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Key Points The predominant mode of growth of most bacteria in natural and engineered environments is as a surface-associated community encased in an extracellular matrix, called a biofilm. When conditions within the biofilm become unfavourable, bacteria must be able to disperse to escape and colonize new habitats. The dispersal response of bacterial biofilms is regulated through the production and perception of extracellular and intracellular signalling molecules and in response to environmental cues such as changes in nutrient concentrations. Such signals and cues are translated into changes in gene expression that induce effectors, such as enzymes and surfactants, which break down the biofilm matrix and prepare bacteria for planktonic growth. In addition to releasing bacteria to colonize new sites, dispersal is associated with the formation of genetic variants that may be altered in traits which are important for colonization of and competition in new habitats. The sessile (biofilm) and motile (dispersal) phases of bacterial growth have close analogies to the lifestyles of colonial and holometabolous eukaryotes, including the generation of variants in the dispersal propagules. Biofilms may therefore be useful experimental tools to further explore ecological and evolutionary theories surrounding organisms with sessile and motile life phases. Dispersal is an essential stage in the 'life cycle' of many bacterial biofilms and is carefully regulated. McDougald et al . describe the factors that control this step, and place this process in an ecological context with a comparison to analogous eukaryotic life cycles. 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.

The failure of antibiotic therapies to clear Pseudomonas aeruginosa lung infection, the key mortality factor for cystic fibrosis (CF) patients, is partly attributed to the high tolerance of P. aeruginosa biofilms. Mannitol has previously been found to restore aminoglycoside sensitivity in Escherichia coli by generating a proton-motive force (PMF), suggesting a potential new strategy to improve antibiotic therapy and reduce disease progression in CF. Here, we used the commonly prescribed aminoglycoside tobramycin to select for P. aeruginosa persister cells during biofilm growth. Incubation with mannitol (10-40 mM) increased tobramycin sensitivity of persister cells up to 1,000-fold. Addition of mannitol to pre-grown biofilms was able to revert the persister phenotype and improve the efficacy of tobramycin. This effect was blocked by the addition of a PMF inhibitor or in a P. aeruginosa mutant strain unable to metabolise mannitol. Addition of glucose and NaCl at high osmolarity also improved the efficacy of tobramycin although to a lesser extent compared to mannitol. Therefore, the primary effect of mannitol in reverting biofilm associated persister cells appears to be an active, physiological response, associated with a minor contribution of osmotic stress. Mannitol was tested against clinically relevant strains, showing that biofilms containing a subpopulation of persister cells are better killed in the presence of mannitol, but a clinical strain with a high resistance to tobramycin was not affected by mannitol. Overall, these results suggest that in addition to improvements in lung function by facilitating mucus clearance in CF, mannitol also affects antibiotic sensitivity in biofilms and does so through an active, physiological response.

Despite aggressive antibiotic therapy, bronchopulmonary colonization by Pseudomonas aeruginosa causes persistent morbidity and mortality in cystic fibrosis (CF). Chronic P. aeruginosa infection in the CF lung is associated with structured, antibiotic-tolerant bacterial aggregates known as biofilms. We have demonstrated the effects of non-bactericidal, low-dose nitric oxide (NO), a signaling molecule that induces biofilm dispersal, as a novel adjunctive therapy for P. aeruginosa biofilm infection in CF in an ex vivo model and a proof-of-concept double-blind clinical trial. Submicromolar NO concentrations alone caused disruption of biofilms within ex vivo CF sputum and a statistically significant decrease in ex vivo biofilm tolerance to tobramycin and tobramycin combined with ceftazidime. In the 12-patient randomized clinical trial, 10 ppm NO inhalation caused significant reduction in P. aeruginosa biofilm aggregates compared with placebo across 7 days of treatment. Our results suggest a benefit of using low-dose NO as adjunctive therapy to enhance the efficacy of antibiotics used to treat acute P. aeruginosa exacerbations in CF. Strategies to induce the disruption of biofilms have the potential to overcome biofilm-associated antibiotic tolerance in CF and other biofilm-related diseases. This paper reports the first example of targeted anti-biofilm therapy in human disease. We have demonstrated that using low-dose nitric oxide as a non-bactericidal signaling molecule to induce biofilm dispersal may be useful as a novel adjunctive therapy to treat chronic pseudomonal biofilm infection in cystic fibrosis.

