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Bacillus spp. as source of Natural Antimicrobial Compounds to control aquaculture bacterial fish pathogens

2018; Frontiers Media; Volume: 5; Linguagem: Inglês

10.3389/conf.fmars.2018.06.00129

2296-7745

Rafaela A. Santos, Aires Oliva‐Teles, María José Saavedra, Paula Enes, Cláudia R. Serra,

Identification and Quantification in Food

Event Abstract Back to Event Bacillus spp. as source of Natural Antimicrobial Compounds to control aquaculture bacterial fish pathogens Rafaela A. Santos1, 2, 3, 4*, Aires Oliva-Teles1, 2, Maria J. Saavedra2, 3, 4, 5, Paula Enes2 and Cláudia R. Serra2, 3, 4 1 Universidade do Porto, Biologia, Portugal 2 Centro Interdisciplinar de Pesquisa Marine e Ambiental (CIIMAR), Portugal 3 Centro de Investigação e Tecnologias Agroambientais e Biológicas, Universidade de Trás-os-Montes e Alto Douro (CITAB-UTAD), Portugal 4 Centro de Ciência Animal e Veterinária (CECAV), Portugal 5 Departamento de Ciências Veterinárias, Universidade de Trás os Montes e Alto Douro, Portugal Aquaculture industry is the world’s fastest growing food protein producer. It is indispensable to satisfy the world’s fish demand, being responsible for 50% of the global seafood consumption. Aquaculture sustainable development is however constantly defeated by bacterial disease outbreaks, a major constraint to the economical profitability of the industry according to the United Nations (FAO, 2016). The emergence of bacterial diseases is also associated with a misuse of antibiotics, posing a serious threat to the public health (WHO, 2014). Therefore, it is urgent to find new and natural alternatives that limit the occurrence of bacterial diseases, improving human and animal health. One promising alternative to promote global health is the use of probiotics, “live organisms which when administrated in the adequate amounts can confer a health benefit on the host” (Food and Agriculture Organization of the United Nations, 2001). Among the probiotics commonly used, Bacillus species are the most attractive organisms in the aquaculture field due to their capacity to produce endospores (highly resistant structures) which represents an advantage for industrial applications (such as fish feeds processing) without losing their characteristics (Hong et al., 2005; Cutting, 2011; Hai, 2015). Moreover, Bacillus are also known to produce Natural Antimicrobial Compounds (NACs) capable of antagonizing the bacterial growth and communication of Gram+ and Gram- pathogens, and when produced inside the animal’s intestine might constitute a barrier against pathogens proliferation (Barbosa et al., 2005; Abriouel et al., 2011; Sumi et al., 2014). Taking in consideration the Bacillus ubiquitous nature, in this work we aimed at isolating, identifying and characterizing different Bacillus spp. capable of producing NACs with antimicrobial activity against the major aquaculture bacterial fish pathogens. For that purpose, Sparus aurata, Diplodus sargus, and Dicentrarchus labrax were fed with the same commercial diet and their heat-treated intestinal contents were used to obtain the aerobic sporeforming intestinal bacteria. After selection and storage, all isolates were screened for antimicrobial activities against the major bacterial pathogens that include: Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas veronii, Aeromonas bivalvium, Vibrio anguillarum, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio vulnificus, Photobacterium damselae subsp. damselae, Photobacterium damselae subsp. piscicida, Tenacibaculum maritimum, Edwardsiella tarda, Shigella sonnei, Staphylococcus aureus. The potential production of NACs by Bacillus spp. was accessed through different antagonistic assays: (i) colony overlay assay to screen the isolate’s collection for presence of NACs; (ii) well-diffusion assay to evaluate the extra-cellular nature of the NACs; (iii) 96 microplate assay to analyse the growth performance of pathogenic bacteria in the presence of such compounds; and finally (iv) anti-biofilm assay to evaluate the potential of NACs in reducing biofilm formation, one of the mechanisms associated with pathogens virulence. Sporeformers with the most promising activities were identified by 16S rRNA gene sequencing followed by on-line phylogenetic analysis using Sequence Match software package through the Ribosomal Database Project 10 (http://rdp.cme.msu.edu/). A total of 176 isolates representing different colony morphologies and samples were obtained from the fish intestinal contents. Before the antimicrobial tests, spore production of each isolate was confirmed by phase-contrast microscopy, with approximately 98% of the isolates producing endospores of different sizes and shapes. Screening the entire collection of isolates for NACs production revealed that 52% displayed antimicrobial activity against at least one pathogen tested. From the colony overlay assay, the most promising 6 isolates capable of inhibiting