Abstract
As the human body's most extensive and exposed organ, the skin is pivotal in safeguarding overall health and well-being. Its significance necessitates vigilant protection against skin diseases, which affect an estimated 1.9 billion individuals globally. This widespread prevalence has positioned skin and subcutaneous diseases among the top 25 primary causes of global morbidity. A substantial portion of this burden stems from Skin and Soft Tissue Infections (SSTIs), which manifest in various forms, from parasitic afflictions, such as scabies, demodicosis, headlice, and tungiasis to bacterial infections, predominantly caused by Staphylococcus aureus and Streptococcus pyogenes (commonly referred to as Group A beta-haemolytic Streptococcus, GAS). Children in developing nations, particularly those in resource-limited settings, bear a disproportionate burden of these infections.Two SSTIs, impetigo and tungiasis, warrant particular attention. Impetigo, a bacterial condition, has a significant global impact, affecting over 162 million children, with northern and central Australia being notable hotspots. In contrast, tungiasis, a parasitic skin condition, is endemic across 88 countries, with sub-Saharan Africa, South America, and the Caribbean experiencing epidemic levels. Both diseases, deeply intertwined with socio-economic disparities, impede developmental progress in affected regions. The challenge is further exacerbated by the escalating threat of antimicrobial resistance (AMR), identified by the World Health Organization (WHO) as a top public health concern, undermining the efficacy of current antibiotics, including impetigo treatments.
In light of rapidly emerging and evolving AMR, particular attention is directed towards plant-derived bioactive phytochemicals. Totarol (SJ), for instance, which is isolated from the New Zealand native Podocarpus totara, has demonstrated substantial antibacterial efficacy. Simultaneously, tea tree oil (TTO), procured through the distillation of leaves from the Australian indigenous Melaleuca alternifolia, has been recognised for its comprehensive antimicrobial properties, specifically its antiparasitic and antibacterial activity. The potential of SJ and TTO to act as therapeutic agents presents a promising avenue for developing novel treatments targeting bacterial infections such as impetigo and parasitic conditions like tungiasis. However, the transition from promising compounds to validated treatments requires extensive empirical research, including but not limited to clinical trials, to verify their therapeutic efficacy and safety profiles and determine the most effective application methods.
This doctoral dissertation adopts a dual-pronged structure to rigorously investigate the potential therapeutic applications of SJ-based skin formulations for treating impetigo and TTO-based topical applications for tungiasis. The primary aim of this thesis is to methodically explore and address the identified gaps in the current treatments for these dermatological conditions, with the intent to contribute to advancing the treatment landscape of these skin conditions.
Part I of the thesis is dedicated to impetigo, addressing the thesis objectives i-vii. Chapter 2 begins the examination with a comprehensive review of the literature published to date, focusing on the therapeutic challenges impetigo presents, particularly in regions of Australia where it is endemic. The increasing incidence of methicillin-resistant S. aureus (MRSA) infections has led to re-evaluating topical antibiotics, highlighting the necessity for novel treatment modalities. Within this review, SJ is identified as a candidate with antibacterial properties that could be effective against the pathogens responsible for impetigo, including MRSA. Building upon this initial review, Chapters 3 through 5 detail original research findings, which include data from industry-standard antimicrobial assays. The promise shown by these findings, particularly their prospective role in advancing antibacterial treatment options, has prompted the active pursuit of intellectual property (IP) protection to preserve this research's potential commercial and therapeutic applications. Chapter 6 complements these findings with a comprehensive review, further contextualising them within the broader field of study.
Chapter 3 embarks on in vitro studies conducted against impetigo-causing bacterial strains to assess SJ's antibacterial efficacy in comparison to current topical and systemic impetigo antibiotics, including fusidic acid (FA), mupirocin (MUP), ozenoxacin (OZN), retapamulin (RETAP), and vancomycin (VAN) as well as antiseptics recommended for impetigo, such as hydrogen peroxide (H2O2), manuka oil (MO), and TTO. The evaluation included minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time- and concentration-dependent killing rates. The findings spotlight SJ's commendable bactericidal activity, especially against methicillin-susceptible S. aureus (MSSA) and MRSA strains, with both MIC and MBC values of 4 μg/ml against MSSA and MRSA strains and 64 μg/ml against GAS strains. SJ proves to be the most potent bactericidal agent, maintaining a consistent MBC-to-MIC ratio of 1, unlike the comparator antibiotic and antiseptic agents, which exhibited MIC-to-MBC ratios ranging from 1 to 4096. This strong bactericidal activity of SJ was further affirmed through time-kill studies, with SJ demonstrating a remarkable swift killing rate that effectively eliminated the targeted initial bacterial load within 30–90 minutes. This represents a significant improvement when contrasted with the performances of the comparator antibiotic and antiseptic agents. Notably, OZN achieved bacterial elimination at 24 h, while the remaining antibiotic and antiseptic agents exhibited a bacteriostatic effect at the same concentration compared to SJ.
