Nicolas Barraud

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, DeltanirS) did not disperse, whereas a NO reductase mutant (DeltanorCB) 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.

Bacteria in biofilms often undergo active dispersal events and revert to a free-swimming, planktonic state to complete the biofilm life cycle. The signaling molecule nitric oxide (NO) was previously found to trigger biofilm dispersal in the opportunistic pathogen Pseudomonas aeruginosa at low, nontoxic concentrations (N. Barraud, D. J. Hassett, S. H. Hwang, S. A. Rice, S. Kjelleberg, and J. S. Webb, J. Bacteriol. 188:7344-7353, 2006). NO was further shown to increase cell motility and susceptibility to antimicrobials. Recently, numerous studies revealed that increased degradation of the secondary messenger cyclic di-GMP (c-di-GMP) by specific phosphodiesterases (PDEs) triggers a planktonic mode of growth in eubacteria. In this study, the potential link between NO and c-di-GMP signaling was investigated by performing (i) PDE inhibitor studies, (ii) enzymatic assays to measure PDE activity, and (iii) direct quantification of intracellular c-di-GMP levels. The results suggest a role for c-di-GMP signaling in triggering the biofilm dispersal event induced by NO, as dispersal requires PDE activity and addition of NO stimulates PDE and induces the concomitant decrease in intracellular c-di-GMP levels in P. aeruginosa. Furthermore, gene expression studies indicated global responses to low, nontoxic levels of NO in P. aeruginosa biofilms, including upregulation of genes involved in motility and energy metabolism and downregulation of adhesins and virulence factors. Finally, site-directed mutagenesis of candidate genes and physiological characterization of the corresponding mutant strains uncovered that the chemotaxis transducer BdlA is involved in the biofilm dispersal response induced by NO.

In drinking water distribution systems (DWDS), biofilms are the predominant mode of microbial growth, with the presence of extracellular polymeric substance (EPS) protecting the biomass from environmental and shear stresses. Biofilm formation poses a significant problem to the drinking water industry as a potential source of bacterial contamination, including pathogens, and, in many cases, also affecting the taste and odor of drinking water and promoting the corrosion of pipes. This article critically reviews important research findings on biofilm growth in DWDS, examining the factors affecting their formation and characteristics as well as the various technologies to characterize and monitor and, ultimately, to control their growth. Research indicates that temperature fluctuations potentially affect not only the initial bacteria-to-surface attachment but also the growth rates of biofilms. For the latter, the effect is unique for each type of biofilm-forming bacteria; ammonia-oxidizing bacteria, for example, grow more-developed biofilms at a typical summer temperature of 22 °C compared to 12 °C in fall, and the opposite occurs for the pathogenic Vibrio cholerae. Recent investigations have found the formation of thinner yet denser biofilms under high and turbulent flow regimes of drinking water, in comparison to the more porous and loosely attached biofilms at low flow rates. Furthermore, in addition to the rather well-known tendency of significant biofilm growth on corrosion-prone metal pipes, research efforts also found leaching of growth-promoting organic compounds from the increasingly popular use of polymer-based pipes. Knowledge of the unique microbial members of drinking water biofilms and, importantly, the influence of water characteristics and operational conditions on their growth can be applied to optimize various operational parameters to minimize biofilm accumulation. More-detailed characterizations of the biofilm population size and structure are now feasible with fluorescence microscopy (epifluorescence and CLSM imaging with DNA, RNA, EPS, and protein and lipid stains) and electron microscopy imaging (ESEM). Importantly, thorough identification of microbial fingerprints in drinking water biofilms is achievable with DNA sequencing techniques (the 16S rRNA gene-based identification), which have revealed a prevalence of previously undetected bacterial members. Technologies are now moving toward in situ monitoring of biomass growth in distribution networks, including the development of optical fibers capable of differentiating biomass from chemical deposits. Taken together, management of biofilm growth in water distribution systems requires an integrated approach, starting from the treatment of water prior to entering the networks to the potential implementation of "biofilm-limiting" operational conditions and, finally, ending with the careful selection of available technologies for biofilm monitoring and control. For the latter, conventional practices, including chlorine-chloramine disinfection, flushing of DWDS, nutrient removal, and emerging technologies are discussed with their associated challenges.

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.