissions are required to obtain water and sediment samples from open public areas.

Analysis of environmental and physicochemical parameters

Determinations of the environmental parameters were performed with a Hach 5465000 model 156 multi-parameter measuring instrument. The Lorenzen method was used to determine chlorophyll-a [21] with 90% acetone and the concentration was calculated according to the formula: Where, OD665o: absorbance at 665 nm before acidification; OD665a: absorbance at 665 nm after acidification; VA: volume (ml) of acetone for extraction; VM: volume (ml) of filtered water; L: length (cm) of the photometric cell.

Determinations of the physicochemical parameters (silicates, phosphates, nitrates, nitrites and ammonia) were performed using the spectrophotometric techniques described and modified by Strickland and Parsons [21].

Enrichment

The water samples were cultured in trypticase soy broth (TCS) (BD-Bioxon) containing 0.5% NaCl. Additionally, the samples enriched in salt medium were cultured in TCS supplemented with 3% NaCl. All cultures were incubated at 37°C with agitation at 150 rpm for 48 hours.

Total (metagenomic) DNA extraction

0.5 ml of overnight culture was poured into a 1.7 ml microcentrifuge tube with the addition of 1 ml of TEN buffer (100 mM Tris– HCI, 50 mM EDTA, 500 mM NaCl, pH 8.0) and vortexed for 1 min. After centrifugation for 10 min at 10,000 xg at RT, the pellet was resuspended in 1 ml of TEN buffer with 0.2 mg of added lysozyme and incubated for 1 h at 37°C under agitation. Then three cycles of freezing/defrosting were done by incubation 10 min in an ice/alcohol bath and then 5 min at 65°C. 100 μl of 20% SDS was added and vortexed for 1 min and incubated for 30 min at RT. After centrifugation for 10 min at 10,000 xg at RT, the supernatant was transferred into a fresh microcentrifuge tube and 500 μl of 5 M potassium acetate was added and then incubated at 65°C for 5 min. Tubes were placed in an ice bath for 20 min and centrifuged at 20,000 xg for 30 min at 4°C. The supernatant was transferred into a fresh microcentrifuge tube and 200 μl of a 4% silica suspension was added and incubated at RT for 2 min with gentle agitation. Silica-DNA complex was recovered by centrifugation at 16,000 xg for 2 min at RT and the pellet was washed by adding 1 ml of 70% ethanol. DNA was eluted with 50 μl of sterile distilled water and incubated at 55°C for 5 min and recovered by centrifugation at 16,000 x g for 5 min at RT [22]. DNA samples were quantified with a NanoDropND-1000 spectrophotometer (NanoDrop Technologies,Wilmington, DE, USA).

Sample pooling

Since a great number of subsamples were generated per lagoon (20 from each) we decide to prescreen samples to reduce the total number of sequencing reactions. For that we used a DGGE approach based on the technique described by Muyzer et al. [23], using specific primers for the genus Vibrio, GC567f and 680r [24], to visually analyze if meaningful differences existed among different samples from the same lagoon. After DGGE experiments it was shown there were not major differences among enriched subsamples of the same lagoon (data not shown), so we decided to pool together five DNA samples of each enrichment condition per lagoon what resulted in two composite samples for every lagoon. Data presented in this paper correspond to those composite samples.

Massively parallel bTEFAP

Purified metagenomic DNAs were submitted to the Research and Testing Laboratory (RTL) (Lubbock, TX, USA) for 16S rRNA tag-pyrosequencing. Bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) was performed as described previously using Gray28F (5’-TTTGATCNTGGCTCAG-3’) and Gray519r (5’-GTNTTACNGCGGCKGCTG-3’) were used for amplification of the variable regions V1-V3 [25]. Initial generation of the sequencing library utilized a one-step PCR with a total of 30 cycles, a mixture of HotStart and HotStar high fidelity Taq polymerases, and amplicons originating and extending from the 28F for bacterial diversity. Tag-encoded FLX amplicon pyrosequencing analyses utilized Roche 454 FLX instrument with Titanium reagents; Titanium procedures were based on RTL protocols (www.researchandtesting.com).

