Plasma activated water as a pre-treatment strategy in the context of biofilm-infected chronic wounds.

Healing and treatment of chronic wounds are often complicated due to biofilm formation by pathogens. Here, the efficacy of plasma activated water (PAW) as a pre-treatment strategy has been investigated prior to the application of topical antiseptics polyhexamethylene biguanide, povidone iodine, and MediHoney, which are routinely used to treat chronic wounds. The efficacy of this treatment strategy was determined against biofilms of Escherichia coli formed on a plastic substratum and on a human keratinocyte monolayer substratum used as an in vitro biofilm-skin epithelial cell model. PAW pre-treatment greatly increased the killing efficacy of all the three antiseptics to eradicate the E. coli biofilms formed on the plastic and keratinocyte substrates. However, the efficacy of the combined PAW-antiseptic treatment and single treatments using PAW or antiseptic alone was lower for biofilms formed in the in vitro biofilm-skin epithelial cell model compared to the plastic substratum. Scavenging assays demonstrated that reactive species present within the PAW were largely responsible for its anti-biofilm activity. PAW treatment resulted in significant intracellular reactive oxygen and nitrogen species accumulation within the E. coli biofilms, while also rapidly acting on the microbial membrane leading to outer membrane permeabilisation and depolarisation. Together, these factors contribute to significant cell death, potentiating the antibacterial effect of the assessed antiseptics.

Synthesis and Evaluation of Functionalized Polyurethanes for pH-Responsive Delivery of Compounds in Chronic Wounds.

Chronic wounds, depending on the bacteria that caused the infection, can be associated with an extreme acidic or basic pH. Therefore, the application of pH-responsive hydrogels has been instigated for the delivery of therapeutics to chronic wounds. Herein, with the aim of developing a flexible pH-responsive hydrogel, we functionalized hydrophilic polyurethanes with either cationic (polyethylene imine) or anionic (succinic anhydride) moieties. A comprehensive physicochemical characterization of corresponding polymers was carried out. Particularly, when tested in aqueous buffers, the surface charge of hydrogel films was closely correlated with the pH of the buffers. The loading of the cationic and anionic hydrogel films with various compound models (bromophenol blue; negatively charged or Pyronin Y; positively charged) showed that the electrostatic forces between the polymeric backbone and the compound model will determine the ultimate release rate at any given pH. The potential application of these films for chronic wound drug delivery was assessed by loading them with an antibiotic (ciprofloxacin). In vitro bacterial culturing was performed using Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Results showed that at the same drug dosage, different release profiles achievable from cationic and anionic polyurethanes can yield different degrees of an antibacterial effect. Overall, our results suggest the potential application of cationic and anionic hydrophilic polyurethanes as flexible pH-responsive materials for the delivery of therapeutics to chronic wounds.

An Effective Sanitizer for Fresh Produce Production: In Situ Plasma-Activated Water Treatment Inactivates Pathogenic Bacteria and Maintains the Quality of Cucurbit Fruit.

The effect of plasma-activated water (PAW) generated with a dielectric barrier discharge diffusor (DBDD) system on microbial load and organoleptic quality of cucamelons was investigated and compared to the established sanitizer, sodium hypochlorite (NaOCl). Pathogenic serotypes of Escherichia coli, Salmonella enterica, and Listeria monocytogenes were inoculated onto the surface of cucamelons (6.5 log CFU g-1) and into the wash water (6 log CFU mL-1). PAW treatment involved 2 min in situ with water activated at 1,500 Hz and 120 V and air as the feed gas; NaOCl treatment was a wash with 100 ppm total chlorine; control treatment was a wash with tap water. PAW treatment produced a 3-log CFU g-1 reduction of pathogens on the cucamelon surface without negatively impacting quality or shelf life. NaOCl treatment reduced the pathogenic bacteria on the cucamelon surface by 3 to 4 log CFU g-1; however, this treatment also reduced fruit shelf life and quality. Both systems reduced 6-log CFU mL-1 pathogens in the wash water to below detectable limits. The critical role of superoxide anion radical (·O2-) in the antimicrobial power of DBDD-PAW was demonstrated through a Tiron scavenger assay, and chemistry modeling confirmed that ·O2- generation readily occurs in DBDD-PAW generated with the employed settings. Modeling of the physical forces produced during plasma treatment showed that bacteria likely experience strong local electric fields and polarization. We hypothesize that these physical effects synergize with reactive chemical species to produce the acute antimicrobial activity seen with the in situ PAW system. IMPORTANCE Plasma-activated water (PAW) is an emerging sanitizer in the fresh food industry, where food safety must be achieved without a thermal kill step. Here, we demonstrate PAW generated in situ to be a competitive sanitizer technology, providing a significant reduction of pathogenic and spoilage microorganisms while maintaining the quality and shelf life of the produce item. Our experimental results are supported by modeling of the plasma chemistry and applied physical forces, which show that the system can generate highly reactive ·O2- and strong electric fields that combine to produce potent antimicrobial power. In situ PAW has promise in industrial applications as it requires only low power (12 W), tap water, and air. Moreover, it does not produce toxic by-products or hazardous effluent waste, making it a sustainable solution for fresh food safety.

