Evaluation of Safety and Probiotic Traits from a Comprehensive Genome-Based In Silico Analysis of Ligilactobacillus salivarius P1CEA3, Isolated from Pigs and Producer of Nisin S

Abstract

Ligilactobacillus salivarius is an important member of the porcine gastrointestinal tract (GIT). Some L. salivarius strains are considered to have a beneficial effect on the host by exerting different probiotic properties, including the production of antimicrobial peptides which help maintain a healthy gut microbiota. L. salivarius P1CEA3, a porcine isolated strain, was first selected and identified by its antimicrobial activity against a broad range of pathogenic bacteria due to the production of the novel bacteriocin nisin S. The assembled L. salivarius P1CEA3 genome includes a circular chromosome, a megaplasmid (pMP1CEA3) encoding the nisin S gene cluster, and two small plasmids. A comprehensive genome-based in silico analysis of the L. salivarius P1CEA3 genome reveals the presence of genes related to probiotic features such as bacteriocin synthesis, regulation and production, adhesion and aggregation, the production of lactic acid, amino acids metabolism, vitamin biosynthesis, and tolerance to temperature, acid, bile salts and osmotic and oxidative stress. Furthermore, the strain is absent of risk-related genes for acquired antibiotic resistance traits, virulence factors, toxic metabolites and detrimental metabolic or enzymatic activities. Resistance to common antibiotics and gelatinase and hemolytic activities have been discarded by in vitro experiments. This study identifies several probiotic and safety traits of L. salivarius P1CEA3 and suggests its potential as a promising probiotic in swine production.

Keywords: Ligilactobacillus; bacteriocin; megaplasmid; nisin S; probiotic.

Figure 1

Chromosome and plasmids map of L. salivarius P1CEA3 created using Proksee v1.0.0a6 server (not to scale). This map shows the spread of CDS, tRNA, rRNA, ncRNA, MGE (mobile genetic elements), CRISPR-Cas systems, antibiotic resistance, and the GC content shift.

Figure 2

Comparison of megaplasmids of different L. salivarius. A BLAST atlas diagram of seven megaplasmids of L. salivarius was generated using BLAST Ring Image Generator (BRIG), employing pMP1CEA3 as the reference replicon (the inner orange ring). Working outwards from pMP1CEA3, the next six rings represent query plasmids of the L. salivarius strains named as pLS2102-15_1, p612A, pR1, unnamed1, pHN3 and pMP118. Regions of diversity between the megaplasmids are shown in light colors (less than 70% identity) and white color (less than 50% identity). The CDS of pMP1CEA3 was projected inside the black ring backbone and outside the kilobase pair (kbp) ruler at the center of the figure. Gene clusters for bacteriocins Abp118 and nisin S are shown within the CDS in garnet and black colors, respectively.

Figure 3

(a) Abp118 and (b) nisin S bacteriocin gene clusters in pMP1CEA3 from L. salivarius P1CEA3 as compared to those in megaplasmids pLS2102-15_1, p612A, pHN3 and pMP118. BAGEL v.4.0 and SnapGene v.7.0.3 programs were used as templates to generate the diagram. ORFs are indicated by arrows and numbers, and gene identity and color denote those with known functions. ORFs marked with a dashed arrow and their gene identity with an asterisk indicate putative non-functional ORFs.