Data analysis, bacteria identification and diversity index

Following sequencing, all failed sequence reads, low quality sequence ends and tags and primers were removed and sequences collections depleted of any non-bacterial ribosome sequences and chimeras using B2C2 [26], as has been described previously [25,27]. To determine the identity of bacteria in the remaining reads, sequences were denoised, assembled into clusters and queried using a distributed BLASTn. NET algorithm [28] against a database of high quality 16S bacterial sequences derived from NCBI. Database sequences were characterized as high quality based upon similar criteria utilized by RDP. Using a.NET and C# analysis pipeline. The resulting BLASTn outputs were compiled, validated using taxonomic distance methods, and data reduction analysis performed as described previously [25,27]. Based upon the above BLASTn derived sequence identity (percent of total query sequence length which aligns with a given database sequence) and validated using taxonomic distance methods, the sequences were classified at the appropriate taxonomic levels based upon the following criteria: sequences with identity scores (relative to known or well characterized 16S sequences) greater than 97% identity (<3% divergence) were resolved at the species level, between 95% and 97% at the genus level, between 90% and 95% at the family and between 85% and 90% at the order level, 80 and 85% at the class and 77% to 80% at the phylum level. In addition, the high-score pair must be at least 75% of the query sequence or it will be discarded, regardless of identity.

Sequencing reads were aligned and clustered following the Ribosomal Database Project (RDP-Release 10) pyrosequencing pipeline (http://pyro.cme.msu.edu/). Shannon, Chao2, and evenness indices, as well as rarefaction curves, Jaccard index and heat map were obtained using the RDP tools.

Accession number

All 16S rRNA gene sequences were deposited in the SRA database at the National Center for Biotechnological Information (http://www.ncbi.nlm.nih.gov/sra) with accession number SRA149316 (Experiments SRX502094 (CECS), SRX502095 (CESS), SRX502096 (CHCS), SRX502097 (CHSS), SRX502098 (LRCS), SRX502099 (LRSS), SRX502100 (SACS) and SRX502101 (SASS)).

Correlation analysis

The Pearson coefficient was used to correlate the environmental and physicochemical parameters with diversity measured in operational taxonomic units (OTUs). They were analyzed by means of multivariate correlation analysis with ρ ≤ 0.05 using the STATGRAPHIC CENTURION XVI package.

Results

Pyrosequencing of the water samples cultured in trypticase soy broth enriched with NaCl yielded a total of 46,827 reads from which 32,807 reads (70.1%) passed the quality control stage. These reads were grouped according to lagoon and culture conditions. The genus Vibrio was present in 43.7% (14,337 reads), the related genera such as Aliivibrio, Photobacterium and Salinivibrio in 7.4% (2,420 reads) and other genera in 48.9% (16,050 reads).

This database permitted the calculation of the alpha diversity of each site, using the number of OTU's (sequence similarity cut-off 0.97) and the Shannon-Weaver and Chao2 diversity indices. Based on these values, the most diverse sites were determined to be Rosada Lagoon and Celestun Lagoon. Celestun Lagoon yielded richness values of 116 OTU's, a Shannon index of 3.0 and a Chao2 index of 126.5. Rosada Lagoon showed a richness of 132 OTU’s, a Shannon index of 3.7 and a Chao2 index of 143.1 (Table 1).

thumbnail
Table 1. Number of reads and diversity indexs of Vibrio spp for each composite sample.

https://doi.org/10.1371/journal.pone.0134953.t001

The analysis of sequences from the lagoons with most diversity index without enrichment showed that the community of Celestun was dominated by Geobacillus and Pseudomonas, representing over 70% of total reads whereas Vibrio represented only 0.04%. On the other hand, in Rosada Lagoon a more structured community was found and prevalent genera were Marinobacterium (26%), Pseudoalteromonas (15.5%), Synechococcus (7.8%), Ruegeria (7.2%), Bacillus (6.7%) and Planktaluna (6.6%) While Vibrio represented the 0.29% of total reads (data not shown).