Clinically relevant in vitro biofilm models: A need to mimic and recapitulate the host environment.

Biofilm-associated infections are difficult to treat and eradicate because of their increased antimicrobial tolerance. In vitro biofilm models have enabled the high throughput testing of an array of differing novel antimicrobials and treatment strategies. However, biofilms formed in these oftentimes basic in vitro systems do not resemble biofilms seen in vivo. As a result, translatability from the lab to the clinic is poor or limited. To improve translatability, in vitro models must better recapitulate the host environment. This review describes and critically evaluates new and innovative in vitro models that better mimic the environments of a variety of clinically important, biofilm-associated infections of the skin, oropharynx, lungs, and infections related to indwelling implants and medical devices. This review highlights that many of these models represent considerable advances in the field of biofilm research and help to translate laboratory findings into the clinical practice.

ToxR is a c-di-GMP binding protein that modulates surface-associated behaviour in Pseudomonas aeruginosa.

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 antimicrobial efficacy of plasma-activated water against Listeria and E. coli is modulated by reactor design and water composition.

AIMS: This study aimed to compare the efficacy of plasma-activated water (PAW) generated by two novel plasma reactors against pathogenic foodborne illness organisms. METHODS AND RESULTS: The antimicrobial efficacy of PAW produced by a bubble spark discharge (BSD) reactor and a dielectric barrier discharge-diffuser (DBDD) reactor operating at atmospheric conditions with air, multiple discharge frequencies and Milli-Q and tap water, was investigated with model organisms Listeria innocua and Escherichia coli in situ. Optimal conditions were subsequently employed for pathogenic bacteria Listeria monocytogenes, E. coli and Salmonella enterica. DBDD-PAW reduced more than 6-log of bacteria within 1 min. The BSD-PAW, while attaining high log reduction, was less effective. Analysis of physicochemical properties revealed that BSD-PAW had a greater variety of reactive species than DBDD-PAW. Scavenger assays designed to specifically sequester reactive species demonstrated a critical role of superoxide, particularly in DBDD-PAW. CONCLUSIONS: DBDD-PAW demonstrated rapid antimicrobial activity against pathogenic bacteria, with superoxide the critical reactive species. SIGNIFICANCE AND IMPACT OF STUDY: This study demonstrates the potential of DBDD-PAW produced using tap water and air as a feasible and cost-effective option for antimicrobial applications, including food safety.

Interactions of plasma-activated water with biofilms: inactivation, dispersal effects and mechanisms of action.

Biofilms have several characteristics that ensure their survival in a range of adverse environmental conditions, including high cell numbers, close cell proximity to allow easy genetic exchange (e.g., for resistance genes), cell communication and protection through the production of an exopolysaccharide matrix. Together, these characteristics make it difficult to kill undesirable biofilms, despite the many studies aimed at improving the removal of biofilms. An elimination method that is safe, easy to deliver in physically complex environments and not prone to microbial resistance is highly desired. Cold atmospheric plasma, a lightning-like state generated from air or other gases with a high voltage can be used to make plasma-activated water (PAW) that contains many active species and radicals that have antimicrobial activity. Recent studies have shown the potential for PAW to be used for biofilm elimination without causing the bacteria to develop significant resistance. However, the precise mode of action is still the subject of debate. This review discusses the formation of PAW generated species and their impacts on biofilms. A focus is placed on the diffusion of reactive species into biofilms, the formation of gradients and the resulting interaction with the biofilm matrix and specific biofilm components. Such an understanding will provide significant benefits for tackling the ubiquitous problem of biofilm contamination in food, water and medical areas.

