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Salmonella enteritidis plays a significant role in zoonotic infections. This pathogen settles in the intestinal tract of poultry and contaminates meat and egg products during production for slaughter. Consequently, it can also be transmitted to humans along the food chain, posing a significant risk to public health. Bacteriophages offer a viable substitute for antibiotics in treating Salmonella enteritidis due to their specific bactericidal effect and safety. This study aimed to investigate the protective effect of bacteriophage against Salmonella enteritidis infection in chicken intestinal epithelial cells. Chicken intestinal epithelial cells were treated with various concentrations of bacterial suspension, and the impact on cell damage was assessed by measuring changes in cell viability and observing structural changes via transmission electron microscopy.In the phage protection experiment, the phages were co-incubated with Salmonella enteritidis for 2, 4, and 6 h. Thereafter, the adhesion rate and invasion rate of bacteria and gene transcription levels of Occludin, Claudin-1, ZO-1, NF-κB p65, TNF-α and IL-6 in cells were determined. The results indicated that the phage could significantly reduce both adhesion and invasion rates of Salmonella enteritidis at MOI = 10 (P < 0.05). Following phage treatment of the co-culture of chicken intestinal epithelial cells and Salmonella enteritidis (CICC10467), there was a significant reduction in the gene expression of pro-inflammatory cytokines NF-κB p65, TNF-α and IL-6, and a significant increase in the gene expression of anti-inflammatory cytokines Occludin, Claudin-1, and ZO-1. This indicates that the phage can inhibit the infection of chicken small intestinal epithelial cells by Salmonella enteritidis. Furthermore, phages were able to significantly alleviate inflammation and barrier integrity disruption caused by the bacteria in the co-culture. These observations suggest that phages are promising candidates for preventing and treating gastrointestinal bacterial infections., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Currently, phage biocontrol is increasingly used as a green and natural technology for treating Salmonella and other infections, but phages exhibit instability and activity loss during storage. Therefore, in this study, the effects of lyophilization on the activity and stability of phage cocktails for the control of multidrug-resistant Salmonella in broiler chickens were determined. Eight serotypes of Salmonella were isolated and identified from broiler chicken farms, and bacteriophages against multidrug-resistant Salmonella enterica subsp. enterica serovar Kentucky, Salmonella enterica subsp. enterica serovar Typhimrium and Salmonella enterica subsp. enterica serovar Enteritidis were isolated. The bacteriophage cocktail was prepared and lyophilized, and it was subjected to in vitro and in vivo examinations. A reconstituted lyophilized bacteriophage cocktail was used for the oral treatment of chicks before and after challenge with multidrug-resistant S. Kentucky. The colonization of cecum by S. Kentucky was detected by using real-time PCR, and the serum levels of IgM, IgA and IL-4 and pathological changes in the different groups were detected. Three Caudovirales phages families were identified including Autographiviridae, Straboviridae and Drexlerviridae against multidrug-resistant S. Kentucky, S. Typhimrium and S. Enteritidis. The groups treated with the bacteriophage cocktail showed no clinical signs, no postmortem lesions, and a mortality rate of 0%, which improved the growth performance parameters. Additionally, the estimated serum levels of IgM, IgA and IL-4 were significantly greater in the bacteriophage cocktail-treated groups. Lyophilization effectively preserves the long-term storage stability of phages. Therefore, lyophilized bacteriophage cocktail therapy is a valuable approach for controlling multidrug-resistant Salmonella infections in broiler chickens., (© 2024. The Author(s).)

