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.
Highlights
Chronic wounds take months, years or may even never heal and present a major biological and financial problem on both individual patients and the broader health system
Several honey samples collected from regional beekeepers were characterized (Supplementary Table S1)
Based on two main characteristics—MGO content, a molecule reported as the major antibacterial agent in honeys (Kilty et al, 2011), and minimum inhibitory concentration (MIC), two honeys were selected for further characterization and antimicrobial evaluation in E. coli biofilms
Summary
Years or may even never heal and present a major biological and financial problem on both individual patients and the broader health system. Biofilms are formed in a sequential cycle of discrete and well-regulated events starting from: (i) adsorption of macro and smaller molecules to surfaces; (ii) bacterial adhesion to the wound surface and expression of extracellular polymeric substances (EPS); (iii) microcolony formation and biofilm maturation. Cell aggregation in these biofilm communities are well known to block antibiotics from reaching bacteria and block host’s immune cells contrarily to their planktonic counterparts which lack structure and are not surrounded by a polymeric matrix (Costerton and Lewandowski, 1995; Stewart and William Costerton, 2001; Fux et al, 2005). Besides protection to antimicrobials and host defenses, the biofilm mode of growth confers protection to the microorganism from mechanical and shear forces (McCarty et al, 2012) and from altered pH, osmolarity, and nutrient limitation (Costerton et al, 1999; Fux et al, 2005)
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