Underwater microplasma bubbles for efficient and simultaneous degradation of mixed dye pollutants.

Complete degradation of mixtures of organic pollutants is a major challenge due to their diverse degradation pathways. In this work, a novel microplasma bubble (MPB) reactor was developed to generate plasma discharges inside small forming bubbles as an effective mean of delivering reactive species for the degradation of the target organic contaminants. The results show that the integration of plasma and bubbles resulted in efficient degradation for all azo, heterocyclic, and cationic dyes, evidenced by the outstanding energy efficiency of 13.0, 18.1 and 22.1 g/kWh with 3 min of processing, in degrading alizarin yellow (AY), orange II (Orng-II) and methylene blue (MB), individually. The MPB treatment also effectively and simultaneously degraded the dyes in their mixtures such as AY + Orng-II, AY + MB and AY + Orng-II + MB. Scavenger assays revealed that the short-lived reactive species, including the hydroxyl (OH) and superoxide anion (O2-) radicals, played the dominant role in the degradation of the pollutants. Possible degradation pathways were proposed based on the intermediate products detected during the degradation process. The feasibility of this proposed strategy was further evaluated using other common water pollutants. Reduced toxicity was confirmed by the observed increases in human cell viability for the treated water. This work could support the future development of high performance- and energy-efficient wastewater abatement technologies.

Cold plasma effect on the proteome of Pseudomonas aeruginosa - Role for bacterioferritin.

Cold atmospheric-pressure plasma (CAP) is a relatively new method used for bacterial inactivation. CAP is ionized gas that can be generated by applying an electric current to air or a feeding gas. It contains reactive species and emits UV radiation, which have antibacterial activity. Previous data suggests that CAP is effective in microbial inactivation and can decontaminate and sterilize surfaces, but its exact mode of action is still under debate. This study demonstrates the effect of CAP on the whole proteome of Pseudomonas aeruginosa PAO1 biofilms, which is a dominant pathogen in cystic fibrosis and medical device-related infections. Liquid chromatography-mass spectrometry (LC-MS) was used to identify differentially regulated proteins of whole cell P. aeruginosa extracts. A total of 16 proteins were identified to be affected by plasma treatment compared to the control. Eight of the identified proteins have functions in transcription and translation and their expression changes are likely to be part of a general physiological response instead of a CAP-specific adaptation. However, CAP also affected bacterioferritin (Bfr), Isocitrate dehydrogenase (Idh), Trigger factor (Tig) and a chemotaxis protein, which may be involved in P. aeruginosa's specific response to CAP. We confirm that bacterioferritin B plays a role in the bacterial response to CAP because ΔbfrB mutants of both PAO1 and PA14 are more susceptible to plasma-induced cell-death than their corresponding wild-type strains. To our knowledge, this is the first study showing the effect of plasma on the whole proteome of a pathogenic microorganism. It will help our understanding of the mode of action of CAP-mediated bacterial inactivation and thus support a safe and effective routine use of CAP in clinical and industrial settings.

Cold plasma treatment for cotton seed germination improvement.

Adverse environmental conditions at planting, such as cold temperature or water limitation, can lead to a reduced level of seed germination and plant establishment for cotton. Cold atmospheric-pressure plasma (CAP) treatment of cotton seeds prior to planting may help alleviate this problem. CAP is ionised gas that has a range of biological activities due to the formation of a mix of reactive oxygen and nitrogen species (RONS), excited molecules, charged particles and UV photons. Our results show that a 27 minutes CAP treatment using air can significantly increase water absorption of the seed, and improve warm germination, metabolic chill test germination and chilling tolerance in cotton. We also observe that the beneficial effect of CAP treatment is long-lasting and stable as improved germination activity is still seen when treatment occurs 4 months before germination testing, suggesting that future large-scale industrial seed plasma treatments may still be effectively applied well (months) before the seed planting. We conclude that CAP treatment is a promising new tool for use in the cotton industry that has the potential to significantly improve plant establishment in a wider range of environmental conditions.

