Honey can inhibit and eliminate biofilms produced by Pseudomonas aeruginosa

Abstract

Chronic wound treatment is becoming increasingly difficult and costly, further exacerbated when wounds become infected. Bacterial biofilms cause most chronic wound infections and are notoriously resistant to antibiotic treatments. The need for new approaches to combat polymicrobial biofilms in chronic wounds combined with the growing antimicrobial resistance crisis means that honey is being revisited as a treatment option due to its broad-spectrum antimicrobial activity and low propensity for bacterial resistance. We assessed four well-characterised New Zealand honeys, quantified for their key antibacterial components, methylglyoxal, hydrogen peroxide and sugar, for their capacity to prevent and eradicate biofilms produced by the common wound pathogen Pseudomonas aeruginosa. We demonstrate that: (1) honey used at substantially lower concentrations compared to those found in honey-based wound dressings inhibited P. aeruginosa biofilm formation and significantly reduced established biofilms; (2) the anti-biofilm effect of honey was largely driven by its sugar component; (3) cells recovered from biofilms treated with sub-inhibitory honey concentrations had slightly increased tolerance to honey; and (4) honey used at clinically obtainable concentrations completely eradicated established P. aeruginosa biofilms. These results, together with their broad antimicrobial spectrum, demonstrate that manuka honey-based wound dressings are a promising treatment for infected chronic wounds, including those with P. aeruginosa biofilms.

Figure 1

Effect of honey and sugar solution on P. aeruginosa biofilm formation. P. aeruginosa PAO1 (A) and PA14 (B) biofilms were allowed to form in the presence of four NZ honeys (manuka, Medihoney, manuka-kanuka, or clover) or a sugar solution. Biofilm formation was assessed using a static biofilm formation assay with crystal violet staining to quantify biomass. Biofilm formation is expressed as % relative to that produced by the untreated control (Control), which is set at 100%. Error bars represent ± SD of three biological samples, all performed in triplicate. *Indicates statistically significant difference (p < 0.05) relative to the untreated control (Control; 0%).

Figure 2

Effect of honey or sugar solution on established P. aeruginosa biofilms and on cell viability within the biofilms. Established P. aeruginosa PAO1 (left panels) and PA14 (right panels) biofilms were treated with four NZ honeys (manuka, Medihoney, manuka-kanuka, and clover), or a sugar solution. Biofilm biomass remaining post-treatment (coloured lines) was quantified using crystal violet staining and expressed as OD₅₉₅ (left y-axis). The corresponding cell viability (black line) within remaining biofilms was assessed via ATP production using the BacTitre Glo Viability Kit and CFU/well values were determined from a previously established standard curve (right y-axis). Data represents mean values from three biological replicates, all performed in triplicate ± SD. Coloured (*) indicate statistically significant decrease (p < 0.05) in biofilm biomass and black (*) indicates statistically significant difference (p < 0.05) in ATP production, both relative to the control (at 0% honey concentration).

Figure 3

Visualisation of established P. aeruginosa biofilms treated with different honeys. 3-D images produced by confocal laser scanning microscopy of established P. aeruginosa PAO1 and PA14 biofilms, following treatment with sub-inhibitory (1 and 2%) and inhibitory (16 and 32%) concentrations of NZ honeys (manuka, Medihoney, manuka-kanuka, or clover) or control sugar solution. Biofilms were stained with Syto9 (green = viable cells) and propidium iodine (red = dead cells). Scale bar represents 50 µm.

Figure 4

Effects of high concentrations of manuka and Medihoney on established P. aeruginosa biofilms. 3-D images produced by confocal laser scanning microscopy of established P. aeruginosa PAO1 and PA14 biofilms, following treatment with high concentrations (64 and 80%) of manuka honey and Medihoney. Biofilms were stained with Syto9 (green = viable cells) and propidium iodine (red = dead cells). Scale bar represents 50 µm.

Figure 5

Effect of MGO on biofilm formation by P. aeruginosa. Biofilm formation by P. aeruginosa PAO1 (A) and PA14 (B) in the presence of MGO and MGO plus sugar solution. MGO concentrations correspond to those present in the manuka-type honeys: 100 mg/kg as in manuka-kanuka honey, 700 mg/kg as in Medihoney, and 900 mg/kg as in manuka honey. Biofilm formation is expressed as a percentage relative to the untreated control, which is set at 100%. Results presented are mean values from three biological replicates, all performed in triplicate ± SD.

Figure 6

Effect of MGO on established P. aeruginosa biofilms. P. aeruginosa PAO1 (A) and PA14 (B) biofilms treated with MGO, with and without sugar solution. MGO concentrations used correspond to those in the manuka-type honeys: 100 mg/kg as in manuka-kanuka honey, 700 mg/kg as in Medihoney^M, and 900 mg/kg as in manuka honey. Biofilm formation is expressed as a percentage relative to the untreated control, which is set at 100%. Results presented are mean values from three biological replicates, all performed in triplicate ± SD.