Rarefaction analysis of the study sites (enriched samples) established that the sampling conditions were representative for determining the total diversity of the genus Vibrio. For each site studied, a rarefaction curve reached an asymptote at over 4,000 reads (Fig 1).

thumbnail
Fig 1. Rarefaction curve at 0.97 similarity of water samples from 4 coastal lagoons: Celestun (CE), Chelem (CH), Laguna Rosada (LR) and Sabancuy (SA); in the two culture conditions: CS (water samples grown tryptone soy broth + 3% NaCl) and SS (water samples cultured in tryptone soy broth).

https://doi.org/10.1371/journal.pone.0134953.g001

It was possible to confirm the halophilicity of the genus Vibrio by enrichment under two different NaCl concentrations. This characteristic allowed for the evaluation of the development of the genus Vibrio, with preferences for sodium, at sites with different salinity conditions (Fig 2).

thumbnail
Fig 2. Halophilicity of Vibrio species in the four coastal lagoons analyzed: Rosada lagoon (LR); Chelem (CH); Celestun (CE) and Sabancuy (SA). + NaCl (T.C.S. broth + 3% NaCl);—NaCl (T.C.S.).

% of OUT’s is referred to the total number of OTU´s classified within the genus Vibrio for each lagoon

https://doi.org/10.1371/journal.pone.0134953.g002

Over 50% of the OTU’s were found to belong to the Vibrionaceae family, which confirms the effectiveness of culturing Vibrio in TCS broth suplemented with NaCl. This effectiveness was clearly observed at sites where salinity was low, such as Celestun Lagoon, where a value of over 60% of OTU’s was obtained (Fig 3). Genera like Fusobacterium (1–20%), Cetobacterium (5–35%) and Clostridium and Shewanella (1–10%) were also identified in the enriched samples.

thumbnail
Fig 3. Effectiveness of the culture medium for the isolation of the family Vibrionaceae in four coastal lagoons: Rosada lagoon (LR); Chelem (CH); Celestun (CE) and Sabancuy (SA).

CS (water samples cultured in tryptone soya broth + 3% NaCl). SS (water samples cultured in tryptone soy broth).

https://doi.org/10.1371/journal.pone.0134953.g003

At the species level, pathogenic species such as V. cholerae (4.9%), V. parahaemolyticus (17.1%), and V. vulnificus (0.7%) were identified; as well as others of ecological importance such as V. mimicus (3.8%), V. harveyi (8.4%), V. proteolyticus (0.8%), V. fischeri (5.5%) and V. algynolyticus (0.1%). Unclassified Vibrio species represented 50.4% (Fig 4).

thumbnail
Fig 4. Prevalence of Vibrio spp in coastal lagoons of the Yucatan Peninsula.

https://doi.org/10.1371/journal.pone.0134953.g004

Different Vibrio species were identified at all sites. Very particular species were identified for each site, such as V. cholerae and V. mimicus in Celestun; V. harveyi in Chelem, and V. fischeri in Rosada Lagoon. However, the prevalence of some species was also observed in more than one lagoon, such as V. parahaemolyticus and V. vulnificus present in Rosada Lagoon and Chelem (Fig 5).

thumbnail
Fig 5. Taxonomic structure of the genus Vibrio in the four coastal lagoons studied: Rosada lagoon (LR) Chelem (CH); Celestun (CE) and Sabancuy (SA).

https://doi.org/10.1371/journal.pone.0134953.g005

In the study of beta diversity, the taxonomic dissimilarity between the different lagoons was determined, and sites that are geographically distant from each other or samples enriched under different conditions were found to present similarities between their Vibrio communities. This was the case with Sabancuy and Chelem lagoons, where a similar structure of Vibrio community was observed, despite the fact that they are very distant in geographical terms. Also in one site (Rosada Lagoon), the species found were observed to be highly similar for both samples enriched under different conditions. This similarity analysis can be seen in the heat map in Fig 6, where the color intensity shows the similarity between the species present.

thumbnail
Fig 6. Heat map based on Jaccard at 0.97 similarity of the prevalence of Vibrio spp in the 4 coastal lagoons: Chelem (CH), Sabancuy (SA), Laguna Rosada (LR) and Celestun (CE) in two culture conditions: tryptone soy + 3% NaCl (CS) and tryptone soya (SS).

https://doi.org/10.1371/journal.pone.0134953.g006

Correlations between the diversity of the genus Vibrio and environmental and physicochemical parameters were performed by multivariate analysis. The Pearson coefficient was obtained, establishing ρ ≤ 0.05 as a significant positive value. Celestun showed a significant positive correlation between the number of OTU’s and salinity (ρ = 0.0449). Likewise, Rosada Lagoon presented a significant positive correlation between the number of OTU’s and chlorophylls (ρ = 0.0255) (S1 Table). (http://doi.pangaea.de/10.1594/PANGAEA.847579).

