Bacterial biofilms at times undergo regulated and coordinated dispersal events where sessile biofilm cells convert to free-swimming, planktonic bacteria. In the opportunistic pathogen Pseudomonas aeruginosa, we previously observed that dispersal occurs concurrently with three interrelated processes within mature biofilms: (i) production of oxidative or nitrosative stress-inducing molecules inside biofilm structures, (ii) bacteriophage induction, and (iii) cell lysis. Here we examine whether specific reactive oxygen or nitrogen intermediates play a role in cell dispersal from P. aeruginosa biofilms. We demonstrate the involvement of anaerobic respiration processes in P. aeruginosa biofilm dispersal and show that nitric oxide (NO), used widely as a signaling molecule in biological systems, causes dispersal of P. aeruginosa biofilm bacteria. Dispersal was induced with low, sublethal concentrations (25 to 500 nM) of the NO donor sodium nitroprusside (SNP). Moreover, a P. aeruginosa mutant lacking the only enzyme capable of generating metabolic NO through anaerobic respiration (nitrite reductase, ⌬nirS) did not disperse, whereas a NO reductase mutant (⌬norCB) exhibited greatly enhanced dispersal. Strategies to induce biofilm dispersal are of interest due to their potential to prevent biofilms and biofilm-related infections. We observed that exposure to SNP (500 nM) greatly enhanced the efficacy of antimicrobial compounds (tobramycin, hydrogen peroxide, and sodium dodecyl sulfate) in the removal of established P. aeruginosa biofilms from a glass surface. Combined exposure to both NO and antimicrobial agents may therefore offer a novel strategy to control preestablished, persistent P. aeruginosa biofilms and biofilm-related infections.

In most environments, bacteria reside primarily in biofilms, which are social consortia of cells that are embedded in an extracellular matrix and undergo developmental programmes resulting in a predictable biofilm 'life cycle'. Recent research on many different bacterial species has now shown that the final stage in this life cycle includes the production and release of differentiated dispersal cells. The formation of these cells and their eventual dispersal is initiated through diverse and remarkably sophisticated mechanisms, suggesting that there are strong evolutionary pressures for dispersal from an otherwise largely sessile biofilm. The evolutionary aspect of biofilm dispersal is now being explored through the integration of molecular microbiology with eukaryotic ecological and evolutionary theory, which provides a broad conceptual framework for the diversity of specific mechanisms underlying biofilm dispersal. Here, we review recent progress in this emerging field and suggest that the merging of detailed molecular mechanisms with ecological theory will significantly advance our understanding of biofilm biology and ecology.

Bacterial biofilms are highly dynamic communities which display a range of differentiated phenotypes during the course of development. By exchange of cell-cell signals, subpopulations of cells can coordinate their activity and undertake particular metabolic tasks or defense strategies (56). At times, the bacterial community releases single cells that escape from the biofilm and revert to a free-swimming, planktonic mode of growth, leaving behind hollow voids in the biofilm architecture (5, 37, 57). This process, referred to as dispersal, completes the biofilm life cycle and is thought to be important for successful colonization of new surfaces. Although the mechanisms underlying these events remain to be fully elucidated, previous studies of various species, including the opportunistic pathogen Pseudomonas aeruginosa, have revealed that dispersal events correlate with the induction of a specific phenotype that involves cellular motility (37, 42).In P. aeruginosa, biofilm dispersal can be triggered by environmental factors, including nutrient (42, 45) and iron (4, 36) availability, and has recently been linked to the intracellular second messenger cyclic di-GMP (c-di-GMP) (45, 47). Numerous studies revealed that decreased c-di-GMP levels are related to a motile mode of growth and to cell dispersal in eubacteria. In this second messenger system, diguanylate cyclases (DGCs) and specific phosphodiesterases (PDEs) are responsible for the biosynthesis and the degradation of c-di-GMP, respectively. DGCs and PDEs contribute to a genetic network that responds to a broad range of environmental cues and/or cell-cell signals and modulate intracellular levels of c-di-GMP, which has been shown to regulate various cellular functions, including biofilm formation, virulence, and dispersal, in many bacterial species (47,(51)(52)(53). Recently, we identified the gas nitric oxide (NO) as an important factor in the regulation of dispersal in P. aeruginosa biofilms (5). Exogenous addition of nontoxic concentrations of NO, typically in the low nanomolar range, was found to stimulate motility and biofilm dispersal in P. aeruginosa. A role for anaerobic metabolism and NO in biofilm dispersal and survival was further supported by other studies of P. aeruginosa (54, 61), Staphylococcus aureus (44), and various single and multispecies biofilms (6).NO is a water-soluble, hydrophobic free radical that can freely diffuse in biological systems. At high concentrations (micromolar to millimolar range), NO

SummaryStrategies to induce biofilm dispersal are of interest due to their potential to prevent biofilm formation and biofilm‐related infections. Nitric oxide (NO), an important messenger molecule in biological systems, was previously identified as a signal for dispersal in biofilms of the model organism Pseudomonas aeruginosa. In the present study, the use of NO as an anti‐biofilm agent more broadly was assessed. Various NO donors, at concentrations estimated to generate NO levels in the picomolar and low nanomolar range, were tested on single‐species biofilms of relevant microorganisms and on multi‐species biofilms from water distribution and treatment systems. Nitric oxide‐induced dispersal was observed in all biofilms assessed, and the average reduction of total biofilm surface was 63%. Moreover, biofilms exposed to low doses of NO were more susceptible to antimicrobial treatments than untreated biofilms. For example, the efficacy of conventional chlorine treatments at removing multi‐species biofilms from water systems was increased by 20‐fold in biofilms treated with NO compared with untreated biofilms. These data suggest that combined treatments with NO may allow for novel and improved strategies to control biofilms and have widespread applications in many environmental, industrial and clinical settings.