Evaluation of Dietary Probiotic mixtures to improve Physiology and Health of L. rohita through gut microbiome Modulation under Mimic Aquaculture conditions

Abstract

Aquaculture is one of the fastest growing industries providing a great deal of high quality dietary protein worldwide. The aim of the present study was to beneficial modulation of fish (Labeo rohita) gut microbial communities by potential probiotics in mimic aquaculture conditions. This biobased solution is the eco-friendly alternative approach for enhancing productivity and infection control. With the perspective of Pakistan, although aquaculture is in infancy, but in future it seems to be a source of safe food and profitable economy. The present study was executed in three phases, during the first phase of study, the morphological and biochemical characterization of the isolates from gut of Labeo rohita were examined, followed by identification of selected strains. All the tested isolates were catalase negative and oxidase negative. The identified strains were Enterococcus hirae QAUF01, Bacillus cereus QAUBC02, Enterococcus faecium QAUF18 and E.mundtii QAUF20. The in vitro analysis of selected isolates (E. faecium QAUF18, E. mundtii QAUF20, B. cereus QAUBC02, E. faecium QAUEF01, E. hirae QAUEH01, G. candidum QAUGC01, F7, F8, F19, F20, O1, O2, O14 and O29) and combination of selected identified bacterial strains with G. candidum QAUGC01, (co-culutre of E. faecium QAUEF01 and G. candidum QAUGC01, co-culture of E. hirae QAUEH01 and G. candidum QAUGC01, co-culture of B. cereus QAUBC02 and G. candidum QAUGC01) was evaluated to check their efficacy as potential probiotic candidates. During the present study for the first time, co-culture of Geotrichum candidum QAUGC01 and bacterial strains were evaluated for synergistic probiotic characteristics. The studied parameters were acid tolerance, percentage survival in bile salts, hydrophobicity, antibiotic sensitivity and antibiotic resistance. The maximum adherence ability was observed in case of G. candidum QAUGC01 and E. faecium QAUEF01 co-culture (40.35±3.45). The B. cereus QAUBC02 and its co-culture with G. candidum QAUGC01 showed more than 50 % tolerance to bile salts at different time intervals. Maximum tolerance to acid at pH 2 and 5 was observed by E. faecium QAUEF01. G. candidum QAUGC01 exhibited antimicrobial activity against selected pathogens. All the strains were sensitive to vacomycin and varied results were obtained for other antibiotics. In the second phase of the study, the selected probiotics were applied as a feed supplement in trial of 90 days to evaluate their impact on physiology of L. rohita . The treatments used were G. candidum QAUGC01 (T1), E. faecium QAUEF01 (T2), E. hirae QAUEH01 (T3), B. cereus QAUBC02 (T4), combination of G. candidum QAUGC01 and E. faecium QAUEF01 (T5), combination of G. candidum QAUGC01 and E. hirae QAUEH01 (T6), combination of G. candidum QAUGC01 and B. cereus QAUBC02 and commercial probiotic (T8) were added to fish feed for ninety days . The control group was fed on basal diet (T0). All the probiotic treatments had significant impact on physiological parameters (growth, hematology, intestinal enzymes , body composition of the fish as compared to control fed group. Moreover, fishes fed on (T7) combination of G. candidum QAUGC01 and B. cereus QAUBC02 significantly grew faster and showed improved hematology. However, higher protease activity in gastrointestinal content was observed in fishes fed on G. candidum QAUGC01 and E. faecium QAUEF01, whereas significant higher activity of amylase and cellulase was observed in group fed (T4) B. cereus QAUBC02. Probiotics also improved the blood profile in comparison to the control fishes fed on basal diet. Higher protein content was found in fish carcasses fed on G. candidum QAUGC01 while fishes fed on G. candidum QAUGC01 and E. hirae QAUEH01 co-culture showed significantly higher fat content. The fishes fed were challenged with Staphylococcus aureus survived as compared to control fed fishes which exhibited mortality after pathogen exposure. The third phase was based on investigation of gut microbiology of L. rohita by culture dependent and culture independent techniques at the end of feeding trial. It was observed that feeding on probiotics had significantly alter the fish gut microbiology as compare to control fishes. Proteobacteria was dominant bacterial phylum in all treatments including control except E. faecium QAUEF01 that promoted dominance of Firmicutes. Fishes fed (T1) G. candidum QAUGC01 as single culture and (T7) G. candidum QAUGC01 co-culture with B. cereus QAUBC02 were dominated by Galactomyces geotrichum (30.18%). Higher reads of G. candidum QAUGC01 showed the survival and persistence capacity of G. candidum QAUGC01 in gut which consequently modulated the microbial communities. Furthermore, preliminary fish gut proteomic analysis revealed the presence of regulatory, stress and metabolic proteins produced by G.candidum QAUGC01. This work is the base line study for use of G. candidum QAUGC01 probiotic for commercial aquaculture based on the idea of probiotic based modulation of microbiome for healthy fish.