Lin, Shih-Min (Sam)2010-06-082010-06-082010Lin, S.-M. (Sam). (2010). The effect of manuka honey on enterobacteria (Thesis, Doctor of Philosophy (PhD)). The University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/3972https://hdl.handle.net/10289/3972Manuka honey (Leptospermum scoparium) produced in New Zealand has been shown to exhibit substantial antibacterial activity against a broad range of pathogens causing wound infection, and is being used in wound management with excellent results. This activity is due to both hydrogen peroxide and non-peroxide components. Manuka honey, however, may not be useful for treating bacterial gastroenteritis because the gastrointestinal environment may be unfavourable to the antibacterial action, and because a sufficiently high concentration for effectiveness may not be achieved. The research in this thesis is set out to evaluate in vitro the efficacy of manuka honey as an antibacterial agent against enterobacteria, taking into consideration some factors that may be involved in the gastrointestinal environment. Because some gastrointestinal bacteria (Campylobacter spp., Helicobacter pylori, Lactobacillus spp. and Bifidobacterium animalis subsp. lactis) are not aerophilic, a cheap yet acceptable gas generating system alternative to the commercial gaspack counterpart was sought for use in this study. Various alternatives were compared for their performance. The spirits burn method was chosen for cultivating microaerobes and some anaerobes because of its comparable performance to that of commercial systems in terms of the growth of bacterial species, and because of the ease of use and the low cost. In the first part of this thesis, the susceptibility of gastrointestinal bacteria against manuka honey was investigated by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using a standardised manuka honey. Throughout the research, a manuka honey with median level non-peroxide antibacterial activity (equivalent to that of 16.5% phenol) was used, except that Campylobacter spp. were assayed with a more potent manuka honey equivalent to 29.4% phenol. The measured sensitivity of bacteria showed that manuka honey is significantly more effective than artificial honey (a mixture sugars as in honey), indicating that osmolarity is not the only factor that is responsible for the antibacterial activity of the honey. It was found that some species of bacteria e.g. Campylobacter spp. are exceptionally sensitive to manuka honey (both MIC and MBC are about 1% honey solution), whereas most other gastrointestinal pathogens have MIC and MBC values in the range 5-10% honey other than Enterobacter and Pseudomonas which were in the range 10-17%. Bifidobacterium, lactobacilli and enterococci appear to be more tolerant to the honey (MIC: 9.36-14.29%; MBC: greater than or equal to 13.3%) than most other species are. The difference in efficacy between the honey with and without hydrogen peroxide removed was also studied, and it was found that both hydrogen peroxide and the non-peroxide components contribute to the bacteriostatic and bactericidal activity of the honey. Because oxygen is required for hydrogen peroxide to be produced in honey, the role that oxygen plays in the antibacterial activity of manuka honey was investigated by analysing the susceptibility data obtained under both aerobic and anaerobic conditions using facultative anaerobes. Manuka honey appeared to be a more potent bacteriostatic agent against most species of bacteria in the absence of oxygen, whereas a relatively higher concentration of manuka honey solution was required to kill some bacteria under anaerobic conditions. This may partially be due to the atmosphere having also affected the metabolism, and hence the growth, of bacteria. Therefore, the activity of manuka honey would not necessary decline in the intestinal environmental atmosphere. To investigate how long it takes for manuka honey to kill bacteria, time-to-kill studies were conducted by monitoring the survival of bacteria in manuka honey. It is found that it takes a 20% solution of manuka honey with a medium-level activity more than 6 h to kill 90% of the cells of most of the species tested if the bacterial cells are kept in contact with the honey. This suggests that manuka honey is not rapidly bactericidal, and that it is unlikely to be possible to fully eradicate a bacterial gut infection by ingesting a small amount of manuka honey for a short period. It was found that probiotics can survive in the 20% honey solution for more than 12 h. The pharmacodynamics of the antibacterial activity of manuka honey were studied to investigate the survival and the re-growth of bacteria after they had been treated with honey. It was revealed that after being exposed to manuka honey for a short term (1 h), the growth of most enteropathogens is slowed for approximately 2-4 h before it gets back to a full rate. The assays of this postantibiotic effect also showed that the latency in the re-growth after being exposed to honey is not proportional to the MIC, MBC or time-to-kill profiles. Finally, the efficacy of manuka honey on bacteria was studied under conditions simulating the environment in the stomach and intestines. The tested bacteria were unable to grow under the acidic conditions as in the stomach, so whether or not the honey had any antibacterial activity under these conditions could not be determined. Under the conditions simulating the intestinal environment, the results demonstrated that the antibacterial activity of manuka honey is slightly decreased in the mildly alkaline conditions of the intestine (pH 7.5). In the presence of pancreatin and bile at the same pH, the activity of manuka honey was found to decrease by more than 50%. This suggests that pancreatin and bile in the gut may negatively affect the efficacy of the antibacterial activity of manuka honey in vivo. This indicates that although ingested manuka honey may still have some antibacterial action when in the gut, the antibacterial activity would be different from that which is usually examined with sensitivity studies in vitro. Gastroenteritis has generally been treated with oral rehydration solution (ORS) that consists of carbohydrates and electrolytes. Manuka honey could be used instead of the usual carbohydrate component of ORS and would provide additional bioactivities such as antibacterial activity and stimulation of growth of probiotics, which would make the honey rehydration solution more beneficial to patients with gastroenteritis than is the traditional ORS. After some initial investigation to find the most appropriate dosage and frequency of doses, a clinical trial may be warranted.application/pdfenAll items in Research Commons are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.manuka honeyenterobacteriaantibacterial activityenvironmental effectgastrointestinalMICsusceptibilitypharmacodynamicsgastroenteritisThesis