16S amplicon sequencing of the gastrointestinal microbiota of Cirrhinus reba and isolation of an autochthonous probiotic using culture based approaches

Abstract

The microbiome in the gastrointestinal tract (GIT) of fish influences host health in both beneficial and detrimental ways and understanding the gut microbiota requires 16S amplicon sequencing. Although, Cirrhinus reba (Hamilton, 1822) has achieved widespread recognition for its nutritional and commercial value, the carp are currently confronting production limits. Probiotic-based carp captive breeding may be an effective method for enhancing the production and health condition of the carp and it requires conventional culture-based analysis. Hence, a comprehensive study was performed on the intestinal microbiota of C. reba by both 16S amplicon sequencing and culture-based approaches. At the phylum level, Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Fusobacteria were dominating the GIT of the fish. Importantly, Gram-negative pathogenic genera like Pseudomonas, Serratia, Aeromonas, and Unclassified Clostridiales are found to be most abundant. Only Bacillus and Lactobacillus are found as the Gram positive bacterial genera among the top ten dominating bacteria in the GIT of the fish. The total load of culturable bacteria in the gut of the carp was 1.12 ± 0.26 × 10⁸ cfu/g comprising twenty-four different types of colonies. The Gram negative bacterial load in the GIT of the carp is 6.9 × 10⁷ cfu/g. Only 37.5% (4.2 × 10⁷ cfu/g) of the isolates were Gram-positive and rod-shaped. Among them, one intestinal isolate (PKS9A) had shown significant antagonism against four common fish pathogens. The isolate was positive for the production of extracellular digestive enzymes like Protease, Amylase, Cellulase, Xylanase, Lipase, Phytase. The isolate PKS9A exhibited sufficient probiotic properties like bile salt hydrolase activity, cell surface hydrophobicity, antibiotic sensitivity, and is non-hemolytic in nature. 16S rDNA sequencing and phylogeny analysis identified the isolate as Bacillus paramycoides PKS9A (Accession no.-OR003914). Hence, the isolate may be utilized as a potential autochthonous probiotic for the cultivation of C. reba to enhance the nutritional and health status of the carp and to protect it from further deterioration.