Vitamin C Enhances the Antibacterial Activity of Honey against Planktonic and Biofilm-Embedded Bacteria

Multifactorial antibacterial action is an important feature of honey; however, its bactericidal efficacy against biofilm-embedded bacteria is limited. The aim of this study was to investigate the impact of vitamin C (Vit C) on the antibacterial activity of natural honeys against planktonic as well as biofilm-embedded bacterial pathogens. The antibacterial activity of four honey samples supplemented with Vit C was expressed as the minimum inhibitory concentration (MIC). At sub-MICs, Vit C significantly increased the antibacterial activity of the tested honeys against Pseudomonas aeruginosa in planktonic cultures. However, after supplementation, honeydew honey, the most active honey, was ineffective against Staphylococcus aureus. On the other hand, when 100% honeydew honey was supplemented with Vit C (100 mg/g of honey) in a multispecies wound biofilm model, complete eradication of almost all bacterial isolates, including S. aureus, was observed. Furthermore, a mixture of honey and Vit C was partially effective against Enterococcus faecalis, whereas honey alone exhibited no antibacterial activity against this bacterium. Vit C counteracted hydrogen peroxide in honey solution and, thus, eliminated the major antibacterial compound present in honey. It is likely that a combination of honey with Vit C may trigger the intracellular production of reactive oxygen species in bacterial cells, but the exact cellular mechanisms warrant further investigations.


Introduction
Honey has been successfully used as a topical wound care product, with pronounced antibacterial, antibiofilm, and anti-inflammatory properties. Numerous randomized controlled clinical studies and case reports have been conducted in order to prove its wound-healing efficacy. A meta-analysis investigating the efficacy of honey in different types of wounds indicated that the relative effect of honey on wounds was unclear because the trials performed so far have been small, clinically heterogeneous, short, and highly biased [1][2][3][4][5]. However, some systematic reviews and meta-analyses have indicated that honey dressings are superior in the treatment of diabetic foot ulcers or burns when compared to conventional dressings [6][7][8]. Further clinical trials using honey as a topical wound care product are highly needed to clarify this ambiguity.

Effect of Vit C on Honey Antibacterial Activity Against Different Bacterial Isolates
The following four different honey samples were used in the study: acacia, sunflower, honeydew, and manuka. The minimum inhibitory concentration (MIC) values of the honey samples and Vit C against Pseudomonas aeruginosa were 10-15% of honey solution and 4 mg/mL, respectively ( Figure 1). The antibacterial activities of the honey solutions supplemented with sub-MICs of Vit C (1, 1.5, and 2 mg/mL) increased significantly in a dose-dependent manner. At a sub-MIC of 2 mg/mL, Vit C decreased the MIC values of all honey samples against P. aeruginosa to 4% (Figure 1). Honeydew honey, the most effective honey against P. aeruginosa with a MIC value of 10%, was used for further testing after its supplementation with sub-MICs of Vit C against a battery of different bacteria (Serratia marcescens, Escherichia coli, Staphylococcus aureus, and Bacillus subtilis) (Figure 2A,B). Honeydew honey exhibited even higher inhibitory activity, with MIC values ranging from 6% to 8% depending on the bacterial species, in comparison to its activity against P. aeruginosa ( Figure 2). On the other hand, supplementation of honeydew honey with sub-MICs of Vit C did not augment the antibacterial potential of the supplemented honey, particularly against Gram-positive bacteria. Furthermore, in the case of S. aureus, supplementation with Vit C resulted in an unwanted significant increase in honey MIC values. A significant increase in the antibacterial activity of the supplemented honeydew honey was only achieved against E. coli (Figure 2A). The resistance of Gram-positive bacteria to Vit C-supplemented honey may be attributed to differences in the cell wall structure or in cell metabolism.
The fractional inhibitory concentration (FIC) index for the combined application of honey samples and Vit C was 1.0 for P. aeruginosa, suggesting an additive interaction of Vit C against this bacterium.
Molecules 2020, 25, x FOR PEER REVIEW 3 of 13 The fractional inhibitory concentration (FIC) index for the combined application of honey samples and Vit C was 1.0 for P. aeruginosa, suggesting an additive interaction of Vit C against this bacterium.