Phage therapy is gaining momentum as an alternative to antibiotics in the treatment of salmonellosis caused by Salmonella. In this study, a novel Salmonella phage, vB&#95;SalS&#95;JNS02, was isolated successfully from poultry farms in Shandong, China. The biological characteristics of vB&#95;SalS&#95;JNS02 were analysed, which revealed a short latent period of approximately 10 min and a burst size of 110 PFU/cell. Moreover, vB&#95;SalS&#95;JNS02 exhibited remarkable stability across a wide pH range (pH 3-12) and temperatures ranging from 30 to 80°C. Genome sequencing analysis provided valuable insights into the genetic composition of vB&#95;SalS&#95;JNS02, which consists of a double-stranded DNA genome that spans 42,450 base pairs and has a G + C content of 49.4%. Of significant importance, the genomic sequence of vB&#95;SalS&#95;JNS02 did not contain any genes related to lysogenicity, virulence, or antibiotic resistance. The phage's efficacy was evaluated in a larval challenge study. Treatment with the phage resulted in increased survival of Galleria mellonella larvae (100, 70, and 85%) (MOI 0.1) in the prophylactic treatment, co-infection treatment, and remedial treatment experiments, respectively. Another in vivo experiment investigated the potential application of the phage in broiler chickens and revealed that a single oral dose of vB&#95;SalS&#95;JNS02 (10 8 PFU/mL, 100 µL/chick) administered 3 h after S. enteritidis oral administration provided effective protection. The introduction of bacteriophage not only enhances the production of secretory immunoglobulin A (sIgA), but also induces alterations in the composition of the gut microbial community. Phage therapy increases the relative abundance of beneficial bacteria, which helps to maintain intestinal barrier homeostasis. However, it is unable to fully restore the disrupted intestinal microbiome caused by S. enteritidis infection. Importantly, no significant adverse effects were observed in the animal subjects following oral administration of the phage, and our findings highlight vB&#95;SalS&#95;JNS02 is a hopeful candidate as a promising tool to target Salmonella infections in poultry., Competing Interests: DISCLOSURES The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Lulu Li reports financial support was provided by Ministry of Science and Technology of the People's Republic of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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 10 7 CFU of S. Typhimurium strain ST149 were administrated to chicks by intraperitoneal injection, the phage combination (∼10 8 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., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Salmonella Enteritidis remains a leading cause of human foodborne disease, mostly associated with the consumption of contaminated poultry products. To more strategically implement a phage therapy scheme for S. Enteritidis control in broilers, a cocktail containing three wild-type lytic bacteriophages (LBs) previously isolated from chickens was evaluated shortly and later after a challenge. Genomic characterization, lytic spectrum and in vitro efficacy were determined for each studied LB. In independent trials, broilers challenged with S. Enteritidis on day of hatch received phage therapy from 6 to 10 days of age (early treatment), and from 31 to 35 days of age (later treatment). S. Enteritidis analyses were performed before treatment and at 1, 4, 7 and 10 days post-treatment (dpt) in both trials. Partial DNA sequence analysis of each LB revealed close similarity to the Ackermannviridae family. LBs lysed different Salmonella enterica serovars, while other tested bacteria were refractory. An in-vitro reduction of 1.49, 0.65 and 0.58 log 10 CFU/mL in S. Enteritidis number was obtained after co-incubation for 3 h with each LB. Both in vivo trials showed a significant reduction in the average number of intestinal S. Enteritidis calculated after phage therapy compared with controls. However, the highest efficiency was found in the later therapy, which resulted in a reduction of 1.08 log 10 CFU/g in the average from 4 to 10 dpt, showing potential for future use as a pre-harvest strategy to reduce the S. Enteritidis intestinal colonization in broilers on farms., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Salmonella pullorum is the major pathogen that is harmful to the poultry industry in developing countries, and the treatment of chicken diarrhea caused by S. pullorum has become increasingly difficult. In this study, a virulent bacteriophage YSP2, which was able to specifically infect Salmonella, was isolated and characterized. Phage YSP2 was classified in the Siphoviridae family and had a short latent period of 10 min. No bacterial virulence- or lysogenesis-related ORF is present in the YSP2 genome, making it eligible for use in phage therapy. Experiments in vivo investigated the potential use of phages as a therapy against diarrhea in chickens caused by S. pullorum in a chicken diarrhea model, demonstrating that a single oral administration of YSP2 (1 × 10 10  PFU/mL, 80 μL/chicken) 2 h after S. pullorum oral administration at a double median lethal dose was sufficient to protect chickens against diarrhea. Gross inspection showed that YSP2 can effectively reduce organ damage and significantly relieve hemorrhage in the intestine and liver tissue. Moreover, YSP2 can maintain a high curative effect when diluted to 10 8 PFU/mL. In light of its therapeutic effect on chicken diarrhea, YSP2 may serve as an alternative treatment strategy for infections caused by S. pullorum.

Bacteriophages UAB&#95;Phi20, UAB&#95;Phi78, and UAB&#95;Phi87 were encapsulated in liposomes, and their efficacy in reducing Salmonella in poultry was then studied. The encapsulated phages had a mean diameter of 309 to 326 nm and a positive charge between +31.6 and +35.1 mV (pH 6.1). In simulated gastric fluid (pH 2.8), the titer of nonencapsulated phages decreased by 5.7 to 7.8 log units, whereas encapsulated phages were significantly more stable, with losses of 3.7 to 5.4 log units. The liposome coating also improved the retention of bacteriophages in the chicken intestinal tract. When cocktails of the encapsulated and nonencapsulated phages were administered to broilers, after 72 h the encapsulated phages were detected in 38.1% of the animals, whereas the nonencapsulated phages were present in only 9.5%. The difference was significant. In addition, in an in vitro experiment, the cecal contents of broilers promoted the release of the phages from the liposomes. In broilers experimentally infected with Salmonella, the daily administration of the two cocktails for 6 days postinfection conferred similar levels of protection against Salmonella colonization. However, once treatment was stopped, protection by the nonencapsulated phages disappeared, whereas that provided by the encapsulated phages persisted for at least 1 week, showing the enhanced efficacy of the encapsulated phages in protecting poultry against Salmonella over time. The methodology described here allows the liposome encapsulation of phages of different morphologies. The preparations can be stored for at least 3 months at 4°C and could be added to the drinking water and feed of animals., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

