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 log₁₀ 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 KH₂PO₄ 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 log₁₀ CFU mL^-1 after 10 h at 37 °C under simulated intestinal conditions compared to a maximal reduction of 5.1 log₁₀ 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.

Keywords: Bacteriophage; Biocontrol; E. coli; Phage encapsulation.

Fig. 1

Representation of the bead encapsulation components in cross-section. The blue color refers to the chitosan, purple the Ca-alginate, green the internal matrix and yellow represents the bacteriophage

Fig. 2

Optical micrographs of beads 1 (0.3% honey, 0.25% gelatin) in fresh form (a), beads 1 after 1-h incubation (a1), beads 2 (3% honey, 2.5% gelatin) in fresh form (b), beads 2 after 1-h incubation (b1), beads 3 (50 mM Tris-HCl pH 7.4) in fresh form (c), beads 3 after 1-h incubation (c1) and beads 4 (0.01% gelatin, 0.05% honey, 0.15 M NaCl and 10 mM MgSO₄·7H₂O) in fresh form (d), each bead was loaded with bacteriophage ZCEC5 in simulated intestinal juice

Fig. 3

In vitro Log₁₀ PFU mL⁻¹ release of phages from chitosan–alginate capsules during incubation in gastrointestinal fluid for 6 h

Fig. 4

a Log₁₀ reductions of E. coli O157:H7 incubated with encapsulated and non-encapsulated bacteriophages in gastrointestinal fluid at 37 °C for 6 h and 10 h. Nt stands for the number of E. coli O157:H7 after treatment with encapsulated and non-encapsulated bacteriophages at MOI = 1 and Nc represents the number of E. coli O157:H7 at the control state. All phage treatments produced significant falls in the viable count of E. coli O157:H7 (p value < 0.01). b Bacteriophage titers (Log₁₀ PFU) of non-capsulated and encapsulated phages after infecting E. coli O157:H7 at MOI = 1 in gastrointestinal fluid for 6 h and 10 h

Fig. 5

a The stability of non-encapsulated and encapsulated bacteriophages against high temperature (80 °C) for 3 min. b Low pH stability of the non-encapsulated and encapsulated bacteriophages 1 h at 37 °C. Limit of detection is < 10³ PFU mL⁻¹