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

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.

Salmonella Phage CKT1 Effectively Controls the Vertical Transmission of Salmonella Pullorum in Adult Broiler Breeders.

Phage therapy is widely being reconsidered as an alternative to antibiotics for the treatment of multidrug-resistant bacterial infections, including salmonellosis caused by Salmonella. As facultative intracellular parasites, Salmonella could spread by vertical transmission and pose a great threat to both human and animal health; however, whether phage treatment might provide an optional strategy for controlling bacterial vertical infection remains unknown. Herein, we explored the effect of phage therapy on controlling the vertical transmission of Salmonella enterica serovar Gallinarum biovar Pullorum (S. Pullorum), a poultry pathogen that causes economic losses worldwide due to high mortality and morbidity. A Salmonella phage CKT1 with lysis ability against several S. enterica serovars was isolated and showed that it could inhibit the proliferation of S. Pullorum in vitro efficiently. We then evaluated the effect of phage CKT1 on controlling the vertical transmission of S. Pullorum in an adult broiler breeder model. The results demonstrated that phage CKT1 significantly alleviated hepatic injury and decreased bacterial load in the liver, spleen, heart, ovary, and oviduct of hens, implying that phage CKT1 played an active role in the elimination of Salmonella colonization in adult chickens. Additionally, phage CKT1 enabled a reduction in the Salmonella-specific IgG level in the serum of infected chickens. More importantly, the decrease in the S. Pullorum load on eggshells and in liquid whole eggs revealed that phage CKT1 effectively controlled the vertical transmission of S. Pullorum from hens to laid eggs, indicating the potential ability of phages to control bacterial vertical transmission.

A Combination of Virulent and Non-Productive Phages Synergizes the Immune System against Salmonella Typhimurium Systemic Infection.

Effective phage cocktails consisting of multiple virus types are essential for successful phage therapy against pandrug-resistant pathogens, including Salmonella enterica serovar (S.) Typhimurium. Here we show that a Salmonella phage, F118P13, with non-productive infection and a lytic phage, PLL1, combined to inhibit pandrug-resistant S. Typhimurium growth and significantly limited resistance to phages in vitro. Further, intraperitoneal injection with this unique phage combination completely protected mice from Salmonella-induced death and inhibited bacterial proliferation rapidly in various organs. Furthermore, the phage combination treatment significantly attenuated the inflammatory response, restored the generation of CD4+ T cells repressed by Salmonella, and allowed macrophages and granulocytes to participate in immunophage synergy to promote bacterial clearance. Crucially, the non-productive phage F118P13 is less likely to be cleared by the immune system in vivo, thus providing an alternative to phage cocktail against bacterial infections.

Salmonella phage CKT1 significantly relieves the body weight loss of chicks by normalizing the abnormal intestinal microbiome caused by hypervirulent Salmonella Pullorum.

Pullorum disease caused by Salmonella Pullorum remains an important disease for the poultry industry due to high morbidity and mortality in many countries. Phage therapy is becoming an alternative strategy to control multidrug-resistant Salmonella infections in young chicks. However, how bacteriophages affect the growth performance of chicks infected with S. Pullorum remains poorly understood. Herein, we assessed the therapeutic efficacy of Salmonella phage CKT1 against hypervirulent arthritis-causing S. Pullorum. The results showed that single phage treatment after hypervirulent S. Pullorum infection significantly improved body weight loss of chicks. Compared with enlarged liver and spleen in only Salmonella challenged group, phage administration substantially reduced the liver/body and spleen/body weight ratios, bacterial loads in organs and the degree of hepatic sinusoidal dilatation and congestion. Moreover, phage CKT1 can enter the organs of chicks and stay for at least 3 d in liver and spleen, and promote higher serum levels of IL-6 production within 6 d postinfection, indicating phage-induced bacterial lysis may be involved in inflammatory immune response to S. Pullorum infection. Analysis of the microbiome of gastrointestinal tract of chicks demonstrated that Salmonella challenge significantly reduced the relative abundances of Lachnoclostridium and Blautia, resulting in remarkably increased Escherichia-Shigella and Klebsiella becoming the predominant bacterial taxa. In contrast, the use of phage CKT1 significantly reduced Escherichia-Shigella and Klebsiella populations in intestine, permitting the proliferation of beneficial microbiota in Firmicutes including Lachnoclostridium, Ruminococcus, Lactobacillus, and Pseudoflavonifractor. In addition, phage alone treatments did not affect the normal gut microbiota structure of chicks, and phage therapy on Salmonella infected chicks increased bacteria species richness in the cecum. These results suggest that Salmonella phage CKT1 could improve growth performance of chicks challenged with S. Pullorum by normalizing the abnormal intestinal microbiome.