Cells dispersed from Marinobacter hydrocarbonoclasticus SP17 biofilm exhibit a specific protein profile associated with a higher ability to reinitiate biofilm development at the hexadecane-water interface

Biofilm formation by marine hydrocarbonoclastic bacteria is commonly observed and has been recognized as an important mechanism for the biodegradation of hydrocarbons. In order to colonize new oil-water interfaces, surface-attached communities of hydrocarbonoclastic bacteria must release cells into the environment. Here we explored the physiology of cells freshly dispersed from a biofilm of Marinobacter hydrocarbonoclasticus developing at the hexadecane-water interface, by combining proteomic and physiological approaches. The comparison of the dispersed cells' proteome with those of biofilm, logarithmic- and stationary-phase planktonic cells indicated that dispersed cells had lost most of the biofilm phenotype and expressed a specific proteome. Two proteins involved in cell envelope maturation, DsbA and CtpA, were exclusively detected in dispersed cells, suggesting a reshaping of the cell envelopes during biofilm dispersal. Furthermore, dispersed cells exhibited a higher affinity for hexadecane and initiated more rapidly biofilm formation on hexadecane than the reference planktonic cells. Interestingly, storage wax esters were rapidly degraded in dispersed cells, suggesting that their observed physiological properties may rely on reserve mobilization. Thus, by promoting oil surface colonization, cells emigrating from the biofilm could contribute to the success of marine hydrocarbonoclastic bacteria in polluted environments.     

A novel in vitro model for haematogenous spreading of S. aureus device biofilms demonstrating clumping dispersal as an advantageous dissemination mechanism

Staphylococcus aureus is able to disseminate from vascular device biofilms to the blood and organs, resulting in life‐threatening infections such as endocarditis. The mechanisms behind spreading are largely unknown, especially how the bacterium escapes immune effectors and antibiotics in the process. Using an in vitro catheter infection model, we studied S. aureus biofilm growth, late‐stage dispersal, and reattachment to downstream endothelial cell layers. The ability of the released biofilm material to resist host response and disseminate in vivo was furthermore studied in whole blood and phagocyte survival assays and in a short‐term murine infection model. We found that S. aureus biofilms formed in flow of human plasma release biofilm thromboemboli with embedded bacteria and bacteria‐secreted polysaccharides. The emboli disseminate as antibiotic and immune resistant vehicles that hold the ability to adhere to and initiate colonisation of endothelial cell layers under flow. In vivo experiments showed that the released biofilm material reached the heart similarly as ordinary broth‐grown bacteria but also that clumps to some extend were trapped in the lungs. The clumping dispersal of S. aureus from in vivo‐like vascular biofilms and their specific properties demonstrated here help explain the pathophysiology associated with S. aureus bloodstream infections.     

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.     

Breeding dispersal of Eurasian kestrels Falco tinnunculus under temporally fluctuating food abundance

Costs and benefits of dispersal can vary in space and time, depending on environmental factors and individual state. Plastic, condition‐dependent dispersal strategies, in which individuals rely on external cues such as food abundance to adjust their dispersal distances, are therefore expected to evolve in temporally fluctuating environments. We examined factors affecting breeding dispersal distances in Eurasian kestrels Falco tinnunculus subsisting on multi‐annually and cyclically fluctuating voles as their main food. We attempted to avoid traditional bias in dispersal studies by having large study areas and by taking detection probabilities into account. We observed 320 dispersal events of male and 215 events of female kestrels from our study areas in western Finland during 24 yr. After correcting for distance‐specific detection probability, the estimates of mean dispersal distances increased two‐fold being still clearly higher for females than males. Vole abundance in the spring of settlement was more important in determining average dispersal distances than vole abundance in the previous autumn. At the population level (cross‐sectional model), both males and females dispersed longer distances when the spring abundance of their main food (voles) was low compared to when it was abundant, as predicted by the food depletion hypothesis. At the individual level (longitudinal model), only females responded to the food situation by dispersing more when food abundance was low in the spring of settlement. Females also dispersed longer when vole abundance in the previous autumn had been high. Individual males did not respond to vole abundance, which implies that the population level response in males might have been caused by long‐distance dispersers, which breed in the study area only in good vole years. Our results show that the dispersal distances of kestrels at northern latitudes depend both on individual properties (gender, age, and possibly individual tendency to disperse) and environmental conditions (temporal variation in main food abundance).     

