Research Note: Therapeutic effect of a Salmonella phage combination on chicks infected with Salmonella Typhimurium

Abstract

Antibiotic treatment failure is increasingly encountered for the emergence of pandrug-resistant isolates, including the prototypical broad-host-range Salmonella enterica serovar (S.) Typhimurium, which mainly transmitted to humans through poultry products. In this study we explored the therapeutic potential of a Salmonella phage composition containing a virulent phage and a nonproductive phage that does not produce progeny phage against chicks infected with a pandrug-resistant S. Typhimurium strain of avian origin. After approximately 10⁷ CFU of S. Typhimurium strain ST149 were administrated to chicks by intraperitoneal injection, the phage combination (∼10⁸ PFU) was gavaged at 8-h, 32-h, and 54-h postinfection. At d 10 postinfection, phage treatment completely protected chicks from Salmonella-induced death compared to 91.7% survival in the Salmonella challenge group. In addition, phage treatment also greatly reduced the bacterial load in various organs, with Salmonella colonization levels decreasing more significantly in spleen and bursa than in liver and cecal contents, possibly due to higher phage titers in these immune organs. However, phages could not alleviate the decreased body weight gain and the enlargement of spleen and bursa of infected chicks. Further examination of the bacterial flora in the cecal contents of chicks found that S. Typhimurium infection caused a remarkable decrease in abundance of Clostridia vadin BB60 group and Mollicutes RF39 (the dominant genus in chicks), making Lactobacillus the dominate genus. Although phage treatment partially restored the decline of Clostridia vadin BB60 group and Mollicutes RF39 and increased abundance of Lactobacillus caused by S. Typhimurium infection, Fournierella that may aggravate intestinal inflammation became the major genus, followed by increased Escherichia-Shigella as the second dominate bacterial genus. These results suggested that successive phage treatment modulated the structural composition and abundance of bacterial communities, but failed to normalize the intestinal microbiome disrupted by S. Typhimurium infection. Phages need to be combined with other means to control the spread of S. Typhimurium in poultry.

Keywords: Salmonella Typhimurium; bacteriophage; chick; growth performance; intestinal microbiome.

Figure 1

Effect of phages on SPF chicks infected with S. Typhimurium. (A) Survival rate of infected chicks in the Salmonella-challenged group and the phage treatment group. n = 12. Approximately 10⁷ CFU of S. Typhimurium strain ST149 were administrated to chicks by intraperitoneal injection (IP), then 10⁸ PFU of the phage combination in 1 mL SM buffer were orally gavaged (OG) at 8-h, 32-h, and 54-h postinfection. Chicks were maintained for 10 d and monitored daily for clinical signs and mortalities. (B) Comparison of weight gain of chicks among different groups. (C) The spleen-to-body weight ratio. (D) The bursa-to-body weight ratio on d 10 postinfection. *, P < 0.05 (Kruskal-Wallis test); *** P < 0.001; **** P < 0.0001; ns, no significance. (E) Phage titer in vivo. (F) Bacterial load in various organs. * P < 0.05; ** P < 0.01 (Mann-Whitney test); ns, no significance. Con, the control group; Pha, the phage alone group; Sal, the only Salmonella challenge group; Sal+pha, the phage-treated group.

Figure 2

Effect of phages on cecal microbiomes of chicks infected with S. Typhimurium. Abundance of bacteria in the ceca of chicks from different groups at phylum (A) and genus level (B), respectively. The relative abundances of the top 10 genera were displayed. (C) Comparison of alpha diversity including the Shannon index and Chao1 index among different groups. (D) Beta diversity analysis visualized via NMDS analysis on Bray-Curtis distance. (E) Differences of chick microbial community abundance via LEfSe analysis using Kruskal-Wallis test (P < 0.05) with LDA score > 4. St, the only Salmonella challenge group; SP, the phage-treated group; Ph, the phage alone group; Control, the control group.