Figure 1.
Antibacterial activities of honey samples (acacia, sunflower, honeydew, and manuka honey) supplemented with Vit C at sub-inhibitory concentrations against Pseudomonas aeruginosa isolates. The activity was determined with a MIC assay. The MIC was defined as the lowest concentration of honey solution (%) that inhibited bacterial growth. The data are expressed as mean values with standard deviations. **P  0.01 and ***P  0.001. Figure 1. Antibacterial activities of honey samples (acacia, sunflower, honeydew, and manuka honey) supplemented with Vit C at sub-inhibitory concentrations against Pseudomonas aeruginosa isolates. The activity was determined with a MIC assay. The MIC was defined as the lowest concentration of honey solution (%) that inhibited bacterial growth. The data are expressed as mean values with standard deviations. ** P < 0.01 and *** P < 0.001. The data are expressed as mean values with standard deviations. *P  0.05, **P  0.01, and ***P  0.001.

Effect of Vit C on the Antibacterial Activity of Heat-and Catalase-Treated Honey Samples
Honeydew and manuka honey samples were heat-treated in order to denature the major compounds responsible for direct (defensin-1) and indirect (GOX and polyphenols) antibacterial activity. Heated honey samples were supplemented with Vit C at final sub-MICs, and the overall antibacterial activity against P. aeruginosa was determined. Heat treatment of honeydew honey caused a significant loss of antibacterial activity; however, at sub-MICs, Vit C was able to restore the antibacterial activity of honeydew honey to its original level before heat treatment ( Figure 3A). As expected, manuka honey was resistant to heat treatment, and its antibacterial activity with/without Vit C supplementation remained unchanged after exposure to heat ( Figure 3B).
Catalase-treated honeydew honey showed weak antibacterial activity due to the neutralization of H2O2, and this reduced activity was comparable to the heat-affected antibacterial activity. At a concentration of 2 mg/mL, Vit C in the presence of catalase was able to increase the antibacterial activity of honeydew honey, as documented by a change of the MIC value from 30% to 15% ( Figure  3C).

Effect of Vit C on the Antibacterial Activity of Heat-and Catalase-Treated Honey Samples
Honeydew and manuka honey samples were heat-treated in order to denature the major compounds responsible for direct (defensin-1) and indirect (GOX and polyphenols) antibacterial activity. Heated honey samples were supplemented with Vit C at final sub-MICs, and the overall antibacterial activity against P. aeruginosa was determined. Heat treatment of honeydew honey caused a significant loss of antibacterial activity; however, at sub-MICs, Vit C was able to restore the antibacterial activity of honeydew honey to its original level before heat treatment ( Figure 3A). As expected, manuka honey was resistant to heat treatment, and its antibacterial activity with/without Vit C supplementation remained unchanged after exposure to heat ( Figure 3B).
Catalase-treated honeydew honey showed weak antibacterial activity due to the neutralization of H 2 O 2 , and this reduced activity was comparable to the heat-affected antibacterial activity. At a concentration of 2 mg/mL, Vit C in the presence of catalase was able to increase the antibacterial activity of honeydew honey, as documented by a change of the MIC value from 30% to 15% ( Figure 3C). Molecules 2020, 25, x FOR PEER REVIEW 5 of 13 Honeydew honey was also subjected to catalase treatment, and the antibacterial activity against P. aeruginosa was determined after its supplementation with Vit C at sub-inhibitory concentrations. The activity was determined with a MIC assay. The MIC was defined as the lowest concentration of honey solution (%) that inhibited bacterial growth. The data are expressed as mean values with standard deviations. *P  0.05, and ***P  0.001.

