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Metagenomic analysis of the bacterial microbiota associated with cultured oysters (Crassostrea sp.) in estuarine environments

Abstract

In this work, we identified the bacterial microbiota associated with farmed oystersin estuarine regions of four states in the north eastern region of Brazil. During the drought and rainy seasons, for eight months, twenty oysters were sampled seasonally from seven different marine farms. In the laboratory, DNA extraction, amplification, and sequencing of the 16S rRNA gene were performed to establish the taxonomic units. We identified 106 genera of bacteria belonging to 103 families, 70 orders, 39 classes, and 21 phyla. Out of the total, 40 of the genera represented bacteria potentially pathogenic to humans; of these, nine are known to cause foodborne diseases and six are potentially pathogenic to oysters. The most prevalent genera were Mycoplasma, Propionigenium, Psychrilyobacter, and Arcobacter. The results indicate the need for more systematic monitoring of bacteria of the genus Mycoplasma in oyster farming operations in the Brazilian north eastern region. Currently, Mycoplasma is not one of the microorganisms analysed and monitored by order of Brazilian legislation during the oyster production and/or commercialization process, even though this genus was the most prevalent at all sampling points and presents pathogenic potential both for oysters and for consumers.

Key words
contamination; cultivation; oyster farming; pathogenicity

INTRODUCTION

The close relationship between vital functions of bivalve molluscs and the environment in which they live makes these animals recognized as bioindicators for monitoring environmental quality, and their health can affect the food safety of consumers (Kim & Powell 2006KIM Y & POWELL EN. 2006. Relationships among parasites and pathologies in sentinel bivalves: Noaa status and trends “mussel watch” program. Bull Mar Sci 79: 83-112.).

Oysters, in addition to naturally inhabiting estuarine environments, are widely cultivated in these places where food is plentiful for its development (Dame 2012DAME RF. 2012. Bivalve filter feeders: in estuarine and coastal ecosystem processes. n. 33, New York, 579 p.). Oyster feeding takes place through filtration and capture of suspended particles present in the water. The phytoplankton and microzooplankton organisms, dissolved organic and inorganic material, and microorganisms such as bacteria are retained in the gills (Kach & Ward 2008KACH D & WARD JE. 2008. The role of marine aggregates in the ingestion of picoplankton size particles by suspension feeding molluscs. Mar Biol 153: 797-805.). Such a feeding mechanism causes the entire microbiota present in the oysters to be directly associated with the inhabited aquatic environment, and this microbiota varies according to environmental factors, such as salinity, bacterial load in water, temperature, feed, and anthropic activities and management during production (Prieur et al. 1990PRIEUR D, MEVEL G, NICOLAS JL, PLUSQUELLEC A & VIGNEULLE M. 1990. Interactions between bivalve molluscs and bacteria in the marine environment. Oceanogr Mar Biol 28: 277-352.).

Oysters naturally harbour a diverse bacterial microbiota, often composed of pathogenic bacteria, mainly by the species belonging to the genera Vibrio, Pseudomonas, Alcaligenes, Aeromonas, Flavobacterium, Bacillus, and Micrococcus (Paillard et al. 2004PAILLARD C, LE ROUX F & BORREGO JJ. 2004. Bacterial disease in marine bivalves, a review of recent studies: Trends and evolution. Aquat Living Resour 17: 477-498.). However, these bacterial species may also include some pathogens that are naturally present in cultured water, such as Vibrio parahaemolyticus and V. vulnificus, while other species are generally associated with the presence of faecal contamination in waters such as V. cholerae, Salmonella sp., Escherichia coli, Shigella sp., Campylobacterium fasci and Yersinia enterocolitica (USA 2020USA. 2020. Fish and fishery products hazards and controls guidance. Department of Health and Human Services - Food and Drug Administration - University of Florida. Florida, 498 p.).

