Isolation of putative probionts from cod rearing environment
Introduction
Proliferation of opportunistic and pathogenic microorganisms in intensive rearing systems is known to cause poor larval growth and high mortality rates (Munro et al., 1995). Antibiotics have been used to prevent and control bacterial diseases in aquaculture, but they can lead to the emergence of resistant bacteria and environmental problems (Serrano, 2005). Effective commercial vaccines against some fish pathogens are available, but not applicable to larvae due to their small size and immature immune system. There is an urgent need to control the microbiota in hatching facilities by using alternative approaches, like probiotics. “Probiotics” traditionally refers to a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance (Fuller, 1989). Such oral treatments may provide the most effects but, in aquaculture, surface treatments through rearing water may also be of importance (Gatesoupe, 1999). Also, a probiotic should have a proven safety and efficacy in the host (http://www.who.int/foodsafety/fs_management/en/probiotic_guidelines.pdf).
Available commercial probiotic products for aquaculture use are dedicated to warmwater species while none has been developed for coldwater species. Atlantic cod (Gadus morhua L.) is gaining importance as a farmed fish. It is important to ensure the sustainability of cod aquaculture by supporting its safe and effective development. In the search for probiotic candidates, the following criteria should be considered (Gatesoupe, 1999, Verschuere et al., 2000): the microorganisms should (1) be non-pathogenic to the host, (2) compete with or hinder the growth of undesirable microbes, (3) adhere to, develop within the host and (4) be indigenous to the environment to which it will be used. Many studies have demonstrated that the indigenous microbiota of fish or rearing environment can inhibit pathogen growth (Fjellheim et al., 2007, Joborn et al., 1997, Robertson et al., 2000, Spanggaard et al., 2001, Vijayan et al., 2006, Vine et al., 2004).
The purpose of this study was to isolate and characterize bacteria from healthy cod aquacultural environment for possible use as probiotics at early stages of cod rearing. A multi-level screening approach was selected to evaluate the inhibitory potential towards three fish pathogens, growth behaviour and metabolite production of inhibitory isolates as well as adhesion capacity to fish cell lines. To our knowledge, this is the first study reporting on a cod rearing microbiota screening for probiotics.
Section snippets
Sampling of cod rearing systems
Sampling of cod rearing systems was performed during seven visits to the Aquaculture Centre of the Marine Research Institute (Grindavik, Iceland) during the pre- and post-hatching period of 2004. The pre- and post-hatching periods consisted of 12 days at 8 °C in 25 l incubators and up to 56 additional days at 8–14 °C in hatchery silos/tanks (150 and 3400 l), respectively. Algal supplement (Nannochloropsis oculata, Instant Algae® Nannochloropsis, Reed Mariculture, USA) was used to shade the rearing
Bacterial isolation and determination of inhibitory activity towards pathogens
Phenotypic analysis of cod rearing microbiota discerned 185 groups out of the 447 isolates, among which 158 isolates were selected for partial 16S rRNA gene sequencing. This analysis resulted in an identification of 60 bacterial groups, of which 33 were singletons, while the rest of the isolates (n = 125) were divided into 27 contigs. Representative isolates, i.e. at least one isolate per genotypic group, were selected for inhibition studies and also 17 of the 185 phenotypic groups that were not
Discussion
In this study, aquacultural environment during pre- and post-hatching periods of cod was screened for putative probiotic bacteria. The approach chosen was based on a set of criteria focusing on competitive exclusion of pathogens, active metabolite production, strain identification, growth characteristics and adhesion capacity of putative probionts.
The inhibitory spectrum of representative isolates towards fish pathogens, a widely used characteristic for probiont determination, was verified. The
Conclusion
A multi-level screening of cod aquacultural isolates, based on several in vitro tests, was conducted in this study. Four strains that could be used as a mixed supplement to rearing water were identified as putative probiotic bacteria. Future in vivo trials will ascertain their safety in cod hatchery and determine their effects on larval survival, growth, development and composition of microbiota. This study stresses the importance of lactic acid bacteria in aquaculture.
Acknowledgements
This work was supported by a grant from the AVS Research Fund of The Ministry of Fisheries in Iceland, being part of the PhD studies of Mrs. Lauzon funded by Matis ohf. We would like to thank Agnar Steinarsson and the staff of the Aquaculture Centre of the Marine Research Institute for their assisstance during the sampling of cod rearing systems, Eyjólfur Reynisson for the preparation of PCR products for taxonomic identification and Sólveig K. Pétursdóttir for the 16S rRNA partial sequencing
References (34)
- et al.