In both natural and artificial environments, bacteria predominantly grow in biofilms, and bacteria often disperse from biofilms as freely suspended single-cells. In the present study, the formation and dispersal of planktonic cellular aggregates, or 'suspended biofilms', by Pseudomonas aeruginosa in liquid batch cultures were closely examined, and compared to biofilm formation on a matrix of polyester (PE) fibers as solid surface in batch cultures. Plankton samples were analyzed by laser-diffraction particle-size scanning (LDA) and microscopy of aggregates. Interestingly, LDA indicated that up to 90% of the total planktonic biomass consisted of cellular aggregates in the size range of 10-400 microm in diameter during the growth phase, as opposed to individual cells. In cultures with PE surfaces, P. aeruginosa preferred to grow in biofilms, as opposed to planktonicly. However, upon carbon, nitrogen or oxygen limitation, the planktonic aggregates and PE-attached biofilms dispersed into single cells, resulting in an increase in optical density (OD) independent of cellular growth. During growth, planktonic aggregates and PE-attached biofilms contained densely packed viable cells and extracellular DNA (eDNA), and starvation resulted in a loss of viable cells, and an increase in dead cells and eDNA. Furthermore, a release of metabolites and infective bacteriophage into the culture supernatant, and a marked decrease in intracellular concentration of the second messenger cyclic di-GMP, was observed in dispersing cultures. Thus, what traditionally has been described as planktonic, individual cell cultures of P. aeruginosa, are in fact suspended biofilms, and such aggregates have behaviors and responses (e.g. dispersal) similar to surface associated biofilms. In addition, we suggest that this planktonic biofilm model system can provide the basis for a detailed analysis of the synchronized biofilm life cycle of P. aeruginosa.

Carbon starvation has been shown to induce a massive dispersal event in biofilms of the opportunistic pathogen Pseudomonas aeruginosa; however, the molecular pathways controlling this dispersal response remain unknown. We quantified changes in the proteome of P. aeruginosa PAO1 biofilm and planktonic cells during glucose starvation by differential peptide-fingerprint mass-spectrometry (iTRAQ). In addition, we monitored dispersal photometrically, as a decrease in turbidity/opacity of biofilms pre-grown and starved in continuous flow-cells, in order to evaluate treatments (e.g. inhibitors CCCP, arsenate, chloramphenicol, L-serine hydroxamate) and key mutants altered in biofilm development and dispersal (e.g. nirS, vfr, bdlA, rpoS, lasRrhlR, Pf4-bacteriophage and cyaA). In wild-type biofilms, dispersal started within five minutes of glucose starvation, was maximal after 2 h, and up to 60% of the original biomass had dispersed after 24 h of starvation. The changes in protein synthesis were generally not more than two fold and indicated that more than 100 proteins belonging to various classes, including carbon and energy metabolism, stress adaptation, and motility, were differentially expressed. For the different treatments, only the proton-ionophore CCCP or arsenate, an inhibitor of ATP synthesis, prevented dispersal of the biofilms. For the different mutants tested, only cyaA, the synthase of the intracellular second messenger cAMP, failed to disperse; complementation of the cyaA mutation restored the wild-type phenotype. Hence, the pathway for carbon starvation-induced biofilm dispersal in P. aeruginosa PAO1 involves ATP production via direct ATP synthesis and proton-motive force dependent step(s) and is mediated through cAMP, which is likely to control the activity of proteins involved in remodeling biofilm cells in preparation for planktonic survival.

Summary Strategies 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.

Mixed species biofilm communities that include ammonia oxidising organisms as well as heterotrophic bacteria can mediate chloramine decay through a combination of biochemical degradation through ammonia oxidation, nitrite mediated decay as well as direct effects of the biofilm matrix. Additionally, biofilm formation by heterotrophic bacteria may support biofilm formation by AOB, while the AOB supply organic carbon for the heterotrophs. Summary As water distribution centres increasingly switch to using chloramine to disinfect drinking water, it is of paramount importance to determine the interactions of chloramine with potential biological contaminants, such as bacterial biofilms, that are found in these systems. For example, ammonia‐oxidizing bacteria (AOB) are known to accelerate the decay of chloramine in drinking water systems, but it is also known that organic compounds can increase the chloramine demand. This study expanded upon our previously published model to compare the decay of chloramine in response to alginate, Pseudomonas aeruginosa, Nitrosomonas europaea and a mixed‐species nitrifying culture, exploring the contributions of microbial by‐products, heterotrophic bacteria and AOBs to chloramine decay. Furthermore, the contribution of AOBs to biofilm stability during chloramination was investigated. The results demonstrate that the biofilm matrix or extracellular polymeric substances (EPS), represented by alginate in these experiments, as well as high concentrations of dead or inactive cells, can drive chloramine decay rather than any specific biochemical activity of P. aeruginosa cells. Alginate was shown to reduce chloramine concentrations in a dose‐dependent manner at an average rate of 0.003 mg l−1 h−1 per mg l−1 of alginate. Additionally, metabolically active AOBs mediated the decay of chloramine, which protected members of mixed‐species biofilms from chloramine‐mediated disinfection. Under these conditions, nitrite produced by AOBs directly reacted with chloramine to drive its decay. In contrast, biofilms of mixed‐species communities that were dominated by heterotrophic bacteria due to either the absence of ammonia, or the addition of nitrification inhibitors and glucose, were highly sensitive to chloramine. These results suggest that mixed‐species biofilms are protected by a combination of biofilm matrix‐mediated inactivation of chloramine as well as the conversion of ammonia to nitrite through the activity of AOBs present in the community.

