The Mycobacterium avium (Mav) 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... Show moreThe Mycobacterium avium (Mav) 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 in vitro Mav infection models using the human MelJuSo cell line as well as primary human macrophages and a fluorescently labeled Mav 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 Mav 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 Mav 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 Mav infection models are reliable tools to determine the intracellular loads of Mav, which will enable researchers to investigate host-pathogen interactions and to evaluate the efficacy of (host-directed) therapeutic strategies against Mav. Show less
Upon infection, mycobacteria, such as Mycobacterium tuberculosis (Mtb) and nontuberculous mycobacteria (NTM), are recognized by host innate immune cells, triggering a series of intracellular... Show moreUpon infection, mycobacteria, such as Mycobacterium tuberculosis (Mtb) and nontuberculous mycobacteria (NTM), are recognized by host innate immune cells, triggering a series of intracellular processes that promote mycobacterial killing. Mycobacteria, however, have developed multiple counter-strategies to persist and survive inside host cells. By manipulating host effector mechanisms, including phagosome maturation, vacuolar escape, autophagy, antigen presentation, and metabolic pathways, pathogenic mycobacteria are able to establish long-lasting infection. Counteracting these mycobacteria-induced host modifying mechanisms can be accomplished by host-directed therapeutic (HDT) strategies. HDTs offer several major advantages compared to conventional antibiotics: (a) HDTs can be effective against both drug-resistant and drug-susceptible bacteria, as well as potentially dormant mycobacteria; (b) HDTs are less likely to induce bacterial drug resistance; and (c) HDTs could synergize with, or shorten antibiotic treatment by targeting different pathways. In this review, we will explore host-pathogen interactions that have been identified for Mtb for which potential HDTs impacting both innate and adaptive immunity are available, and outline those worthy of future research. We will also discuss possibilities to target NTM infection by HDT, although current knowledge regarding host-pathogen interactions for NTM is limited compared to Mtb. Finally, we speculate that combinatorial HDT strategies can potentially synergize to achieve optimal mycobacterial host immune control. Show less