Bacteriocins: modes of action and potentials in food preservation and control of food poisoning
Int. J. Food Microbiol.
(1995) - et al.
Basic local alignment search tool
J. Mol. Biol.
(1990) - et al.
Characterisation of bacterial communities associated with early stages of intensively reared cod (Gadus morhua) using Denaturing Gradient Gel Electrophoresis (DGGE)
Aquaculture
(2007) - et al.
Vibrionaceae dominates the microflora antagonistic towards Listonella anguillarum in the intestine of cultured Atlantic cod (Gadus morhua L.) larvae
Aquaculture
(2007) The use of probiotics in aquaculture
Aquaculture
(1999)- et al.
Effects of supplementing the feed to Atlantic cod (Gadus morhua) fry with lactic acid bacteria and immuno-stimulating peptides during a challenge trial with Vibrio anguillarum
Aquaculture
(1998) - et al.
Selection and identification of autochthonous potential probiotic bacteria from turbot larvae (Scophthalmus maximus) rearing units
Syst. Appl. Microbiol.
(2004) - et al.
Identification of potential probiotic starter cultures for Scandinavian-type fermented sausages
Int. J. Food Microbiol.
(2005) - et al.
Evidence for 2 bacteriocins produced by Carnobacterium piscicola and Carnobacterium divergens isolated from fish and active against Listeria monocytogenes
J. Food Protect.
(1995) - et al.
Absence of host specificity for in vitro adhesion of probiotic lactic acid bacteria to intestinal mucus
Vet. Microbiol.
(2003)
Use of Carnobacterium sp. as a probiotic for Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss, Walbaum)
Aquaculture
Universal chemical assay for the detection and determination of siderophores
Anal. Biochem.
Random amplification of polymorphic DNA (RAPD) typing of carnobacteria isolated from hindgut chamber and large intestine of Atlantic cod (Gadus morhua L.)
Syst. Appl. Microbiol.
A brackishwater isolate of Pseudomonas PS-102, a potential antagonistic bacterium against pathogenic vibrios in penaeid and non-penaeid rearing systems
Aquaculture
In vitro growth characteristics of five candidate aquaculture probiotics and two fish pathogens grown in fish intestinal mucus
FEMS Microbiol. Lett.
Medium for selective enumeration of lactic acid bacteria from foods
Appl. Microbiol.
Probiotics in man and animals
J. Appl. Bacteriol.
Cited by (35)
Selection of New Probiotics: The Case of Streptomyces
2018, Therapeutic, Probiotic, and Unconventional FoodsIsolation of TDA-producing Phaeobacter strains from sea bass larval rearing units and their probiotic effect against pathogenic Vibrio spp. in Artemia cultures
2016, Systematic and Applied MicrobiologyCitation Excerpt :Many studies have addressed the potential use of probiotics in aquaculture [22,72], however, a reproducible reduction of mortalities has only occasionally been achieved [49]. Bacteria belonging to the marine Roseobacter clade have been isolated and/or detected in marine larviculture systems [38,42,55], and Phaeobacter inhibens, P. gallaeciensis and Ruegeria mobilis have been isolated due to their antagonism against pathogenic V. anguillarum. This antagonism is partially due to production of the antibacterial compound tropodithietic acid (TDA) [17].
Vibrio lentus protects gnotobiotic sea bass (Dicentrarchus labrax L.) larvae against challenge with Vibrio harveyi
2016, Veterinary MicrobiologyCitation Excerpt :Comparable results were obtained by several other studies for Japanese flounder (Paralichthys olivaceus) and Atlantic cod (Gadus morhua) (Sugita et al., 2002; Fjellheim et al., 2007). A study by Lauzon et al. (2008) indicates that this is not always the case, as they found that 80% of the antagonistic bacteria obtained from cod rearing systems were Gram-positive, most of them belonging to the genus Lactobacillus. The use of 16S rRNA gene allows an accurate identification of Vibrios at the family and genus level.
Atlantic cod in the dynamic probiotics research in aquaculture
2014, Aquaculture
- 1
Present address: William Cook Europe ApS, Sandet 6, 4632 Bjaeverskov, Denmark.