Search

Sugars, in particular glucose and fructose, are the main constituents of honey, comprising up to 85% of its total weight. A high-performance thin-layer chromatography (HPTLC) method to identify and quantify common sugars (glucose, fructose and sucrose) in honey has been developed and fully validated according to the International Conference on Harmonisation guidelines. It allows to determine a honey’s fructose-to-glucose ratio, which is not only an important authentication and quality control parameter, but also an indication of its tendency to crystallise. The HPTLC analysis is easy to perform, accurate, precise, specific and sensitive and requires only minimal sample preparation. With a limit of detection/limit of quantification of 21.98 ng/66.62 ng for fructose, 33.00 ng/100.00 ng for glucose and 21.17 ng/64.15 ng for sucrose, the sensitivity of the method has been greatly improved compared to other HPTLC-based approaches. An additional advantage is the method’s simplicity and fast processing time as it only requires a single development step without plate or sample pre-treatment.

Using honey as a model sample, the aim of this study is to develop and validate a simple, rapid screening tool that allows for the visualization of constituents that contribute to the antioxidant activity of a complex natural product and to quantify their individual antioxidant effects even if their chemical identity is unknown. The method employs a validated analysis, which is based on the separation of constituents using high-performance thin-layer chromatography (HPTLC) followed by their visualization using DPPH* (2,2-diphenyl-1-picrylhydrazyl) as derivatizing reagent and the quantification of the antioxidant activity of individual bands expressed as gallic acid equivalents.

The antibacterial activity of honeys derived from the endemic flora of the southwest corner of Western Australia, including the trees Jarrah (Eucalyptus marginata) and Marri (Corymbia calophylla), remains largely unexplored. Investigation of these honeys showed minimum inhibitory concentrations (MICs) of 6.7–28.0% (w/v) against Gram positive and negative bacteria. Honey solutions showed enhanced antibacterial activity after hydrogen peroxide was allowed to accumulate prior to testing, with a mean MIC after accumulation of 14.3% compared to 17.4% before accumulation. Antibacterial activity was reduced after treatment with catalase enzyme, with a mean MIC of 29.4% with catalase compared to 15.2% without catalase. Tests investigating the role of the Gram negative outer membrane in honey susceptibility revealed increases in activity after destabilisation of the outer membrane. Honeys reduced both the formation of biofilm and the production of bacterial pigments, which are both regulated by quorum sensing. However, these reductions were closely correlated with global growth inhibition. Honey applied to existing biofilms resulted in decreased metabolic activity and minor decreases in viability. These results enhance our understanding of the mechanisms of antibacterial action of Jarrah and Marri honeys, and provide further support for the use of honey in the treatment of infected wounds.

The physicochemical parameters and antibacterial activity of 10 Western Australian (WA) and two comparator honeys were determined. Honeys showed a pH range of 4.0–4.7, colour range of 41.3–470.7 mAU, methylglyoxal levels ranging from 82.2 to 325.9 mg kg−1 and hydrogen peroxide levels after 2 h of 22.7–295.5 µM. Antibacterial activity was assessed by the disc diffusion assay, phenol equivalence assay, determination of minimum inhibitory and bactericidal concentrations and a time-kill assay. Activity was shown for all honeys by one or more method, however, activity varied according to which assay was used. Minimum inhibitory concentrations for WA honeys against 10 organisms ranged from 4.0 to >32.0% (w/v). Removal of hydrogen peroxide activity by catalase resulted in decreased activity for several honeys. Overall, the data showed that honeys in addition to those derived from Leptospermum spp. have antimicrobial activity and should not be overlooked as potential sources of clinically useful honey.

The spectrum of activity and mode of action of a novel antibacterial agent, 135C, was investigated using a range of microbiological and genomic approaches. Compound 135C was active against Gram-positive bacteria with MICs for Staphylococcus aureus ranging from 0.12–0.5 μg/ml. It was largely inactive against Gram-negative bacteria. The compound showed bacteriostatic activity in time-kill studies and did not elicit bacterial cell leakage or cell lysis. Checkerboard assays showed no synergy or antagonism when 135C was combined with a range of other antibacterials. Multi-step serial passage of four S. aureus isolates with increasing concentrations of 135C showed that resistance developed rapidly and was stable after drug-free passages. Minor differences in the fitness of 135C-resistant strains and parent wildtypes were evident by growth curves, but 135C-resistant strains did not show cross-resistance to other antibacterial agents. Genomic comparison of resistant and wildtype parent strains showed changes in genes encoding cell wall teichoic acids. 135C shows promising activity against Gram-positive bacteria but is currently limited by the rapid resistance development. Further studies are required to investigate the effects on cell wall teichoic acids and to determine whether the issue of resistance development can be overcome.

This study investigated the hypothesis that the salt adaptation response of Enterococcus faecalis alters susceptibility to tea tree oil (TTO). Six E. faecalis isolates were adapted to 6.5 % NaCl, and then exposed to TTO in phosphate-buffered saline (PBS). One isolate was also exposed to TTO in Brain Heart Infusion Broth (BHIB). The viability of salt-adapted and non-adapted control cells was determined at 0, 45 and 90 min and compared. MICs for several antibiotics and TTO were also determined by E test and broth microdilution, respectively. Results showed that susceptibility to TTO in PBS was significantly reduced after salt adaptation for five isolates (83 %) (P < 0.05). Mean differences between salt-adapted and non-adapted cell counts were 2.51 log at 45 min and 2.13 log at 90 min. However, when E. faecalis ATCC 19433 was exposed to TTO in BHIB, no significant differences were seen. In conclusion, salt adaptation resulted in reduced susceptibility to TTO in PBS for the majority of isolates, indicating that cross protection had occurred. This effect was absent in BHIB, suggesting that the uptake of compatible solutes from the growth medium protected non-adapted cells from TTO. Whether this has implications for the clinical effectiveness of TTO remains to be determined.