Development of Human Cell-Based In Vitro Infection Models to Determine the Intracellular Survival of Mycobacterium avium
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 vary in different regions, currently reaching 0.3–9.8 per 100,000 individuals. Poor clinical outcomes,...
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| Format: | Article |
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Frontiers Media S.A.
2022-06-01
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| Series: | Frontiers in Cellular and Infection Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fcimb.2022.872361/full |
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| author | Gül Kilinç Kimberley V. Walburg Kees L. M. C. Franken Merel L. Valkenburg Alexandra Aubry Mariëlle C. Haks Anno Saris Tom H. M. Ottenhoff |
| author_facet | Gül Kilinç Kimberley V. Walburg Kees L. M. C. Franken Merel L. Valkenburg Alexandra Aubry Mariëlle C. Haks Anno Saris Tom H. M. Ottenhoff |
| author_sort | Gül Kilinç |
| collection | DOAJ |
| description | 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 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. |
| first_indexed | 2024-04-13T19:25:00Z |
| format | Article |
| id | doaj.art-da295c0ae99748708f62ed7e974666b5 |
| institution | Directory Open Access Journal |
| issn | 2235-2988 |
| language | English |
| last_indexed | 2024-04-13T19:25:00Z |
| publishDate | 2022-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cellular and Infection Microbiology |
| spelling | doaj.art-da295c0ae99748708f62ed7e974666b52022-12-22T02:33:24ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882022-06-011210.3389/fcimb.2022.872361872361Development of Human Cell-Based In Vitro Infection Models to Determine the Intracellular Survival of Mycobacterium aviumGül Kilinç0Kimberley V. Walburg1Kees L. M. C. Franken2Merel L. Valkenburg3Alexandra Aubry4Mariëlle C. Haks5Anno Saris6Tom H. M. Ottenhoff7Department of Infectious Diseases, Leiden University Medical Center, Leiden, NetherlandsDepartment of Infectious Diseases, Leiden University Medical Center, Leiden, NetherlandsDepartment of Infectious Diseases, Leiden University Medical Center, Leiden, NetherlandsDepartment of Infectious Diseases, Leiden University Medical Center, Leiden, NetherlandsSorbonne Université, INSERM, Centre d’Immunologie et des Maladies Infectieuses, U1135, AP-HP, Hôpital Pitié-Salpêtrière, Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux, Paris, FranceDepartment of Infectious Diseases, Leiden University Medical Center, Leiden, NetherlandsDepartment of Infectious Diseases, Leiden University Medical Center, Leiden, NetherlandsDepartment of Infectious Diseases, Leiden University Medical Center, Leiden, NetherlandsThe 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.https://www.frontiersin.org/articles/10.3389/fcimb.2022.872361/fullMycobacterium aviumprimary human macrophagesinfection modelsdrug susceptibility assaysMGIT 960 system |
| spellingShingle | Gül Kilinç Kimberley V. Walburg Kees L. M. C. Franken Merel L. Valkenburg Alexandra Aubry Mariëlle C. Haks Anno Saris Tom H. M. Ottenhoff Development of Human Cell-Based In Vitro Infection Models to Determine the Intracellular Survival of Mycobacterium avium Frontiers in Cellular and Infection Microbiology Mycobacterium avium primary human macrophages infection models drug susceptibility assays MGIT 960 system |
| title | Development of Human Cell-Based In Vitro Infection Models to Determine the Intracellular Survival of Mycobacterium avium |
| title_full | Development of Human Cell-Based In Vitro Infection Models to Determine the Intracellular Survival of Mycobacterium avium |
| title_fullStr | Development of Human Cell-Based In Vitro Infection Models to Determine the Intracellular Survival of Mycobacterium avium |
| title_full_unstemmed | Development of Human Cell-Based In Vitro Infection Models to Determine the Intracellular Survival of Mycobacterium avium |
| title_short | Development of Human Cell-Based In Vitro Infection Models to Determine the Intracellular Survival of Mycobacterium avium |
| title_sort | development of human cell based in vitro infection models to determine the intracellular survival of mycobacterium avium |
| topic | Mycobacterium avium primary human macrophages infection models drug susceptibility assays MGIT 960 system |
| url | https://www.frontiersin.org/articles/10.3389/fcimb.2022.872361/full |
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