Ex vivo infection of murine precision-cut lung tissue slices with Mycobacterium abscessus: a model to study antimycobacterial agents

Abstract

Background Multidrug-resistant infections due to Mycobacterium abscessus often require complex and prolonged regimens for treatment. Here, we report the evaluation of a new ex vivo antimicrobial susceptibility testing model using organotypic cultures of murine precision-cut lung slices, an experimental model in which metabolic activity, and all the usual cell types of the organ are found while the tissue architecture and the interactions between the different cells are maintained. Methods Precision cut lung slices (PCLS) were prepared from the lungs of wild type BALB/c mice using the Krumdieck® tissue slicer. Lung tissue slices were ex vivo infected with the virulent M. abscessus strain L948. Then, we tested the antimicrobial activity of two drugs: imipenem (4, 16 and 64 μg/mL) and tigecycline (0.25, 1 and 4 μg/mL), at 12, 24 and 48 h. Afterwards, CFUs were determined plating on blood agar to measure the surviving intracellular bacteria. The viability of PCLS was assessed by Alamar Blue assay and corroborated using histopathological analysis. Results PCLS were successfully infected with a virulent strain of M. abscessus as demonstrated by CFUs and detailed histopathological analysis. The time-course infection, including tissue damage, parallels in vivo findings reported in genetically modified murine models for M. abscessus infection. Tigecycline showed a bactericidal effect at 48 h that achieved a reduction of > 4log10 CFU/mL against the intracellular mycobacteria, while imipenem showed a bacteriostatic effect. Conclusions The use of this new organotypic ex vivo model provides the opportunity to test new drugs against M. abscessus, decreasing the use of costly and tedious animal models.

Precision cut lung slices: an integrated ex vivo model for studying lung physiology, pharmacology, disease pathogenesis and drug discovery

“…Some bacteria, like Trueperella pyogenes, only appear to infect animals but not humans, with their pathogenic effects having been studied in PCLS [48]. Additional studies in PCLS studied Mycobacterium tuberculosis [49], Mycobacterium abscessus [50], Yersinia pestis [51], and Staphylococcus aureus [52] to understand the pathologic features of infection and agents used to treat these infections. Additionally, co-infection of PCLS with influenza and a Mycobacterium strain showed that the influenza infection increased the susceptibility to Mycobacterium infection by attenuating responses to the bacteria that would otherwise allow the animal to clear the bacteria [53].…”

Application of Precision-Cut Lung Slices as an In Vitro Model for Research of Inflammatory Respiratory Diseases

“…It was found that in Mycobacterium abscessus infections, lesions in the tissue structure or inflammatory infiltration in the alveolar lumen were observed 24–48 h after infection, while animal models required at least 10 to 60 days for lung injury to occur [ 48 , 61 ]. Different types of mice have different infectivity and relative drug resistance, leading to inconsistent results in vivo [ 62 , 63 ].…”

Precision‐cut lung slices: A powerful ex vivo model to investigate respiratory infectious diseases

“…The process of PCLS generation preserves most features of the native lung tissue, that is, its architecture and mechanical properties (Hiorns et al., 2016 ; Pybus et al., 2021 ) as well as a complex cellular composition, including resident lung cells, for example, fibroblasts, ATI and ATII epithelial cells, and immune cells, for example, macrophages, monocytes, and NK and T cells (Stegmayr et al., 2021 ). Neutrophils have been detected in murine PCLS (mPCLS) (Akram et al., 2019 ; Molina‐Torres et al., 2020 ) but considering that they are short‐lived ex vivo and that the PCLS are disconnected from blood and lymphatic circulation, their number is highly dependent on the way PCLS are generated and for how long slices are kept in culture. The absence of immune cell recruitment can be exploited to define the response of resident cells to infection and treatment.…”

“…Mycobacterium abscessus (MAb) is a fast growing NTM and imminent multidrug‐resistant health threat for patients with respiratory conditions such as COPD and cystic fibrosis (CF) (Molina‐Torres et al., 2020 ). Upon infection of mPCLS, progressive tissue damage occurred, for example, alveolar edema, vascular congestion, and extravasion of lymphocytes and erythrocytes in the septa and alveolar spaces with rupture and thickening of alveolar septa by 48 hr post infection, concomitantly with infiltration of histiocytes, aggregates of foamy macrophages, and fragmentation of polymorphonuclear cells (Molina‐Torres et al., 2020 ). MAb were found in macrophages and type I and II pneumocytes.…”