Gram positive and Gram negative bacteria differ in their sensitivity to cold plasma.

Cold atmospheric-pressure plasma (CAP) is a relatively new method being investigated for antimicrobial activity. However, the exact mode of action is still being explored. Here we report that CAP efficacy is directly correlated to bacterial cell wall thickness in several species. Biofilms of Gram positive Bacillus subtilis, possessing a 55.4 nm cell wall, showed the highest resistance to CAP, with less than one log10 reduction after 10 min treatment. In contrast, biofilms of Gram negative Pseudomonas aeruginosa, possessing only a 2.4 nm cell wall, were almost completely eradicated using the same treatment conditions. Planktonic cultures of Gram negative Pseudomonas libanensis also had a higher log10 reduction than Gram positive Staphylococcus epidermidis. Mixed species biofilms of P. aeruginosa and S. epidermidis showed a similar trend of Gram positive bacteria being more resistant to CAP treatment. However, when grown in co-culture, Gram negative P. aeruginosa was more resistant to CAP overall than as a mono-species biofilm. Emission spectra indicated OH and O, capable of structural cell wall bond breakage, were present in the plasma. This study indicates that cell wall thickness correlates with CAP inactivation times of bacteria, but cell membranes and biofilm matrix are also likely to play a role.

Draft Genome Sequence of Pseudomonas aeruginosa ATCC 9027 (DSM 1128), an Important Rhamnolipid Surfactant Producer and Sterility Testing Strain.

Pseudomonas aeruginosa ATCC 9027 (DSM1128) is often used as a quality-control strain for sterility and microbial contamination testing and is an important biosurfactant producer. Here, we present the 6.4-Mb draft genome sequence and highlight some genomic differences to its closest relative, P. aeruginosa strain PA7.

Protein retention on plasma-treated hierarchical nanoscale gold-silver platform.

Dense arrays of gold-supported silver nanowires of about 100 nm in diameter grown directly in the channels of nanoporous aluminium oxide membrane were fabricated and tested as a novel platform for the immobilization and retention of BSA proteins in the microbial-protective environments. Additional treatment of the silver nanowires using low-temperature plasmas in the inductively-coupled plasma reactor and an atmospheric-pressure plasma jet have demonstrated that the morphology of the nanowire array can be controlled and the amount of the retained protein may be increased due to the plasma effect. A combination of the neutral gold sublayer with the antimicrobial properties of silver nanowires could significantly enhance the efficiency of the platforms used in various biotechnological processes.

Pseudomonas aeruginosa Biofilm Response and Resistance to Cold Atmospheric Pressure Plasma Is Linked to the Redox-Active Molecule Phenazine.

Pseudomonas aeruginosa is an important opportunistic pathogen displaying high antibiotic resistance. Its resistance is in part due to its outstanding ability to form biofilms on a range of biotic and abiotic surfaces leading to difficult-to-treat, often long-term infections. Cold atmospheric plasma (CAP) is a new, promising antibacterial treatment to combat antibiotic-resistant bacteria. Plasma is ionized gas that has antibacterial properties through the generation of a mix of reactive oxygen and nitrogen species (RONS), excited molecules, charged particles and UV photons. Our results show the efficient removal of P. aeruginosa biofilms using a plasma jet (kINPen med), with no viable cells detected after 5 min treatment and no attached biofilm cells visible with confocal microscopy after 10 min plasma treatment. Because of its multi-factorial action, it is widely presumed that the development of bacterial resistance to plasma is unlikely. However, our results indicate that a short plasma treatment (3 min) may lead to the emergence of a small number of surviving cells exhibiting enhanced resistance to subsequent plasma exposure. Interestingly, these cells also exhibited a higher degree of resistance to hydrogen peroxide. Whole genome comparison between surviving cells and control cells revealed 10 distinct polymorphic regions, including four belonging to the redox active, antibiotic pigment phenazine. Subsequently, the interaction between phenazine production and CAP resistance was demonstrated in biofilms of transposon mutants disrupted in different phenazine pathway genes which exhibited significantly altered sensitivity to CAP.

Plasma treatment for next-generation nanobiointerfaces.

