Enhanced tenacity of mycobacterial aerosols from necrotic neutrophils

Abstract

The tuberculosis agent Mycobacterium tuberculosis is primarily transmitted through air, but little is known about the tenacity of mycobacterium-containing aerosols derived from either suspensions or infected neutrophils. Analysis of mycobacterial aerosol particles generated from bacterial suspensions revealed an average aerodynamic diameter and mass density that may allow distant airborne transmission. The volume and mass of mycobacterial aerosol particles increased with elevated relative humidity. To more closely mimic aerosol formation that occurs in active TB patients, aerosols from mycobacterium-infected neutrophils were analysed. Mycobacterium-infected intact neutrophils showed a smaller particle size distribution and lower viability than free mycobacteria. In contrast, mycobacterium-infected necrotic neutrophils, predominant in M. tuberculosis infection, revealed particle sizes and viability rates similar to those found for free mycobacteria, but in addition, larger aggregates of viable mycobacteria were observed. Therefore, mycobacteria are shielded from environmental stresses in multibacillary aggregates generated from necrotic neutrophils, which allows improved tenacity but emphasizes short distance transmission between close contacts.

Figure 1

Biophysical properties of mycobacteria: (a) Representative 3D reconstruction of a mycobacterial cell using Igor Pro software to calculate the volume of a single mycobacterium in air. (b) Volume of a single mycobacterium in air under different RH. The RH was adjusted by deliquescence salts inside a closed fluid chamber (n = 3 per humidity, p = 0.0008). (c) Gravimetric measurement of changes in the mass of mycobacterial pellets upon a decrease in the RH from 90% to 0% RH at 25 °C (n = 2). (d,e) Ability of soluble mycobacteria from broth cultures to survive for different periods of drying. (d) Live/dead staining using a LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen), depicted as the ratios of live vs. dead mycobacteria out of 100 mycobacteria analysed (n = 3). (e) Independent determination of CFU counts (n = 3).

Figure 2

Properties of mycobacterium-containing aerosols: (a) CFU of mycobacteria collected from the different levels of the Andersen impinger upon aerosolization in PBS at a 28 l/min airflow (n = 5; p > 0.001). (b) Percentages of live vs. dead mycobacteria as determined by live/dead staining upon aerosolization in PBS assessed immediately after settling for 10 min at the different impinger levels in comparison to control samples directly transferred from culture into PBS for viability staining before nebulization (n = 3, out of 100 mycobacteria, p > 0.0015). (c) Frequency of different mycobacterial cell numbers within aerosol particles, as depicted by electron microscopy analysis counted on levels 1–8 (n = 3, p > 0.0001). (d) Representative 3D reconstruction of airborne mycobacteria collected at different levels and analysis by AFM (the mean and standard deviation of three independent experiments are shown).

Figure 3

Aerodynamic and optical diameters of empty PBS aerosols and mycobacterium-containing aerosol particles determined by and aerodynamic particle spectrometer (APS): (a) Left: Distribution of aerodynamic diameters of jet-nebulized buffer and mycobacterium-containing aerosols (OD 0.6, 580 nm). Right: Distribution of optical diameters of jet-nebulized buffer and mycobacterium-containing aerosols (n = 3). (b) Left: Frequency of aerosol particles with a certain aerodynamic diameter upon ultrawave nebulization. Right: Optical diameters of ultrawave-nebulized buffer and mycobacterium-containing aerosols (n = 3).

Figure 4

Tenacity of mycobacterium-containing aerosols. (a) Distribution of aerodynamic diameters of aerosols generated by jet nebulization from either buffer (PBS) or mycobacterial suspensions in PBS in ACD air at different time points after aerosol generation (n = 3). (b) Settling time of jet-nebulized aerosols vs ultrawave-nebulized aerosols (n = 3, **p > 0.001, ***p > 0.0001). (c) CFU of airborne mycobacteria collected from the different levels of an Andersen impinger at the starting time point of jet nebulization and 30 min later (n = 3, *p > 0.01, **p > 0.001).

Figure 5

Mimicking mycobacterial-infected neutrophil necrosis that occurs in active TB patients. (a) Human neutrophils isolated from peripheral blood were infected with M. bovis BCG and either left untreated or treated with H₂O₂ to induce necrotic cell death. Necrotic cell death was assessed by measuring the activity of the cytoplasmic enzyme LDH in culture supernatants (n = 3 *p > 0.01**p > 0.001). (b) Viability assessed by CFU of mycobacteria recovered from M. bovis BCG-infected neutrophils that were either treated with H₂O₂ to induce necrotic cell death or left untreated and succumbed to default apoptotic cell death. CFU were determined upon exposure to ambient air for the indicated time periods (n = 3, **p > 0.001, ***p > 0.0001). (c) Morphological analysis of human neutrophils either left uninfected or infected with M. bovis BCG without or with H₂O₂ treatment by H&E staining and SEM (n = 3).

Figure 6

Aerosols from mycobacterium-infected neutrophils. (a) Frequency of aerosol particles with certain aerodynamic diameters upon ultrawave nebulization of free mycobacterial cells in RPMI, RPMI alone, and mycobacterium-infected but untreated or H₂O₂-treated necrotic neutrophils (n = 3). (b) Distribution of optical diameters of aerosol particles generated by ultrawave nebulization from a mycobacterial suspension in RPMI, RPMI alone, or mycobacterium-infected but untreated or H₂O₂-treated necrotic neutrophils (n = 3). (c) Mycobacterial viability was assessed by CFU in aerosols collected from different levels of an Andersen impinger after nebulization using the ultrawave nebulizer for either mycobacterial cells alone or mycobacterium-infected but untreated or H₂O₂-treated necrotic neutrophils. Selected fractions containing airborne mycobacteria from necrotic neutrophils (PMNs) were further analysed by SEM (below) (n = 3, *p > 0.001).

Figure 7

Schematic overview of the aerosol collection device (ACD). (a) The device consists of an acrylic glass chamber of 27 l, including a HEPA filter to remove particles from incoming air. The ACD is connected to nebulizers, which use either jets or ultrawaves to generate aerosols. The air containing these aerosols is actively pumped through the multi-stage impinger. The airflow is measured by a flowmetre. The HEPA filter in front of the pump and flowmetre protects the equipment. (b) A photograph of level 1 of the impinger equipped with three different surfaces to collect settling aerosols, i.e., (I) glass coverslip for viability testing as appraised with CFU and live/dead microscopy staining, (II) copper grid for EM analysis, and (III) mica for atomic force microscopy analysis.