Liposome-Encapsulated Bacteriophages for Enhanced Oral Phage Therapy against Salmonella spp

Abstract

and UAB_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. Foodborne diseases are a global health problem and include infections with nontyphoidal Salmonella, which is among the most common zoonotic pathogens that affect humans. In fact, this bacterium was responsible for more than 1 million annual cases of food-related illnesses in the United States from 2000 to 2008 (1) and for more than 90,000 cases of salmonellosis (mainly from Salmonella enterica serovars Enteritidis and Typhimurium) diagnosed in the European Union in 2012 (2). The major source of Salmonella infections in humans is poultry products (2), and poultry is the major reservoir of this bacterium. The health risk is exacerbated in the case of broilers, which are asymptomatic carriers that house Salmonella in their gut. In fact, several Salmonella strains persistently colonize chickens but without causing any signs of illness (3). This lack of clinical symptoms facilitates the dissemination of Salmonella within flocks (4), thereby increasing the probability of cross-contamination during the transport, slaughter, and processing of broilers.Salmonella infection in broilers has been controlled mainly through the use of vaccines (5, 6), probiotics, prebiotics, and synbiotics (7), and antibiotics (8), although the effectiveness of these treatments is limited. Importantly, antibiotics can ultimately increase the severity and frequency of colonization by certain resistant strains of Salmonella (9).Phages are viruses ...

Microencapsulation with alginate/CaCO3: A strategy for improved phage therapy

“…While our results are similar to those achieved with liposome encapsulation24, preparation of the alginate/CaCO 3 capsules is simpler and provides much higher encapsulation rates. Although treatment with the alginate/CaCO 3 cocktail did not remove Salmonella totally, the concentration of Salmonella used in the experimental poultry infection was very high (~6 log 10 /g of caecum).…”

“…Thus, bacteriophages were detected in 71.4% of the chickens 72 h after oral administration of a single dose of the cocktail of encapsulated bacteriophages compared to 9.5% of the chickens treated with the non-encapsulated phages ( p  < 0.001). The percentage obtained with the alginate/CaCO 3 capsules was better than obtained in a previous in vivo study of liposome-encapsulated bacteriophages24, perhaps due to the higher encapsulation efficiency achieved with the former.…”

“…One such method is encapsulation, which in farm animals protects orally administered bacteriophages from the harsh environment of the stomach and facilitates their retention during passage through the intestinal tract to ensure a successful therapeutic effect242526. By maintaining bacteriophage stability, micro- or nano-encapsulation enables not only their oral administration through feed or water but also their administration in other forms, such as inhalation, thereby assuring an adequate dose of the therapeutic phage.…”

“…To our knowledge, the present study is the first in which wet bacteriophage-containing alginate/CaCO 3 microcapsules <150 μm in size were obtained and then used as oral therapy to protect broiler chickens against Salmonella infection, under conditions mimicking those of poultry production farms. In particular, a cocktail composed by three virulent bacteriophages (UAB_Phi20, UAB_Phi78, and UAB_Phi87) was prepared according to previous promising results of phage-therapy studies112324.…”

Genetically manipulated phages with improved pH resistance for oral administration in veterinary medicine

“…Phage encapsulation in natural biopolymer-derived matrices have been used as a strategy to improve phage stability2728293031. An increased survival of phages has been obtained with this technology in the GIT of cattle, pigs and poultry273233. However, for this type of strategy, the selection of the biopolymer is critical, and some important features have to be met, such as the ability to be synthesized under mild environmental conditions, and to be easily tailored, non-toxic and environmental friendly (e.g.…”