Biofilm Maintenance as an Active Process: Evidence that Biofilms Work Hard to Stay Put

Abstract

Biofilm formation represents a critical strategy whereby bacteria can tolerate otherwise damaging environmental stressors and antimicrobial insults. While the mechanisms bacteria use to establish a biofilm and disperse from these communities have been well-studied, we have only a limited understanding of the mechanisms required to maintain these multicellular communities. Indeed, until relatively recently, it was not clear that maintaining a mature biofilm could be considered an active, regulated process with dedicated machinery. Using Pseudomonas aeruginosa as a model system, we review evidence from recent studies that support the model that maintenance of these persistent, surface-attached communities is indeed an active process. Biofilm maintenance mechanisms include transcriptional regulation and second messenger signaling (including the production of extracellular polymeric substances). We also discuss energy-conserving pathways that play a key role in the maintenance of these communities. We hope to highlight the need for further investigation to uncover novel biofilm maintenance pathways and suggest the possibility that such pathways can serve as novel antibiofilm targets.

Keywords: Pseudomonas aeruginosa; biofilm; c-di-GMP; maintenance; metabolism; peroxide.

FIG 1

Biofilm maintenance as a discrete step in the biofilm life cycle. The progression of the biofilm life cycle begins with the initial attachment of a cell (blue) to a surface. During subsequent microcolony growth, surface-attached cells aggregate and produce EPS (green), which provides intercellular adhesion and protection from environmental challenges. Microcolonies can transition into mature biofilms, which exhibit a complex 3-dimensional morphology and take on classic features of a biofilm, including high-level tolerance to antimicrobial agents. We propose that after biofilms are established, active processes of biofilm maintenance are employed to respond to internal cues and external environmental perturbations and thereby facilitate long-term surface persistence. During biofilm dispersal, cells within the aggregate can respond to environmental stimuli, resulting in enhanced motility and a transition to the planktonic mode of growth, with the potential for the dispersed cells to eventually adhere and form biofilms at new locations.

FIG 2

Inactivation of bfiS (PA4197), bfmR (PA4101), and mifR (PA5511) expression in mature biofilms result in biofilm architectural collapse and biomass loss. P. aeruginosa mutants complemented with plasmid-borne copies of the respective genes placed under the regulation of the arabinose-inducible P_BAD were grown under continuous flow conditions in glutamate minimal medium in the presence of 0.1% arabinose for 144 h after which time the biofilms were visualized by confocal microscopy (0 h). Arabinose was then eliminated from the growth medium, and the biofilm architecture was monitored post arabinose removal at the times indicated. PAO1 strain harboring the empty pJN105 vector was used as control. Biofilms were stained with the LIVE/DEAD BacLight viability stain (Invitrogen Corp.). White bars = 100 μm. This figure and legend are reproduced in their entirety from Petrova and Sauer (17), with permission.

FIG 3

Mutations in the ΔrmcA and ΔmorA genes result in a biofilm maintenance defect. Shown are biofilms grown under static conditions then stained with the BacLight Live/Dead kit. Live cells are green (Syto9-stained), and cells with compromised membranes are red (propidium iodide-stained). All three strains show robust biofilm formation with few red-staining cells at 16 h, when there is still a carbon/energy source available to the cells. At 48 h, when the cells have exhausted their carbon/energy source, marked cell death is apparent in the mutants but not in the WT parent. This figure is taken from a published report (53) with the permission of the authors.