A CONTRIBUTION TO THE STUDY OF BIOCONTROL AGENTS APITHERAPY AND OTHER POTENTIAL ALTERNATIVES TO ANTIBIOTICS

We are living in what has been termed the “post antibiotics era’ where the emergence of multidrug-resistant pathogen strains is rapidly growing and where the introduction of and the development of novel antimicrobial agents, which are effective against these pathogens has declined significantly. As a result, we urgently need effective alternatives to the use of antibiotics. The overall aim of the study was to add further our knowledge of some of these alternatives to antibiotics, concentrating on maggot therapy, apitherapy and to a lesser extent mycotherapy. The nature of the exceptionally high non-peroxide antibacterial activity of Leptospermum scoparium Manuka honey was further investigated. It was shown that this activity is unlikely to be due to accumulated residual hydrogen peroxide and the microflora of bees and honey probably act as a source of any non–hydrogen peroxide inhibitory components. Tregothnan English Manuka did not show any exceptionally high non-peroxide antibacterial activity compared to New Zealand Manuka. Studies on the mechanisms of the antibacterial activity of high medical grade New Zealand Manuka (UMF 20+,25+) against methicillin-resistant Staphylococcus aureus (MRSA) and (SEM) and TEM studies on the morphological effects on (MRSA) and Escherichia coli were conducted. It was observed that Gram-positive and Gram-negative cells were not uniformly affected by Manuka honey, Gram-positive bacteria were more sensitive and had less MICs and MBCs values. Time killing assays confirmed the bactericidal effect of Manuka. 16S rRNA sequencing technique confirmed that that C. botulinum spores are not likely to be detectable in tested medical grade New Zealand Manuka honey samples. Leptospermum scoparium essential oil showed a marked inhibitory activity against Gram-positive bacteria, including MRSA, M. phlei and B. subtilis;significant antifungal activity against Trichophyton terrestre was also observed. The in vitro antibacterial activity of tamarind (Tamarindus indica) was evaluated and compared with 25+ Manuka honey. Data obtained from the agar diffusion, MICs, MBCs values and time to kill study show that tamarind exhibits a stronger antimicrobial effect against the six tested bacteria and C. albicans than do high medical grade manuka honeys. It is suggested that an autoclaved, sterilized, commercially available tamarind paste should be clinically evaluated for the treatment of wounds and indolent ulcers which are infected with bacteria, notably MRSA. In relation to maggot therapy, evidence is provided to support the anti-Mycobacterium activity of Calliphora quadrimaculata maggot gases; unfortunately, attempts to isolate these gases were unsuccessful. For first time, in vitro evidence ( such as antifungal and yeast (S. cerevisiae (BY4742) GFP ingestion ability), was provided to support the use of Lucilia sericata maggot to control fungi involved in superficial fungal infections, i.e. fungal burn wound infections and dermatomycoses. A study was made to determine if Mycoplasmas can be detected in the hemolymph of maggots of Calliphora quadrimaculata and Lucilia sericata maggots by using Mycoplasma, EZ-PCR Mycoplasma Test Kit. Mycoplasmas were not detected in any of the maggot samples tested. These observations suggest that Calliphora quadrimaculata and Lucilia sericata larvae are unlikely to act as transmission vectors for Mycoplasmas. Another antibiotic-alternative called mycotherapy (an approach which as yet has not be reinstated in medicine) was also studied. The results presented here show that mycelium of all Penicillium cultures, isolated from foods, and inhibited both MSSA and MRSA. SNAP* MRL Beta-Lactam Kit has been used to test for penicillin and TLC for patulin showed that penicillin was found in culture filtrates obtained from all of the individual fungi, while patulin was produced by only two samples. Clearly then, a pure culture of a Penicillium, known to produce penicillin and not patulin or other toxicant (like the two IMI strains used here) could be safely used to treat wound infections in hospitals at the present time.