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Characterization of novel double-reporter strains of Mycobacterium abscessus for drug discovery: a study in mScarlet

C M Bento(1,2,3,4) K V Calster(5) T Piller(5) G S Oliveira(1,4) L D Vooght(5) P Cos(5) M S Gomes(1,4) T Silva(1,4)

1:i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal; 2:IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal; 3:Programa Doutoral em Biologia Molecular e Celular (MCBiology), Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto, Portugal; 4:ICBAS – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto, Portugal; 5:Laboratory for Microbiology, Parasitology and Hygiene (LMPH), Wilrijk, Belgium

Treatment of nontuberculous mycobacteria (NTM) disease is based on a long multidrug regimen associated with severe side effects and increased antibiotic resistance. Moreover, in vitro antibiotic susceptibility does not reflect clinical effectiveness, hampering the treatment. Therefore, there is an urgency to find new treatments for NTM infections and better infection models to test them. We have been developing in vitro models for drug susceptibility testing to better recreate the environment the bacteria experience in the host. In this context, we are developing new strains of Mycobacterium abscessus which simultaneously express the gene for a fluorescent protein, mScarlet, and the gene for luciferase, which converts D-luciferin in oxyluciferin in the presence of ATP, generating light. The fluorescent signal can be used as a marker of bacterial load and the bioluminescent signal to monitor bacterial metabolism. The strains characterization was made by comparing their growth with the non-transformed strain, measuring, simultaneously, optical density, fluorescence, and luminescence, and correlating those parameters with the bacterial load assessed by colony-forming units assay. We also evaluated the in vitro antibiotic susceptibility of the reporter strains (when compared with the non-transformed), their ability to form biofilms and infect host cells, and validated them for in vivo infection using bioluminescence imaging technology. The results show that these new reporter strains can be an essential tool to aid in the discovery of new drugs against mycobacterial infections.

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