Microbial interactions in aquaculture - Probiotic roseobacters as a sustainable means to control fish pathogens

Abstract

Aquaculture is the fastest growing protein production sector in the world. A major bottleneck in aquaculture is bacterial infections, which can lead to great losses. Fish larvae of several commercially important finfish are fed live feed due to the lack of appropriate artificial feed formulations. Live feed cultures are high in nutrition, which promote growth of bacteria including fast growing opportunistic pathogens. Live feed can function as infection vectors for pathogenic bacteria. Thus, fish larvae are especially susceptible to bacterial infections. Antibiotics are still used to combat bacterial infections in fish larvae cultures as the larvae cannot be efficiently vaccinated, however, the risk of resistance development and the associated health risk have led to the search for sustainable alternatives. Two types of promising alternative biocontrol are the use of probiotic bacteria and bacteriophages (virus that infect bacteria) the socalled phage therapy. The probiotic Phaeobacter spp. are able to inhibit the growth of a range of pathogens including Vibrio spp. due to the production of the antimicrobial compound tropodithietic acid (TDA). Phaeobacter spp. have successfully been shown to inhibit Vibrio spp. in axenic, i.e., without background microbiota, as well as few non-axenic live feed systems. Furthermore, Phaeobacter spp. are able to protect the live feed Artemia as well as fish larvae of cod and turbot against the Vibrio spp. induce disease vibriosis. The purpose of this PhD project was to investigate if probiotic Phaeobacter spp. were able to inhibit fish pathogenic bacteria in non-axenic live feed cultures either alone or in combination with bacteriophages (the latter in collaboration with researchers from aquaculture and in the area of phage therapy). Furthermore, this PhD sought to examine the possible practical application of Phaeobacter based probiotics in commercial aquaculture via upscaling in live feed microalgae. The present PhD study has shown that Phaeobacter spp. are able to antagonise pathogenic vibrios in a range of non-axenic live feed systems, i.e., the microalgae Rhodomonas salina and Tetraselmis suecica and the zooplankton Artemia and copepod species Acartia tonsa with two log units or more. Furthermore, Phaeobacter spp. significantly increased survival of vibrio-challenged, as well as, non-challenged Artemia in non-axenic systems. We found that inhibition of pathogens by Phaeobacter spp. varied between different non-axenic systems and we speculated that this is due to the composition of microbiota in the non-axenic systems, which may or may not protect the pathogens. In collaboration with researchers from aquaculture and the phage therapy field, we tested the broad host-range bacteriophage KVP40s abilities to inhibit four different Vibrio anguillarum strains and protect turbot and cod against vibriosis. Mortality of Vibrio challenged turbot and cod larvae were decreased or delayed when treated with KVP40. Growth of indigenous pathogenic bacteria resulted in high mortality in the non-challenged larvae controls. However, treatment with KVP40 reduced larvae mortality in these cultures. KVP40 was also able to inhibit V. anguillarum in live feed cultures T. suecica and Artemia, however, regrowth of V. anguillarum was seen in the Artemia cultures. By combining the probiotic Phaeobacter inhibens with KVP40 as treatment in T. suecica and Artemia cultures, faster inhibition of V. anguillarum was seen compared to P. inhibens only and lower V. anguillarum counts compared to KVP40 only. A marginal improvement was observed in the protection of Artemia against vibriosis compared to P. inhibens only. We were to some extend able to upscale Phaeobacter spp. in T. suecica culture, however, as the Phaeobacter spp. did not grow well when changing from nonaerated cultures, with large surface area to volume ratio, to aerated cultures, with small surface area to volume ratio. In order to test if the upscaled Phaeobacter spp. retained their probiotic abilities, Artemia were fed the Phaeobacter infused algae and challenged with V. anguillarum. Only Phaeobacter piscinae S26 was able to inhibit V. anguillarum and no protection of Artemia was observed, however, we ascribe this to the low levels of probiont, not the loss of inhibitory ability. Summed up, this study demonstrates that Phaeobacter spp. can inhibit pathogenic vibrios in a range of non-axenic live feed systems, where they will establish themselves and can be scaled up if conditions favour it. Furthermore, if combined with phage therapy slightly improved inhibition profiles are seen.