In addition to representing risks to public health when oysters are consumed, these bacteria can also cause the death of farmed oysters, drastically affecting commercial scale enterprises (Fernandez et al. 2014FERNANDEZ NT, MAZON-SUASTEGUI JM, VAZQUEZ-JUAREZ R, ASCENCIO-VALLE F & ROMERO J. 2014. Changes in the composition and diversity of the bacterial microbiota associated with oysters (Crassostrea corteziensis, Crassostrea gigas and Crassostrea sikamea) during commercial production. FEMS Microbiol Ecol 88: 69-83.). The main bacterial genera that is present in some species of oysters (mainly Crassostrea) and known to cause mortality are Vibrio (Le Roux et al. 2002LE ROUX F, GAY M, LAMBERT C, WAECHTER M, POUBALANNE S, CHOLLET B, NICOLAS JL & BERTHE F. 2002. Comparative analysis of Vibrio splendidus related strains isolated during Crassostrea gigas mortality events. Aquat Living Resour 15: 251-258.); Nocardia (Friedman et al. 1991FRIEDMAN CS, BEATTIE JH, ELSTON RA & HEDRICK RP. 1991. Investigation of the relationship between the presence of a Gram positive bacterial infection and summer mortality of the Pacific oyster, Crassostrea gigas Thunberg. Aquaculture 94: 1-15.); Mycoplasma (Azevedo 1993AZEVEDO C. 1993. Occurrence of an unusual branchial mycoplasma-likeinfection in cockle Cerastoderma edule (Mollusca, Bivalvia). Dis Aquat Org 16: 55-59.); Rickettsia (Azevedo & Villalba 1991AZEVEDO C & VILLALBA A. 1991. Extracellular giant rickettsiae associated with bacteria in the gill of Crassostrea gigas (Mollusca, Bivalvia). J Invertebr Pathol 58: 75-81.) and Chlamydia (Renault & Cochennec 1995RENAULT T & COCHENNEC N. 1995. Chlamydia-like organisms in ctenidia and mantle cells of the Japanese oyster Crassostrea gigas from the French Atlantic coast. Dis Aquat Org 23: 153-159.).

One of the main problems in identifying bacteria is related to the need for bacterial culture in laboratory conditions. It is estimated that less than 0.1% of all known bacteria are cultured by traditional methods (Nocker et al. 2004NOCKER A, LEPO JE & SNYDER RA. 2004. Influence of an oyster reef on development of the microbial heterotrophic community of an estuarine biofilm. Appl Environ Microbiol 70: 6834-6845.). In this context, methods based on the sequencing of bacterial genes have favored studies of bacterial communities in organisms and in marine environments, since it allows the complete mapping of the bacteria present in a certain sample from direct sample analysis (Postollec et al. 2011POSTOLLEC F, FALENTIN H, PAVAN S, COMBRISSON J & SOHIER D. 2011. Recent advances in quantitative PCR (qPCR) applications in food microbiology. Food Microbiol 28: 848-861.), without the need to culture bacteria.

One of the most advanced analytical methods for this purpose is metagenomics, which can be used in the further characterization of complex bacterial communities. The method allows the identification of bacteria in samples obtained directly from the environment, thus eliminating the need for isolation and cultivation; the results are fast, selective and high sensitive since the methods are able to detect specific gene fragments (Petrosino et al. 2009PETROSINO JS, HIGHLANDER S, LUNA RA, GIBBS RA & VERSALOVIC J. 2009. Metagenomic pyrosequencing and microbial identification. Clin Chem 55: 5856-5866.).

This work aimed to identify and characterize the bacterial microbiota of oysters grown in estuarine regions of four states of north eastern Brazil using next-generation sequencing as an analytical tool.

MATERIALS AND METHODS

Study area

The study was carried out at seven oyster farms of four states of the Brazilian north eastern region. These properties were considered the most representative oyster farms in each state by the Brazilian Service of Support to Micro and Small Companies (SEBRAE), as they presented know-how, level of technology and volume of production in accordance with the average values from each state. All the sampling sites were identified by the city and state that they are located in (Figure 1): city of Macau (Macau-RN) and city Tibau do Sul (Tibau do Sul-RN), in Rio Grande do Norte state; city of Marcação (Marcação-PB), in Paraíba state; city of Passo de Camaragibe (Passo de Camaragibe-AL) and city of Barra de São Miguel (Barra de São Miguel-AL), in Alagoas state; and city of Brejo Grande (Brejo Grande-SE) and city of Indiaroba (Indiaroba-SE), in Sergipe state. Among the selected sampling sites, the adopted oyster farming model was based on three types of fixed suspended systems: 1) racks - plastic “pillows” positioned on a PVC “table” (Macau-RN, Tibau do Sul-RN, Marcação-PB, Passo de Camaragibe-AL, Barra de São Miguel-AL); 2) floating baskets - plastic baskets attached to a guide wire that was fixed to a set of stakes buried in the bottom (Brejo Grande-SE and Indiaroba-SE); and 3) BST®- adjustable long-line oyster system (Indiaroba-SE).

Figure 1
Location of the sampling sites of the farmed oyster used in the analysis of the bacterial microbiota.

