A novel biosafety level 2 compliant tuberculosis infection model using a Δ<italic>leuD</italic>Δ<italic>panCD</italic> double auxotroph of <italic>Mycobacterium tuberculosis</italic> H37Rv and <italic>Galleria mellonella</italic>.

Mammalian infection models have contributed significantly to our understanding of the host-mycobacterial interaction, revealing potential mechanisms and targets for novel antimycobacterial therapeutics. However, the use of conventional mammalian models such as mice, are typically expensive, high maintenance, require specialised animal housing, and are ethically regulated. Furthermore, research using <italic>Mycobacterium tuberculosis</italic> (MTB), is inherently difficult as work needs to be carried out at biosafety level 3 (BSL3). The insect larvae of <italic>Galleria mellonella</italic> (greater wax moth), have become increasingly popular as an infection model, and we previously demonstrated its potential as a mycobacterial infection model using <italic>Mycobacterium bovis</italic> BCG. Here we present a novel BSL2 complaint MTB infection model using <italic>G. mellonella</italic> in combination with a bioluminescent <italic>ΔleuDΔpanCD</italic> double auxotrophic mutant of MTB H37Rv (SAMTB <italic>lux</italic>) which offers safety and practical advantages over working with wild type MTB. Our results show a SAMTB <italic>lux</italic> dose dependent survival of <italic>G. mellonella</italic> larvae and demonstrate proliferation and persistence of SAMTB <italic>lux</italic> bioluminescence over a 1 week infection time course. Histopathological analysis of <italic>G. mellonella</italic>, highlight the formation of early granuloma-like structures which matured over time. We additionally demonstrate the drug efficacy of first (isoniazid, rifampicin, and ethambutol) and second line (moxifloxacin) antimycobacterial drugs. Our findings demonstrate the broad potential of this insect model to study MTB infection under BSL2 conditions. We anticipate that the successful adaptation and implementation of this model will remove the inherent limitations of MTB research at BSL3 and increase tuberculosis research output. [ABSTRACT FROM AUTHOR]