Potential applications of nonthermal plasmas against biofilm‐associated micro‐organisms in vitro

Biofilms as complex microbial communities attached to surfaces pose several challenges in different sectors, ranging from food and healthcare to desalination and power generation. The biofilm mode of growth allows microorganisms to survive in hostile environments and biofilm cells exhibit distinct physiology and behaviour in comparison with their planktonic counterparts. They are ubiquitous, resilient and difficult to eradicate due to their resistant phenotype. Several chemical‐based cleaning and disinfection regimens are conventionally used against biofilm‐dwelling micro‐organisms in vitro. Although such approaches are generally considered to be effective, they may contribute to the dissemination of antimicrobial resistance and environmental pollution. Consequently, advanced green technologies for biofilm control are constantly emerging. Disinfection using nonthermal plasmas (NTPs) is one of the novel strategies having a great potential for control of biofilms of a broad spectrum of micro‐organisms. This review discusses several aspects related to the inactivation of biofilm‐associated bacteria and fungi by different types of NTPs under in vitro conditions. A brief introduction summarizes prevailing methods in biofilm inactivation, followed by introduction to gas discharge plasmas, active plasma species and their inactivating mechanism. Subsequently, significance and aspects of NTP inactivation of biofilm‐associated bacteria, especially those of medical importance, including opportunistic pathogens, oral pathogenic bacteria, foodborne pathogens and implant bacteria, are discussed. The remainder of the review discusses majorly about the synergistic effect of NTPs and their activity against biofilm‐associated fungi, especially Candida species.     

Should we stay or should we go: mechanisms and ecological consequences for biofilm dispersal

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.     

Biofilm formation and dispersal and the transmission of human pathogens

Several pathogenic bacterial species that are found in the environment can form complex multicellular structures on surfaces known as biofilms. Pseudomonas aeruginosa, Vibrio cholerae and certain species of nontuberculous mycobacteria are examples of human pathogens that form biofilms in natural aquatic environments. We suggest that the dynamics of biofilm formation facilitates the transmission of pathogens by providing a stable protective environment and acting as a nidus for the dissemination of large numbers of microorganisms; both as detached biofilm clumps and by the fluid-driven dispersal of biofilm clusters along surfaces. We also suggest that emerging evidence indicates that biofilm formation conveys a selective advantage to certain pathogens by increasing their ability to persist under diverse environmental conditions.     

Antibiofilm activity of sodium bicarbonate, sodium metaperiodate and SDS combination against dental unit waterline-associated bacteria and yeast

Aim: To determine the effect of sodium bicarbonate (SB), sodium metaperiodate (SMP) and sodium dodecyl sulfate (SDS) combination on biofilm formation and dispersal in dental unit waterline (DUWL)-associated bacteria and yeast. Methods and Results: The in vitro effect of SB, SMP and SDS alone and in combination on biofilm formation and dispersal in Pseudomonas aeruginosa, Klebsiella pneumoniae, Actinomyces naeslundii, and Candida albicans was investigated using a 96-well microtitre plate biofilm assay. The combination showed a broad-spectrum inhibitory effect on growth as well as biofilm formation of both gram-negative and gram-positive bacteria, and yeast. In addition, the SB + SMP + SDS combination was significantly more effective in dispersing biofilm than the individual compounds. The combination dispersed more than 90% of P. aeruginosa biofilm whereas the commercial products, Oxygenal 6, Sterilex Ultra, and PeraSafe showed no biofilm dispersal activity. Conclusion: The composition comprising SB, SMP, and SDS was effective in inhibiting as well as dispersing biofilms in DUWL-associated bacteria and yeast. Significance and Impact of the Study: This study shows that a composition comprising environmentally friendly and biologically safe compounds such as SB, SMP, and SDS has a potential application in reducing DUWL-associated acquired infections in dental clinics.     

The pathogenic actinobacterium Rhodococcus equi: what's in a name?

A key regulatory decision for many bacteria is the switch between biofilm formation and motile dispersal, and this dynamic is well illustrated in the light‐organ symbiosis between the bioluminescent bacterium Vibrio fischeri and the Hawaiian bobtail squid. Biofilm formation mediated by the syp gene cluster helps V. fischeri transition from a dispersed planktonic lifestyle to a robust aggregate on the surface of the nascent symbiotic organ. However, the bacteria must then swim to pores and down into the deeper crypt tissues that they ultimately colonize. A number of positive and negative regulators control syp expression and biofilm formation, but until recently the environmental inputs controlling this clash between opposing regulatory mechanisms have been unclear. Thompson et al. have now shown that Syp‐mediated biofilms can be repressed by a well‐known host‐derived molecule: nitric oxide. This regulation is accomplished by the NO sensor HnoX exerting control over the biofilm regulator HahK. The discoveries reported here by Thompson et al. cast new light on a critical early stage of symbiotic initiation in the V. fischeri‐squid model symbiosis, and more broadly it adds to a growing understanding of the role(s) that NO and HnoX play in biofilm regulation by many bacteria.     

