Abstract
Anaerobic bacteria constitute most of the mammalian gastrointestinal microbiota and are essential to human and animal health. However, imbalances in this microbial community that favour opportunistic anaerobic pathogens can lead to dysbiosis and disease. Like aerobic bacteria, anaerobic bacteria can become resistant to antibiotics and are often associated with polymicrobial infections, making anaerobic infections challenging to treat. The work presented here focused on two key anaerobic pathogens; Fusobacterium nucleatum and Clostridiodes difficile. F. nucleatum has become the focus of numerous studies due to its association with cancer disease progression. Identifying novel inhibitors of F. nucleatum has been proposed as a key strategy in treating colorectal cancer (CRC). Clostridiodes difficile infection (CDI) is the leading cause of healthcare-associated diarrhoea, and novel antibiotics are required to target C. difficile but not the resident host microbiota. Despite the clinical importance of these bacteria, our knowledge of their physiology is lacking. We have focused on two areas to understand their physiology further; targeting metal ion homeostasis as an antibacterial mechanism and identifying novel natural antimicrobials.
Metal ions are essential bacterial micronutrients; therefore, exploiting bacterial metal ion homeostasis (metallostasis) holds great promise for developing novel anaerobic inhibitors. Ionophores represent one such class of inhibitors, reversibly binding metal ions and catalysing rapid ion movement across bacterial membranes. The Zn ionophore and 8-hydroxyquinoline derivative, PBT2, has been demonstrated to have potent antibacterial activity and the ability to resensitise both Gram-positive and Gram-negative drug-resistant bacteria in vivo. However, the antibacterial mechanism of action of PBT2 under strictly anaerobic conditions is unknown. Using bacterial cell physiology, genetics, biochemistry, and chemical biology, we characterised the response to PBT2-Zn in two Fusobacterium species, Fusobacterium nucleatum ATCC 25586 and Fusobacterium necrophorum ATCC 25286.
Antibiotics are still the clinical mainstay of CDI treatment; however, they can indiscriminately target other bacteria; further, C. difficile has already developed resistance to the recently developed antibiotic, fidaxomicin. Therefore, to identify a new chemical target space for antimicrobial development and to explore the physiology of C. difficile, we performed high-throughput screening of a natural product library against vegetative cells of the non-toxigenic isolate C. difficile ATCC 700057 and identified 91 total ‘hit’ compounds. From this, the selected plant-derived compounds anacardic acid, acetyl-11-keto-ß-boswellic acid (AKBA), and isobavachalcone were investigated for their antimicrobial activity against C. difficile in vitro.
This work provides new insights into the genetic and physiological capabilities of clinically relevant anaerobes in response to metal stress and novel plant-derived compounds. Finally, it explores both human and agricultural applications of novel inhibitors for combating obligate anaerobic pathogens.