Abiotic variables

The water temperature (°C), dissolved oxygen concentration (mg/L) (YSI® Pro 20, USA), salinity (g/L) (Instrutemp®, Brazil) and pH (Sensoglass® pH meter SP1400, Brazil) were measured along with the oyster sampling procedures. Precipitation data were obtained from the National Institute of Meteorology database (INMET 2016INMET. 2016. Instituto Nacional de Meteorologia [Online]. Brasil. Available: http://www.inmet.gov.br/portal/ 2016].
http://www.inmet.gov.br/portal/...
).

Sampling

Samples of Crassostrea gasar (Tibau do Sul-RN, Marcação-PB, Passo de Camaragibe-AL, Barra de São Miguel-AL, Brejo Grande-SE, and Indiaroba-SE) and Crassostre arhizophorae (Macau-RN) were collected during drought (Sep. and Oct. of 2015) and rainy (Mar. and Jun. of 2015) seasonal periods. Twenty oysters [height = 7.5 cm ± 1.8 (mean ± SD)] were collected at each sampling location during each sampling period. The sample size (n) was established through statistical calculation using the following formula:

n = N . Z 2 . p . ( 1 p ) Z 2 . p . ( 1 p ) + e 2 . ( N 1 )

Where N is the population of oysters, Z is the normalized standard variable associated with the level of confidence, p is the probability of the event, and e is the sampling error.

The collections were always carried out during low tides (ebb). At some sampling locations (Passo de Camaragibe-AL, Barra de São Miguel-AL, Brejo Grande-SE, and Indiaroba-SE), the oysters were occasionally submitted to air exposure. In others (Macau-RN, Tibau do Sul-RN, and Marcação-PB), they were always submerged during sampling.

The samples were sent by air to the Laboratory of Histology and Microbiology (LHM), belonging to the Integrated Group of Aquaculture and Environmental Studies, located at the Universidade Federal do Paraná (UFPR), in Curitiba, Paraná, Brazil. The transport period did not exceed 48 hours, as recommended by the Codex Alimentarius Manual (Codex Alimentarius 1978) and the National Plan for Hygienic-Sanitary Control of Bivalve Mollusks (PNCMB) (Brasil 2012BRASIL. 2012. Instrução Normativa Interministerial n° 07, 08 de maio de 2012. In: MINISTÉRIO DA PESCA E AQUICULTURA E MINISTÉRIO DA AGRICULTURA, PEA (Ed), Brasília: Ministério da Pesca e Aquicultura, p. 56.). For transportation, the oysters were placed in plastic bags and kept inside Styrofoam boxes (20 L) containing synthetic ice to ensure temperature maintenance (5.0 ± 1.5 °C). The temperature was monitored during the total period of transportation using portable temperature meters (Datalogger TagTemp Stick – Novus®, Brazil) that were set to measure temperature every five minutes. A cardboard foil was placed between the oysters and the synthetic ice to avoid direct contact between the samples and the ice.

Sample processing

Prior to DNA extraction, the oysters were externally cleaned with a sterile brush in running tap water. The valves were opened for the removal of the tissues and the intervalvar liquor. After homogenization of the sample in a stomacher (Marconi MA440, Brazil), 200 μL the homogenate was taken and stored in Eppendorf tubes for later extraction of total DNA using the Invitrogen kit (PureLink® Genomic DNA). The quality and quantity of the total DNA were evaluated by spectrophotometry at 260 nm and 280 nm of absorbance with a NanoDrop® 2000 spectrophotometer (ThermoScientific, USA).

The amplification and expression of the 16S rRNA gene were performed based on the methodology proposed by Caporaso et al. (2011)CAPORASO JG, LAUBER CL, WALTERS WA, BERG-LYONS D, LOZUPONE C, TURNBAUGH P, FIERER N & KNIGHT R. 2011. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci USA 108: 4516-4522., with adaptations described below. Briefly, the 16S rRNA gene amplification was performed by PCR analysis of the samples in a 10 μL system containing 5 μL of DNA (10 ng/L), 1 μL of universal primer mix (515F/806r), 1 μL of the first “adapter” and 5 μL of the Klentaq DV Readymix enzyme. Gene expression levels of the 16S gene were analysed in a thermocycler (Veriti 96 well Applied Biosystems, USA). The reactions were carried out at 94 °C for 3 minutes for initial denaturation of the DNA, followed by the amplification process (25 cycles at 94 °C for 45 seconds for final denaturation; 50 °C for 30 seconds for annealing; 68 °C for 1 minute for the initial extension and 10 minutes for 72 °C for the final extension) and kept in temperature-controlled conditions (0 - 4 °C).