Reversible control of biofilm formation by Cellulomonas spp. in response to nitrogen availability

The microbial degradation of cellulose contributes greatly to the cycling of carbon in terrestrial environments and feedbacks to the atmosphere, a process that is highly responsive to nitrogen inputs. Yet how key groups of cellulolytic microorganisms adaptively respond to the global conditions of nitrogen limitation and/or anthropogenic or climate nitrogen inputs is poorly understood. The actinobacterial genus Cellulomonas is of special interest because it incorporates the only species known to degrade cellulose aerobically and anaerobically. Furthermore, despite their inability to fix nitrogen, they are active decomposers in nitrogen-limited environments. Here we show that nitrogen limitation induced biofilm formation in Cellulomonas spp., a process that was coupled to carbon sequestration and storage in a curdlan-type biofilm matrix. The response was reversible and the curdlan matrix was solubilized and used as a carbon and energy source for biofilm dispersal once nitrogen sources became available. The biofilms attached strongly to cellulosic surfaces and, despite the growth limitation, produced cellulases and degraded cellulose more efficiently. The results show that biofilm formation is a competitive strategy for carbon and nitrogen acquisition and provide valuable insights linking nitrogen inputs to carbon sequestration and remobilization in terrestrial environments.     

Biofilm formation and proteolytic activities of Pseudoalteromonas bacteria that were isolated from fish farm sediments

In order to save natural resources and supply good fishes, it is important to improve fish‐farming techniques. The survival rate of fish fry appears to become higher when powders of foraminifer limestone are submerged at the bottom of fish‐farming fields, where bacterial biofilms often grow. The observations suggest that forming biofilms can benefit to keep health status of breeding fishes. We employed culture‐based methods for the identification and characterization of biofilm‐forming bacteria and assessed the application of their properties for fish farming. Fifteen bacterial strains were isolated from the biofilm samples collected from fish farm sediments. The 16S rRNA gene sequences indicated that these bacteria belonged to the genera, Pseudoalteromonas (seven strains), Vibrio (seven strains) and Halomonas (one strain). It was found that Pseudoalteromonas strains generally formed robust biofilms in a laboratory condition and produced extracellular proteases in a biofilm‐dependent manner. The results suggest that Pseudoalteromonas bacteria, living in the biofilm community, contribute in part to remove excess proteineous matters from the sediment sludge of fish farms.     

Evolution of dispersal traits along an invasion route in the wind‐dispersed Senecio inaequidens (Asteraceae)

In introduced organisms, dispersal propensity is expected to increase during range expansion. This prediction is based on the assumption that phenotypic plasticity is low compared to genetic diversity, and an increase in dispersal can be counteracted by the Allee effect. Empirical evidence in support of these hypotheses is however lacking. The present study tested for evidence of differentiation in dispersal‐related traits and the Allee effect in the wind‐dispersed invasive Senecio inaequidens (Asteraceae). We collected capitula from individuals in ten field populations, along an invasion route including the original introduction site in southern France. In addition, we conducted a common garden experiment from field‐collected seeds and obtained capitula from individuals representing the same ten field populations. We analysed phenotypic variation in dispersal traits between field and common garden environments as a function of the distance between populations and the introduction site. Our results revealed low levels of phenotypic differentiation among populations. However, significant clinal variation in dispersal traits was demonstrated in common garden plants representing the invasion route. In field populations, similar trends in dispersal‐related traits and evidence of an Allee effect were not detected. In part, our results supported expectations of increased dispersal capacity with range expansion, and emphasized the contribution of phenotypic plasticity under natural conditions.     

Environmentally controlled bacterial vesicle‐mediated export

Over the past two decades, researchers studying both microbial and host cell communities have gained an appreciation for the ability of bacteria to produce, regulate, and functionally utilize outer membrane vesicles (OMVs) as a means to survive and interact with their cellular and acellular environments. Common ground has emerged, as it appears that vesicle production is an environmentally controlled and specific secretion process; however, it has been challenging to discover the principles that govern fundamentals of vesicle‐mediated transport. Namely, there does not appear to be a single mechanism modulating OMV export, nor universal “markers” for OMV cargo incorporation, nor particular host cell responses common to treatment with all OMVs. Given the diversity of species studied, their differences in envelope architecture and composition, the diversity of environmentally regulated bacterial processes, and the variety of interactions between bacteria and their abiotic and biotic environments, this is hardly surprising. Nevertheless, the ability of bacteria to control exported material in the context of a packaged insoluble particle, a vesicle, is emerging as a significant contribution to bacterial viability, biofilm communities, and bacterial‐host interactions. In this review, we focus on detailing important, recent findings regarding the content and functional differences in bacterially secreted vesicles that are influenced by growth conditions.     