the bacterial growth of Aeromonas spp., Vibrio spp., Photobacterium spp., Tenacibaculum sp., Staphylococcus sp., and Edwardsiella sp. were selected. By characterizing the localization (intra- or extra-cellular) of the inhibitory molecules, it was observed that cell-free supernatants of three out of the six selected isolates inhibited or reduced the growth of all pathogens tested, except for A. salmonicida. Additionally, these three isolates were capable of interfering with biofilm formation of Aeromonas spp., Vibrio spp., Photobacterium spp., Tenacibaculum sp., Staphylococcus sp., and Shigella sp.. Interestingly, the cell-free supernatants of those three promising strains were not capable of interfering with bacterial growth, but significantly decreased the biofilm production in A. salmonocida. The results here presented reveal that a great diversity of sporeformers can be found in association with the gastrointestinal tract of fish. Moreover, more than 50% of the sporeforming community is capable of producing antimicrobial substances with activity against major aquaculture fish pathogens, known to cause huge economic losses in the aquaculture sector. These in vitro tests, allowed the selection of three strains with important characteristics including antimicrobial and anti-biofilm extracellular compounds, and the absence of antimicrobial resistances. From the 16S rRNA identification the three strains are Bacillus subtilis, a species generally regarded as safe by the European Food Safety Authority (EFSA-FEEDAP, 2012). These strains and their NACs are being further studied to identify and characterize the disease-preventive molecules. Acknowledgements R. A. Santos, P. Enes and C. R. Serra were supported by the grants SFRH/BD/ 131069/2017, SFRH/BPD/101012/2014, SFRH/BPD/101038/2014, respectively from FCT (Foundation for Science and Technology), under the POCH program. The authors thank F. Tavares (CIBIO-InBIO), MA Morinigo (Univ. Málaga) for the gift of pathogenic bacterial strains, and A. Henriques (ITQB-NOVA) for the gift of B. subtilis 168. References Abriouel, H., Franz, C.M., Ben Omar, N., and Galvez, A. (2011). Diversity and applications of Bacillus bacteriocins. FEMS Microbiol Rev 35(1), 201-232. doi: 10.1111/j.1574-6976.2010.00244.x. Barbosa, T.M., Serra, C.R., La Ragione, R.M., Woodward, M.J., and Henriques, A.O. (2005). Screening for Bacillus Isolates in the Broiler Gastrointestinal Tract. Applied and Environmental Microbiology 71(2), 968-978. doi: 10.1128/AEM.71.2.968-978.2005. Cutting, S.M. (2011). Bacillus probiotics. Food Microbiology 28(2), 214-220. doi: http://dx.doi.org/10.1016/j.fm.2010.03.007. EFSA-FEEDAP (2012). Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance, European Food Safety Authority Panel on Additives and Products or Substances used in Animal Feed (EFSA-FEEDAP). EFSA Journal 10(6), 2740. doi: 10.2903/j.efsa.2012.2740. FAO (2016). The State of World Fisheries and Aquaculture. Rome. Food and Agriculture Organization of the United Nations, W.H.O. (2001). "Report of the Joint FAO/WHO Expert Consultation on Evaluation of Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria". (Córdoba, Argentina). Hai, N.V. (2015). The use of probiotics in aquaculture. J Appl Microbiol 119(4), 917-935. doi: 10.1111/jam.12886. Hong, H.A., Duc, L.H., and Cutting, S.M. (2005). The use of bacterial spore formers as probiotics. FEMS Microbiology Reviews 29(4), 813-835. doi: 10.1016/j.femsre.2004.12.001. Sumi, C.D., Yang, B.W., Yeo, I.-C., and Hahm, Y.T. (2014). Antimicrobial peptides of the genus Bacillus: a new era for antibiotics. Canadian Journal of Microbiology 61(2), 93-103. doi: 10.1139/cjm-2014-0613. WHO (2014). Antimicrobial Resistance: Global Report on Surveillance Keywords: Aquaculture, Fish Diseases, Sporeformers, Bacillus spp., Natural antimicrobial compounds Conference: IMMR'18 | International Meeting on Marine Research 2018, Peniche, Portugal, 5 Jul - 6 Jul, 2018. Presentation Type: Oral Presentation Topic: Aquaculture Citation: Santos RA, Oliva-Teles A, Saavedra MJ, Enes P and Serra CR (2019). Bacillus spp. as source of Natural Antimicrobial Compounds to control aquaculture bacterial fish pathogens. Front. Mar. Sci. Conference Abstract: IMMR'18 | International Meeting on Marine Research 2018. doi: 10.3389/conf.FMARS.2018.06.00129 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 May 2018; Published Online: 07 Jan 2019. * Correspondence: Miss. Rafaela A Santos, Universidade do Porto, Biologia, Porto, 4169-007, Portugal, rafaela.santos@ciimar.up.pt Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Rafaela A Santos Aires Oliva-Teles Maria J Saavedra Paula Enes Cláudia R Serra Google Rafaela A Santos Aires Oliva-Teles Maria J Saavedra Paula Enes Cláudia R Serra Google Scholar Rafaela A Santos Aires Oliva-Teles Maria J Saavedra Paula Enes Cláudia R Serra PubMed Rafaela A Santos Aires Oliva-Teles Maria J Saavedra Paula Enes Cláudia R Serra Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.