Dispersed cells represent a distinct stage in the transition from bacterial biofilm to planktonic lifestyles

Abstract

Bacteria assume distinct lifestyles during the planktonic and biofilm modes of growth. Increased levels of the intracellular messenger c-di-GMP determine the transition from planktonic to biofilm growth, while a reduction causes biofilm dispersal. It is generally assumed that cells dispersed from biofilms immediately go into the planktonic growth phase. Here we use single-nucleotide resolution transcriptomic analysis to show that the physiology of dispersed cells from Pseudomonas aeruginosa biofilms is highly different from those of planktonic and biofilm cells. In dispersed cells, the expression of the small regulatory RNAs RsmY and RsmZ is downregulated, whereas secretion genes are induced. Dispersed cells are highly virulent against macrophages and Caenorhabditis elegans compared with planktonic cells. In addition, they are highly sensitive towards iron stress, and the combination of a biofilm-dispersing agent, an iron chelator and tobramycin efficiently reduces the survival of the dispersed cells.

PslG, a self-produced glycosyl hydrolase, triggers biofilm disassembly by disrupting exopolysaccharide matrix

“…As previously described [63], 5 × 10 5 RAW264.7 macrophages were grown in 24-well plates (Nunc, Denmark) and incubated at 37 °C, 5% CO 2 for 16 h. The macrophages were then washed twice with phosphate-buffered saline (PBS) and incubated with 0, 50, 100, 200, and 500 nM PslG in DMEM at 37 °C, 5% CO 2 for 24 h. The toxicity of the PslG was determined by monitoring cell viability with 20 µM propidium iodide. Cells that were observed unstained or stained red under the inverted epifluorescent microscope (Zeiss) were counted as live or dead, respectively.…”

“…The assay was performed as previously described with modifications [63]. The PslG-dispersed bacterial cells were collected from PAO1 pellicles grown in Jensen's medium for 24 h. PslG was used at 50 nM.…”

“…The assay was done as previously described with modifications [63]. Briefly, P. aeruginosa PAO1 biofilm was grown on sterile glass coverslips in minimal medium at 37 °C for 24 h. The biofilms were washed twice in 0.9% NaCl to remove unattached cells and aggregates and then treated with 0 or 50 nM PslG in minimal medium at 37 °C for 1 h. The treated biofilms were then washed twice in 0.9% NaCl and further incubated with 5 × 10 5 RAW264.7 macrophages in DMEM at 37 °C, 5% CO 2 for 2 h. After washing away the dispersed bacterial cells in 0.9% NaCl, the remaining biofilms were scraped off the glass slides and re-suspended in 1 ml 0.9% NaCl.…”

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in <em>Pseudomonas aeruginosa</em>

“…In line with this, it has been experimentally demonstrated that artificial decrease of intracellular c-di-GMP in in vitro-grown P. aeruginosa leads to decreased biofilm formation and increased susceptibility to antimicrobials, while P. aeruginosa-developed biofilms on silicone implants, located in the peritoneal cavity of mice, can be dispersed in a similar fashion [8][9][10][11] .…”

The anti-cancerous drug doxorubicin decreases the c-di-GMP content in Pseudomonas aeruginosa but promotes biofilm formation

“…In P. aeruginosa, the biofilm dispersal effect mediated by low c-di-GMP levels has been shown to be multifactorial. Low c-di-GMP levels have been shown to reduce the production of biofilm matrix components such as Psl, Pel, CdrA and Cup fimbria, and activate bacterial motility (Chua et al, 2014;Hickman et al, 2005). Evidence has also been provided that low c-di-GMP levels lead to upregulation of the activity of the periplasmic LapG protease that cleaves off the surface-associated protein CdrA, which tethers the cells to the Psl matrix during biofilm formation (Cooley et al, 2015;Rybtke et al, 2015).…”