Identifying mechanisms associated with metronidazole resistance in Trichomonas vaginalis and investigating newer therapeutics against this pathogen.

Abstract

Introduction Trichomonas vaginalis, the etiological agent of trichomoniasis, a prevalent non-viral sexually transmitted infection, presents a growing public health concern due to the emergence of metronidazole resistance, which compromises the effectiveness of this frontline treatment. To address this challenge, this PhD thesis comprises four research manuscripts aimed at understanding the mechanisms associated with metronidazole resistance in T. vaginalis and investigating innovative therapeutic approaches against this pathogen. T. vaginalis, a flagellated protozoan parasite, has long been susceptible to metronidazole. However, the alarming increase in drug-resistant strains necessitates a comprehensive examination of this resistance. This study explored the interaction of T. vaginalis with endosymbionts, particularly T. vaginalis viruses (TVVs) and Mycoplasma hominis, delving into their association with drug resistance. Additionally, alternative treatments such as plant-based nanoemulsions and nanotechnology-mediated approaches were investigated. This thesis serves as a beacon of knowledge and innovation in the ongoing battle against metronidazole resistant T. vaginalis isolates, offering new insights and promising strategies for the future of trichomoniasis management. Methods This research encompassed a multifaceted methodological approach, systematically examining metronidazole resistance in T. vaginalis and exploring alternative therapeutic avenues. The initial set of investigations utilized a combination of in vitro studies and molecular assays to analyse clinical isolates of T. vaginalis. This entailed scrutinizing the presence of T. vaginalis viruses and intracellular M. hominis within these isolates and delving into the intricate associations between these endosymbionts and metronidazole resistance. Further studies conducted in vitro susceptibility assays, gene expression analyses, and investigated the influence of iron supplementation on metronidazole resistance in T. vaginalis isolates. This multifaceted approach aimed to uncover the role of iron in regulating genes associated with metronidazole resistance and to assess how alterations in iron levels might affect the susceptibility of T. vaginalis isolates to the drug. In separate investigations, plant based nanoemulsions were meticulously prepared from select medicinal plants and rigorously tested for efficacy against metronidazole-resistant T. vaginalis isolates, exploring their potential as alternative therapeutic agents. Additionally, our research ventured into the realm of nanotechnology, focusing on the synthesis of iron nanoparticles, modified with chitosan and small interfering RNA (siRNA). This novel approach aimed to explore the potential of these nanocomplexes for gene silencing and targeted therapy in the battle against T. vaginalis infections. Results The research findings have unveiled valuable insights into the mechanisms associated with metronidazole resistance in T. vaginalis. Twenty-one clinical isolates of T. vaginalis were included in the endosymbiosis analysis. The prevalence of TVV and M. hominis were 76% (16/21) and 86% (18/21), respectively. The presence of any TVV was significantly associated with metronidazole susceptibility patterns (p=0.012). No significant associations were noted between the coinfection of both endosymbionts and metronidazole resistance. This study went on to show that iron concentrations of 30 μM and 60 μM had led to a loss of resistance in certain isolates of T. vaginalis. Furthermore, gene expression analysis indicated that iron played a role in modulating the expression of resistance-associated genes. Additionally, the antimicrobial properties of iron on T. vaginalis growth was also evident, which potentially influenced the significance of these gene expression alterations. The efficacy of plant-based nanoemulsions from medicinal plants against metronidazole-resistant T. vaginalis isolates was highlighted. The nanoemulsions shifted resistant isolates towards susceptibility in a concentration-dependent manner, with minimum inhibitory concentrations (MICs) decreasing from 4 μg/ml to 1 μg/ml, promising the restoration of plant-based treatments' effectiveness. Finally, the synthesis of iron nanoparticles modified with chitosan and small interfering RNA (siRNA) for potential gene silencing and targeted therapy against T. vaginalis was presented. These nanoparticles were successfully synthesized and efficiently bound with siRNA, offering a potential for innovative therapeutic interventions. Concentration-dependent effects on cell viability were observed, highlighting the need for precise optimization in therapeutic strategies. These comprehensive results collectively signify alternative strategies for addressing metronidazole resistance and mark a significant step in advancing precision medicine and targeted therapies for T. vaginalis infections. Conclusion The collective findings from this research provides compelling evidence for alternative approaches to address metronidazole resistance in T. vaginalis. The multifaceted nature of this thesis illustrates the complexity of metronidazole resistance and the potential for innovative therapies. These discoveries pave the way for a more comprehensive understanding of T. vaginalis infections and resistance mechanisms. They hold the promise of precision medicine, drug delivery, gene silencing, and targeted therapies. However, for these research outcomes to translate into effective clinical therapies, further exploration, in vivo validation, and rigorous clinical studies are essential. These steps are crucial for addressing the growing challenges posed by T. vaginalis infections and metronidazole resistance while ensuring the continued efficacy of treatment options in the field of public health.