For confirmation of the 16S gene amplification, the samples were tested on agarose gel (1%) in Tris / Borate / 1X EDTA buffer (0.09 M Tris-HCl, 0.09 M boric acid and 0.002 M EDTA). In the gel, 3 μL of the mixture with 2 μL of sample and 2 μL of FSUDS dye (bromophenol blue) were added to each well. After loading the gel, the samples were subjected to electrophoresis (one hour) at a constant electric voltage of 70 volts under 1X TBE buffer. Together with the samples, 3 μL of 1 Kb molecular weight marker was applied to the gel. The gel was stained with ethidium bromide (1%) (15 minutes), washed in water (10 minutes) and visualized in a UVP 3UV Transilluminator Imaging System.

The sequencing of the samples was performed on the next-generation Illumina MiSeq platform, which is capable of generating information on thousands of base pairs in a single run. The reagent kit used for sample sequencing was the MiSeq v2 500 cycle kit.

Data analysis

Sequence-generated 16S rRNA gene sequences were analysed according to the Quantitative Insights Into Microbial Ecology (QIIME®) protocol developed by Caporaso et al. (2010)CAPORASO JG ET AL. 2010. QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7: 335-336.. Shannon-Winner diversity and dominance analyses were performed using the Past software, version 2.17, for the measurement of the diversity of species at each sampling site through the use of categorical data and for the estimation of the predominance of bacterial species in each sample, respectively

The results obtained in the genetic analyses were also tabulated, ordered and classified according to their prevalence. It was established as a criterion of analysis that the prevalent genera would be those found in amounts equal to or greater than 5% in each sample analysed. The potential pathogenic role of the identified bacterial communities to humans and oysters was performed through scientific bibliographic review. A systematic search for articles was performed in scientific databases (i.e., Science Direct, Wiley, Springer Link, Google Scholar) using the IP of the UFPR.

After selection of the prevalent groups, the influence of the abiotic variables on the percentage of prevalent bacteria found in the samples was evaluated through multiple regression. Subsequently, Kruskal-Wallis test was performed to evaluate the occurrence of significant differences between the prevalent bacteria in each of the four sampling sites, between sampling sites and between seasons. All analyses were performed using Statistica 10.0 software (StatSoft®).

RESULTS

Bacterial microbiota

In the set of sample points evaluated, 106 genera of bacteria belonging to 103 families, 70 orders, 39 classes and 21 phyla were identified. The phyla with the highest prevalence were Tenericutes (21.7%), Spirochaetes (6.6%), Cyanobacteria (2.6%), and Fusobacteria (2.1%) (Table I). The prevalence of the other 17 phyla cumulatively reached 2.4% of the prevalence. Of all the genera found, 40 were of bacteria potentially pathogenic to humans, and only nine belonged to the group known to cause foodborne diseases (FD). Six genera [Mycoplasma (38.8%), Photobacterium (0.2%), Vibrio (0.2%), Pseudomonas (0.1%), Oceanospirillum (0.1%), and Alteromonas (0.1%)] were identified as potentially pathogenic to oysters (Table I).

Table I
The average percentage of phyla and genera of bacteria found in oysters cultivated in north eastern Brazil.

A large percentage of the 16S gene sequences (BD2-6, WH1-8, SC3-56, for example) were also observed for bacteria not yet specifically identified (named here as “unclassified”). Bacteria that were not adequately identified by the method were considered herein as “undefined” (Table I). After statistically comparing the data presented in Table I that presented a prevalence above 5%, no differences were detected among sampling sites (p = 0.86) or between drought and rainy seasons (p = 0.24).

When analysing the diversity index of Shannon-Winner for each sampling location during all sampling periods, it was observed that Tibau do Sul-RN presented higher bacterial diversity, followed by Marcação-PB and Macau-RN. On the other hand, the lowest diversity indexes were recorded in Indiaroba-SE, Camaragibe-AL, and Barra de São Miguel-AL. Indiaroba-SE presented greater genotype dominance due to the high percentage of Mycoplasma identified during the four sampling collections. For example, the prevalence of this genus was 71% in sample 2, while the other genera did not exceed 3.7% (Table II).

Table II
Dominance and Shannon-Winner diversity indexes obtained for bacteria present in oysters cultivated in north eastern Brazil.

Of all the genera identified, those with the highest prevalence (equal to or greater than 5%) in the analysed samples were Mycoplasma, Propionigenium, Psychrilyobacter and Arcobacter. The last three bacterial genera occur naturally in marine environments, while Mycoplasma is part of the microbiota normally found in oysters (Table III).