It is essential to highlight that the tested antiseptics exhibited promising antibacterial activity against GAS strains compared to S. aureus strains. Considering the higher activity of SJ against S. aureus strains, coupled with the strong antibacterial potential of antiseptic agents against GAS strains, the exploration of combining SJ with these antiseptics presents a compelling avenue. This combined approach has the potential to enhance antibacterial efficacy, enabling the use of reduced dosages while simultaneously minimising the risk of side effects. This hypothesis warranted further investigation as it may open new horizons for innovative impetigo treatment strategies. Chapter 4, therefore, further investigates the synergistic potential of SJ when combined with other agents, such as H2O2, MO, or TTO. The assessment was based on Fractional Inhibitory Concentration Indexes (FICIs) and killing rates, revealing pronounced interactions, especially between SJ and TTO. FICI values for the combinations of SJ with H2O2, MO, or TTO ranged from 0.5 to 1. 0. Two distinct synergistic interactions were observed between SJ and MO and SJ and TTO, with the remaining interactions demonstrating additive effect. The time-kill synergy findings further confirmed the synergistic interaction between SJ with H2O2 and TTO against MRSA strains and SJ with MO and TTO against GAS strains.
Chapter 5 synthesises the garnered insights to advance the development of SJ-based gel formulations, with initial assessments indicating promising efficacy for impetigo treatment. Comparative in vitro antibacterial assays have shown that the SJ-based formulation exhibits a more rapid action against MRSA and GAS strains in contrast to the standard MUP topical application, which is currently the first-line treatment for impetigo in Australia. Furthermore, skin permeation studies employing a Franz diffusion cell apparatus revealed that the SJ-based formulation exhibited a lower transdermal release and permeation rate than the MUP topical application. This indicates that the SJ formulation may provide a more controlled release of the active compounds, potentially enhancing therapeutic efficacy while minimising systemic exposure. A subsequent accelerated stability assessment indicated that the SJ-based gel formulation consistently maintained its physical and chemical stability over 60 days, under standard room temperature and the accelerated conditions of 40°C. This finding suggests the formulation's robust shelf-life. The safety and tolerability profile of the SJ topical gel was rigorously assessed and corroborated through a Human Repeat Insult Patch Test (HRIPT). This evaluation confirmed the gel's non-irritant properties upon application to the epidermis of 56 healthy volunteers, substantiating its suitability for potential clinical use. Chapter 6 features a published critical review of the animal models employed in impetigo research alongside a detailed protocol ratified by the University of Canberra’s Animal Ethics Committee (UC AEC) to assess the efficacy of the SJ-based gel using a mouse model. The commencement of this empirical evaluation, however, was unavoidably postponed due to the extensive disruptions to research activities caused by the global COVID-19 pandemic.
Part II of the thesis transitions to an in-depth examination of tungiasis, methodically addressing the thesis objectives viii through xi. It begins with Chapter 7, presenting a systematic review published in the Lancet Infectious Diseases journal, marking the first comprehensive analysis of existing treatment options for tungiasis in endemic areas, thereby identifying a critical need for new topical therapies and proceeding to Chapter 8, provides a narrative review that discusses the potential of TTO as a therapeutic agent for tungiasis, as published in a peer-reviewed journal, contributing to the ongoing scholarly conversation on this subject. It underscores TTO's comprehensive parasiticidal efficacy, anti-bacterial and anti-inflammatory properties, essential for managing this parasitic skin disease.
Drawing on these foundational insights, Chapter 9 presents a protocol for a Randomised Controlled Trial (RCT) to evaluate the effectiveness of a TTO-based treatment for tungiasis in Kenya. This protocol addresses the methodological limitations observed in prior studies and aims to establish a scalable and economically viable therapeutic option for tungiasis. The RCT protocol was designed and prepared in compliance with recognised best practices for RCT design and implementation. It was developed through a collaborative effort with experts in tungiasis research, ensuring a comprehensive and rigorous approach. The dissemination of this protocol in BMJ Open marks a significant step forward, as it constitutes the very first peer-reviewed, published RCT protocol for tungiasis research in the scientific literature. Should the TTO-based gel formulation prove effective in treating tungiasis, it holds the potential to provide evidence supporting a simple, affordable, and accessible treatment option for this condition, for which there is currently no approved standardised treatment. The COVID-19 pandemic's unforeseen disruptions, particularly border closures, halted the protocol's implementation. Chapter 10 addresses the challenges posed by the COVID-19 pandemic that impeded the execution of the planned RCT by presenting a foundational systematic review. This review systematically evaluates the therapeutic efficacy and safety of TTO against a selection of clinically relevant ectoparasites, providing an empirical assessment of TTO's antiparasitic properties. The review is the first of its kind, systematically collating and critically evaluating the fragmented body of literature on TTO, consolidating knowledge, and informing future research directions in ectoparasitic interventions. The findings consolidate the role of TTO as a potential therapeutic agent for tungiasis and furnish the scientific community with data that may be pivotal in developing treatment protocols, especially in the absence of RCT data.
Throughout these chapters, this thesis explores plant-based bioactives, specifically SJ and TTO, presenting a novel, dual-pronged approach to combat impetigo and tungiasis against the backdrop of escalating AMR. It underscores SJ's efficacy against bacteria such as MRSA and its amplified effectiveness when combined with TTO, including developing an SJ-based topical gel. Despite research setbacks due to the pandemic, TTO's potential in treating impetigo and tungiasis is highlighted.
Future efforts aim to extend these findings into animal models, bridging the gap between laboratory and practical applications, particularly in resource-scarce regions. While acknowledging challenges in biological variability and scalability, the study significantly contributes to developing plant-based alternatives for childhood skin conditions. In conclusion, this thesis presents innovative solutions for childhood skin conditions, marking a significant step in creating effective treatments, particularly vital in high-burden settings where conventional options are limited.
| Date of Award | 2025 |
|---|---|
| Original language | English |
| Supervisor | Gabrielle COOPER (Supervisor), Ashraf GHANEM (Supervisor), Jackson THOMAS (Supervisor) & Sam KOSARI (Supervisor) |