Communities of bacteria called biofilms are characterized by reduced diffusion, steep oxygen, and redox gradients and specific properties compared to individualized planktonic bacteria. In this study, we investigated whether signaling via nitrosylation of protein cysteine thiols (S-nitrosylation), regulating a wide range of functions in eukaryotes, could also specifically occur in biofilms and contribute to bacterial adaptation to this widespread lifestyle. We used a redox proteomic approach to compare cysteine S-nitrosylation in aerobic and anaerobic biofilm and planktonic Escherichia coli cultures and we identified proteins with biofilm-specific S-nitrosylation status. Using bacterial genetics and various phenotypic screens, we showed that impairing S-nitrosylation in proteins involved in redox homeostasis and amino acid synthesis such as OxyR, KatG, and GltD altered important biofilm properties, including motility, biofilm maturation, or resistance to oxidative stress. Our study therefore revealed that S-nitrosylation constitutes a physiological basis underlying functions critical for E. coli adaptation to the biofilm environment.

Pseudomonas aeruginosa uses multiple protein regulators that work in tandem to control the production of a wide range of virulence factors and facilitate rapid adaptation to diverse environmental conditions. In this opportunistic pathogen, ToxR was known to positively regulate the production of the major virulence factor exotoxin A and now, through analysis of genetic changes between two sublines of P. aeruginosa PAO1 and functional complementation of swarming, we have identified a previously unknown role of ToxR in surface-associated motility in P. aeruginosa. Further analysis revealed that ToxR had an impact on swarming motility by regulating the Rhl quorum sensing system and subsequent production of rhamnolipid surfactants. Additionally, ToxR was found to tightly bind cyclic diguanylate (c-di-GMP) and negatively affect traits controlled by this second messenger including reducing biofilm formation and the expression of Psl and Pel exopolysaccharides, necessary for attachment and sessile communities matrix scaffolding, in P. aeruginosa. Moreover, a link between the post-transcriptional regulator RsmA and toxR expression via the alternative sigma factor PvdS, induced under iron-limiting conditions, is established. This study reveals the importance of ToxR in a sophisticated regulation of free-living and biofilm-associated lifestyles, appropriate for establishing acute or chronic P. aeruginosa infections.

The dispersal phase that completes the biofilm lifecycle is of particular interest for its potential to remove recalcitrant, antimicrobial tolerant biofilm infections. Here we found that temperature is a cue for biofilm dispersal and a rise by 5 °C or more can induce the detachment of Pseudomonas aeruginosa biofilms. Temperature upshifts were found to decrease biofilm biomass and increase the number of viable freely suspended cells. The dispersal response appeared to involve the secondary messenger cyclic di-GMP, which is central to a genetic network governing motile to sessile transitions in bacteria. Furthermore, we used poly((oligo(ethylene glycol) methyl ether acrylate)- block -poly(monoacryloxy ethyl phosphate)-stabilized iron oxide nanoparticles (POEGA- b -PMAEP@IONPs) to induce local hyperthermia in established biofilms upon exposure to a magnetic field. POEGA- b -PMAEP@IONPs were non-toxic to bacteria and when heated induced the detachment of biofilm cells. Finally, combined treatments of POEGA- b -PMAEP@IONPs and the antibiotic gentamicin reduced by 2-log the number of colony-forming units in both biofilm and planktonic phases after 20 min, which represent a 3.2- and 4.1-fold increase in the efficacy against planktonic and biofilm cells, respectively, compared to gentamicin alone. The use of iron oxide nanoparticles to disperse biofilms may find broad applications across a range of clinical and industrial settings.