Table III
Prevalent bacteria (≥5%) in the samples of oyster cultivated in north eastern Brazil.

Abiotic variables

There was great variation between the results of measurements of abiotic variables in the water in all the collections, as would be expected in an estuarine environment. The oxygen concentration was below the limit considered tolerable by oysters (> 3 mg/L) (Mello 2007MELLO GL. 2007. Policultivo de ostras e camarões marinhos em viveiros de aquicultura. In: SEBRAE (Ed), FAEPE, Recife, p. 23.), during the first collection in Tibau do Sul-RN (1.61 mg/L) and the second in Brejo Grande-SE (1.08 mg/L). Some of the sampling time points at Brejo Grande-SE (collection 1, 2 and 4) and Indiaroba-SE (collection 1) presented temperatures above & range considered optimal (23-31 °C) (Ansa & Bashir 2007ANSA EJ & BASHIR RM. 2007. Fishery and culture potentials of the mangrove oyster (Crassostrea gasar) in Nigeria. Res J Biol Sci 2: 392-394.). Some of the sample points had lower salinity than those considered optimal for oysters (10-50 g/L) (Funo et al. 2015FUNO ICSA, ANTONIO IG, MARINHO YF & GÁLVEZ AO. 2015. Influência da salinidade sobre a sobrevivência e crescimento de Crassostrea gasar. Bol Inst Pesca 41: 837-847.). For these cases, during the first sampling collection, Tibau do Sul-RN presented the lowest value (1 g/L), followed by Marcação-PB (5 g/L), Passo de Camaragibe-AL (7 g/L) and Indiaroba-SE (7 g/L). The pH, in turn, was within the tolerance limits of oysters (6.7-8.7) (Morales 1986MORALES JC. 1986. Acuicultura marina animal. 3 ed., Madrid: Madrid Mundi-Prensa.) during all sampling collections (Table IV).

Table IV
Abiotic variables measured during the four sampling periods from oyster-farms in north eastern Brazil.

There were no multiple correlations (r2 = 0.02, p = 0.23) between the prevalent bacteria (> 5%) in the samples and the abiotic variables measured. However, when the genera were analysed separately for abiotic variables, a negative correlation of pH with Mycoplasma was observed (r2 = 0.25, p = 0.001).

DISCUSSION

With 96% sensitivity, the use of the molecular analysis tools adopted in the present work allowed the mapping of the bacterial microbiota of farmed oysters in the Brazilian north eastern region. This type of analysis has been carried out worldwide for several purposes, among which include the studies by King et al. (2012)KING GM, JUDD C, KUSKE C & SMITH C. 2012. Analysis of stomach and gut microbiomes of the eastern oyster (Crassostrea virginica) from coastal Louisiana, USA. PLoS ONE 7: 1-11. to evaluate the bacteria found in the stomachs of farmed oysters (C. virginica) in Louisiana, USA; the studies by Chauhan et al. (2014)CHAUHAN A, WAFULA D, LEWIS D & PATHAK A. 2014. Metagenomic assessment of the Eastern Oyster-associated microbiota. Genome Announc 2: 1-2., to describe the farmed oyster microbiota (C. virginica) in the Apalachicola Bay, USA; and the studies by Trabal et al. (2012)TRABAL N, MAZÓN-SUÁSTEGUI JM, VÁZQUEZ-JUÁREZ R, ASENCIO-VALLE F, MORALES-BOJÓRQUEZ E & ROMERO J. 2012. Molecular analysis of bacterial microbiota associated with oysters (Crassostrea gigas and Crassostrea corteziensis) in different growth phases at two cultivation sites. Microb Ecol 64: 555-569. to differentiate the bacterial microbiota present in juvenile and adult oysters of C. gigas and C. corteziensis farmed on the coast of Mexico. In Brazil, the only work that used the same methodological analysis to assess the bacterial microbiota in oysters held in different storage conditions was recently published by Ostrensky et al. (2018)OSTRENSKY A, HORODESKY A, FAORO H, BALSANELLI E, SFEIR MZT, COZER N, PIE MR, DAL PONT G & CASTILHO-WESTPHAL GG. 2018. Metagenomic evaluation of the effects of storage conditions on the bacterial microbiota of oysters Crassostrea gasar (Adanson, 1757). J Appl Microbiol 0: 1-9..