Effect of Vit C on H2O2 Production in Honey Samples
H2O2 concentration was assessed in freshly prepared 40% honey solutions supplemented with Vit C at a sub-MIC of 1 mg/mL. As shown in Figure 4A, Vit C neutralized H2O2 in all honey samples, including manuka honey. Furthermore, when the 40% honeydew solution with/without Vit C supplementation at concentrations of 1 and 2 mg/mL was incubated for a certain period of time (up to 24 h) at 37 °C, a significant decrease in H2O2 production occurred ( Figure 4B). Manuka honey, as a non-peroxide honey type, did not generate H2O2 in the 40% honey solution due to the methylglyoxalinduced loss of GOX activity ( Figure 4C). (C) Honeydew honey was also subjected to catalase treatment, and the antibacterial activity against P. aeruginosa was determined after its supplementation with Vit C at sub-inhibitory concentrations. The activity was determined with a MIC assay. The MIC was defined as the lowest concentration of honey solution (%) that inhibited bacterial growth. The data are expressed as mean values with standard deviations. * P < 0.05, and *** P < 0.001.

Effect of Vit C on H 2 O 2 Production in Honey Samples
H 2 O 2 concentration was assessed in freshly prepared 40% honey solutions supplemented with Vit C at a sub-MIC of 1 mg/mL. As shown in Figure 4A, Vit C neutralized H 2 O 2 in all honey samples, including manuka honey. Furthermore, when the 40% honeydew solution with/without Vit C supplementation at concentrations of 1 and 2 mg/mL was incubated for a certain period of time (up to 24 h) at 37 • C, a significant decrease in H 2 O 2 production occurred ( Figure 4B). Manuka honey, as a non-peroxide honey type, did not generate H 2 O 2 in the 40% honey solution due to the methylglyoxal-induced loss of GOX activity ( Figure 4C).

Effect of Honeydew Honey Supplemented with Vit C Against Bacterial Wound Pathogens Grown in a Multi-Species Biofilm
Honeydew honey, exhibiting strong antibacterial activity, was used for evaluating honey antibiofilm activity. A multi-species biofilm was formed by four bacterial species (S. aureus, Streptococcus agalactiae, P. aeruginosa, and Enterococcus faecalis) and remained stable during the experimental time period. The mature biofilm was treated with honeydew honey at a concentration of 100%, Vit C at a concentration of 100 mg/mL, and a mixture of honeydew honey with Vit C at a concentration of 100 mg/g of honey, and the number of surviving cells was determined by plate counting on different media ( Figure 5). Vit C exhibited the weakest antibiofilm activity, and a significant reduction in bacterial viability in the biofilm was documented only for S. aureus. Honeydew honey showed strong antibiofilm activity and was able to significantly reduce the cell viability of S. aureus, S. agalactiae, and P. aeruginosa in the mature biofilm within 48 h. On the other hand, in the multi-species biofilm, E. faecalis was rather resistant to the antibacterial effect of honeydew honey. The antibiofilm effect of honeydew honey supplemented with Vit C was stronger, and the mixture significantly lowered the counts of all bacterial wound isolates, including E. faecalis, within 24 h. Complete eradication of S. aureus, S. agalactiae, and P. aeruginosa in the biofilm was achieved after 48 h of incubation ( Figure 5).

Effect of Honeydew Honey Supplemented with Vit C Against Bacterial Wound Pathogens Grown in a Multi-Species Biofilm
Honeydew honey, exhibiting strong antibacterial activity, was used for evaluating honey antibiofilm activity. A multi-species biofilm was formed by four bacterial species (S. aureus, Streptococcus agalactiae, P. aeruginosa, and Enterococcus faecalis) and remained stable during the experimental time period. The mature biofilm was treated with honeydew honey at a concentration of 100%, Vit C at a concentration of 100 mg/mL, and a mixture of honeydew honey with Vit C at a concentration of 100 mg/g of honey, and the number of surviving cells was determined by plate counting on different media ( Figure 5). Vit C exhibited the weakest antibiofilm activity, and a significant reduction in bacterial viability in the biofilm was documented only for S. aureus. Honeydew honey showed strong antibiofilm activity and was able to significantly reduce the cell viability of S. aureus, S. agalactiae, and P. aeruginosa in the mature biofilm within 48 h. On the other hand, in the multi-species biofilm, E. faecalis was rather resistant to the antibacterial effect of honeydew honey. The antibiofilm effect of honeydew honey supplemented with Vit C was stronger, and the mixture significantly lowered the counts of all bacterial wound isolates, including E. faecalis, within 24 h. Complete eradication of S. aureus, S. agalactiae, and P. aeruginosa in the biofilm was achieved after 48 h of incubation ( Figure 5). Figure 5. The antibiofilm effects of Vit C (100 mg/mL), honeydew honey, and honeydew honey supplemented with Vit C at a concentration of 100 mg/g of honey against particular bacterial species forming a modified Lubbock chronic wound biofilm after 24 h and 48 h of treatment. The data are expressed as mean values with standard deviations. **P  0.01; ***P  0.001; ns: non-significant.