Discussion

The Yucatan Peninsula is characterized by having estuary-lagoon systems that are shallow, with highly variable water residence times and salinity, generating high species diversity, especially of microorganisms [20]. This work comprises the first study of the composition of the taxonomic structure of the Vibrio community in the coastal lagoons of the Yucatan Peninsula. This genus is considered to be of great epidemiological and ecological importance.

Species diversity was determined by massive sequencing of metagenomics DNA obtained from enriched water samples. This tool produced a database that allowed to identifying the species that inhabit each of the lagoons and describing the taxonomic structure of the Vibrio community dwelling in these bodies of water. This information revealed that Rosada Lagoon was the most diverse site, yielding the highest values for the number of OTU’s and the Shannon and Chao2 diversity indices under the two culture conditions. This lagoon is a system characterized by having the highest salinity levels and being the least polluted of the studied sites and receives little influence from humans [20]. The high diversity of OTU’s can be caused by the fact that it is a hypersaline system, therefore providing many Vibrio species with the appropriate conditions for development. A large number of species from this genus need Na+ to grow and develop, and usually display optimum growth rates between 0.5 and 3.5% NaCl [2,8,16,29]. An example of this is V. parahaemolyticus. Its presence in this lagoon is because this system meets the salinity conditions required for its development, which are above 3.0% salt (S1 Table). This species is of great epidemiological importance, given that it causes severe gastroenteritis in humans if raw or undercooked contaminated seafood is consumed [4].

Species of great ecological importance were also identified in Rosada Lagoon, such as V. fischeri, which is a species that presents an interesting symbiosis with the squid Euprymna scolopes, providing it with bioluminescence thanks to the expression of the lux operon [29]. Another species of ecological as well as pathogenic importance is V. harveyi, a bioluminescent bacterium that produces a large quantity of degradative enzymes. However, pathogenic strains of this species have caused severe economic losses in aquaculture through the contamination of oysters and shrimp [19].

In Rosada Lagoon, a positive correlation was observed between OTU’s and chlorophyll-a. These data agree with other studies that show positive correlations between the diversity of the genus Vibrio and chlorophylls, in which the genus Vibrio has been observed to attach to phytoplankton, algae and zooplankton present in bodies of water, establishing a symbiosis between these organisms [3,5,8,30].

Not all of the species are halophiles, however, and some of them can develop under very low salinity conditions. These conditions are met by Celestun Lagoon, which is a mesohaline system caused by the effect of its geographical location on the “ring of cenotes” on the salinity gradients in its different zones [31]. This lagoon also yielded high values for species richness and diversity indices. V. cholerae was identified, which is the species that is the etiological agent of cholera and has serotypes and biotypes that have caused 7 epidemics around the world [32]. The highest percentages of this species from among the four study sites were found in this lagoon. The same applied to V. mimicus, which is the species that shares the greatest number of genotypic and phenotypic characteristics with V. cholerae and can cause severe gastroenteritis [33]. The hemolysins that it produces are very similar to those from pathogenic strains such as V. cholerae El Tor and the thermostable direct hemolysin (TDH) of V. parahaemolyticus. For this reason it is thought that V. mimicus could become an emerging pathogen [34]. V. cholerae and V. mimicus were identified only in this lagoon, given that their salinity requirements are minimal and they can even develop in fresh water. Celestun is also a lagoon subject to a strong anthropogenic influence as a result of touristic activities, causing it to become polluted [20]. Other species of epidemiological importance identified in this lagoon, were V. parahaemolyticus, V. fluvialis, V. harveyi and V. porteresiae in samples enriched in TCS + 3% NaCl. OTU’s identified in this sample were positively correlated to salinity, which agree with other studies that show positive correlations between salinity and the diversity of the genus Vibrio [15,16,35,36]. Other species, such as V. natriegens, which is capable of fixing atmospheric nitrogen into more available forms such as ammonia [37] were also identified. V. fluvialis was detected at a very low percentages in this lagoon; this bacterium has been reported to cause extraintestinal infections such as peritonitis, hemorrhagic cellulitis, bacteremias, cerebritis and otitis [38]. Other species found were V. vulnificus, present in oysters and reported as a human pathogen [39]; and V. shilonii, synonymous with V. mediterranei, and recognized as the causal agent of bleaching of the coral Oculina patagonica [40].

