Synergistic Antimicrobial Interaction between Honey and Phage against Escherichia coli Biofilms

Abstract

Chronic wounds afford a hostile environment of damaged tissues that allow bacterial proliferation and further wound colonization. Escherichia coli is among the most common colonizers of infected wounds and it is a prolific biofilm former. Living in biofilm communities, cells are protected, become more difficult to control and eradicate, and less susceptible to antibiotic therapy. This work presents insights into the proceedings triggering E. coli biofilm control with phage, honey, and their combination, achieved through standard antimicrobial activity assays, zeta potential and flow cytometry studies and further visual insights sought by scanning electron microscopy and transmission electron microscopy. Two Portuguese honeys (PF2 and U3) with different floral origin and an E. coli-specific phage (EC3a), possessing depolymerase activity, were tested against 24- and 48-h-old biofilms. Synergic and additive effects were perceived in some phage-honey experiments. Combined therapy prompted similar phenomena in biofilm cells, visualized by electron microscopy, as the individual treatments. Honey caused minor membrane perturbations to complete collapse and consequent discharge of cytoplasmic content, and phage completely destroyed cells leaving only vesicle-like structures and debris. Our experiments show that the addition of phage to low honey concentrations is advantageous, and that even fourfold diluted honey combined with phage, presents no loss of antibacterial activity toward E. coli. Portuguese honeys possess excellent antibiofilm activity and may be potential alternative therapeutic agents in biofilm-related wound infection. Furthermore, to our knowledge this is the first study that assessed the impacts of phage-honey combinations in bacterial cells. The synergistic effect obtained was shown to be promising, since the antiviral effect of honey limits the emergence of phage resistant phenotypes.

Keywords: E. coli; bacteriophage; biofilms; honey; synergy.

FIGURE 1

Characteristics of EC3a. (a) Plaque morphology (black arrows indicate diameter of EC3a plaque and diameter of EC3a plaque and the surrounding halo. Scale bar 1 cm), (b) virion particle, and (c) circular view of phage vB_EcoS_CEB-EC3a and TBLASTX comparison with the two closest E. coli phage homologs and T1. The outer ring represents EC3a CDSs. The other three outer rings represent TBLASTX homologies with phages M12, RTP, and T1, respectively. The GC content appears in the black ring, and the inner rings are the GC skew⁺ (green) and the GC skew^- (pink). Some important EC3a genes are indicated.

FIGURE 2

Phage EC3a viability. (a) PFU counts after EC3a exposure to PF2 and U3 honeys at 25% (w/v) and 50% (w/v) concentrations, TEM micrographs of EC3a phage tails after 6 h of contact with honeys with (b) U3_25% and (c) PF2_25%. Scale bar in TEM micrographs is 100 nm.

FIGURE 3

Antibiofilm effect of phage EC3a, honey, and of the phage–honey combination on 24-h-old E. coli biofilms. (A) PF2_25%, (B) PF2_50%, (C) U3_25%, (D) U3_50%. ^†Values present no reductions compared to control samples. ^∗Indicates that the difference is statistically significant at the p-value < 0.05 level.

FIGURE 4

Antibiofilm effect of phage EC3a, honey, and of the phage–honey combination on 48-h-old E. coli biofilms. (A) PF2_25%, (B) PF2_50%, (C) U3_25%, (D) U3_50%. ^†Values present no reductions compared to control samples. ^∗Indicates that the difference is statistically significant at the p-value < 0.05 level.

FIGURE 5

SEM micrographs showing the effect of honey, phage, and honey–phage combination treatments in E. coli cells. (a) Control E. coli, (b–i) honey treatment, (j,k) phage treatment, (l–p) phage–honey treatment. The honey used in these experiments was PF2.

FIGURE 6

TEM micrographs showing the effect of honey, phage, and honey–phage combination treatments in E. coli cells. (a) Control E. coli, (b) PF2_25%, (c) U3_25%, (d) EC3a phage at a MOI 10, (e) phage–PF2_25% at a MOI 10, (f) phage–U3_25% at a MOI 10 (arrows indicate membrane detachment and dashed arrows point cell debris).

FIGURE 7

Flow cytometric analysis after 12 h application of single and combined treatments to 24-h-old E. coli biofilms. Representative dot plot FL1 (x-axis) vs FL4 channel (y-axis) showing E. coli cells stained with SYTO BC (250 nM) and PI (20 μg.mL^-1). (A) E. coli control, (B) PF2_25%, (C) U3_25%, (D) U3_50%, (E) EC3a phage at a MOI 10, (F) phage–PF2_25% at a MOI 10, (G) phage–U3_25% at a MOI 10, (H) phage–U3_50% at a MOI 10. Results are a representative example of two independent experiments.