Salmonella Phages Affect the Intestinal Barrier in Chicks by Altering the Composition of Early Intestinal Flora: Association With Time of Phage Use

Abstract

Phages show promise in replacing antibiotics to treat or prevent bacterial diseases in the chicken breeding industry. Chicks are easily affected by their environment during early growth. Thus, this study investigated whether oral phages could affect the intestinal barrier function of chicks with a focus on the cecal microbiome. In a two-week trial, forty one-day-old hens were randomly divided into four groups: (1) NC, negative control; (2) Phage 1, 10⁹ PFU phage/day (days 3-5); (3) Phage 2, 10⁹ PFU phage/day (days 8-10); and (4) AMX, 1 mg/mL amoxicillin/day (days 8-10). High-throughput sequencing results of cecal contents showed that oral administration of phages significantly affected microbial community structure and community composition, and increased the relative abundance of Enterococcus. The number of different species in the Phage 1 group was much higher than that in the Phage 2 group, and differences in alpha and beta diversity also indicated that the magnitude of changes in the composition of the cecal microbiota correlated with the time of phage use. Particularly in the first stage of cecal microbiota development, oral administration of bacteriophages targeting Salmonella may cause substantial changes in chicks, as evidenced by the results of the PICRUSt2 software function prediction, reminding us to be cautious about the time of phage use in chicks and to avoid high oral doses of phages during the first stage. Additionally, the Phage 2 samples not only showed a significant increase in the relative abundance of Bifidobacterium and Subdoligranulum, but also improved the intestinal morphology (jejunum) and increased the mRNA expression level of occludin and ZO-1. We concluded that phages do not directly interact with eukaryotic cells. The enhancement of intestinal barrier function by phages in chicks may be related to changes in the intestinal flora induced by phages. This implies that phages may affect intestinal health by regulating the intestinal flora. This study provides new ideas for phage prevention of intestinal bacterial infections and promotes large-scale application of phages in the poultry industry.

Keywords: Enterococcus; cecal microbiota; chicks; intestinal barrier; phage.

FIGURE 1

Effect of phages on the morphology of the duodenum and jejunum in chicks. (A) H&E staining of duodenum in the NC, Phage 1, Phage 2, and AMX groups under 40 × (a-d) and 100 × (e-h) magnification. (B) H&E staining of jejunum under 40 × (a-d) and 100 × (e-h) magnification. NC, negative control; Phage 1, 10⁹PFU GRNsp6 (day 3-5); Phage 2, 10⁹PFU GRNsp6 (day 8-10); AMX, 1 mg/ml amoxicillin (day 8-10).

FIGURE 2

Effect of phages on (A) villus height, (B) crypt depth, and (C) villus height to crypt depth ratio of the duodenum and jejunum of chicks in the NC, Phage 1, Phage 2, and AMX groups, respectively. Values are means (n = 5). NC, negative control; Phage 1, 10⁹PFU GRNsp6 (day 3-5); Phage 2, 10⁹PFU GRNsp6 (day 8-10); AMX, 1 mg/ml amoxicillin (day 8-10). *p < 0.05; ****p < 0.0001.

FIGURE 3

Effect of phages on the relative mRNA expression of the jejunal tight junction. Fold induction of relative mRNA expressions of claudin-1, occludin, and ZO-1 (n = 5). NC, negative control; Phage 1, 10⁹PFU GRNsp6 (day 3-5); Phage 2, 10⁹PFU GRNsp6 (day 8-10); AMX, 1 mg/ml amoxicillin (day 8-10). *p < 0.05.

FIGURE 4

Effect of phage on serum endotoxin in chicks (n = 5). The serum endotoxin level is an important biomarker of intestinal permeability and was detected using an ELISA kit. NC, negative control; Phage 1, 10⁹PFU GRNsp6 (day 3-5); Phage 2, 10⁹PFU GRNsp6 (day 8-10); AMX, 1 mg/ml amoxicillin (day 8-10).

FIGURE 5

Effect of phages on the relative mRNA expression of jejunal cytokines. Fold induction of relative mRNA expressions of IL-1β, TNF-α, and IFN-γ (n = 5). NC, negative control; Phage 1, 10⁹PFU GRNsp6 (day 3-5); Phage 2, 10⁹PFU GRNsp6 (day 8-10); AMX, 1 mg/ml amoxicillin (day 8-10).*p < 0.05.

FIGURE 6

Effect of phages on the cecal microbiota of chicks. (A) Venn diagram generated to compare operational taxonomic units (OTUs) between different groups. (B) Diversity and richness indexes of cecal microbiota in each group. Figure a-d represents ACE, Chao1, Shannon and Simpson indices, respectively. *p < 0.05. (C) Beta-diversity analysis of cecal microbiota among groups. NMDS analysis based on Bray–Curtis distances. Points of different colors or shapes represent samples of different groups. The closer the two sample points are, the more similar the species composition of the two samples. NC, negative control; Phage 1, 10⁹PFU GRNsp6 (day 3-5); Phage 2, 10⁹PFU GRNsp6 (day 8-10); AMX, 1 mg/ml amoxicillin (day 8-10).

FIGURE 7

Effect of phages on the microbiota composition in the cecal digesta at different taxonomic levels (A) at phylum levels, (B) at family levels, and (C) at genus levels). Less than 1% abundance at the phylum, family, or genus level was merged with others. NC, negative control; Phage 1, 10⁹PFU GRNsp6 (day 3-5); Phage 2, 10⁹PFU GRNsp6 (day 8-10); AMX, 1 mg/ml amoxicillin (day 8-10).

FIGURE 8

Effect of phages on the relative bacterial abundance in chicks. Analysis of the differential flora of cecal microbiota at (A) family level and (B) genus level. The Kruskal–Wallis H test was used for multi-group comparison of cecal microorganisms, and Scheffe post hoc tests were used to test the difference between any two groups. NC, negative control; Phage 1, 10⁹PFU GRNsp6 (day 3-5); Phage 2, 10⁹PFU GRNsp6 (day 8-10); AMX, 1 mg/ml amoxicillin (day 8-10). *p < 0.05; ^**p < 0.01; and p < 0.1 trend.

FIGURE 9

LEfSe analysis of cecal microbiota. Cladogram (A) and LDA scores (B) show taxa that best characterize each biological class. Different-colored regions represent different constituents. Circles indicate phylogenetic level from domain to genus. The diameter of each circle is proportional to the abundance of the group. Species with significant difference that have an LDA score greater than the estimated value (3.0). The length of the histogram represents the LDA score. NC, negative control; Phage 1, 10⁹PFU GRNsp6 (day 3-5); Phage 2, 10⁹PFU GRNsp6 (day 8-10); AMX, 1 mg/ml amoxicillin (day 8-10).

FIGURE 10

Functional prediction of cecal microbiota (A) KEGG Pathway level 2 function classification; (B) Differential analysis of the KEGG pathway level 3 of cecal microbiota. The Kruskal–Wallis H test was used for differential analysis. NC, negative control; Phage 1, 10⁹PFU GRNsp6 (day 3-5); Phage 2, 10⁹PFU GRNsp6 (day 8-10); AMX, 1 mg/ml amoxicillin (day 8-10). *p < 0.05.