This Is AuburnElectronic Theses and Dissertations

Isolation and Characterization of Bacillus spp. as Potential Probiotics for Channel Catfish, Ictalurus punctatus




Ran, Chao

Type of Degree



Biological Sciences


Enteric septicemia of catfish (ESC), caused by the bacterium Edwardsiella ictaluri, is considered the most important bacterial disease of cultured channel catfish Ictalurus punctatus and is estimated to cost the industry $40 to $60 million yearly in economic losses. In addition to ESC, the recent epidemic outbreak of motile Aeromonas septicemia (MAS) of catfish caused by a highly virulent and emerging strain of Aeromonas hydrophila is a major threat to the catfish industry in southeastern United States. Control of ESC and MAS has been elusive using available treatment agents. A promising alternative approach for controlling catfish diseases is the use of probiotics. Bacillus strains were isolated and tested as potential probiotics for channel catfish in this research. Bacillus strains isolated from soil or the intestine of channel catfish were screened for their antagonism against E. ictaluri and A. hydrophila as well as their capacity to survive and persist in the intestine of channel catfish. Bacillus strains AP79, AP143, AP193, AP254 and AB01 that showed good antimicrobial activity and intestinal survival were incorporated into feed in spore form and fed to channel catfish for 14 days before they were challenged by E. ictaluri in replicates. They all conferred benefit in reducing catfish mortality. Four of the five Bacillus strains were tested in Vietnam and protection of striped catfish against E. ictaluri was also observed. The safety of the four strains exhibiting strongest in vivo protection activity was investigated in terms of the presence of plasmids or antibiotics resistance. A long term feeding experiment was conducted to investigate the effect of selected Bacillus strains on growth and innate immune response of channel catfish. Bacillus strain AP79, AP143, AP193 and AB01 were supplemented in feed and given to fish for 10 weeks. All the Bacillus feeding groups showed improved weight gain and feed conversion ratio, however the difference was not significant. Similarly, a marginal increase in the serum lysozyme and respiratory burst activity were observed in the Bacillus fed groups. Fish were subjected to E. ictaluri challenge after the 10 weeks feeding regime, and the AP193 fed group had a 12% reduction in mortality compared with control (P = 0.07). As an important parameter, the effect of Bacillus dosage on the probiotic effect was also evaluated. Fish were fed with AP193 supplemented diets at doses from 4 × 106 CFU/g to 4 × 108 CFU/g for two weeks and challenged with E. ictaluri. Decreased mortality was observed as the quantity of AP193 increased in the diet. The genome of Bacillus strain AP193 was determined by next-generation DNA sequencing. Genome analysis identified gene clusters for the production of polyketides (bacillaene, macrolactin and difficidin), lipopeptiedes (surfactin, bacillomycin D and fengycin) and a dipeptide antibiotic bacilycin. The recalcitrance of AP193 to uptake exogenous DNA made it very difficult to construct mutants deficient in the synthesis of specific antibiotics to determine their contribution to the biocontrol activity of AP193. Low efficiency transformation of AP193 was accomplished by isolation of plasmid from an E. coli ER2925 (dam dcm) strain followed by in vitro methylation of plasmid with cell–free extracts of AP193. A gene knock-out procedure was conducted that involved a temperature sensitive shuttle plasmid vector based on the pWV01 origin of replication, a two-step replacement recombination procedure and PCR screening of mutants in order to construct marker-free deletion mutants deficient in the biosynthesis of specific antibiotic(s). Using an agar diffusion test, the major secondary metabolites responsible for in vitro antagonism of AP193 against E. ictaluri and A. hydrophila were identified as difficidin, one of the three polyketides. A comparative genomic analysis was conducted on thirteen biocontrol strains from the Bacillus subtilis group. Phylogenetic analysis indicated that 6 of the Bacillus strains belong to Bacillus amyloliquefaciens subsp. plantum, a subspecies with Bacillus amyloliquefaciens FZB42 as the type strain, which was also included in the 13 strains. More than 11,000 pan-gene families comprising full sets of non-orthologous gene families were found within the genomes of the 13 strains, while the conserved core gene families were around 1,800. A pairwise BLASTp Matrix was generated to determine similarities between each two strains in terms of number and percentage of conserved gene families. The matrix revealed that the gene family similarity between any two Bacillus genomes ranges from 30% to 90%. Comparative analysis of the thirteen biocontrol strains with reference strains from Bacillus subtilis group with no biocontrol activity was carried out to identify unique genes present in the biocontrol strains that may be responsible for biocontrol activity. Fourteen unique genes were found present among all the 13 Bacillus strains. They express functions including signaling, transportation, secondary metabolite production, and carbon source utilization. Specifically, 70 unique genes were identified as shared by the 7 strains from Bacillus amyloliquefaciens subsp. plantum, including the complete difficidin biosynthetic pathway that was critical for the antibacterial activity of Bacillus strain AP193.