Genomic and Functional Evaluation of Two Lacticaseibacillus paracasei and Two Lactiplantibacillus plantarum Strains, Isolated from a Rearing Tank of Rotifers (Brachionus plicatilis), as Probiotics for Aquaculture

Abstract

Aquaculture plays a crucial role in meeting the increasing global demand for food and protein sources. However, its expansion is followed by increasing challenges, such as infectious disease outbreaks and antibiotic misuse. The present study focuses on the genetic and functional analyses of two Lacticaseibacillus paracasei (BF3 and RT4) and two Lactiplantibacillus plantarum (BF12 and WT12) strains isolated from a rotifer cultivation tank used for turbot larviculture. Whole-genome sequencing (WGS) and bioinformatics analyses confirmed their probiotic potential, the absence of transferable antibiotic resistance genes, and the absence of virulence and pathogenicity factors. Bacteriocin mining identified a gene cluster encoding six plantaricins, suggesting their role in the antimicrobial activity exerted by these strains. In vitro cell-free protein synthesis (IV-CFPS) analyses was used to evaluate the expression of the plantaricin genes. The in vitro-synthesized class IIb (two-peptide bacteriocins) plantaricin E/F (PlnE/F) exerted antimicrobial activity against three indicator microorganisms, including the well-known ichthyopathogen Lactococcus garvieae. Furthermore, MALDI-TOF MS on colonies detected the presence of a major peptide that matches the dimeric form of plantaricins E (PlnE) and F (PlnF). This study emphasizes the importance of genome sequencing and bioinformatic analysis for evaluating aquaculture probiotic candidates. Moreover, it provides valuable insights into their genetic features and antimicrobial mechanisms, paving the way for their application as probiotics in larviculture, which is a major bottleneck in aquaculture.