Abstract:
Tuberculosis (TB) caused by the bacterium Mycobacterium tuberculosis remains a major cause of mortality worldwide. In addition, non-tuberculous mycobacterial (NTM) infections are emerging as a public health problem globally. Antibiotic-resistance has worsened the situation and there is an urgent need for new and more effective anti-mycobacterial agents. The aim of this thesis was to test the feasibility of using the larvae of the Greater wax moth Galleria mellonella (wax worms) to study the pathogenicity and virulence of mycobacteria using bioluminescent/fluorescent labelled strains. Light expression was used as a non-invasive surrogate of bacterial numbers in vivo. Our findings indicated that G. mellonella is highly susceptible to infection with M. marinum; wax worms were unable to clear the bacteria, even when they were infected with doses as low as 10³ colony forming units (CFU). Death occurred when light levels reached approximately 105 relative light units (RLU) per wax worm, equating to between 109 to 1010 CFU. By histological examination we observed that, at the early stages of infection, wherever M. marinum bacilli were present within the body of the wax worm, they could be seen in lesions surrounded by groups of immune cells. At later time points, we observed that the early lesions formed distinct nodule-like pigmented lesions with less haemocytes around them and more acid fast bacilli inside. Within dead wax worms, nodules appeared full of acid fast bacilli with almost no immune cells present. As disease progressed, destruction of the insect’s fat body was observed. Wax worms were also found to succumb to infection another pathogenic NTM, M. abscessus, but not after inoculation with the saprophytic NTM M. smegmatis. In a preliminary study to investigate the suitability of using wax worms for anti-mycobacterial susceptibility testing, we observed that treated wax worms survived a day longer than untreated wax worms after injection of a single dose of either rifampicin (10 mg/kg) or streptomycin (15 mg/kg) within 30 minutes of being infection. In conclusion, we have successfully established G. mellonella wax worms as an alternative in vivo model system for investigating mycobacterial pathogenicity, which could potentially be used to evaluate anti-mycobacterial compounds.