The genus Vibrio is a group of very cosmopolitan bacteria that develop in both freshwater systems (rivers and lakes) and in saline systems (seas or coastal bodies of water) [8]. At our studied sites, the levels of the environmental and physicochemical parameters are conditions that can vary depending on the season and geographical location, and these conditions influence the development of the different species of this genus.

Chelem Lagoon is considered the most polluted lagoon as a result of anthropogenic influences and is currently undergoing an eutrophication process [20,31]. It is surrounded by human settlements, and is also subject to significant touristic and fishing activity. In this lagoon, it was observed that the addition of 3% NaCl favored the most halophilic species, such as V. parahaemolyticus, for which it yielded the highest value of all of the studied sites. As mentioned previously, this species causes severe gastroenteritis in humans, and a large number of infections caused by this species have been reported on the coast of the Gulf of Mexico [41]. V. vulnificus another species of pathogenic importance for humans was also identified in this lagoon. This bacteria has a very fast-acting pathogenic mechanism, and in the United States 303 cases of infections were reported from 2000 to 2009, 148 of which were fatal [39]. Although low percentages of this species were reported at Chelem, its presence should not be ignored given the riverine fishing activity that takes place in this site. The same applies to V. parahaemolyticus. V. harveyi was detected in high number under both enrichment conditions. This species is considered a pathogen of some species of mollusks and fishes, especially shrimp [2,42]. This lagoon showed the highest values among all of the lagoons for OTU’s from this species, as well as for V. natriegens and V. fischeri, Finally, the highest assignation of reads to unclassified Vibrio (Vibrio spp) were for samples obtained from Chelem and enriched in TCS with 3% NaCl.

The fourth studied site, Sabancuy Estuary, is the most geographically distant from the other three sites and is located in the state of Campeche. It is considered an estuary, fed by the Terminos Lagoon and the sea [43]. It has variable salt concentrations. It yielded high OTU values for samples enriched in TCS with 3% NaCl which might indicate that these salinity variations may favored the development of species that are salt-dependent. In this lagoon, species such as V. parahaemolyticus, V. vulnificus, V. fischeri and V. shilonii were detected and a very high percentage of Vibrio spp obtained. V. cholerae and V. mimicus were not found in these samples explainable by reasons mentioned above. Other species such as V. neptunius, V. proteolyticus, V. sinaloensis were also observed in very small percentages.

Rarefaction and cluster analysis

Three main approximations for quantifying diversity were used to analyze Vibrio communities: species richness estimators, diversity indices and rarefaction analysis [44]. The latter was used to evaluate the “sampling effort” to quantify the diversity of the genus Vibrio at each site. This analysis related the number of base sequences with the number of OTU’s obtained, and from this relationship, a curve is obtained indicating that the total diversity has been sampled when it reaches an asymptote. These asymptotes were found to be greater than 4,000 for all of the sampled sites which means that sampling was representative in order to quantify the diversity of this genus at all the sites.

Cluster analysis showed the similarities and differences between the different Vibrio communities, based on the site and culture condition. This analysis was performed with the Jaccard index set at 0.97 similarity. The communities of vibrios of Sabancuy and Chelem (samples enriched in TCS with no added salt), despite the fact that the two sites are very different and geographically distant, are quite similar perhaps because these two lagoons have very similar salinity and other ecological parameters caused by the anthropogenic influence (both lagoons are surrounded by human settlements).

In the same way, Vibrio community of Rosada Lagoon showed very little differences in samples enriched with or without salt. It would seem that the addition of NaCl does not make a difference in terms of species proliferation maybe due to the fact that it is a hypersaline lagoon and autochthonous species are halophilic, such as V. parahaemolyticus and V. harveyi.

