Abstract
Streptococcosis is a bacterial disease in tilapia that produces economic losses, caused mainly by Streptococcus agalactiae and S. iniae. It is treated using oxytetracycline and florfenicol, which when inappropriately used promotes the selection of antibiotic resistance genes (ARGs). The disease has been mainly associated with stress events such as variations in temperature. The aims of the present study were (1) to detect by molecular methods two species of Streptococcus sp. in a tilapia farm, (2) to relate their presence to physicochemical parameters in the culture system, and (3) to detect the presence of ARGs in tilapia tissues and/or ponds. Tilapia grow-out ponds (n = 30) were sampled, collecting 15 individuals per pond. The physicochemical parameters of water were measured in each pond. Per pond, organs such as the liver, spleen, brain, and eyes were collected from each individual. Then, each organ type was pooled with the respective organ of the other individuals, processed for DNA extraction, and used for PCR analyses to determine the presence of S. agalactiae and S. iniae and for the detection of ARGs (tetM, tetO, fexA, and ermB). The correlations between the presence of S. agalactiae and water physicochemical parameters were determined. Sixty percent of the ponds and 46% of the organ pools were positive for S. agalactiae, whereas S. iniae was not detected. The positive samples showed the following resistance genes: tet(O) (29.1%), tet(M) (12.7%), and erm(B) (1.8%). A moderate but significant positive correlation was found between temperature and the presence of S. agalactiae. This work reported the molecular detection of two species of Streptococcus and ARGs, providing information that allows fast and effective control of these pathogens in tilapia farming. In addition, a future complementary study on Streptococcus sp. serotype distribution and antibiotic resistance genes from tilapia cultured in Costa Rica could also contribute to increase the knowledge of S. agalactiae infections in tilapia farming worldwide.
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Acknowledgements
To Hanzel Sanabria Arroyo and Karolina Ramos Jiménez for their assistance in molecular assays. Also to Rebeca Rojas Alfaro for her support in microbiological approach. To the Vicerrectoría de Investigación of Universidad Nacional for their support in the English language editing.
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The data that support the findings of this study are available from the corresponding author upon reasonable request.
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This work was financed by Fittacori funds # F33-17: “Determination of water quality and the presence of opportunistic pathogenic bacteria that affect the productivity of aquaculture systems in Costa Rica.”
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The authors contributed to the study as follows:
Karen Oviedo-Bolaños: designed study, collected material, analyzed data, and drafted paper.
Jorengeth Abad Rodríguez-Rodríguez: collected material, analyzed data, and drafted paper.
Carolina Sancho-Blanco: obtained economic funds, collected material, analyzed data, and drafted paper.
Juan Esteban Barquero-Chanto: obtained economic funds, collected material, and drafted paper.
Nelson Peña Navarro: obtained economic funds, analyzed data, and drafted paper.
Cesar Marcial Escobedo-Bonilla: designed study, analyzed data, and drafted paper.
Rodolfo Umaña-Castro: obtained economic funds, designed study, analyzed data, and drafted paper.
The first draft of the manuscript was written by Karen Oviedo-Bolaños, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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The animals used for this study were handled according to the Animal Welfare Law # 7451 of the Republic of Costa Rica.
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Supplementary information
ESM 1
Endpoint PCR sensitivity assay for the 16S rRNA region of DNA. Visualization using 1.5% agarose gel. a) Total DNA extracted from a field sample positive for S. agalactiae. b) DNA from S. agalactiae ATCC® 12386 ™ + negative field sample. Lanes show different concentrations of total DNA: 1 = 60 ng, 2 = 30 ng, 3 = 15 ng, 4 = 1 ng, 5 = 0.5 ng, 6 = 0.1 ng, 7 = 0.01 ng, 8 = 0.001 ng, E = negative control of PCR reaction (water). GeneRuler 1 kb DNA Ladder (Thermo Scientific). (JPG 65 kb)
ESM 2
Endpoint PCR sensitivity assay for the 16S rRNA region of DNA from different tilapia organs infected with S. agalactiae. Visualization using 1.5% agarose gel. A) Liver, B) Spleen, C) Brain, D) Eyes. Lanes show different total DNA concentrations for each organ tested: 1 = 30 ng, 2 = 15 ng, 3 = 1 ng, 4 = 0.5 ng, 5 = 0.1 ng, 6 = 0.01 ng, 7 = 0.001 ng. GeneRuler 100 bp DNA Ladder (Thermo Scientific). (JPG 92 kb)
ESM 3
Duplex PCR optimization assay for detection of S. agalactiae and S. iniae, with positive controls. Lanes show different annealing temperatures tested: 50, 50.8, 51.8, 53.2, 54.9, 56.8, 58.6, 60.0, 61.1, 61.7 and 62 °C. GeneRuler 1 kb DNA Ladder (Thermo Scientific). (JPG 48 kb)
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Endpoint PCR assay for detection of antimicrobial resistance genes Tet (M) (samples 1, 2, 3 and 4) and Tet (O) (samples 4 and 5). Lanes show some representative samples: 1 = pond 133 brain, 2 = pond 51 eyes, 3 = pond 133 eyes, 4 = pond 136A spleen, 5 = pond 49 eyes. GeneRuler 100 bp Plus DNA Ladder (Thermo Scientific). (JPG 30 kb)
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Oviedo-Bolaños, K., Rodríguez-Rodríguez, J.A., Sancho-Blanco, C. et al. Molecular identification of Streptococcus sp. and antibiotic resistance genes present in Tilapia farms (Oreochromis niloticus) from the Northern Pacific region, Costa Rica. Aquacult Int 29, 2337–2355 (2021). https://doi.org/10.1007/s10499-021-00751-0
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DOI: https://doi.org/10.1007/s10499-021-00751-0