Targeted dose delivery of Mycobacterium tuberculosis in mice using silicon antifoaming agent via aerosol exposure system

Abstract

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that forms aggregates (clumps) on solid agar plates and in liquid media. Detergents such as Tween 80/Tyloxapol are considered the gold standard to disrupt clump formation in Mtb cultures. The presence of detergent, however, may generate foam and hinder Mtb aerosolization thus requiring addition of an antifoam agent for optimal Mtb aerosol-based procedures. Aerosol inhalation can be technically challenging, in particular to achieve a reproducible inhaled target dose. In this study, the impact of an antifoam, the silicon antifoaming agent (SAF), on Mtb aerosolization and whole-body mouse aerosol infection was investigated. A comparative study using SAF in a liquid suspension containing Mycobacterium bovis BCG (M. bovis BCG) or Mtb H37Rv did not cause any adverse effect on bacterial viability. Incorporation of SAF during mycobacteria inhalation procedures revealed that aerosolized mycobacterial strains were maintained under controlled environmental conditions such as humidity, temperature, pressure, and airflow inside the aerosol chamber. In addition, environmental factors and spray factors were not affected by the presence of SAF in mycobacterial cultures during aerosolization. Spray factor was significantly less during aerosol procedures with a low-input dose of mycobacteria in comparison to high-dose, as predicted. The mycobacterial load recovered in the biosampler (AGI) was ~2-3 logs lower than nebulizer or input bacterial load. A consistent Mtb bacillary load determined in mouse lungs indicates that SAF does not affect mycobacteria aerosolization during the aerosol generation process. These data confirmed that 1) SAF prevents formation of excessive foam during aerosolization, 2) SAF had no negative impact on mycobacterial viability within aerosol droplets, 3) Mtb droplets within aerosol-generated particles are well within the range required for reaching and depositing deep into lung tissue, and 4) SAF had no negative impact on achieving a target dose in mice exposed to Mtb aerosol.

Fig 1. Testing SAF and foam formation.

Tubes containing PBSTy showing foam suppressed (with SAF) or retained (without SAF) at 5 minute interval for the duration of 0–20 min (panels A-F). Graph in panel ‘F’ extrapolated from panels ‘A-E’. Viability testing of M. bovis BCG and Mtb H37Rv by CFU assay without SAF and with SAF in contact with mycobacteria for 20 min and 24 hr (day 1) for two CFU titers tested; ~4e6 (G), ~6e4 (H).

Fig 2. Environmental factors measured in chamber during mock aerosol experiment.

Environmental factors measured for mock1 and mock2 during aerosol procedures by AeroMP system; range of relative humidity plotted with respect to time (0–1200 sec) (A), mean relative humidity (RH) mock1 (60.05%), mock2 (58.12%) plotted versus time (0–1200 sec) (B), temperature recorded for mock1 and mock 2 every 5 sec up to 1200 sec (20 min) and plotted with respect to time (C), mean temperature recorded for mock1 (22.8°C), mock2 (22.9°C) plotted versus time up to 20 min (0–1200 sec) (D), range of relative humidity plotted versus temperature for mock1 and their linear regression (r² = 0.0035, P = 0.359) (E), relative humidity plotted versus temperature measured for mock2 and their linear regression (r² = 0.012, P = 0.089) (F).

Fig 3. Bacterial burden in aerosol assembly during mock experiment.

Bacterial burdens obtained in nebulizer (NEB) and biosampler (AGI) plotted as total CFUs; mock1 (A), mock2 (B). Spray factor for each mock1 and mock2 aerosol procedures (C). Data are mean ± SEM, unpaired t-Test, **P = 0.0016 for mock1, **P = 0.0090 for mock2 using Graphpad prism 9.

Fig 4. Environmental factors measured in chamber during Mtb-aerosol exposure of mice.

Range of environmental factors measured during mouse aerosol procedures using the AeroMP system; range of relative humidity plotted with respect to time (0–1200 sec) (A), mean relative humidity plotted versus time (B), range of relative humidity plotted versus the temperature measured over time (0–1200 sec) (C), range of temperature recorded every 5 sec plotted with respect to time (D), mean temperatures plotted versus time (0–1200 sec) and their linear regression (r² = 0.0012, P = 0.597) (E).

Fig 5. Bacterial burden in aerosol assembly during Mtb-aerosol exposure of mice and post Mtb-infection.

Bacterial burdens obtained in nebulizer (NEB) and biosampler (AGI) plotted as total CFUs (A), spray factor measured for Mtb-aerosol exposure of mice (B), average body weight (C) and temperature (D) of mice measured up to 6 weeks; closed shapes (Mtb-infected), open shapes (control mice that did not receive Mtb). Bacterial burdens day-1 and week-6 post infection of mice; lung (E), liver (F). CFU data are from duplicate plating; results in panels E and F are expressed as CFUs in the entire tissue; data are means ± SEM. For panel A, ** P = 0.0069, unpaired t-Test using GraphPad prism 9.