Antimicrobial resistance (AMR) is considered by the World Health Organization as one of the top 10 global public health threats facing humanity. It is estimated that there are 700,000 annual global deaths due to resistant infections, which are predicted to rise to 10 million, alongside a cumulative cost of $100 trillion by 2050. Human health, animal health, as well as food and environment security are interlinked, therefore cross-sector approaches are needed to accelerate progress in the battle against organisms resistant to antimicrobials. Understanding precisely how antimicrobials work inside the human or animal body is necessary to develop novel treatments and to make sure the existing ones are used optimally. My research focuses on an understudied mechanism of antibiotic evasion, called L-form switching. Almost all bacteria are surrounded by a structure called the cell wall, which protects them against environmental stresses and helps with regular division. Many of our most commonly used antibiotics, such as penicillin, target this structure. During treatment with these antibiotics, bacteria usually burst and die, but some can survive in a wall-less state referred to as an L-form, if the surrounding environment protects them from bursting. Without the wall, bacteria are fragile and slow growing, but resistant to all types of antibiotics that target this structure. Once antibiotic treatment is finished, bacteria that survived in an L-form state can rebuild the wall and start dividing rapidly, potentially contributing to recurrent infections. Importantly, many pathogens, including E. coli and S. aureus, can undergo L-form switching and over the years it has been speculated that the host can provide a supportive environment for the process. L-form switching has been implicated in several recurrent diseases in human and animals, such as sepsis, mastitis, urinary tract and gastrointestinal infections. However, the fragile nature of L-forms and their low numbers in the host's body make them difficult to study, and convincing evidence for their role in disease was historically slow to emerge. I have developed novel approaches using cutting-edge technologies to study L-form switching. Using advanced fluorescent microscopy, I previously demonstrated the presence of cell wall-deficient bacteria in the urine of patients with recurrent urinary tract infections and that, paradoxically, they can survive inside the cells of our immune system. Following on from these significant discoveries, further work is urgently needed to answer the many questions about L-forms that remain. What are the precise locations within a host that are favourable for L-form survival, and do they provide additional protection for L-forms against environmental changes? Do L-forms prefer intracellular or extracellular locations? And most importantly, how can they be eradicated? The aim of my fellowship proposal is to overcome this major impediment to progress by combining microscopy and genetics to study L-form switching using various models, including organoids. These 'mini-organs' can be readily grown and mimic real organ structures, such as the bladder or intestine. I will follow L-form switching of various bacterial species and test their susceptibility to antibiotic treatment in the context of a specific tissue. I will use mutagenesis to identify bacterial genes that allow efficient L-from switching in the host. I will also expand my current focus on human health to animal health and study if L-form switching also plays a role in farm animal disease, such as bovine mastitis. My study will provide a transformation in our understanding of recurrent infection by L-forms. Although studying L-form switching in the host is challenging, it brings novel insights into bacterial pathogenicity. My research has the potential to influence how to use currently available treatments better, as well as develop novel strategies for tackling infection.

Antimicrobial resistance (AMR) is a growing threat to global health, and the excessive use of antimicrobials in agriculture is a major contributor to its development. In particular, plant pathogenic fungi pose a significant problem for food safety and quality, and with fewer fungicides available due to stricter regulations, the risk of AMR development is increasing. Current surveillance methods are slow and laborious. This project aims to develop a new approach combining the latest technological advances in sampling fungal spores from air and high-throughput long-read Oxford Nanopore Technology (ONT) sequencing to track the development and spread of AMR in fungal pathogens of cereal crops and the wider environment, which includes the opportunistic human pathogen Aspergillus fumigatus whose spores are widespread in the air we breathe. This new approach for the detection of fungal species from air and detection of the fungicide resistance alleles will be validated with the laboratory based conventional fungicide resistance tests of the fungi isolated from naturally infected plants sampled from the same locations as the air. Early season sampling and diagnosis of the status of AMR at the start of the growing season, will lead to improved better cereal disease management practices based on choice and optimal minimal use of fungicide inputs as part of IPM and reduce the risk for further AMR development in plant pathogens. Ultimately, this project will help to safeguard our food supply while improving human health and sustaining biodiversity in the environment.