Competitive Exclusion Bacterial Culture Derived from the Gut Microbiome of Nile Tilapia (Oreochromis niloticus) as a Resource to Efficiently Recover Probiotic Strains: Taxonomic, Genomic, and Functional Proof of Concept

Abstract

This study aims to mine a previously developed continuous-flow competitive exclusion culture (CFCEC) originating from the Tilapia gut microbiome as a rational and efficient autochthonous probiotic strain recovery source. Three isolated strains were tested on their adaptability to host gastrointestinal conditions, their antibacterial activities against aquaculture bacterial pathogens, and their antibiotic susceptibility patterns. Their genomes were fully sequenced, assembled, annotated, and relevant functions inferred, such as those related to pinpointed probiotic activities and phylogenomic comparative analyses to the closer reported strains/species relatives. The strains are possible candidates of novel genus/species taxa inside Lactococcus spp. and Priestia spp. (previously known as Bacillus spp.) These results were consistent with reports on strains inside these phyla exhibiting probiotic features, and the strains we found are expanding their known diversity. Furthermore, their pangenomes showed that these bacteria have indeed a set of so far uncharacterized genes that may play a role in the antagonism to competing strains or specific symbiotic adaptations to the fish host. In conclusion, CFCEC proved to effectively allow the enrichment and further pure culture isolation of strains with probiotic potential.

Keywords: Aeromonas hydrophila; Bacillus; Lactococcus lactis; Nile tilapia; Streptococcus agalactiae; continuos-flow competitive exclusion culture (CFCEC); freshwater fishes; microbiome; probiotics; whole-genome sequencing.

Figure 1

Core-genome maximum-likelihood phylogeny of Lactococcus lactis subsp. lactis (a), Priestia megaterium (b), and Priestia aryabhattai (c) strain from NCBI (RefSeq Database, https://www.ncbi.nlm.nih.gov/refseq/ (accessed on 9 January 2022)) and Lactococcus lactis A12, Priestia megaterium M4, and Priestia sp. M10, respectively. A core-genome phylogenetic representation using IQ-Tree. The tree was built from core genes identified by Roary per each strain. The log-likelihood score for the consensus tree was constructed from 1000 bootstrap.

Figure 1

Core-genome maximum-likelihood phylogeny of Lactococcus lactis subsp. lactis (a), Priestia megaterium (b), and Priestia aryabhattai (c) strain from NCBI (RefSeq Database, https://www.ncbi.nlm.nih.gov/refseq/ (accessed on 9 January 2022)) and Lactococcus lactis A12, Priestia megaterium M4, and Priestia sp. M10, respectively. A core-genome phylogenetic representation using IQ-Tree. The tree was built from core genes identified by Roary per each strain. The log-likelihood score for the consensus tree was constructed from 1000 bootstrap.

Figure 2

UpSetR plot of Roary genes presence/absence file from the closest related genome to Lactococcus lactis A12 (a), Priestia megaterium M4 (b), and Priestia sp. M10 (c).

Figure 2

UpSetR plot of Roary genes presence/absence file from the closest related genome to Lactococcus lactis A12 (a), Priestia megaterium M4 (b), and Priestia sp. M10 (c).

Figure 2

UpSetR plot of Roary genes presence/absence file from the closest related genome to Lactococcus lactis A12 (a), Priestia megaterium M4 (b), and Priestia sp. M10 (c).

Figure 3

Antibacterial activity of the cell-free extracellular products (CFECP) of L. lactis A12, P. megaterium M4, and Priestia sp. M10 against S. agalactiae (SA) and A. hydrophila (AH). Capital letters indicate significant differences between each probiotic against the control in Streptococcus agalactiae treatment (p < 0.05). Lower-case letters indicate significant differences of each probiotic against the control in Aeromonas hydrophila treatment (p < 0.05). Data represent mean ± SEM (n = 3).

Figure 4

Hemolysis analysis picture results for (a) L. lactis A12, (b) P. megaterium M4, (c) Priestia sp. M10.

Figure 5

Evaluation of the survival to gastrointestinal conditions such as low pH (2 and 3) and bile salts of L. lactis A12, P. megaterium, Priestia sp. M10. (a): % survival pH 2 L. lactis A12, (b): % survival pH 2 P. megaterium M4, (c): % survival pH 2 Priestia sp. M10, (d): % survival pH 3 L. lactis A12, (e): % survival pH 3 P. megaterium M4, (f): % survival pH 3 Priestia sp. M10, (g): % survival bile salts L. lactis A12, (h): % survival bile salts P. megaterium M4, (i): % survival bile salts Priestia sp. M10.

Figure 5

Evaluation of the survival to gastrointestinal conditions such as low pH (2 and 3) and bile salts of L. lactis A12, P. megaterium, Priestia sp. M10. (a): % survival pH 2 L. lactis A12, (b): % survival pH 2 P. megaterium M4, (c): % survival pH 2 Priestia sp. M10, (d): % survival pH 3 L. lactis A12, (e): % survival pH 3 P. megaterium M4, (f): % survival pH 3 Priestia sp. M10, (g): % survival bile salts L. lactis A12, (h): % survival bile salts P. megaterium M4, (i): % survival bile salts Priestia sp. M10.