Development of Human Cell-Based Infection Models to Determine the Intracellular Survival of .

The () complex accounts for more than 80% of all pulmonary diseases caused by non-tuberculous mycobacteria (NTM) infections, which have an alarming increase in prevalence and vary in different regions, currently reaching 0.3-9.8 per 100,000 individuals. Poor clinical outcomes, as a result of increasing microbial drug resistance and low treatment adherence due to drug-toxicities, emphasize the need for more effective treatments. Identification of more effective treatments, however, appears to be difficult, which may be due to the intracellular life of NTM and concomitant altered drug sensitivity that is not taken into account using traditional drug susceptibility testing screenings. We therefore developed human cell-based infection models using the human MelJuSo cell line as well as primary human macrophages and a fluorescently labeled strain. By testing a range of multiplicity of infection (MOI) and using flow cytometry and colony-forming unit (CFU) analysis, we found that an MOI of 10 was the most suitable for infection in primary human macrophages, whereas an MOI of 50 was required to achieve similar results in MelJuSo cells. Moreover, by monitoring intracellular bacterial loads over time, the macrophages were shown to be capable of controlling the infection, while MelJuSo cells failed to do so. When comparing the MGIT system with the classical CFU counting assay to determine intracellular bacterial loads, MGIT appeared as a less labor-intensive, more precise, and more objective alternative. Next, using our macrophage infection models, the drug efficacy of the first-line drug rifampicin and the more recently discovered bedaquiline on intracellular bacteria was compared to the activity on extracellular bacteria. The efficacy of the antibiotics inhibiting bacterial growth was significantly lower against intracellular bacteria compared to extracellular bacteria. This finding emphasizes the crucial role of the host cell during infection and drug susceptibility and highlights the usefulness of the models. Taken together, the human cell-based infection models are reliable tools to determine the intracellular loads of , which will enable researchers to investigate host-pathogen interactions and to evaluate the efficacy of (host-directed) therapeutic strategies against .