Celestun showed significant differences with respect to the other sites and grouped separately. Many of the species found at this site are very particular. For example, we found V. cholerae in very high percentages and V. mimicus in a lower abundance; none of these species were found in any other sample from other lagoon.

Conclusions

Knowledge of epidemiologically relevant bacterial groups such as Vibrio, in coastal environments where activities of fishing and tourism take place, is of crucial importance. As it was demonstrated in this study, the taxonomical structure of Vibrio communities in lagoons of the Yucatan Peninsula seems to be quite complex and variable and no significant correlation was found with environmental parameters except salinity and chlorophyll in particular lagoons. Besides, Vibrio species that may pose a threat to people who eat fish and shellfish extracted from these water bodies were detected. Moreover, the presence of species of biotechnological interest, as well as other species that might play a role in the ecosystem was evidenced. In this respect, the presence of species such as V. harveyi, which produce a large amount of degradative enzymes, may be indicative of an increase in the levels of contamination of these bodies of water, or because of their status of marine heterotrophic bacteria they could be acting as regulators of the major biogeochemical cycles that develop in these environments. These studies are particularly important for regions like the Yucatan Peninsula, given the vastness of coastal lagoons (ca. 19000 km2) and the influence they may have on extensive marine areas of the Gulf of Mexico.

Supporting Information

S1 Table. Physicochemical parameters of water sample the four coastal lagoons.

https://doi.org/10.1371/journal.pone.0134953.s001

(DOCX)

Acknowledgments

This work partially was supported by a grant from FOMIX Yucatán project 0165026 “Aprovechamiento sustentable del acuífero de Yucatán mediante tecnología metagenómica para la búsqueda de productos biotecnológicos con potencial de alto impacto económico”. Ortiz-Carrillo, I. was supported during this work by a doctoral fellowship (78008) from CONACyT (Consejo Nacional de Ciencia y Tecnología, Mexico). Authors are deeply indebted to Thomas Haverkamp and an anonymous reviewer as well as the Academic Editor whose pertinent comments have greatly improved the quality of this paper.

Author Contributions

Conceived and designed the experiments: IOC NEEG RRH MZM. Performed the experiments: IOC NEEG RRH MZM. Analyzed the data: IOC NEEG RRH MZM. Contributed reagents/materials/analysis tools: IOC NEEG RRH MZM. Wrote the paper: IOC NEEG RRH MZM. Technique ADN extraction: RRH.