Discussion
One of the important features of honey is its multifactorial antibacterial action, with no risk of development of bacterial resistance. However, the bactericidal efficacy of honey is limited, and some bacterial pathogens could be more resistant to honey than others. In general, honey is more effective against Gram-positive bacteria, except for E. faecalis, than Gram-negative bacteria in a planktonic form. In the case of biofilm-embedded bacteria, honey becomes less effective, and a high Figure 5. The antibiofilm effects of Vit C (100 mg/mL), honeydew honey, and honeydew honey supplemented with Vit C at a concentration of 100 mg/g of honey against particular bacterial species forming a modified Lubbock chronic wound biofilm after 24 h and 48 h of treatment. The data are expressed as mean values with standard deviations. ** P < 0.01; *** P < 0.001; ns: non-significant.

Discussion
One of the important features of honey is its multifactorial antibacterial action, with no risk of development of bacterial resistance. However, the bactericidal efficacy of honey is limited, and some bacterial pathogens could be more resistant to honey than others. In general, honey is more effective against Gram-positive bacteria, except for E. faecalis, than Gram-negative bacteria in a planktonic form. In the case of biofilm-embedded bacteria, honey becomes less effective, and a high concentration of a honey solution is needed to eradicate the bacteria within the biofilm. To facilitate improvements in the antibacterial efficacy of honey, particularly against bacterial biofilms, honey has been supplemented with several distinct substances, including antimicrobial peptides [15], bacteriophages [26,27], and essential oils [17]. In the present study, we showed that supplementation of different types of honey with sub-MICs of Vit C resulted in a significant enhancement of the antibacterial activity against P. aeruginosa and E. coli. On the other hand, Vit C did not improve the antibacterial efficacy of honey against B. subtilis or it significantly reduced the antibacterial activity of the tested honey against S. aureus. Taking into account the state of the bacteria and the fact that biofilm-embedded bacteria are more resistant to antibacterial compounds, Vit C at a higher concentration (100 mg per g of honey) improved the antibacterial efficacy of honeydew honey against all bacteria including S. aureus and E. faecalis. Interestingly, the honeydew honey supplemented with Vit C significantly decreased the viability of E. faecalis, which was highly resistant to 100% honeydew honey.
E. faecalis together with Enterococcus faecium demonstrate an intrinsic resistance to common antibiotics and they readily acquire resistance to other novel classes of antibiotics due to a plastic genome [28]. In our previous study [29], within a multispecies biofilm, E. faecalis was shown to be resistant to honeydew honey and manuka honey, as well as to the antibacterial peptide defensin-1. These results are in agreement with those of other studies, where E. faecalis exhibited the highest resistance among the nosocomial and foodborne pathogens against Corsican and Italian honeys [30,31]. The difficulty of E. faecalis treatment has been attributed to the lack of anti-infective strategies to eradicate its biofilm and to the frequent emergence of multidrug-resistant strains. Therefore, effective options are needed to eradicate E. faecalis, both in planktonic and in biofilm forms. One potential effective option is a combined treatment where individual agents may act synergistically. Recently, an anti-E. faecalis phage combined with vancomycin effectively eliminated an E. faecalis biofilm [32].
Vit C, a regular but concentration-variable component of honey, is an important antioxidant agent that significantly decreases the adverse effects of ROS. A surprising observation regarding the pro-oxidant effect of Vit C was reported [20]. Vit C acts as an antimicrobial agent against different bacterial pathogens, including S. aureus [23], E. coli [21], and Klebsiella pneumoniae [22]. It has been proposed that the antibacterial effects of Vit C might be both bacterial strain-and concentration-dependent. The most probable Vit C antibacterial mechanism of action is based on oxidative stress due to the generation of ROS. According to a very recent study [33], the combination of Vit C with an essential oil and copper resulted in free radical generation through the Fenton reaction. Honey, particularly honeydew honey, is rich in minerals including transition metal ions (Fe and Cu) and polyphenols, and its combination with Vit C may trigger the intracellular production of ROS in bacterial cells. At sub-MICs, Vit C in a honey solution rapidly neutralizes the generated H 2 O 2 , which is a major antibacterial factor of honey. The antibacterial activity of the honey/Vit C combination against certain Gram-positive bacteria in planktonic cultures is less effective or ineffective, which can be attributed to differences in the bacterial cell wall structure, ROS-protective enzymes, or metabolism.
Discrepancies in the antibacterial efficacy of honey supplemented with Vit C against planktonic and biofilm cultures of S. aureus were reported in this study. There could be several reasons for these discrepancies, but the most important is the fact that a multi-bacterial wound biofilm consisting of four different bacterial isolates, including S. aureus, was utilized. From a clinical point of view, honey supplemented with Vit C mediated the eradication of all species in the multispecies biofilm, which was achieved after 24 h. Therefore, the honey/Vit C combination represents a very promising therapy for the treatment of chronically infected wounds, which needs to be evaluated clinically.
A combination of honey and Vit C has already been pre-clinically, topically tested in an animal, non-infected, burn wound model [34]. It was shown that the combination of honey with Vit C promoted rapid debridement, and an advanced proliferative phase was observed with respect to unsupplemented honey. Unfortunately, no clinical data are currently available for the topical application of the combined therapy (honey and Vit C) in wound care management. Further clinical studies are warranted in order to determine the potential therapeutic effect of Vit C-supplemented honey.

