Nicolas Barraud

Pseudomonas aeruginosauses 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 ofP. aeruginosaPAO1 and functional complementation of swarming, we have identified a previously unknown role of ToxR in surface-associated motility inP. 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, inP. aeruginosa. Moreover, a link between the post-transcriptional regulator RsmA andtoxRexpression 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 chronicP. aeruginosainfections.

Nitric oxide (NO), which induces biofilm dispersal, is a promising strategy for biofilm control in both clinical and industrial contexts. However, competing environmental signals may reduce the efficacy of NO. The results presented here suggest that the presence of iron represents one such environmental cue that antagonizes the activity of NO as a biofilm-dispersing agent. Based on this understanding, we developed a strategy to enhance dispersal by combining NO with an iron-scavenging agent. Overall, this study links two important environmental signals, iron and NO, with their roles in biofilm development and suggests new ways for improving the use of NO in biofilm control strategies.

The biological signal molecule nitric oxide (NO) was found to induce biofilm dispersal across a range of bacterial species, which led to its consideration for therapeutic strategies to treat biofilms and biofilm-related infections. However, biofilms are often not completely dispersed after exposure to NO. To better understand this phenomenon, we investigated the response of Pseudomonas aeruginosa biofilm cells to successive NO treatments. When biofilms were first pretreated with a low, noneffective dose of NO, a second dose of the signal molecule at a concentration usually capable of inducing dispersal did not have any effect. Amperometric analysis revealed that pretreated P. aeruginosa cells had enhanced NO-scavenging activity, and this effect was associated with the production of the flavohemoglobin Fhp. Further, quantitative real-time reverse transcription-PCR (qRT-PCR) analysis showed that fhp expression increased by over 100-fold in NO-pretreated biofilms compared to untreated biofilms. Biofilms of mutant strains harboring mutations in fhp or fhpR , encoding a NO-responsive regulator of fhp , were not affected in their dispersal response after the initial pretreatment with NO. Overall, these results suggest that FhpR can sense NO to trigger production of the flavohemoglobin Fhp and inhibit subsequent dispersal responses to NO. Finally, the addition of imidazole, which can inhibit the NO dioxygenase activity of flavohemoglobin, attenuated the prevention of dispersal after NO pretreatment and improved the dispersal response in older, starved biofilms. This study clarifies the underlying mechanisms of impaired dispersal induced by repeated NO treatments and offers a new perspective for improving the use of NO in biofilm control strategies.

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.

One common feature of biofilm development is the active dispersal of cells from the mature biofilm, which completes the biofilm life cycle and allows for the subsequent colonization of new habitats. Dispersal is likely to be critical for species survival and appears to be a precisely regulated process that involves a complex network of genes and signal transduction systems. Sophisticated molecular mechanisms control the transition of sessile biofilm cells into dispersal cells and their coordinated detachment and release in the bulk liquid. Dispersal cells appear to be specialized and exhibit a unique phenotype different from biofilm or planktonic bacteria. Further, the dispersal population is characterized by a high level of heterogeneity, reminiscent of, but distinct from, that in the biofilm, which could potentially allow for improved colonization under various environmental conditions. Here we review recent advances in characterizing the molecular mechanisms that regulate biofilm dispersal events and the impact of dispersal in a broader ecological context. Several strategies that exploit the mechanisms controlling biofilm dispersal to develop as applications for biofilm control are also presented.

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 ofPseudomonas aeruginosabiofilms. 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.

Colistin is often the only effective antibiotic against the respiratory infections caused by multidrug-resistant Gram-negative bacteria. However, colistin-resistant multidrug-resistant isolates have...

The rise of hospital-acquired infections, also known as nosocomial infections, is a growing concern in intensive healthcare, causing the death of hundreds of thousands of patients and costing billions of dollars worldwide every year. In addition, a decrease in the effectiveness of antibiotics caused by the emergence of drug resistance in pathogens living in biofilm communities poses a significant threat to our health system. The development of new therapeutic agents is urgently needed to overcome this challenge. We have developed new dual action polymeric nanoparticles capable of storing nitric oxide, which can provoke dispersal of biofilms into an antibiotic susceptible planktonic form, together with the aminoglycoside gentamicin, capable of killing the bacteria. The novelty of this work lies in the attachment of NO-releasing moiety to an existing clinically used drug, gentamicin. The nanoparticles were found to release both agents simultaneously and demonstrated synergistic effects, reducing the viability of Pseudomonas aeruginosa biofilm and planktonic cultures by more than 90% and 95%, respectively, while treatments with antibiotic or nitric oxide alone resulted in less than 20% decrease in biofilm viability.

In this study, catalytic generation of nitric oxide by a copper(II) complex embedded within a poly(vinyl chloride) matrix in the presence of nitrite (source of nitric oxide) and ascorbic acid (reducing agent) was shown to effectively control the formation and dispersion of nitrifying bacteria biofilms. Amperometric measurements indicated increased and prolonged generation of nitric oxide with the addition of the copper complex when compared to that with nitrite and ascorbic acid alone. The effectiveness of the copper complex–nitrite–ascorbic acid system for biofilm control was quantified using protein analysis, which showed enhanced biofilm suppression when the copper complex was used in comparison to that with nitrite and ascorbic acid treatment alone. Confocal laser scanning microscopy (CLSM) and LIVE/DEAD staining revealed a reduction in cell surface coverage without a loss of viability with the copper complex and up to 5 mM of nitrite and ascorbic acid, suggesting that the nitric oxide generated from the system inhibits proliferation of the cells on surfaces. Induction of nitric oxide production by the copper complex system also triggered the dispersal of pre-established biofilms. However, the addition of a high concentration of nitrite and ascorbic acid to a pre-established biofilm induced bacterial membrane damage and strongly decreased the metabolic activity of planktonic and biofilm cells, as revealed by CLSM with LIVE/DEAD staining and intracellular adenosine triphosphate measurements, respectively. This study highlights the utility of the catalytic generation of nitric oxide for the long-term suppression and removal of nitrifying bacterial biofilms.

Pseudomonas aeruginosa is an opportunistic pathogen causing a variety of life-threatening diseases such as cystic fibrosis and nosocomial infections in burn victims. The ability of P. aeruginosa to cause infection is attributed to the production of virulence factors such as pyocyanin and elastases. These virulence factors are under the control of quorum sensing (QS) a cell to cell communication process controlled by small diffusible signalling molecules based on N-acyl-homoserine lactones (AHLs) known as autoinducers. The inhibition of QS and thereby virulence factors is seen as a potential new anti-infective strategy. Additionally, the role of nitric oxide (NO) in downstream processes in bacteria such as biofilm dispersal, motility, virulence and antimicrobial defence systems is gaining attention and could be used to control bacterial. Herein we report the design and synthesis of hybrid compounds based on AHL signalling molecules and NO donors as anti-infective agents. A series of AHL-NO hybrids were synthesised and potent inhibitors of QS and virulence factors of P. aeruginosa were identified. This research has led to conversion of agonist AHLs to antagonist AHLs with dual properties of QS inhibition and NO release.