A New Isolate of Pediococcus pentosaceus (SL001) With Antibacterial Activity Against Fish Pathogens and Potency in Facilitating the Immunity and Growth Performance of Grass Carps

Abstract

Probiotic-feeding continues to be a promising strategy to control the bacterial pathogens in aquaculture. A new Pediococcus pentosaceus strain (SL001) was isolated from 1000s of soil samples, which exhibited wide antimicrobial spectrum of against fish pathogens, involving Aeromonas hydrophila, Aeromonas veronii, Aeromonas sobria, Edwardsiella tarda, Lactococcus garvieae, and Plesiomonas shigelloide. The challenge test against A. hydrophila showed that the survival rate of SL001-supplemented group was significantly higher than that of control group (P < 0.05). Moreover, SL001 could stably colonize in gut of grass carp and increased mucus-secreting goblet cells and extended intestinal villi could be observed in SL001-supplemented group (P < 0.05). Feeding with SL001 supplemented diet could significantly enhance the growth rate (P < 0.05) and markedly affect gut microbiota structure of grass carps, resulting in reduced potential pathogens and increased potential probiotics. Furthermore, feeding grass carps with SL001 caused the up-regulated expression of insulin-like growth factor (IGF-1 and IGF-2) and down-regulated expression of myostatin (MSTN-1 and MSTN-2) (P < 0.05), which probably also account for the increased growth rate of SL001-fed group. Meanwhile, relative mRNA expression levels of immune-related genes in liver, spleen, and head kidney were analyzed in grass carps after feeding for 30 days with SL001 supplemented diets. In all three immune organs, the expression levels of immunoglobulin M (IgM) and complement 3 (C3) were significantly increased (P < 0.05), whereas the interleukin-8 (IL-8) was down-regulated (P < 0.05). Besides, whole genome sequencing revealed several probiotics properties of SL001, including organic acid synthesis, bacteriocin synthesis (coagulin), superoxide dismutase, and digestive enzymes. In conclusion, P. pentosaceus SL001 which could enhance immunity and promoter growth rate of grass carps, is prospective to be used as a dietary probiotic in freshwater fish aquaculture.

Keywords: Pediococcus pentosaceus; antibacterial activity; fish immunity; grass carps; growth rate; gut microbiota.

FIGURE 1

The characterization and antimicrobial activity of Pediococcus pentosaceus SL001. (A) Scanning electron microscope (SEM) of SL001. (B) The inhibition zones of SL001 against fish pathogens. CFS, cell-free supernatant (pH 3.48); MRS, Man Ragosa Sharpe medium; PL, phosphate-buffered saline added lactic acid (pH 3.48). (C) The phylogenetic tree based on 16S rRNA gene sequences inferred evolutionary relationships of strain SL001 by neighbor-joining method.

FIGURE 2

The morphology of grass carp hepatic L8824 cell line after treatment with SL001. Medium or bacterial culture applied to L8824 cells and incubated for 12, 24, and 36 h, respectively. MRS, Man Ragosa Sharpe medium (negative control); Ah CFS, cell free culture supernatant of Aeromonas hydrophila (positive control); SL001 CFS, cell free culture supernatant of SL001; SL001, cell pellet of SL001.

FIGURE 3

Challenge test to detect the protection of SL001 against A. hydrophila. The survival rates of grass carp after A. hydrophila infection were measured. Each value represents mean ± SEM (n = 3).

FIGURE 4

Photomicrographs of the different intestinal site of grass carp after 30 days of feeding with SL001-supplemented diet (n = 6, data from one fish is presented). Enterocytes (white arrow), goblet cells (red arrow), lamina propria (indicated by yellow lines) are shown in the figure. PA, SL001-supplemented group; DB, without SL001 supplemented group.

FIGURE 5

The expression levels of genes involved in muscle growth in grass carp. (A) Genes involved in restraining muscle growth (MSTN-1 and MSTN-2). (B) Genes involved in promoting muscle growth (IGF-1 and IGF-2). Values represented mean ± SEM. Each gene expression level in control group was regarded as 1. ^∗P < 0.05, ^∗∗P < 0.01 (n = 4). PA, SL001-supplemented group; DB, without SL001 supplemented group.

FIGURE 6

The influence of SL001 on gut microbiota of grass carps. V3–V4 region of bacterial 16S rRNA genes were sequenced and analyzed on 30 dpf grass carps. (A) The rarefaction curves plot of all samples. (B) Relative abundance in phyla level. (C) Stacked bar chart about the relative abundance in genus level. (D) Two dimensional principal coordinates analysis (PCoA) graph based on weighted UniFrac distances. (E) Venn diagram on shared and unique OTUs in SL001-supplemented group and without SL001 supplemented group. (F) The phylogenetic tree was constructed using unweighted pair group method with arithmetic mean (UPGMA). PA, SL001-supplemented group; DB, without SL001 supplemented group.

FIGURE 7

The effect of SL001-feeding on the gene expression of immune-related cytokines in the grass carp. The gene expression levels of immune-related cytokines in (A) liver and (B) spleen were measured by qRT-PCR. Values represented mean ± SEM. Each gene expression level in control group was regarded as 1. ^∗P < 0.05, ^∗∗P < 0.01 (n = 4). PA, SL001-supplemented group; DB, without SL001 supplemented group.

FIGURE 8

The relative gene expression level of immune-related cytokines in grass carps in challenge test. The grass carp were fed with or without SL001-supplemented diet for 30 days and then infected with A. hydrophila. The gene expression levels of immune-related cytokines in liver (A,D) and spleen (B,E) and head-kidney (C,F) were analyzed (A–C) were at 6 dpi (D–F) were 12 dpi. Values represented mean ± SEM. Each gene expression level in control group was regarded as 1. ^∗P < 0.05, ^∗∗P < 0.01 (n = 4). PA, SL001-supplemented group; DB, without SL001 supplemented group.

FIGURE 9

The enzyme activity of non-specific immunological factors from serum of grass carp. Values represented mean ± SEM. ^∗P < 0.05, ^∗∗P < 0.01 (n = 3). PA, SL001-supplemented group; DB, without SL001 supplemented group.

FIGURE 10

The annotation and comparison of P. pentosaceus SL001 genome. (A) The distribution of predicted CDSs of SL001 in different categories of metabolic function. (B) A full genome comparison analysis of P. pentosaceus SL001 with P. pentosaceus strains. P. pentosaceus wikim20; P. pentosaceus SL4; P. pentosaceus SRCM100892; P. pentosaceus KCCM 40703; P. pentosaceus ATCC 25745. (C) Bacteriocin synthesis gene clusters identified in the genome of P. pentosaceus SL001. (D) The homology analysis of the gene cluster 1 in genome of SL001 and coagulin biosynthetic gene cluster from B. coagulans.