The effects of New Zealand manuka-type honeys on bacterial growth and morphology, biofilm formation and biofilm eradication
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Bacterial pathogenesis is a major threat to human health due to the increase antibiotic resistance among disease-causing bacteria. Effective and alternative therapeutics are urgently required to combat this problem. Honey is a natural product that has been used for over 2,000 years, as an effective topical chronic wound treatment. Numerous studies in the last 30 years have revealed its potent antibacterial properties (due to high sugar content, low pH and hydrogen peroxide production upon dilution). Honeys sourced from the Leptospermum scoparium bush in New Zealand (NZ), also referred to as manuka-type honeys, have been known to contain additional 'non-peroxide' antibacterial components (including methylglyoxal (MGO) and various phenolic compounds). However, for honey to be considered as a mainstream wound treatment by medical professionals, the mechanism behind its antibacterial activity needs to be determined. Moreover, bacteria produce biofilms that is a matrix of extracellular polymeric substance and allow cells to adhere to a surface such as a wound. Biofilms are the preferred mode of life in wounds because it also offers protection from antibiotic treatment. It is therefore essential to evaluate honeys' effects on bacterial biofilms. Unfortunately, almost all previous studies have utilized honeys that are ill-defined chemically. Thus, the objectives of this work were to use a range of well-defined NZ manuka-type honeys and their specific antibacterial components (such as methylglyoxal and sugars) to firstly examine their antibacterial effects on bacterial cell growth and cellular morphology, across a range of different bacteria. Subsequently, the antibiofilm activities on different strains of the same organism were also investigated on preventing biofilm formation and eradicating the pre-established biofilms. The bacterial cell growth and cellular morphology of three clinically relevant bacteria; the Gram-positive Staphylococcus aureus, and, the Gram-negative organisms Escherichia coli and Pseudomonas aeruginosa were examined against the selected range of NZ honeys, by cell growth assays and fluorescent microscopy. In addition, a Gram-positive organism, Bacillus subtilis, was also studied because it is a model organism where the functions of many genes associated with cellular growth and morphology have been documented. Moreover, B. subtilis is often used as a Gram-positive representative organism, typically in drug discovery studies in the industry. Results presented in this work indicate that different bacterial species are susceptible to different components or concentrations of honey and therefore respond in different ways. It is proposed that the complexity of honey makes it hard for bacteria to become resistant to honey's antibacterial effects. The second and third parts of this work examined the effectiveness of manuka-type honeys in preventing and eradicating preformed bacterial biofilms in S. aureus and P. aeruginosa. This was performed by using a crystal violate based static biofilm formation assay in combination with Confocal Laser Scanning Microscopy (CLSM) to visualise the integrity of the biofilms after honey treatment. It was found that very low levels of NZ manuka-honey enhanced both S. aureus and P. aeruginosa biofilm formation, which could possibly due to the evoke of a stress response similar to that seen with some conventional antibiotics. When higher concentrations of honey were used, NZ manuka-honeys were able to prevent or eliminate biofilms. This appears to be influenced by MGO levels and the presence of sugar. However, MGO and sugar content alone does not account for all of the antibiofilm properties observed. Finally, an ATP-based viability assay suggested that both S. aureus and P. aeruginosa planktonic cells, which were released after honey treatment of pre-formed biofilms were significantly reduced. The development of resistance or tolerance from these recovered planktonic cells was also determined by exposing these cells to the same previously exposed honey agents. Results indicated that the recovered S. aureus planktonic cells did not display any resistance to honey. However, the recovered P. aeruginosa planktonic cells had an increased tolerance to the same honey treatment. Altogether, these results show that at an appropriate level of manuka-type honey as a whole agent, can be used to kill P. aeruginosa and S. aureus when present in the biofilm, thereby supporting the use of this honey as an effective topical treatment for chronic wound infections. Lastly, this work also provided guidelines and strategies for new formulation of wound treatment managements and products, respectively.
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