The impact of agarose immobilization on the activity of lytic Pseudomonas aeruginosa phages combined with chemicals

Abstract

The implementation of non-traditional antibacterials is currently one of the most intensively explored areas of modern medical and biological sciences. One of the most promising alternative strategies to combat bacterial infections is the application of lytic phages combined with established and new antibacterials. The presented study investigates the potential of agarose-based biocomposites containing lytic Pseudomonas phages (KT28, KTN4, and LUZ19), cupric ions (Cu²⁺), strawberry furanone (HDMF), and gentamicin (GE) as antibacterials and anti-virulent compounds for novel wound dressings. Phages (KT28, KTN4, LUZ19, and triple-phage cocktail) alone and in combination with a triple-chemical mixture (Cu + GE + HDMF) when applied as the liquid formulation caused a significant bacterial count reduction and biofilm production inhibition of clinical P. aeruginosa strains. The immobilization in the agarose scaffold significantly impaired the bioavailability and diffusion of phage particles, depending on virion morphology and targeted receptor specificity. The antibacterial potential of chemicals was also reduced by the agarose scaffold. Moreover, the Cu + GE + HDMF mixture impaired the lytic activity of phages depending on viral particles' susceptibility to cupric ion toxicity. Therefore, three administration types were tested and the optimal turned out to be the one separating antibacterials both physically and temporally. Taken together, the additive effect of phages combined with chemicals makes biocomposite a good solution for designing new wound dressings. Nevertheless, the phage utilization should involve an application of aqueous cocktails directly onto the wound, followed by chemicals immobilized in hydrogel dressings which allow for taking advantage of the antibacterial and anti-virulent effects of all components. KEY POINTS: • The immobilization in the agarose impairs the bioavailability of phage particles and the Cu + GE + HDMF mixture. • The cupric ions are toxic to phages and are sequestrated on phage particles and agarose matrix. • The elaborated TIME-SHIFT administration effectively separates antibacterials both physically and temporally.

Keywords: Agarose immobilization; Cupric ions; Gentamicin; HDMF furanone; Phages; Pseudomonas aeruginosa.

Fig. 1

Scheme of phage various administrations in the presence of Cu + GE + HDMF in agarose medium to test the activity against P. aeruginosa

Fig. 2

Different aspects of the interactions between the individual components of the designed biocomposite influencing anti-P. aeruginosa activity

Fig. 3

The antibacterial activity of KT28, KTN4, and LUZ19 phages separately, and as a triple-phage cocktail against selected P. aeruginosa strains. Results are presented as cumulative OD₆₀₀ after 20 h of incubation in the presence of a single/triple-phage cocktail without chemicals, and b single/triple-phage cocktail combined with Cu + GE + HDMF. Statistical analyses of tested variants are presented in Supplementary Materials (Table S2). Only the principal statistically significant differences (p < 0.05) if compared to a TSB or b TSB + Cu + GE + HDMF control without phages were presented in the graph as brackets (black represents significantly better antibacterial effect; red represents significantly worse antibacterial effect). The significant antibacterial effect of Cu + GE + HDMF alone against P. aeruginosa strains is marked as a green asterisk next to the strain name (panel b). The results displayed are the mean of three independent experiments

Fig. 4

The anti-biofilm activity of KT28, KTN4, and LUZ19 phages separately, and as a triple-phage cocktail against selected P. aeruginosa strains. Results are presented as absorbance of CV-stained biofilm biomass after 20 h of incubation in the presence of a single/triple-phage cocktail without chemicals, and b single/triple-phage cocktail combined with Cu + GE + HDMF. Statistical analyses of tested variants are presented in Supplementary Materials (Table S2). Only the principal statistically significant differences (p < 0.05) if compared to a TSB or b TSB + Cu + GE + HDMF control without phages were presented in the graph as brackets (black represents significantly better anti-biofilm effect, red represents significantly worse anti-biofilm effect). The significant anti-biofilm effect of Cu + GE + HDMF alone against P. aeruginosa strains is marked as a green asterisk next to the strain name (panel b). The results displayed are the mean of three independent experiments

Fig. 5

The release of KT28, KTN4, or LUZ19 phages from agarose gel measured by a biological method with the PFU/ml determination during 20 h, and b physical method (laser interferometry) for 60 min with time interval Δt = 2 min. The results displayed are the mean of three independent experiments

Fig. 6

The representative microscopy images of a mature P. aeruginosa PAO1 biofilm treated for 24 h with KT28, KTN4, or LUZ19 phages released from agarose gel. Biofilm was stained using Filmtracer™ LIVE/DEAD™ Biofilm Viability Kit, where green bacterial cells are alive, and red cells are dead (magnification 100 ×)