Anti-Infective Effect of Adhesive Probiotic Lactobacillus in Fish is Correlated With Their Spatial Distribution in the Intestinal Tissue

Abstract

In this study, we tested the distribution of 49 Lactobacillus strains in the mucus and mucosa of the intestine tissue of zebrafish. We observed a progressive change in the spatial distribution of Lactobacillus strains, and suggested a division of the strains into three classes: mucus type (>70% in mucus), mucosa type (>70% in mucosa) and hybrid type (others). The hybrid type strains were more efficient in protection of zebrafish against Aeromonas hydrophila infection. Three strains representing different distribution types (JCM1149, CGMCC1.2028, and JCM 20300) were selected. The mucosa type strain JCM1149 induced higher intestinal expression of inflammatory cytokines and Hsp70 than the other strains. Furthermore, we used L. rhamnosus GG and its mutant (PB22) lacking SpaCBA pili to investigate the influence of pili on spatial distribution. LGG showed a mucosa type distribution, while PB22 revealed a hybrid distribution and the disease protection was accordingly improved. The different protection ability between LGG and PB22 did not involve the intestinal microbiota, however, LGG induced injury to the mucosa of zebrafish. Collectively, the disease protection activity of Lactobacillus in zebrafish is correlated with their spatial distribution in the intestinal tissue, with strains showing a balanced distribution (hybrid type) more efficient in protection.

Figure 1

The spatial distribution of 49 Lactobacillus strains in zebrafish gut. The fish were immersed in Lactobacillus-inoculated water for 14 days. n = 8.

Figure 2

Protective ability of selected Lactobacillus strains representing different spatial distribution types. (A) Abundance of A. hydrophila NJ-1 in the gut of zebrafish 24 h after challenge (B) IAP activity of zebrafish 24 h after challenge with A. hydrophila NJ-1. (C) Cumulative survival of zebrafish after infection. (D) Final survival of zebrafish. All data are presented as mean ± SEM, ^* P < 0.05, ^** P < 0.01.

Figure 3

Protection of zebrafish against A. hydrophila NJ-1 by the three Lactobacillus strains after 14 days immersion treatment. (A) Intestinal alkaline phosphatase (IAP) activity after A. hydrophila NJ-1 infection for 24 h. (B) Cumulative mortality of zebrafish after A. hydrophila NJ-1 infection. Data were presented as mean ± SEM. ^*P < 0.05, ^**P < 0.01. Means sharing a common letter (a,b,c) were not significantly different (P > 0.05). LP, L. plantarum JCM1149; LB, L. brevis CGMCC1.2028; LR, L. rhamnosus JCM 20300; CON, control.

Figure 4

Effect of the three Lactobacillus strains treatment on the intestinal microbiota of zebrafish. Heatmap of the 10 most abundant family (A) and genus (B) in the intestinal microbiota of zebrafish from different treatments. LP, L. plantarum JCM1149; LB, L. brevis CGMCC1.2028; LR, L. rhamnosus JCM 20300; CON, control.

Figure 5

Effect of the three Lactobacillus strains on the intestinal expression of immunity-related genes in zebrafish. (A–D) nuclear factor kappa B (NF-kB), tumor necrosis factor (TNF)-α, interleukin IL-1β, and transforming growth factor-β (TGF-β) expression levels in gut of zebrafish after Lactobacillus immersion for 5 h and 14 days; (E) Hsp70 expression levels in gut of zebrafish after Lactobacillus immersion for 14 days. All data are presented as mean ± SEM, ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001. LP, L. plantarum JCM1149; LB, L. brevis CGMCC1.2028; LR, L. rhamnosus JCM 20300; CON, control.

Figure 6

Spatial distribution of LGG and LGG mutant PB22 in the gut of zebrafish. (A) The overall colonization level of LGG or PB22 in fish gut at 10⁶ cfu/ml immersion. (B) Spatial distribution of LGG or PB22 at 10⁶ cfu/ml immersion. (C) The overall colonization level of LGG or PB22 in fish gut at 10⁷ cfu/ml immersion. (D) Spatial distribution of LGG or PB22 at 10⁷ cfu/ml immersion. LGG, L. rhamnosus GG; PB22, LGG mutant PB22. PB22 is pilus deficient as it has lost the pilus island and flanking sequences (75 kb DNA) and has 51 other SNPs.

Figure 7

Protection ability of LGG and mutant PB22 in adult zebrafish. Zebrafish were immersed for 14 days in water inoculated with each strain at 10⁷ cfu/ml and were challenged with A. hydrophila NJ-1. (A) Cumulative survival of zebrafish after infection, (B) The final survival of zebrafish after infection. Data were presented as mean ± SEM. ^*P < 0.05, ^**P < 0.01. CON, control; LGG, L. rhamnosus GG; PB22, LGG mutant PB22.

Figure 8

Protective effects of the gut microbiotas transplanted from adult zebrafish in recipient GF zebrafish against A.hydrophila NJ-1 infection. (A) Cumulative survival for different groups. (B) The final survival of GF zebrafish colonized with gut microbiotas for 3 days. All values are presented as mean ± SEM, ^* P < 0.05. GF, germ-free zebrafish; LGG, L. rhamnosus GG; PB22, LGG mutant PB22.

Figure 9

Protection ability of LGG and PB22 in GF zebrafish larvae. GF zebrafish at 3 dpf were immersed in water inoculated with each strain (10⁷ cfu/ml) and were challenged with NJ-1 at 6 dpf. (A) Cumulative survival of zebrafish larvae after infection. (B) The final survival of zebrafish larvae after infection. Data were presented as mean ± SEM. ^*P < 0.05. CON, control; LGG, L. rhamnosus GG; PB22, LGG mutant PB22.

Figure 10

Histology (H&E staining) of intestine of zebrafish after immersion treatment with Lactobacillus strains for 14 days. CON, control; LGG, L. rhamnosus GG; PB22, LGG mutant PB22; LR, L. rhamnosus JCM 20300 Asterisk, edema and floating of the intestinal mucous membrane. Arrow, degeneration and focal necrosis of intestinal villus.

Figure 11

Cumulative mortality of NJ-1 challenged zebrafish after immersion in LB or equal mixture of LP and LR. Zebrafish were immersed with LB (10⁷ cfu/ml), LP + LR (0.5 × 10⁷ cfu/ml LP, 0.5 × 10⁷ cfu/ml LR; low dose), LP + LR (10⁷ cfu/ml LP, 10⁷ cfu/ml LR; high dose) for 14 days, then challenge with 10⁸ cfu/ml A. hydrophila NJ-1. Data marked with different letters (a,b) were significantly different (P < 0.05).