Real-time monitoring of the dynamics and interactions of bacteria and the early-stage formation of biofilms

Abstract

Bacterial biofilms are complex colonies of bacteria adhered to a static surface and/or one to another. Bacterial biofilms exhibit high resistance to antimicrobial agents and can cause life-threatening nosocomial infections. Despite the effort of the scientific community investigating the formation and growth of bacterial biofilms, the preliminary interaction of bacteria with a surface and the subsequent early-stage formation of biofilms is still unclear. In this study, we present real-time, label-free monitoring of the interaction of Escherichia coli and Pseudomonas aeruginosa bacteria with untreated glass control surfaces and surfaces treated with benzalkonium chloride, a chemical compound known for its antimicrobial properties. The proof of principle investigation has been performed in a standard inverted optical microscope exploiting the optical phenomenon of caustics as a tool for monitoring bacterial diffusion and early adhesion and associated viability. The enhanced resolving power of the optical set-up allowed the monitoring and characterization of the dynamics of the bacteria, which provided evidence for the relationship between bacterial adhesion dynamics and viability, as well as the ability to form a biofilm. Viable bacteria adhered to the surface exhibited noticeable sliding or rotary dynamics while bacteria killed by surface contact remained static once adhered to the surface. This difference in dynamics allowed the early detection of biofilm formation and offers the potential to quantify the efficiency of antimicrobial surfaces and coatings.

Figure 1

Schematic representation of the five stages of biofilm formation.

Figure 2

Comparison between the same E. coli bacteria population imaged with an inverted optical microscope in caustics mode (top) and fluorescence mode (bottom). E. coli bacteria were stained with the fluorescent dyes SYTO9 and propidium iodide (PI) from the LIVE/DEAD BacLight kit. There are no significant differences between the two imaging techniques. The three bacteria highlighted by the black arrows in the caustics image are not at the surface and have been captured when aligned perpendicularly to the surface during their random diffusion, resulting in an almost spherical optical signature.

Figure 3

Photograph of the optical signature generated by an E. coli bacterium taken with the optical inverted microscope used in this work set-up for: (a) caustics mode; (b) brightfield mode.

Figure 4

Tracks (yellow lines) of the sections (purple circles) of four E. coli bacteria experiencing: (a) random diffusion above the surface; (b) rotary attachment; (c) lateral attachment; (d) static attachment. The dynamics of the four bacteria was monitored for approximately 20 s. The length of the scale bars is 5 μm.

Figure 5

E. coli bacteria stained with trypan blue and statically attached to a BKC surface imaged with an inverted optical microscope in caustics mode (a) and fluorescence mode (b).

Figure 6

Photograph of the optical signature generated by a dead E. coli bacterium.

Figure 7

Population of dead E. coli bacteria dispersed in PBS and exposed to a glass surface treated with BKC for 1 h. The length of the scale bar is 20 μm.

Figure 8

Tracks (red lines) of the sections (purple circles) of a P. aeruginosa bacterium: (a) rotary attached; (b) laterally attached and (c) static (dead). The dead bacteria were statically adhered to the surface, resulting in the absence of any noticeable tracks over time. The dynamics of the two bacteria was monitored for approximately 20 s. The length of the scale bars is 5 μm.

Figure 9

E. coli bacteria clustering together to start biofilms.

Figure 10

Population of E. coli bacteria dispersed in PBS and exposed to a glass surface for 24 h. The length of the scale bar is 20 μm.

Figure 11

Population of P. aeruginosa bacteria dispersed in PBS and exposed to a glass surface for 24 h. The length of the scale bar is 20 μm.

Figure 12

Population of E. coli bacteria dispersed in a solution of 10% LB in PBS and exposed to a glass surface for 24 h. The length of the scale bar is 20 μm.

Figure 13

Population of P. aeruginosa bacteria dispersed in a solution of 10% LB in PBS and exposed to a glass surface for 24 h. The length of the scale bar is 20 μm.

Figure 14

Population of dead E. coli bacteria dispersed in a PBS and exposed to a glass surface treated with BKC for 24 h. The length of the scale bar is 20 μm.

Figure 15

Population of dead P. aeruginosa bacteria dispersed in a PBS and exposed to a glass surface treated with BKC for 24 h. The length of the scale bar is 20 μm.