Energy deficiency, global poverty, chronic hunger, chronic diseases, and environment conservation are among the major problems threatening the whole mankind. Nanostructure-based technologies could be a possible solution. Such techniques are now used for the production of many vitally important products including cultured and fermented food, antibiotics, various medicines, and biofuels. On the other hand, the nanostructure-based technologies still demonstrate low efficiency and controllability, and thus still are not capable to decisively address the global problems. Furthermore, future technologies should ensure lowest possible environmental impact by implementing green production principles. One of the most promising approaches to address these challenges are the sophisticatedly engineered biointerfaces. Here, the authors briefly evaluate the potential of the plasma-based techniques for the fabrication of complex biointerfaces. The authors consider mainly the atmospheric and inductively coupled plasma environments and show several examples of the artificial plasma-created biointerfaces, which can be used for the biotechnological and medical processes, as well as for the drug delivery devices, fluidised bed bioreactors, catalytic reactors, and others. A special attention is paid to the plasma-based treatment and processing of the biointerfaces formed by arrays of carbon nanotubes and graphene flakes.

'Big things in small packages: the genetics of filamentous phage and effects on fitness of their host'.

This review synthesizes recent and past observations on filamentous phages and describes how these phages contribute to host phentoypes. For example, the CTXφ phage of Vibrio cholerae encodes the cholera toxin genes, responsible for causing the epidemic disease, cholera. The CTXφ phage can transduce non-toxigenic strains, converting them into toxigenic strains, contributing to the emergence of new pathogenic strains. Other effects of filamentous phage include horizontal gene transfer, biofilm development, motility, metal resistance and the formation of host morphotypic variants, important for the biofilm stress resistance. These phages infect a wide range of Gram-negative bacteria, including deep-sea, pressure-adapted bacteria. Many filamentous phages integrate into the host genome as prophage. In some cases, filamentous phages encode their own integrase genes to facilitate this process, while others rely on host-encoded genes. These differences are mediated by different sets of 'core' and 'accessory' genes, with the latter group accounting for some of the mechanisms that alter the host behaviours in unique ways. It is increasingly clear that despite their relatively small genomes, these phages exert signficant influence on their hosts and ultimately alter the fitness and other behaviours of their hosts.

Environmental cues and genes involved in establishment of the superinfective Pf4 phage of Pseudomonas aeruginosa.

Biofilm development in Pseudomonas aeruginosa is in part dependent on a filamentous phage, Pf4, which contributes to biofilm maturation, cell death, dispersal and variant formation, e.g., small colony variants (SCVs). These biofilm phenotypes correlate with the conversion of the Pf4 phage into a superinfection (SI) variant that reinfects and kills the prophage carrying host, in contrast to other filamentous phage that normally replicate without killing their host. Here we have investigated the physiological cues and genes that may be responsible for this conversion. Flow through biofilms typically developed SI phage approximately days 4 or 5 of development and corresponded with dispersal. Starvation for carbon or nitrogen did not lead to the development of SI phage. In contrast, exposure of the biofilm to nitric oxide, H2O2 or the DNA damaging agent, mitomycin C, showed a trend of increased numbers of SI phage, suggesting that reactive oxygen or nitrogen species (RONS) played a role in the formation of SI phage. In support of this, mutation of oxyR, the major oxidative stress regulator in P. aeruginosa, resulted in higher level of and earlier superinfection compared to the wild-type (WT). Similarly, inactivation of mutS, a DNA mismatch repair gene, resulted in the early appearance of the SI phage and this was four log higher than the WT. In contrast, loss of recA, which is important for DNA repair and the SOS response, also resulted in a delayed and decreased production of SI phage. Treatments or mutations that increased superinfection also correlated with an increase in the production of morphotypic variants. The results suggest that the accumulation of RONS by the biofilm may result in DNA lesions in the Pf4 phage, leading to the formation of SI phage, which subsequently selects for morphotypic variants, such as SCVs.

Atmospheric pressure plasmas: infection control and bacterial responses.

