Non-typhoidal Salmonella, one of the most common zoonotic pathogens, causes foodborne disease outbreaks in humans. Poultry is the major reservoir of this bacterium. Following the ban on the use of antibiotics as growth promoters in animal production, Salmonella infection in poultry has been mainly controlled through biosafety measures implemented in animal production facilities and through the use of vaccines, probiotics, prebiotics, and synbiotics. Although reduction in the prevalence of several Salmonella serovars was achieved between 2007 and 2012 in the European Union, these methods are insufficient such that phage therapy has gained increasing attention for Salmonella control. In previous work, we demonstrated that oral phage therapy using a cocktail containing three bacteriophages (UAB_Phi20, UAB_Phi78, and UAB_Phi87) very efficiently reduces Salmonella colonization in specific pathogen free chickens. To better understand the efficacy and safety of these phages, we characterized their replication kinetics, genome ends, and transduction capability. The results showed that DNA replication was initiated 10 min after bacterial infection by UAB_Phi78 and 20 min after infection by UAB_Phi20 and UAB_Phi87. UAB_Phi20 and UAB_Phi78 DNA was detected inside the cells for 40 min and that of UAB_Phi87 for >120 min. The genome ends of the three phages differed and were included in specific DNA packaging strategies: P22likevirus headful (UAB_Phi20), SP6likevirus direct terminal repeats (UAB_Phi78), and Felixounalikevirus direct terminal repeats (UAB_Phi87). None was able to promote transduction under the conditions of the assay used. The inherent challenges to the oral administration of bacteriophages as therapeutic agents against intestinal pathogens are related to the lack of phage stability in the stomach and the short residence time in the intestinal tract, which together lower the efficiency of phage therapy. In this work, two methods for phage encapsulation have been developed. The advantages of phage therapy with the encapsulated phages has been studied in an animal model (broiler/Salmonella) developed in this work that mimics the conditions of the farms. Encapsulation in cationic lipids yielded liposome-encapsulated phages with a mean diameter of 308.6 - 325.8 nm, a positive charge between +31.6 and +35.1 mV (pH 6.1), and an encapsulation efficiency yield of ~50%. In comparison, the mean diameter of the alginate-encapsulated phages ranged from 123.7 to 149.3 μm and the encapsulation efficiencies were >90%. The two phage formulations are stable for ≥6 months when stored at 4°C. The titre of non-encapsulated phages in simulated gastric fluid (pH 2.8) decreased by 5.7–8.0 log10 after 60 min, whereas liposome- and alginate-encapsulated phages were significantly more stable, with losses of 3.7–5.4 log10 units and ≤3.5 log10, respectively. Liposome and alginate-CaCO3 encapsulations also significantly improved bacteriophage retention in the chicken intestinal tract compared to the non-encapsulated preparations. In broilers administered cocktails of encapsulated and non-encapsulated phages, the former were detected in 38.1% (liposome) and 71.4% (alginate-CaCO3) of the animals after 72 h whereas the non-encapsulated phages were present in only 9.5%. In in vitro experiments, phage release from the liposomes and alginate-CaCO3 capsules was triggered by the caecal contents of broilers and simulated intestinal fluid, respectively. In commercial broilers experimentally infected with Salmonella, daily administration of the three cocktails for 7 days post-infection conferred similar levels of protection against Salmonella colonization. Although protection by the non-encapsulated phages disappeared once treatment was stopped, the decrease of the Salmonella concentration provided by both encapsulated forms persisted for at least another week, thus showing the enhanced efficacy of encapsulated phages in phage therapy. The work describes the methodology used to obtain liposome and alginate-CaCO3 phage encapsulation, which can be used with bacteriophages of different morphologies, and the advantages of both forms in overcoming the inherent drawbacks of orally administered phage therapy.
Studies of the molecular features of three Salmonella phages for use in phage therapy and of encapsulation methodologies to improve oral phage administration
Colom Comas, J. (Author). 5 Feb 2016
Student thesis: Doctoral thesis
Student thesis: Doctoral thesis