Effect of Aerosolization and Drying on the Viability of Pseudomonas syringae Cells

Abstract

Airborne dispersal of microorganisms influences their biogeography, gene flow, atmospheric processes, human health and transmission of pathogens that affect humans, plants and animals. The extent of their impact depends essentially on cell-survival rates during the process of aerosolization. A central factor for cell-survival is water availability prior to and upon aerosolization. Also, the ability of cells to successfully cope with stress induced by drying determines their chances of survival. In this study, we used the ice-nucleation active, plant pathogenic Pseudomonas syringae strain R10.79 as a model organism to investigate the effect of drying on cell survival. Two forms of drying were simulated: drying of cells in small droplets aerosolized from a wet environment by bubble bursting and drying of cells in large droplets deposited on a surface. For drying of cells both in aerosol and surface droplets, the relative humidity (RH) was varied in the range between 10 and 90%. The fraction of surviving cells was determined by live/dead staining followed by flow cytometry. We also evaluated the effect of salt concentration in the water droplets on the survival of drying cells by varying the ionic strength between 0 and 700 mM using NaCl and sea salt. For both aerosol and surface drying, cell survival increased with decreasing RH (p < 0.01), and for surface drying, survival was correlated with increasing salt concentration (p < 0.001). Imaging cells with TEM showed shrunk cytoplasm and cell wall damage for a large fraction of aerosolized cells. Ultimately, we observed a 10-fold higher fraction of surviving cells when dried as aerosol compared to when dried on a surface. We conclude that the conditions, under which cells dry, significantly affect their survival and thus their success to spread through the atmosphere and colonize new environments as well as their ability to affect atmospheric processes.

Keywords: Pseudomonas syringae; aerosolization; bioaerosols; bubble bursting; drying; ice nucleation activity.

FIGURE 1

Schematic drawing of the three experimental setups for (A) surface drying in air with controlled RH, (B) aerosolization in bubble tank, and (C) aerosolization by SLAG into humidity conditioned flow tube. Black arrows show airflow directions. MFC, mass flow controller; OPS, optical particle sizer; SMPS, scanning mobility particle sizer; SLAG, sparging liquid aerosol generator; RH probe, relative humidity probe; HEPA filter, high efficiency particulate arresting filter.

FIGURE 2

The gating strategy for analysis of flow cytometry results. Dot plots show how live, damaged and dead cell populations were defined based on the fluorescence intensity in the blue laser 1 detector (SYTO9, 530/30 nm) and blue laser 3 detector (PI, 615/24 nm). A representative example was taken from (A) cells suspended in 0.1% NaCl prior to aerosolization, (B) cells after aerosolization at 40% RH, and (C) cells after aerosolization at 60% RH.

FIGURE 3

Fraction of live cells after aerosolization and surface drying in air with different RH. Upper central graph: Average fraction of healthy and damaged cells after aerosolization (n = 29) and surface drying (n = 12). The bar height is the mean of n samples and the error bars represent the standard error of mean (SEM). Left panel: Illustration of bubble bursting together with the flow cytometry results from aerosolization at different RH by SLAG (10–20, 30–40, 60, 90% RH), and a comparison of the SLAG and the bubble tank. Right panel: Illustration of a droplet drying on a surface together with the flow cytometry results from surface drying in air with different RH (15, 30, 60, and 80% RH). ^∗∗ indicates significance between groups with p < 0.001.

FIGURE 4

Calculated evaporation times using (A) equation 3 for airborne droplets of pure water at different relative humidity (at room temperature). All droplets below 20 μm in diameter, which was all of the aerosolized droplets, would have time to evaporate droplet water around the bacterial cell in the aerosolization setup even at high (90%) RH. (B) Calculated drying times for sessile droplets (with a contact angle <90°) on a surface at room temperature using equation 1. The red line indicates the drying times for the surface drying experiment.

FIGURE 5

Results from cultivation showing the fraction of CFU per total cells (total cell concentration from flow cytometry) for (A) aerosolized cells, (B) and surface dried cells. The bar height represents the mean of three replicates and the error bar, the standard error of the mean. ^∗ indicates significance between groups, p < 0.05, using Mann–Whitney U-test.

FIGURE 6

Average fraction of live cells obtained by flow cytometry analysis of the cells suspended in different salt solutions and dried on a surface. Error bars indicate standard error of mean. The colors mark different salt types: black for MilliQ water, yellow for NaCl solutions and gray for sea salt solutions. The red dotted line is an exponential fit to the data points. Note that the y-axis has a logarithmic scale.

FIGURE 7

Number size distribution of the aerosol particles generated by SLAG and dried at 10% RH (left y-axis), and by the bubble tank (right y-axis), measured by an OPS. For SLAG, the concentration is a mean value based on 3 replicates (30 min sampling each), and for the bubble tank, the mean is based on one replicate (3 h sampling). The number size distribution of the background NaCl solution for SLAG and MilliQ water for the bubble tank is included (triangular markers).

FIGURE 8

Transmission electron microscopy images of (A) healthy cells with intact flagella (flagella marked by red arrows) that had not been aerosolized, and of (B–E) cells after aerosolization by SLAG. (B,C) Wrinkled cells surfaces, (D) porous cell wall (pores marked by blue arrows) and shrunk cytoplasm, and (E) detached cytoplasmic membrane and lost flagella.