Identification and Isolation of Novel Probiotic Bacteria for use in Marine Aquaculture

Dennis McIntosh*, Eric J. Schott, Harold J. Schreier and Grant S. Blank
 
Delaware State University
Dover, DE 19901
 
dmcintosh@desu.edu

As disease is a major problem for the fish farming industry, many strategies have been taken to overcome this obstacle, including the addition of antibiotics and chemotherapeutics.  Applying preventive measures may lead to less reliance on the use of chemicals - disinfectants, pesticides and antimicrobials - that treat the symptoms of the problem rather than affecting a cure.  One strategy for controlling disease has been to utilize probiotics, which are live microbial supplements.  Through this project our goal was to identify novel species of probiotic bacteria from a euryhaline fish (Fundulus heteroclitus) that might be useful in a range of commercially important aquaculture species, and to conduct preliminary experiments to see if they had any real world benefits.  

We examined 230 bacterial isolates obtained from gills and intestines of pond-reared and wild-caught fish, which were classified by colony color, morphology, shape and texture.  Screening on selective TCBS agar revealed that all but 22 colonies were Vibrio or Vibrio-related spp.  The non-Vibrio strains were subsequently screened for their ability to inhibit growth of Fundulus pathogens V. damsela or V. harveyi (strain DNO1) and the shellfish pathogen Vibrio sp. B183 by disk diffusion assays.  In addition, we examined whether the isolates were capable of producing extracellular factors able to interfere with biofilm formation by the Fundulus pathogens.  At least three non-Vibrio bacteria from the fishes' GI tract that had the ability to inhibit growth of both DNO1 and V. damsela, and also inhibited growth of B183 as determined by the filter disk assay.  Analysis of these bacteria by 16S rRNA gene sequencing identified them as Shewanella sp. (ISO12), Bacillus sp. (ISO11) (related to B. cereus and B. thuringiensis) and Pseudomonadales sp. (ISO5).  All three genera include representatives that are used as probiotics in agriculture and aquaculture.  

Two trials were undertaken to assess the probiotic potential of these bacteria on F. heteroclitus larvae.  In both instances, we tested each of the four isolates plus a culture medium control.  Fish survival was not impacted by isolate application, but growth appeared to have been beneficially impacted by them.  In trial one, survival over the course of four weeks was not statistically significant between treatments (p = 0.528) but was higher (84% on average) than we had observed in earlier experiments using larval Fundulus.  For this reason, we conducted a second, longer trial.  Similar to trial one, after six weeks there was no difference in larval fish survival between treatments (p = 0.085, average = 86%), though there were differences in animal weight at the end of the study.  Fish in the media control were significantly smaller than fish in the ISO5 treatment (p = 0.019) and ISO12 treatment (p = 0.018) groups.  Our results provide preliminary support for the probiotic activity of these isolates in terms of growth rate; their ability to offer protection against pathogen challenges is the focus of future studies.