The high percentage of bacteria of the taxa Tenericutes, Spirochaetes, and Proteobacteria in the oysters analysed corroborates the data found by Madigan et al. (2014)MADIGAN TL, BOTT NJ, TOROK VA, PERCY NJ, CARRAGHER JF, BARROS LOPES MA & KIERMEIER A. 2014. A microbial spoilage profile of half shell Pacific oysters (Crassostrea gigas) and Sydney rock oysters (Saccostrea glomerata). Food Microbiol 38: 219-227. in oysters farmed in the Camden Haven, Australia. The authors verified that the microbiota of fresh Crassostrea gigas and Saccostrea glomerata comprised 53% Tenericutes, 27% Spirochaetes and 14% Proteobacteria, indicating a certain similarity in terms of the composition of the bacterial community of oysters grown in estuarine environments in regions as far away as north eastern Brazil and Australia.

Since the analysed oysters were cultivated for consumption, the presence of bacteria with pathogenic potential may represent a risk to the health of the consumer. Among the 40 bacterial genera identified, nine are responsible for foodborne diseases in humans. Of these nine genera, only the genus Staphylococcus (0.2%) is among the bacteria whose analysis is required by Brazilian legislation for commercialization and consumption [Resolution RDC N°12 of January 2001 of the Agência Nacional de Vigilância Sanitária (Brasil 2001BRASIL. 2001. Resolução-RDC Nº 12, de 02 de janeiro de 2001. In: SANITÁRIA, ANDV (Ed), Brasília: ANVISA, p. 37.) and Interministerial Normative Instruction MPA / MAPA Nº 07, of May 08, 2012 (Brasil 2012BRASIL. 2012. Instrução Normativa Interministerial n° 07, 08 de maio de 2012. In: MINISTÉRIO DA PESCA E AQUICULTURA E MINISTÉRIO DA AGRICULTURA, PEA (Ed), Brasília: Ministério da Pesca e Aquicultura, p. 56.)].

The presence of Staphylococcus in oysters is usually associated with the manipulation of these animals, as this bacterium is found in the skin and mucosa of humans. This genus is divided into positive and negative coagulase staphylococci, and among the known species, the S. aureus bacterium is associated with the risk of human food poisoning, causing fever and vomiting in patients (Leroy et al. 2016LEROY S, VERMASSEN A & TALON R. 2016. Staphylococcus: Occurrence and Properties. Reference Module in Food Science, França.). Staphylococcus can also cause bacteraemia, endocarditis and cutaneous infections (Tortora et al. 2012TORTORA GJ, FUNKE BR & CASE CL. 2012. Doenças microbianas da pele e dos olhos. Microbiologia, 10a ed., Rio Grande do Sul: Artmed, 967 p.). However, as the method used here does not allow differentiation of negative coagulase Staphylococcus, it was not possible to draw conclusions about the pathogenicity of the bacteria of this group as identified in the analyses performed. Other bacteria genera that cause foodborne disease (Arcobacter, Flavobacterium, Alteromonas, Bacillus, Clostridium, Sphingomonas, Vibrio, and Pseudomonas) have also been registered; these bacteria are capable of causing diseases or physiological disturbances in humans, such as vomiting, fever, diarrhoea and abdominal muscle pains (Tauxe 2002TAUXE RV. 2002. Emerging foodborne pathogens. Int J Food Microbiol 78: 31-41.). According to Kalyoussef & Feja (2014)KALYOUSSEF S & FEJA K. 2014. Foodborne Illnesses. Adv Pediatr 61: 287-312., when identified in foods, follow-up is necessary to determine preventive efforts, especially by including those bacteria genera in the mandatory reporting lists and thereby ensuring consumer food safety.

Of all the bacteria analysed that might present some pathogenicity to oysters (Mycoplasma, Photobacterium, Vibrio, Pseudomonas, Oceanospirillum, and Alteromonas), only Mycoplasma (ranging from 4.5 to 71% of the total number of the bacteria present in each sample) was dominant at all sampling locations, and the other genera did not exceed 0.3%. It is known that Mycoplasma proliferates and predominates in environments with high temperatures (King et al. 2012KING GM, JUDD C, KUSKE C & SMITH C. 2012. Analysis of stomach and gut microbiomes of the eastern oyster (Crassostrea virginica) from coastal Louisiana, USA. PLoS ONE 7: 1-11.). According to Jaffe et al. (2004)JAFFE JD ET AL. 2004. The complete genome and proteome of Mycoplasma mobile. Genome Res 14: 1447-1461., these optimal growth temperatures range from 20-37 °C, depending on the species.

