In Vitro Antibacterial and Antibiofilm Activity of Hungarian Honeys against Respiratory Tract Bacteria

Abstract

Honey is a rich source of carbohydrates, while minor compounds such as amino acids and polyphenols contribute to its health-promoting effects. Honey is one of the oldest traditional remedies applied for microbial infections, due to its antibacterial, anti-inflammatory, and antioxidant properties. The aim of this study was to investigate the antibacterial and antibiofilm effects of Hungarian black locust, linden, and sunflower honeys against the most common biofilm-forming respiratory tract pathogens Haemophilus spp., Pseudomonas aeruginosa, and Streptococcus pneumoniae. The unifloral character of all three honey types was confirmed by melissopalynological analysis. The antibacterial activity of each honey sample against each bacterium strain was proven with agar well diffusion assay and thin layer chromatography-direct bioautography. Kinetics and mechanisms of antibacterial action were clarified with time-kill assay and membrane degradation study. The anti-biofilm activity was evidenced using crystal violet assay. In each assay, linden honey was the most effective, followed by sunflower and black locust honey. In addition, each honey sample had greater potential to suppress respiratory tract bacteria, compared to major sugar components. In conclusion, honey in general and linden honey in particular, can have a role in the treatment of respiratory tract infections caused by biofilm-forming bacteria.

Keywords: Haemophilus spp.; Pseudomonas aeruginosa; Streptococcus pneumoniae; antibacterial effect; antibiofilm activity; honey; respiratory tract.

Figure 1

Analysis of botanical origin of honey samples with light microscopy. Pollen grains of: (a) black locust, (b) linden, and (c) sunflower. Scale bar = 25 μm.

Figure 2

TLC–DB plate in the case of P. aeuruginosa. Negative control: distilled water, Positive control: gentamicin (40 mg/mL) from which 1.0 μL was applied to the plate. The numbers after each honey type refer to Apiary 1 and Apiary 2. The stock solutions from honey samples were 1.0 g/mL, 1.0 μL was applied to the plate. The blue background shows the viable bacteria. The white spots (as inhibition zones) indicated the lack of dehydrogenase activity due to the antibacterial activity of honey samples.

Figure 3

Antibacterial activity of black locust, linden, and sunflower honey samples against respiratory tract bacteria. The diameter of the inhibition zones was expressed in mm. Positive controls: gentamicin against P. aeruginosa, ceftriaxone against Haemophilus spp., and S. pneumoniae. Different letters above bars representing the same bacterial strain indicate significant differences at p ≤ 0.05.

Figure 4

Time-kill assay in case of P. aeruginosa: (A) black locust honey samples, (B) linden honey samples, (C) sunflower honey samples, and (D) sugar (glucose:fructose, 1:1) solutions.

Figure 5

Biofilm inhibition activity of different honey samples against respiratory tract bacteria: (A) Haemophilus influenzae, (B) H. parainfluenzae, (C) Pseudomonas aeruginosa, and (D) Streptococcus pneumoniae. The anti-biofilm formation activity was calculated and demonstrated in terms of inhibitory rate according to the equation: Inhibitory rate = (1 − S/C) × 100% (C and S were defined as the average absorbance of control and sample groups, respectively). Different lower case letters (a, b, c) above boxes in figures (A–D) indicate significant differences at p ≤ 0.05.