References

  1. 1. Ceccarelli D, Colwell RR. Vibrio ecology, pathogenesis, and evolution. Front Microbiol 2014;5.
  2. 2. Thompson FL, Iida T, Swings J. Biodiversity of vibrios. Microbiol Mol Biol Rev 2004 Sep 1;68(3):403–31. pmid:15353563
  3. 3. Asplund ME, Rehnstam-Holm AS, Atnur V, Raghunath P, Saravanan V, Collin B, et al. Water column dynamics of Vibrio in relation to phytoplankton community composition and environmental conditions in a tropical coastal area. Environ Microbiol 2011;13(10):2738–51. pmid:21895909
  4. 4. Martinez . Ecological determinants of the occurrence and dynamics of Vibrio parahaemolyticus in offshore areas. The ISME Journal 2012;6:994–1006. pmid:22094349
  5. 5. Takemura AF, Chien DM, Polz MF. Associations and dynamics of Vibrionaceae in the environment, from the genus to the population level. Front Microbiol 2014;5.
  6. 6. Watnick P, Kolter R. Biofilm, City of Microbes. Journal of Bacteriology 2000 May 15;182(10):2675–9. pmid:10781532
  7. 7. chembri MA, Givskov M, Klemm P. An Attractive Surface: Gram-Negative Bacterial Biofilms. Sci STKE 2002 May 14;2002(132):re6. pmid:12011496
  8. 8. Urukawa H, Rivera N. Aquatic environment. The biology of vibrios. In: Press WDC, editor. The biology of vibrios. 2006. p. 175–89.
  9. 9. Baffone W, Tarsi R, Pane L, Campana R, Repetto B, Mariottini GL, et al. Detection of free-living and plankton-bound vibrios in coastal waters of the Adriatic Sea (Italy) and study of their pathogenicity-associated properties. Environ Microbiol 2006;8(7):1299–305. pmid:16817938
  10. 10. Nichols DS. Prokaryotes and the input of polyunsaturated fatty acids to the marine food web. FEMS Microbiology Letters 2003;219(1):1–7. pmid:12594015
  11. 11. Goecke F, Labes A, Wiese J, Imhoff JF. Chemical interactions between marine macroalgae and bacteria. Marine Ecology Progress Series 2010;409:267–300.
  12. 12. Jay D, Johnson C, Dillon S, Flowers R, Noriea F, Berutti T. What Genomic Sequence Information Has Revealed About Vibrio Ecology in the Ocean, A Review. Microb Ecol 2009;58(3):447–60. pmid:19727929
  13. 13. Riemann L, Azam F. Widespread N-Acetyl-d-Glucosamine Uptake among Pelagic Marine Bacteria and Its Ecological Implications. Appl Environ Microbiol 2002 Nov 1;68(11):5554–62. pmid:12406749
  14. 14. Weal Suginta. An endochitinase A from Vibrio carchariae: cloning, expression, mass and sequence analyses, and chitin hydrolysis. Arch Biochem Biophy 2004 Apr 15;424(2):171–80.
  15. 15. Gilbert JA, Steele JA, Caporaso JG, Steinbruck L, Reeder J, Temperton B, et al. Defining seasonal marine microbial community dynamics. ISME J 2012 Feb;6(2):298–308. pmid:21850055
  16. 16. Soto W, Gutierrez J, Remmenga M, Nishiguchi M. Salinity and Temperature Effects on Physiological Responses of Vibrio fischeri; from Diverse Ecological Niches. Microb Ecol 2009 Jan 1;57(1):140–50. pmid:18587609
  17. 17. Thompson JaPM. Dinamics of Vibrio populations and their role in environmental nutrient cycling. The biology of vibrios. In: Thompson FL, editor. The biology of vibrios.Washington, D.C.: ASM Press; 2006.
  18. 18. Nadell CD, Xavier JB, Levin SA, Foster KR. The Evolution of Quorum Sensing in Bacterial Biofilms. PLoS Biol 2008 Jan 29;6(1):e14. pmid:18232735
  19. 19. Yildiz FH, Visick KL. Vibrio biofilms: so much the same yet so different. Trends in Microbiol 2009 Mar 1;17(3):109–18.
  20. 20. Herrera SJ. Lagunas costeras de Yucatán:Investigación, diagnóstico y manejo. Soc Venez Ecol 2006;Ecot. 19(2):94–108.
  21. 21. Strickland JDH, Parsons TR. A practical handbook of seawater analysis. Fisheries Research Board of Canada; 1972.
  22. 22. Rojas R, Narvaez J, Zamudio M, Mena E. A Simple Silica-based Method for Metagenomic DNA Extraction from Soil and Sediments. Mol Biotechnol 2008;40(1):13–7. pmid:18373226
  23. 23. Muyzer G, de Waal EC, Uitterlinden AG. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 1993 Mar;59(3):695–700. pmid:7683183
