# In Vitro and In Vivo Evaluation of Autochthonous Probiotics and Their Effects on the Mucosal Health of Nile Tilapia (Oreochromis niloticus)

## TL;DR

This study explores the use of naturally occurring gut bacteria from Nile tilapia as probiotics to improve their intestinal and mucosal health.

## Contribution

The study identifies and evaluates autochthonous bacterial strains with probiotic potential for Nile tilapia, focusing on mucosal health effects.

## Key findings

- Two bacterial strains (C61 and T70) showed antagonistic activity against fish pathogens and produced digestive enzymes.
- Probiotic-fed fish exhibited changes in intestinal mucosal structure and increased mucus-secreting cells.
- Despite low Bacillus recovery, the probiotics caused subtle shifts in the intestinal microbial community.

## Abstract

Probiotics, which are beneficial microbes that support digestion and strengthen natural defences, offer a promising approach to support sustainable fish farming. In this study, bacteria naturally found in the gut of Nile tilapia were investigated to see if they could serve as probiotics. Two strains of bacteria were identified that could inhibit relevant fish pathogens and produce digestive enzymes. When these bacteria were added to the diet of the fish, it resulted in subtle shifts in intestinal microbial community composition. The probiotic-fed fish also displayed notable changes in the structure of their intestinal lining and in the production of mucus-secreting cells of the intestine and skin. These findings offer a positive contribution to tilapia probiotic research.

The host microbiome is a promising source of probiotics for aquaculture species including Nile tilapia. In this study, the probiotic potential of autochthonous bacterial isolates from Nile tilapia and carp mid-intestines were screened in vitro. Two isolates (C61 and T70), closely related to Bacillus subtilis, exhibited antagonistic activity against multiple pathogen species and demonstrated multiple digestive enzyme activities. Their antagonistic activity in Aeromonas hydrophila assays remained even under simulated intestinal juice (SIJ) exposure. Subsequently, C61 (PT1) and T70 (PT2) were added to experimental diets at log 7 CFU/g of diet, and fed to Nile tilapia (5.32 ± 0.12 g) for 40 days. There were no significant differences observed in the growth performance across treatments. Despite limited Bacillus intestinal recovery levels, 16S rRNA gene metabarcoding revealed subtle shifts in the intestinal microbial community composition of the probiotic-fed groups. In addition, the PT1 group showed significantly longer mucosal fold length, elevated intestinal and skin goblet cell levels, and higher skin goblet cell coverage compared to the control. These results indicate the potential benefits of the isolates as functional feed additives for enhancing the mucosal health of Nile tilapia, but their benefits were likely achieved through transient activity given the low level of Bacillus recovery in the intestine.