In this study the isolation and characterization of three bacteriophages (ST4, L13, and SG3) infecting Salmonella gallinarum were carried out. They were further tested for their in vivo efficacy in phage therapy. All three phages belong to the Siphoviridae family with isometric heads and non-contractile tails. They have a broad host range among serovars of Salmonella enterica. The burst sizes were observed to be 1670, 80, and 28 for ST4, L13, and SG3, respectively. The in vivo efficacy of the phages was tested in chickens. Layer chickens were challenged with S. gallinarum, whereas contact chickens were cohabited without direct challenge. Each bacteriophage was orally inoculated in the form of feed additives. Mortality was observed and S. gallinarum was periodically re-isolated from the livers, spleens, and cecums of the chickens. Bacterial re-isolation from the organs and mortality decreased significantly in both challenged and contact chickens treated with the bacteriophages compared with untreated chickens serving as the control. The three bacteriophages may be effective alternatives to antibiotics for the control of fowl typhoid disease in chickens.

A Salmonella Gallinarum (SG)-specific bacteriophage isolated from sewage effluent was used to prevent horizontal transmission of SG in commercial layer chickens. Six-week-old chickens, each challenged with 5 x 10(8) colony-forming units of SG, cohabited with contact chickens treated with 10(6) plaque-forming units/kg of bacteriophage, prepared in feed additives, for 7 days before, and 21 days after challenge with SG. Mortality was observed for 3 wk after challenge and SG was periodically reisolated from the liver, spleen, and cecum of chickens. SG re-isolation from organs was decreased and a significant (P < 0.05) reduction in mortality was observed in contact chickens treated with the bacteriophage, as compared to untreated contact chickens, indicating that bacteriophage administration in feed additives significantly prevented the horizontal transmission of SG. These results provide important insights into prevention and control strategies against SG infection and suggest that the use of bacteriophages may be a novel, safe, and effectively plausible alternative to antibiotics for the prevention of SG infection in poultry.

Aims: Salmonella is a worldwide foodborne pathogen causing acute enteric infections in humans. In the recent years, the use of bacteriophages has been suggested as a possible tool to combat this zoonotic pathogen in poultry farms. This work aims to isolate and perform comparative studies of a group of phages active against a collection of specific Salmonella Enteritidis strains from Portugal and England. Also, suitable phage candidates for therapy of poultry will be selected., Methods and Results: The Salm. Enteritidis strains studied were shown to have a significantly high occurrence of defective (cryptic) prophages; however, no live phages were found in the strains. Bacteriophages isolated from different environments lysed all except one of the tested Salm. Enteritidis strains. The bacteriophages studied were divided into different groups according to their genetic homology, RFLP profiles and phenotypic features, and most of them showed no DNA homology with the bacterial hosts. The bacteriophage lytic efficacy proved to be highly dependent on the propagation host strain., Conclusions: Despite the evidences shown in this work that the Salm. Enteritidis strains used did not produce viable phages, we have confirmed that some phages, when grown on particular hosts, behaved as complexes of phages. This is most likely because of the presence of inactive phage-related genomes (or their parts) in the bacterial strains which are capable of being reactivated or which can recombine with lytic phages. Furthermore, changes of the bacterial hosts used for maintenance of phages must be avoided as these can drastically modify the parameters of the phage preparations, including host range and lytic activity., Significance and Impact of the Study: This work shows that the optimal host and growth conditions must be carefully studied and selected for the production of each bacteriophage candidate for animal therapy.

Using a screening procedure, three bacteriophages, ST27, ST29, and ST35, were identified with selective activity for Salmonella enterica serovar Typhimurium (SL1344) but not SL1344 tolC::aph. Overproduction of TolC led to a lower efficiency of plating (EOP), further suggesting that TolC was the target receptor. Activity against other serovars of Salmonella was observed but not against other species of Enterobacteriaceae. This study provides proof of principle that bacteriophages can be active against the outer membrane protein of tripartite resistance-nodulation-division (RND) efflux pumps and so could be used to reduce the numbers of Salmonella cells in animals reared for food production.