phoP maintains the environmental persistence and virulence of pathogenic bacteria in mechanically stressed desiccated droplets

Abstract

Despite extensive studies on kinematic features of impacting drops, the effect of mechanical stress on desiccated bacteria-laden droplets remains unexplored. In the present study, we unveiled the consequences of the impaction of bacteria-laden droplets on solid surfaces and their subsequent desiccation on the virulence of an enteropathogen Salmonella typhimurium (STM). The methodology elucidated the deformation, cell-cell interactions, adhesion energy, and roughness in bacteria induced by impact velocity and low moisture because of evaporation. Salmonella retrieved from the dried droplets were used to understand fomite-mediated pathogenesis. The impact velocity-induced mechanical stress deteriorated the in vitro viability of Salmonella. Of interest, an uninterrupted bacterial proliferation was observed in macrophages at higher mechanical stress. Wild-type Salmonella under mechanical stress induced the expression of phoP whereas infecting macrophages. The inability of STM ΔphoP to grow in nutrient-rich dried droplets signifies the role of phoP in sensing the mechanical stress and maintaining the virulence of Salmonella.

Keywords: Applied physics; Biophysics; Microbiology.

Graphical abstract

Figure 1

Global dynamics of bacterial droplet deposition (A) Profile view Images captured at 10000 fps for Salmonella laden droplet impacted on glass surface with 10 m/s on the glass substrate. The scale bar is 400 μm. (B) High-speed interference images captured at 10000 fps for Salmonella-laden droplet impacted on glass surface with 10 m/s on the glass substrate. The scale bar is 200 μm. (C) Fluorescence intensity captured for dried droplet impacted at different Weber numbers.

Figure 2

Schematic layout of the experimental study (A) The schematic representation to capture the global dynamics of droplet impact involving fluorescence microscopy, high-speed microscopic interferometry, atomic force microscopy, and backlight imaging of bacterial droplet impact. (B) Experimental layout depicting the assessment of in vitro viability of bacteria, infection of macrophages by the bacteria retrieved from desiccated dried droplets, and quantification of the expression of virulent genes of bacteria during infection.

Figure 3

Atomic force microscopy identifies higher stress induction and greater surface damage at a higher impact velocity (A) AFM images for Salmonella in milli-Q in a sessile drop (B) AFM images of the bacteria-laden droplet with milli-Q as the base medium at three different impact velocities in a 5 μm square area. (C and D) (C) AFM images of the bacteria-laden droplet with milli-Q and (D) 0.6 wt % Mucin as the base medium at three different impact velocities in a 5 μm square area for PFA fixed cells. (E) AFM images of the bacterial-laden droplet with milli-Q and (F) 0.6 wt % mucin as a base medium compared with PFA fixed bacteria at three different impact velocities along with their height profiles in 5 μm square area. (G) Adhesion energy in nutrient-neutral (milli-Q) and nutrient-rich (0.6 wt % Mucin) base fluid medium experienced on a dried STM (WT) sessile drop. (H) Roughness measurements presented as RMS roughness at different impact conditions for milli-Q and 0.6 wt % mucins. (P) ∗<0.05, (P) ∗∗<0.005, (P) ∗∗∗<0.0005, (P) ∗∗∗∗<0.0001, ns= non-significant, (Student’s t test-unpaired).

Figure 4

Assessment of in vitro viability of Salmonella retrieved from desiccated droplets of different base solutions impacted on a solid surface with or without mechanical stress induced by impact velocity The viability of Salmonella Typhimurium (in log10 scale) recovered from dried sessile droplets of different base solutions impacted on solid glass surface with or without velocities (5, 7, and 10 m/s). As base solutions. (A–F) milli-Q, (B) glucose 5 wt %, (C) 10% glycerol, (D) LB broth, (E and F) mucin 0.1 and 0.6 wt %, were used (N≥3). Data are represented as mean ± SEM. (P) ∗< 0.05, (P) ∗∗< 0.005, (P) ∗∗∗< 0.0005, (P) ∗∗∗∗< 0.0001, ns= non-significant, (Student’s t test-unpaired).

Figure 5

The virulence of Salmonella Typhimurium retrieved from desiccated mucin droplet (0.1 wt %) impacted on a solid surface at different velocities (A–G) Salmonella Typhimurium recovered from desiccated mucin (0.1 wt %) droplet impacted solid glass surface with or without specific velocities (5, 7, and 10 m/s) were used to infect RAW264.7 cells to determine the (A)percent phagocytosis and (B) intracellular proliferation (n = 3, N = 2). Determining the transcript-level expression of (C)sifA,(D)ssaV, and (E)phoP from intracellular Salmonella by RT-qPCR (n = 3, N = 2). Salmonella retrieved from the bacteria-laden dried droplet (mucin 0.1 wt %) impacted solid glass surface with or without velocities were used to infect C57BL/6 mice. On sixth day post-infection, the mice were sacrificed, and the bacterial burden in the (F) Liver and (G) Spleen was enumerated. Data are represented as mean ± SEM. (P) ∗< 0.05, (P) ∗∗< 0.005, (P) ∗∗∗< 0.0005, (P) ∗∗∗∗< 0.0001, ns= non-significant, (Student’s t test-unpaired).

Figure 6

Deleting phoP reduced the in vitro viability of Salmonella Typhimurium in desiccated droplet of mucin (0.6 wt %) impacted on glass surface impacted with velocity 5, 7, and 10 m/s (A–E) The in vitro viability of (A) STM (WT) and (B)ΔphoP (log10 scale) recovered from dried sessile droplets of mucin (0.6 wt %) impacted solid glass surface with or without velocities (5, 7, and 10 m/s) (N≥3). (C) AFM images of bacteria (STM WT and ΔphoP) laden droplets in mucin (0.6 wt %) on the glass surface. Height profile of (D) STM (WT) and (E)ΔphoP from the desiccated droplets of mucin (0.6 wt %) on the glass surface. Data are represented as mean ± SEM. (P) ∗< 0.05, (P) ∗∗< 0.005, (P) ∗∗∗< 0.0005, (P) ∗∗∗∗< 0.0001, ns = non-significant, (Student’s t test-unpaired).