Genomic Characterization of Bacillus pumilus Sonora, a Strain with Inhibitory Activity against Vibrio parahaemolyticus-AHPND and Probiotic Candidate for Shrimp Aquaculture

Abstract

Acute hepatopancreatic necrosis disease, caused by Vibrio parahaemolyticus strains carrying the pirA and pirB toxin genes (VpAHPND), has been causing great economic losses in Asia and America in the shrimp farming industry. Numerous strains are resistant to antibiotics. However, supplementation with probiotic antagonists has become a more desirable treatment alternative. Fourteen strains of microorganisms were assessed for their potential to inhibit VpAHPND in vitro activity. The bacteria with the highest activity were challenged with VpAHPND-infected Pacific white shrimp Litopenaeus vannamei. Furthermore, the genomic characteristics of probiotic bacteria were explored by whole-genome sequencing. We identified the Sonora strain as Bacillus pumilus, which possesses positive proteolytic and cellulolytic activities that may improve shrimp nutrient uptake and digestion. Challenge trials showed a low cumulative mortality (11.1%). B. pumilus Son has a genome of 3,512,470 bp and 3734 coding sequences contained in 327 subsystems. Some of these genes are related to the biosynthesis of antimicrobial peptides (surfactins, fengycin, schizokinen, bacilibactin, and bacilysin), nitrogen and phosphorus metabolism, and stress response. Our in vitro and in vivo findings suggest that B. pumilus Sonora has potential as a functional probiotic.

Keywords: Bacillus pumilus; Sonora strain; Vibrio parahaemolyticus-AHPND; antimicrobial peptides (AMP); probiotic; whole genome.

Figure 1

Antagonistic activity test of the isolate Sonora (Son) against Vibrio parahaemolyticus (VpAHPND) D11F. Analysis of inhibition zones produced by the well diffusion test.

Figure 2

Inhibition halo (mm) of the antagonist strains vs. VpAHPND. Data represent the means ± SD of triplicate determinations. Columns with different letters indicate that the means were significantly (p < 0.05) different, according to Tukey’s HSD test.

Figure 3

Double layer assay. Inhibition halo (mm) of antagonist strains against Vp AHPND D11F. Data represent the means ± SD of triplicate determinations. Columns with different letters indicate that the means were significantly (p < 0.05) different, according to Tukey’s HSD test.

Figure 4

Hemolytic activity of the isolated Sonora against VPAHPND D11F. Representative γ hemolysis (lack of hemolysis) H3M, H2, and Sonora isolates via the well diffusion test.

Figure 5

Inhibition of preformed biofilm. Data represent the means ± SD of triplicate determinations. ANOVA showed a significance level of p < 0.05. Columns with different letters indicate that the means were significantly (p < 0.05) different, according to Tukey’s HSD test.

Figure 6

Cumulative mortality of the bioassays with the Sonora isolate. Data represent the means ± SD of triplicate determinations. The positive control and Sonora strain showed significant (p < 0.001) differences in Dunnett’s multiple comparisons test.

Figure 7

Bacillus pumilus Sonora genome. Circular graphic visualization of the distribution of genome annotations. External to internal rings, contigs, CDS forward strand (+), CDS reverse strand (−), RNA genes (rRNA and tRNA), and GC content.

Figure 8

Schematic distribution of the categories of subsystems contained in the B. pumilus Sonora genome.

Figure 9

Phylogenetic tree of Bacillus pumilus Sonora (red circle) and its eleven related species based on 16S rDNA sequences. Multiple alignments of amino acids and nucleotides were concatenated into a data matrix via MUSCLE (v.3.8.1551). The maximum likelihood tree was constructed using RAxML v.8.2.12 software with 1000 bootstrap replicates to generate the support values in the tree.