Honey Samples
The following honey sample types were collected across Slovakia and used in this study: acacia, sunflower, linden, and honeydew. Commercially available manuka honey with a unique manuka factor of 15 (UMF 15+), imported from New Zealand, was purchased from Nature's Nectar (Surrey, UK).

Bacterial Strains for Antibacterial and Antibiofilm Activity Testing
The antibacterial activities of honey samples, Vit C, as well as a mixture of honey and Vit C were assessed against P. aeruginosa CCM1960 and S. aureus CCM4223 isolates, obtained from the Department of Medical Microbiology, Slovak Medical University (Bratislava, Slovakia), as well as against E. coli CCM3954, S. marcescencs CCM8587, and B. subtilis CCM2216 isolates obtained from the Institute of Chemistry Slovak Academy of Sciences (Bratislava, Slovakia).
Bacterial strains of S. aureus, S. agalactiae, E. faecalis, and P. aeruginosa which were originally isolated from patients with chronic wounds hospitalized at University Hospital, Hradec Kralove (Hradec Kralove, Czech Republic), were used for multi-species wound biofilms (modified Lubbock chronic wound biofilm model). Sodium chloride peptone broth (buffered peptone bouillon, BPB; Merck, Darmstadt, Germany) was used for dilutions and for normalizing the optical density of the cultures in all wound biofilm experiments.
After 18 h of incubation at 37 • C, bacterial growth inhibition was determined by monitoring the optical density at 490 nm. The MIC was defined as the lowest concentration of honey, Vit C, and mixture of honey with Vit C that inhibited bacterial growth. All tests were performed in triplicate and repeated three times. The

