Assessing the airborne survival of bacteria in populations of aerosol droplets with a novel technology

Abstract

The airborne transmission of infection relies on the ability of pathogens to survive aerosol transport as they transit between hosts. Understanding the parameters that determine the survival of airborne microorganisms is critical to mitigating the impact of disease outbreaks. Conventional techniques for investigating bioaerosol longevity in vitro have systemic limitations that prevent the accurate representation of conditions that these particles would experience in the natural environment. Here, we report a new approach that enables the robust study of bioaerosol survival as a function of relevant environmental conditions. The methodology uses droplet-on-demand technology for the generation of bioaerosol droplets (1 to greater than 100 per trial) with tailored chemical and biological composition. These arrays of droplets are captured in an electrodynamic trap and levitated within a controlled environmental chamber. Droplets are then deposited on a substrate after a desired levitation period (less than 5 s to greater than 24 h). The response of bacteria to aerosolization can subsequently be determined by counting colony forming units, 24 h after deposition. In a first study, droplets formed from a suspension of Escherichia coli MRE162 cells (10⁸ ml^-1) with initial radii of 27.8 ± 0.08 µm were created and levitated for extended periods of time at 30% relative humidity. The time-dependence of the survival rate was measured over a time period extending to 1 h. We demonstrate that this approach can enable direct studies at the interface between aerobiology, atmospheric chemistry and aerosol physics to identify the factors that may affect the survival of airborne pathogens with the aim of developing infection control strategies for public health and biodefence applications.

Keywords: aerosol transport; airborne transmission; bioaerosol; infection; survival.

Figure 1.

Representation of the interplay between biological aerosols and atmospheric factors during aerosol transport. Examples of factors include environmental conditions such as the temperature and relative humidity, day and night-time atmospheric chemistry, and mixing with anthropogenic and other natural aerosols found in the atmosphere. (Online version in colour.)

Figure 2.

(a) Expanded view of the main components of the CELEBS apparatus. (b) Schematic diagram of CELEBS operation. (c,d) Consecutive close-up images for levitation and initial deposition of the same bioaerosol population. The levitated droplets appear as lines due to the slower shutter speed of the camera compared with the oscillatory motion of the droplets driven by the AC waveform applied to the ring electrodes. (Online version in colour.)

Figure 3.

Schematic diagram for determination of BD. In the bioaerosol droplets, green bacteria represent viable cells and red bacteria represent dead cells. Yellow and blue components in the droplets represent media constituents and other organic and inorganic compounds. (Online version in colour.)

Figure 4.

Correlation between the number of cells per droplet (i.e. fluospheres, bacteria and spores) and the cell concentration of the suspension loaded in the DoD dispenser. (Online version in colour.)

Figure 5.

PDF curves, experimental results and confocal microscopy images for particle concentration in aerosol droplets. Scale bar, 30 µm. Diameters of the deposited droplets are larger than the initial droplet sizes due to impaction on the gelatin used to coat the microscope slides. (a) Modelled curves and experimental results for the number of fluospheres per aerosol droplet. The PDFs for the averages of fluospheres per droplet, λ = 0.795, λ = 2.62, λ = 5.70 and λ = 20.6, are shown by the black, yellow, maroon and turquoise curves, respectively. Observed percentages for the number of beads per droplet are (•), (*), (Δ) and (○) at solution concentrations of 8.0 × 10⁶, 2.5 × 10⁷, 3.64 × 10⁷ and 1.14 × 10⁸ cells ml⁻¹, respectively. (b) Modelled curves and experimental results for the number of E. coli MRE-162 cells per aerosol droplet. The PDFs for λ = 1.14, λ = 5.83, λ = 8.96 and λ = 51.3 are shown by the black, yellow, maroon and turquoise curves, respectively. Observed percentages for the number of bacteria cells per droplet are (•), (*), (Δ) and (○) at solution concentrations of 9.32 × 10⁶, 4.66 × 10⁷, 9.32 × 10⁷ and 4.66 × 10⁸ CFU ml⁻¹, respectively. (c) Modelled curves and experimental results for the number of B. atrophaeus spores per aerosol droplet. The PDFs for λ = 0.54, λ = 3.09 and λ = 31.49 are shown by the black, yellow and turquoise curves, respectively. Observed percentages for the number of spores per droplet (•), (*) and (○) at solution concentrations of 3.0 × 10⁶, 3.0 × 10⁷ and 3.0 × 10⁸ cells ml⁻¹, respectively. (d–f) Confocal microscopy images for different particle concentrations in aerosol droplets containing fluospheres beads, E. coli MRE-162 cells and B. atrophaeus spores, respectively. (Online version in colour.)

Figure 6.

Percentage of cells with intact cell membranes obtained by using different aerosolization devices. In consecutive order, bars represent for each set of values: the non-aerosolized control (green) bacterial culture, the bacterial culture aerosolized by using the DoD with a pulse voltage of 3.5 and 8 V (blue), a frequency of 10 and 1000 Hz (pink), a width of 25 and 45 µs (yellow) and an induction voltage of 250 and 1050 V (grey), respectively. Finally, the refluxed bacterial culture after 5- and 20-min nebulization by using the 1-jet refluxing nebulizer, respectively, are shown (maroon). The average and standard deviation for each parameter were calculated by counting at least 200 cells from five different fields of view. (Online version in colour.)

Figure 7.

Effect of suspension in the AC field (2 kV) on the viability of E. coli incorporated in droplets of 27.8 ± 0.08 µm radii. The graph shows the relationship between the predicted number of CFU per droplet (≡) (mean ± s.d.) and the number of CFU per droplet formed after the incubation of bioaerosol populations levitated in the EDT for 5 s (•). (Online version in colour.)

Figure 8.

(a) Sampling efficiency of the CELEBS apparatus. Each data point represents a single experiment showing the correlation between the number of droplets levitated and the number of droplets collected. (b) Images of different sizes of bioaerosol populations levitated inside the EDT (left image 12 and right image 40 bioaerosol droplets). (c) Representative image of droplets containing fluospheres collected on the substrate immediately after aerosolization. The actual size of the particles at generation was measured with the CK-EDB system (27.8 ± 0.08 µm radii) [51]. The enlarged diameter of the impacted droplets provided by the image software is due to droplet spread at impaction on the coated gelatin slide. (Online version in colour.)

Figure 9.

Bioaerosol decay for E. coli MRE162 and B. atrophaeus spores at 33% RH and 24°C temperature. All the longevity data are expressed as the average and standard deviation values for at least three biological replicates (samples from independent E. coli cultures) per experiment. (Online version in colour.)