Encapsulation of E. coli phage ZCEC5 in chitosan–alginate beads as a delivery system in phage therapy

Abstract

Bacteriophages can be used successfully to treat pathogenic bacteria in the food chain including zoonotic pathogens that colonize the intestines of farm animals. However, harsh gastric conditions of low pH and digestive enzyme activities affect phage viability, and accordingly reduce their effectiveness. We report the development of a natural protective barrier suitable for oral administration to farm animals that confers acid stability before functional release of bead-encapsulated phages. Escherichia coli bacteriophage ZSEC5 is rendered inactive at pH 2.0 but encapsulation in chitosan–alginate bead with a honey and gelatin matrix limited titer reductions to 1 log 10 PFU mL −1 . The encapsulated phage titers were stable upon storage in water but achieved near complete release over 4–5 h in a simulated intestinal solution (0.1% bile salt, 0.4% pancreatin, 50 mM KH 2 PO 4 pH 7.5) at 37 °C. Exposure of E. coli O157:H7 to the bead-encapsulated phage preparations produced a delayed response, reaching a maximal reductions of 4.2 to 4.8 log 10 CFU mL −1 after 10 h at 37 °C under simulated intestinal conditions compared to a maximal reduction of 5.1 log 10 CFU mL −1 at 3 h for free phage applied at MOI = 1. Bead-encapsulation is a promising reliable and cost-effective method for the functional delivery of bacteriophage targeting intestinal bacteria of farm animals.

Entrapment of a phage cocktail and cinnamaldehyde on sodium alginate emulsion-based films to fight food contamination by Escherichia coli and Salmonella Enteritidis

“…Most of encapsulation strategies reported for phages were driven by the need to protect them from the adverse conditions found in the digestive tract, such as the low pH and the activity of enzymes. In this context, phages have been encapsulated mostly in alginate-based microspheres (Abdelsattar, Abdelrahman, Dawoud, Connerton, & El-Shibiny, 2019;Colom et al, 2017;Moghtader, Eğri, & Piskin, 2017) and liposomes (Otero et al, 2019). More recently, studies have reported phages encapsulation to be further applied in food products.…”

Local Bacteriophage Delivery for Treatment and Prevention of Bacterial Infections

“…As was shown by Międzybrodzki et al, only a small fraction (<0.1%) of oral administered phage was observed in the intestines, even after neutralizing antacid administration. The rationale behind several reviewed studies is to overcome such problems by protecting bacteriophages against harmful environments through embedding in a biomaterial matrix (Kim et al, 2015;Moghtader et al, 2017;Abdelsattar et al, 2019;Otero et al, 2019;Vinner et al, 2019).…”

“…The presence of organic solvents and their volatility were also observed to be detrimental to phage activity (Salalha et al, 2006). Because of these practical limitations in applying organic solvents, water-soluble biopolymers such as sodium alginate are frequently used for bacteriophage encapsulation (Kim et al, 2015;Abdelsattar et al, 2019;Alves et al, 2019). Agarwal et al (2018) could also prevent exposure of phages to organic solvents by adding them on poly(lactide-co-glycolic acid) (PLGA) porous microspheres, which allowed the phage to be directly absorbed.…”

“…The carboxylic acid groups of alginate are protonated in acidic environments, but at neutral pH levels (for example in the intestine), deprotonation of these carboxylic acids results in a great increase in swelling that allows for diffusion of drug or phages out of the alginate gels where it can be therapeutically active ( Mumper et al, 1994 ). The size of the alginate construct seems to influence phage tolerance of acidic environments, as phage in millimeter-sized beads showed a 1log 10 reduction ( Abdelsattar et al, 2019 ) while phage embedded in microbeads with a diameter of ±150 μm expressed 2–3log 10 reduction in phage titer ( Colom et al, 2017 ).…”

“…The solubility of chitosan is pH dependent as in acidic environments the protonation of its amine groups makes chitosan positively charged and water soluble. A multitude of studies have fabricated polyelectrolyte complexes of chitosan with anionic polymers such as alginate for drug delivery purposes ( Ma et al, 2008 ; Kim et al, 2015 ; Abdelsattar et al, 2019 ). Cationic materials have been recognized as having antiviral properties, with a 1 mg/mL chitosan solution exerting a 2-log reduction of C2 and MS2 bacteriophages after coincubation for 1 min ( Chatain-Ly et al, 2013 ).…”