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.

Star anise (Illicium verum Hook. f.) essential oil: Antioxidant properties and antibacterial activity against Acinetobacter baumannii

“…For scanning electron microscopy (SEM) visualization, biofilms were formed on polystyrene coupons placed on 12‐well plates . Biofilm formation for both A. baumannii strains was performed as described above, adding 500 μL of bacterial suspensions (OD 600nm of 0.02) plus 500 μL of the EO (0.5× MIC), and without the EO (500 μL of LB broth) as a growth control.…”

“…For scanning electron microscopy (SEM) visualization, biofilms were formed on polystyrene coupons placed on 12-well plates. 27 Biofilm formation for both A. baumannii strains was performed as The samples were then allowed to dry overnight in a desiccator.…”

Investigation of different interactions between Staphylococcus aureus phages and pomegranate peel, grape seed, and black cumin extracts

“…The majority of these studies are based on phage-antibiotic synergism and the elimination of biofilms (Chaudhry et al, 2017;Nouraldin et al, 2016) or bacteria such as Acinetobacter baumannii (Jansen et al, 2018), Money, 2017), Pseudomonas aeruginosa (Lin et al, 2018;Uchiyama et al, 2018), S. aureus (Jo, Kim, Ding, & Ahn, 2016). There are also studies examining the interaction of phages with different agents such as honey (Oliveira et al, 2017) or disinfectants (Agún et al, 2018). In these studies, promising results were obtained when phages were used with different concentrations of agents showing antimicrobial activity.…”

Bacteriophages inhibit biofilms formed by multi-drug resistant bacteria isolated from septic wounds

“…Pathogenic bacterial biofilms are associated with both acute and chronic infectious sites and provide protection to the bacteria from antimicrobial agents; since the drugs cannot penetrate this structural barrier (Costerton et al, 2003; Deshpande Kaistha and Kaistha, 2017). Therefore, pathogenic bacteria that form biofilms develop antibiotic resistance and are often found to be very difficult to be eliminated by the conventional antibiotics (Nouraldin et al, 2016; Oliveira et al, 2017; Fang et al, 2018).…”

“…Bacteriophages when delivered at the infection site can replicate to produce a large number of active progeny, which in turn produce elution factors and degrading enzymes that target the polysaccharide components of the host bacteria, leading to bacterial lysis (Ghannad and Mohammadi, 2012; Vieira et al, 2012; Nouraldin et al, 2016). Though many studies have shown bacteriophage mediated mechanism of biofilm degradation, very few studies have used biofilms formed by clinical isolates (Pires et al, 2011; Danis-Wlodarczyk et al, 2015; Deshpande Kaistha and Kaistha, 2017; Oliveira et al, 2017; Ribeiro et al, 2018). Hence, there is an urgent need to identify bacteriophages that can act on the biofilms formed by bacteria isolated from wounds.…”