The genus Mycoplasma was first isolated in fish (Tincatinca) (Kirchhoff & Rosengarten 1984KIRCHHOFF H & ROSENGARTEN R. 1984. Isolation of a motile mycoplasma from fish. J Gen Microbiol 130: 2439-2445.), although it has already been found by King et al. (2012)KING GM, JUDD C, KUSKE C & SMITH C. 2012. Analysis of stomach and gut microbiomes of the eastern oyster (Crassostrea virginica) from coastal Louisiana, USA. PLoS ONE 7: 1-11. in abundance in the stomachs of C. virginica grown in Terrebonne Bay, Louisiana, USA, and in the gills of C. gigas as evaluated by Wegner et al. (2013)WEGNER KM, VOLKENBORN N, PETER H & EILER A. 2013. Disturbance induced decoupling between host genetics and composition of the associated microbiome. BMC Microbiol 13: 1-12. in the bay Sylt-Rømø-Bight and the bay Hörnum Deep in Germany. Paillard et al. (2004)PAILLARD C, LE ROUX F & BORREGO JJ. 2004. Bacterial disease in marine bivalves, a review of recent studies: Trends and evolution. Aquat Living Resour 17: 477-498. reported that in bivalve mollusc larvae, the presence of Mycoplasma can cause infections, resulting in the consequent death of the animal. Azevedo (1993)AZEVEDO C. 1993. Occurrence of an unusual branchial mycoplasma-likeinfection in cockle Cerastoderma edule (Mollusca, Bivalvia). Dis Aquat Org 16: 55-59. associated the presence of Mycoplasma with the mortality of cockles (Cerastodermaedule) in the estuarine region of Aveiro, Portugal.

In humans, however, Mycoplasma is also considered potentially pathogenic and may cause allergic inflammation, pneumonia, diabetes mellitus and multiple sclerosis (Razin 1996RAZIN S. 1996. Mycoplasmas. In: Baron S (Ed), Medical Microbiology, Texas: University of Texas Medical Branch at Galveston.). In this study, no correlation was found between the environmental variables analysed and the bacteria present in oysters. However, it should be stressed that water samples and analyses were performed only at the time of oyster harvesting, with the objective of being used as an indicator of environmental quality. In extremely variable environments such as those that characterize the estuarine regions (Vilanova & Chaves 1988VILANOVA MFV & CHAVES EMB. 1988. Contribuição para o conhecimento da viabilidade do cultivo de ostra-do-mangue, Crassostrea rhizophorae (Guilding, 1828) (Mollusca: Bivalvia), no estuário do rio Ceará, Ceará, Brasil. Arq Ciênc Mar 27: 111-125.), these correlations would require a much larger sampling frequency to be clearly established. Nevertheless, a significant correlation between the pH and the presence of Mycoplasma was identified, with a reduction of the percentage of this microorganism in alkaline waters. Pereira et al. (2009)PEREIRA EL, CAMPOS CMM & MOTERANI F. 2009. Efeitos do pH, acidez e alcalinidade na microbiota de um reator anaeróbio de manta de lodo (UASB) tratando efluentes de suinocultura. Rev Ambient Água 4: 157-168. stated that the optimum pH for the growth of this genus of bacteria is between 6.5 and 7.5; therefore, there is a tendency for the amount of Mycoplasma to decrease at pH levels above 7.5 (the maximum pH reached 8.4 at the monitored collection points).

The pathogenicity of the second most prevalent genus, Propionigenium, is still unknown in both oysters and humans. Propionigenium has been found in marine sediments (Janssen & Liesack 1995JANSSEN PH & LIESACK W. 1995. Succinate decarboxylation by Propionigenium maris sp. nov., a new anaerobic bacterium from an estuarine sediment. Arch Microbiol 164: 29-35.) with an optimum temperature range of 30 to 37 °C (Schink 2006SCHINK B. 2006. The Genus Propionigenium. In: SPRINGER (Ed). The Prokaryotes: A handbook on the biology of bacteria, New York, p. 955-959.) and has been identified in abalones grown in Hokkaido, Japan (Tanaka et al. 2004TANAKA R, OOTSUBO M, SAWABE T, EZURA Y & TAJIMA K. 2004. Biodiversity and in situ abundance of gut microflora of abalone (Haliotis discus hannai) determined by culture-independent techniques. Aquaculture 241: 453-463.); in Mytilus galloprovincialis mussels collected at Lake Faro, Italy (Cappello et al. 2015CAPPELLO S, VOLTA A, SANTISI S, GENOVESE L, MARICCHIOLO G, BONSIGNORE M & YAKIMOV MM. 2015. Study of bacterial communities in mussel Mytilus galloprovincialis (Bivalvia: Mytilidae) by a combination of 16s Crdna and 16s Rdna Sequencing. Int J Microbiol Appl 2: 18-24.); and in marine urochordates (Ciona intestinalis) (Dishaw et al. 2013DISHAW L ET AL. 2013. The gut of geographically disparate Ciona intestinalis harbors a core microbiota. PLoS ONE 9: 1-8.).