Antibiofilm Activity Analysis Using a Polybacterial Biofilm
We used the previously described modified Lubbock chronic wound biofilm model according to Sojka et al. (2016) [29]. First, 6 mL of medium, containing Bolton broth base (Sigma-Aldrich, Germany), 1% gelatin, 50% porcine plasma, and 5% porcine erythrocytes lysed by freeze-thawing, was dispensed into sterile 1.6 × 10 cm glass tubes. Cultures of the selected bacteria, normalized according to the optical density, were mixed, and 10 µL of the mixture containing 10 6 CFU/mL was inoculated into each tube by ejecting the pipette tips along with the bacterial suspension. Inoculated tubes were incubated at 37 • C in an orbital shaker (1500 g) for 48 h, and the pre-formed biofilms were harvested.
Harvested biofilms were washed with BPB. The biofilms were placed into the artificial wound bed in the nutrient medium and covered with a piece of 100% cotton 8-ply gauze sponge (Batist medical, Cerveny Kostolec, Czech Republic), soaked with the test substance (100% honeydew honey or its mixture with Vit C-100 mg of Vit C per g of honey) or BPB (negative control). Biofilms in the artificial wound bed treated with particular substances were incubated at 37 • C for 24 h and 48 h. After treatment, the biofilms were harvested from the artificial wound bed using sterile forceps and a Lang eye spoon and then homogenized. The bacteria per culture were determined using different media, as described before [36], and Brilliance UTI selective medium (Oxoid, Basingstoke, UK).

Determination of Hydrogen Peroxide H 2 O 2 Concentration
The concentration of H 2 O 2 in the honey samples was determined with the Megazyme GOX assay kit (Megazyme International Ireland Ltd.), which is based on the release of H 2 O 2 . For the standard, H 2 O 2 diluted to 9.8-312.5 µM was used. Briefly, 40% (w/w) honey solutions in 0.1 M potassium phosphate buffer (pH 7.0) were prepared and either immediately used for H 2 O 2 measurements or measured after incubation of the prepared solutions for 24 h at 37 • C. Each honey sample and standard were tested in duplicate in a 96-well microplate, and the absorbance was measured at 510 nm using a Synergy HT microplate reader (BioTek Instruments, Winooski, VT, USA).

Heat and Catalase Treatment of Honey
Samples of 100% honeydew honey and manuka honey were heated at 100 • C for 5 min in order to destroy the antibacterial activity mediated by GOX, polyphenols, and defensin-1. The samples were then cooled to room temperature and used alone or supplemented with Vit C at sub-MICs for the determination of the antibacterial activity against P. aeruginosa.
A diluted honeydew honey sample (50% w/w in MHB medium) was treated with catalase (2000-5000 U/mg protein; Sigma-Aldrich) at a final concentration of 1000-2500 U/mL at room temperature for 2 h in order to eliminate H 2 O 2 , the major antibacterial factor of honey. Catalase-treated honeydew honey was then used alone or supplemented with Vit C at sub-MICs in the antibacterial assay to determine MIC values against P. aeruginosa.

Statistical Analysis
All data were statistically analyzed using one-way ANOVA. The data are expressed as mean values with standard deviations (SDs). Data with P values smaller than 0.05 were considered statistically significant. All statistical analyses were performed using GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA).

Conclusions
In conclusion, our present study shows that supplementation of honey with Vit C at sub-MICs significantly enhances the antibacterial activity of honey against P. aeruginosa and E. coli planktonic cultures. On the other hand, the combined therapy was shown to be ineffective against S. aureus planktonic cultures. However, when a 100% honey supplemented with Vit C at concentration of 100 mg/g of honey was used in a multispecies wound biofilm model, complete eradication of all bacterial isolates, including S. aureus, was observed. Furthermore, the combined therapy was effective against E. faecalis, whereas honey alone exhibited no antibacterial activity against this bacterium. The exact mechanism of action of Vit C-supplemented honey is unclear; however, honey polyphenols and mineral contents in the presence of Vit C could be responsible for intracellular free radical generation in bacterial cell.

Patents
Some of the data in this publication have been used in the national utility model application 8435 and are owned by the Institute of Molecular Biology Slovak Academy of Sciences and Slovak Medical University.