Cold atmospheric pressure plasma (APP) is a recent, cutting-edge antimicrobial treatment. It has the potential to be used as an alternative to traditional treatments such as antibiotics and as a promoter of wound healing, making it a promising tool in a range of biomedical applications with particular importance for combating infections. A number of studies show very promising results for APP-mediated killing of bacteria, including removal of biofilms of pathogenic bacteria such as Pseudomonas aeruginosa. However, the mode of action of APP and the resulting bacterial response are not fully understood. Use of a variety of different plasma-generating devices, different types of plasma gases and different treatment modes makes it challenging to show reproducibility and transferability of results. This review considers some important studies in which APP was used as an antibacterial agent, and specifically those that elucidate its mode of action, with the aim of identifying common bacterial responses to APP exposure. The review has a particular emphasis on mechanisms of interactions of bacterial biofilms with APP.

Analysis of the Pseudoalteromonas tunicata genome reveals properties of a surface-associated life style in the marine environment.

BACKGROUND: Colonisation of sessile eukaryotic host surfaces (e.g. invertebrates and seaweeds) by bacteria is common in the marine environment and is expected to create significant inter-species competition and other interactions. The bacterium Pseudoalteromonas tunicata is a successful competitor on marine surfaces owing primarily to its ability to produce a number of inhibitory molecules. As such P. tunicata has become a model organism for the studies into processes of surface colonisation and eukaryotic host-bacteria interactions. METHODOLOGY/PRINCIPAL FINDINGS: To gain a broader understanding into the adaptation to a surface-associated life-style, we have sequenced and analysed the genome of P. tunicata and compared it to the genomes of closely related strains. We found that the P. tunicata genome contains several genes and gene clusters that are involved in the production of inhibitory compounds against surface competitors and secondary colonisers. Features of P. tunicata's oxidative stress response, iron scavenging and nutrient acquisition show that the organism is well adapted to high-density communities on surfaces. Variation of the P. tunicata genome is suggested by several landmarks of genetic rearrangements and mobile genetic elements (e.g. transposons, CRISPRs, phage). Surface attachment is likely to be mediated by curli, novel pili, a number of extracellular polymers and potentially other unexpected cell surface proteins. The P. tunicata genome also shows a utilisation pattern of extracellular polymers that would avoid a degradation of its recognised hosts, while potentially causing detrimental effects on other host types. In addition, the prevalence of recognised virulence genes suggests that P. tunicata has the potential for pathogenic interactions. CONCLUSIONS/SIGNIFICANCE: The genome analysis has revealed several physiological features that would provide P. tunciata with competitive advantage against other members of the surface-associated community. We have also identified properties that could mediate interactions with surfaces other than its currently recognised hosts. This together with the detection of known virulence genes leads to the hypothesis that P. tunicata maintains a carefully regulated balance between beneficial and detrimental interactions with a range of host surfaces.

Hydrogen peroxide linked to lysine oxidase activity facilitates biofilm differentiation and dispersal in several gram-negative bacteria.

The marine bacterium Pseudoalteromonas tunicata produces an antibacterial and autolytic protein, AlpP, which causes death of a subpopulation of cells during biofilm formation and mediates differentiation, dispersal, and phenotypic variation among dispersal cells. The AlpP homologue (LodA) in the marine bacterium Marinomonas mediterranea was recently identified as a lysine oxidase which mediates cell death through the production of hydrogen peroxide. Here we show that AlpP in P. tunicata also acts as a lysine oxidase and that the hydrogen peroxide generated is responsible for cell death within microcolonies during biofilm development in both M. mediterranea and P. tunicata. LodA-mediated biofilm cell death is shown to be linked to the generation of phenotypic variation in growth and biofilm formation among M. mediterranea biofilm dispersal cells. Moreover, AlpP homologues also occur in several other gram-negative bacteria from diverse environments. Our results show that subpopulations of cells in microcolonies also die during biofilm formation in two of these organisms, Chromobacterium violaceum and Caulobacter crescentus. In all organisms, hydrogen peroxide was implicated in biofilm cell death, because it could be detected at the same time as the killing occurred, and the addition of catalase significantly reduced biofilm killing. In C. violaceum the AlpP-homologue was clearly linked to biofilm cell death events since an isogenic mutant (CVMUR1) does not undergo biofilm cell death. We propose that biofilm killing through hydrogen peroxide can be linked to AlpP homologue activity and plays an important role in dispersal and colonization across a range of gram-negative bacteria.