The genus Psychrilyobacter, present in high percentages in the Tibau do Sul-RN and Brejo Grande-SE samples, is found in marine sediment and grows at low temperatures (Zhao et al. 2009ZHAO J, MANNO D & HAWARI J. 2009. Psychrilyobacter atlanticus gen. nov., sp. nov., a marine member of the phylum Fusobacteria that produces H2 and degrades nitramine explosives under low temperature conditions. Int J Syst Evol Microbiol 59: 491-497.). However, the pathogenic potential of Psychrilyobacter for oysters and humans is still unknown. According to Fernandez-Piquer et al. (2012)FERNANDEZ-PIQUER J, BOWMAN JP, ROSS T & TAMPLIN ML. 2012. Molecular analysis of the bacterial communities in the live Pacific oyster (Crassostrea gigas) and the influence of postharvest temperature on its structure. J Appl Microbiol 112: 1134-1143., the occurrence of Psychrilyobacter was associated with post-harvest oyster storage, regardless of the temperature used during that process (between 4 and 30 °C).

Arcobacter was among the most predominant bacteria at the Marcação-PB sampling location. This genus is associated mainly with faecal contamination of marine waters and is considered potentially pathogenic to humans because it causes gastroenteritis, endocarditis, peritonitis and diarrhoea when ingested (Collado & Figueras 2011COLLADO L & FIGUERAS MJ. 2011. Taxonomy, Epidemiology, and Clinical Relevance of the Genus Arcobacter. Clin Microbiol Rev 24: 174-192.). The pathogenic potential of Arcobacter for oysters remains unknown.

In addition to contaminated environments, Arcobacter has been isolated in seafood (fish, oysters, clams, and mussels) (Rathlavath et al. 2016RATHLAVATH S, MISHRA S, KUMAR S & NAYAK BB. 2016. Incidence of Arcobacter spp. in fresh seafood from retail markets in Mumbai, India. Ann Microbiol 66: 165-170.). Fernandez-Piquer et al. (2012)FERNANDEZ-PIQUER J, BOWMAN JP, ROSS T & TAMPLIN ML. 2012. Molecular analysis of the bacterial communities in the live Pacific oyster (Crassostrea gigas) and the influence of postharvest temperature on its structure. J Appl Microbiol 112: 1134-1143. observed high abundance, without dominance, of Arcobacter in the oyster C. gigas. Romero et al. (2002)ROMERO J, GARCÍA-VARELA M, LACLETTE JP & ESPEJO RT. 2002. Bacterial 16S rRNA gene analysis revealed that bacteria related to Arcobacter spp. constitute an abundant and common component of the oyster microbiota (Tiostrea chilensis). Microb Ecol 44: 365-371. reported that this genus is abundant and common in Chilean oysters of the species Tiostrea chilensis.

The results obtained in this study show the importance of next-generation genetic sequencing as an analytical tool for microbiological monitoring studies of and programmes for oysters. These studies show that there is a high diversity of bacteria in cultivated oysters, with a prevalence of those bacterial genera found naturally in the environment itself or in marine/estuarine organisms. Finally, these results recommend the systematic monitoring of bacteria of the genus Mycoplasma in oysters grown and commercialized in the north eastern region of Brazil. Currently, this genus is not on the list of microorganisms whose analysis and monitoring are required by Brazilian legislation during the production and/or commercialization of oysters, although Mycoplasma was the most prevalent genus at all the sampling locations and has pathogenic potential both for oysters and for consumers.

ACKNOWLEGMENTS

We thank the “Conselho Nacional de Desenvolvimento Científico e Tecnológico” (CNPq) for support of this study by awarding the Productivity in Research Scholarship to Dr. Antonio Ostrensky (process 302609/2013-0) and by financing the project-associated processes #403381/2016-9. We also thank “Serviço Brasileiro de Apoio às Micro e Pequenas Empresas” for financial support to make this work possible.

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Publication Dates

  • Publication in this collection
    26 June 2020
  • Date of issue
    2020

History

  • Received
    1 May 2018
  • Accepted
    16 Jan 2019
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