  24. 24. Eiler A, Johansson M, Bertilsson S. Environmental Influences on Vibrio Populations in Northern Temperate and Boreal Coastal Waters (Baltic and Skagerrak Seas). Appl Environ Microbiol 2006 Sep;72(9):6004–11. pmid:16957222
  25. 25. Dowd S, Callaway T, Wolcott R, Sun Y, McKeehan T, Hagevoort R, et al. Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). BMC Microbiology 2008;8(1):125.
  26. 26. Gontcharova V, Youn E, Wolcott RD, Hollister EB, Gentry TJ, Dowd SE. Black Box Chimera Check (B2C2): a Windows-Based Software for Batch Depletion of Chimeras from Bacterial 16S rRNA Gene Datasets. The Open Microbiol J 2010 Aug 11;4:47–52. pmid:21339894
  27. 27. Ishak H, Plowes R, Sen R, Kellner K, Meyer E, Estrada D, et al. Bacterial Diversity in Solenopsis invicta and Solenopsis geminata Ant Colonies Characterized by 16S amplicon 454 Pyrosequencing. Microb Ecol 2011;61(4):821–31. pmid:21243351
  28. 28. Dowd S, Zaragoza J, Rodriguez J, Oliver M, Payton P. Windows.NET Network Distributed Basic Local Alignment Search Toolkit (W.ND-BLAST). BMC Bioinformatics 2005;6(1):93.
  29. 29. Nyholm SV, Nishiguchi MK. The evolutionary ecology of a sepiolid squid Vibrio association: From cell to environment. Vie Milieu Paris 2008;58(2):175–84. pmid:20414482
  30. 30. Soto W, Nishiguchi MK. Microbial Experimental Evolution as a Novel Research Approach in the Vibrionaceae and Squid-Vibrio Symbiosis. Front Microbiol 2014;5.
  31. 31. Tapia FU, Herrera J, Aguirre ML. Water quality variability and eutrophic trends in karstic tropical coastal lagoons of the Yucatan Peninsula. Est Coast Shelf Sci 2008 Jan 20;76(2):418–30.
  32. 32. Chan CH, Tuite AR, Fisman DN. Historical Epidemiology of the Second Cholera Pandemic: Relevance to Present Day Disease Dynamics. PLoS ONE 2013 Aug 22;8(8):e72498. pmid:23991117
  33. 33. Lutz C, Erken M, Noorian P, Sun S, cDougald D. Environmental reservoirs and mechanisms of persistence of Vibrio cholerae. Front Microbiol 2013;4.
  34. 34. Wang D, Wang H, Zhou Y, Zhang Q, Zhang F, et al. Genome Sequencing Reveals Unique Mutations in Characteristic Metabolic Pathways and the Transfer of Virulence Genes between V. mimicus and V. cholerae. PLoS ONE 2011 Jun 22;6(6):e21299. pmid:21731695
  35. 35. Gregoracci GB, Nascimento JR, Cabral AS, Paranhos R, Valentin JL, Thompson CC, et al. Structuring of Bacterioplankton Diversity in a Large Tropical Bay. PLoS ONE 2012 Feb 21;7(2):e31408. pmid:22363639
  36. 36. Nishiguchi MK. Temperature Affects Species Distribution in Symbiotic Populations of Vibrio spp. Appl Environ Microbiol 2000 Aug 1;66(8):3550–5. pmid:10919820
  37. 37. Criminger JD, Hazen TH, Sobecky PA, Lovell CR. Nitrogen Fixation by Vibrio parahaemolyticus and Its Implications for a New Ecological Niche. Appl Environ Microbiol 2007 Sep 15;73(18):5959–61. pmid:17675440
  38. 38. Ramamurthy T, Chowdhury G, Pazhani G, Shinoda S. Vibrio fluvialis: An Emerging Pathogen. Front Microbiol 2014;5.
  39. 39. Morrison S, Williams T, Cain A, Froelich B, Taylor C,. Pyrosequencing-Based Comparative Genome Analysis of Vibrio vulnificus Environmental Isolates. PLoS ONE 2012 May 25;7(5):e37553. pmid:22662170
  40. 40. Koren O, Rosenberg E. Bacteria Associated with Mucus and Tissues of the Coral Oculina patagonica in Summer and Winter. Appl Environ Microbiol 2006 Aug;72(8):5254–9. pmid:16885273
  41. 41. Johnson CN, Flowers AR, Noriea NF, Zimmerman AM, Bowers JC, DePaola A, et al. Relationships between Environmental Factors and Pathogenic Vibrios in the Northern Gulf of Mexico. Appl Environ Microbiol 2010 Nov 1;76(21):7076–84. pmid:20817802
  42. 42. Romalde JL, Dioguez AL, Lasa A, Balboa S. New Vibrio species associated to molluscan microbiota: a review. Front Microbiol 2014;4.
  43. 43. Gonzalez S, Torruco G. La fauna béntica del Estero de Sabancuy, Campeche, México. Rev Biol Trop 2001 Sep 19;49:31–45. pmid:11795160
  44. 44. Bacaro G. The spatial domain matters: Spatially constrained species rarefaction in a Free and Open Source environment. Ecol Complex 2012 Dec;12(0):63–9.