铜绿假单胞菌生物膜分散的研究近况

细菌分泌胞外多糖附着在物体表面组成一个结构性群体即生物膜,导致对抗生素的强抵抗性和感染的迁延不愈。反过来,已形成的生物膜也可以分散为游离菌,许多环境物质能够促进该分散过程,并且这些物质与抗生素合用对生物膜有强大的对抗作用。从生物膜到浮游菌是个复杂的过程,目前关于铜绿假单胞菌生物膜分散的特征、机制、诱导分子等已经引起了学者的强烈兴趣,随着问题的深入研究必然会给人类治疗生物膜所致的难治性感染带来更大的意义。     

Characterization of starvation‐induced dispersion in Pseudomonas putida biofilms: genetic elements and molecular mechanisms

Pseudomonas putida OUS82 biofilm dispersal was previously shown to be dependent on the gene PP0164 (here designated lapG). Sequence and structural analysis has suggested that the LapG geneproduct belongs to a family of cysteine proteinases that function in the modification of bacterial surface proteins. We provide evidence that LapG is involved in P. putida OUS82 biofilm dispersal through modification of the outer membrane‐associated protein LapA. While the P. putida lapG mutant formed more biofilm than the wild‐type, P. putida lapA and P. putida lapAG mutants displayed decreased surface adhesion and were deficient in subsequent biofilm formation, suggesting that LapG affects LapA, and that the LapA protein functions both as a surface adhesin and as a biofilm matrix component. Lowering of the intracellular c‐di‐GMP level via induction of an EAL domain protein led to dispersal of P. putida wild‐type biofilm but did not disperse P. putida lapG biofilm, indicating that LapG exerts its activity on LapA in response to a decrease in the intracellular c‐di‐GMP level. In addition, evidence is provided that associated to LapA a cellulase‐degradable exopolysaccharide is part of the P. putida biofilm matrix.     

Nitric oxide‐mediated dispersal in single‐ and multi‐species biofilms of clinically and industrially relevant microorganisms

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.     

Biofilm formation and the food industry,a focus on the bacterial outer surface

The ability of many bacteria to adhere to surfaces and to form biofilms has major implications in a variety of industries including the food industry, where biofilms create a persistent source of contamination. The formation of a biofilm is determined not only by the nature of the attachment surface, but also by the characteristics of the bacterial cell and by environmental factors. This review focuses on the features of the bacterial cell surface such as flagella, surface appendages and polysaccharides that play a role in this process, in particular for bacteria linked to food‐processing environments. In addition, some aspects of the attachment surface, biofilm control and eradication will be highlighted.     

Dispersion as an Important Step in the Candida albicans Biofilm Developmental Cycle

Biofilms are dynamic microbial communities in which transitions between planktonic and sessile modes of growth occur interchangeably in response to different environmental cues. In the last decade, early events associated with C. albicans biofilm formation have received considerable attention. However, very little is known about C. albicans biofilm dispersion or the mechanisms and signals that trigger it. This is important because it is precisely C. albicans cells dispersed from biofilms that are the main culprits associated with candidemia and establishment of disseminated invasive disease, two of the gravest forms of candidiasis. Using a simple flow biofilm model recently developed by our group, we have performed initial investigations into the phenomenon of C. albicans biofilm dispersion, as well as the phenotypic characteristics associated with dispersed cells. Our results indicate that C. albicans biofilm dispersion is dependent on growing conditions, including carbon source and pH of the media used for biofilm development. C. albicans dispersed cells are mostly in the yeast form and display distinct phenotypic properties compared to their planktonic counterparts, including enhanced adherence, filamentation, biofilm formation and, perhaps most importantly, increased pathogenicity in a murine model of hematogenously disseminated candidiasis, thus indicating that dispersed cells are armed with a complete arsenal of “virulence factors” important for seeding and establishing new foci of infection. In addition, utilizing genetically engineered strains of C. albicans (tetO-UME6 and tetO-PES1) we demonstrate that C. albicans biofilm dispersion can be regulated by manipulating levels of expression of these key genes, further supporting the evidence for a strong link between biofilms and morphogenetic conversions at different stages of the C. albicans biofilm developmental cycle. Overall, our results offer novel and important insight into the phenomenon of C. albicans biofilm dispersion, a key part of the biofilm developmental cycle, and provide the basis for its more detailed analysis.     

Negative frequency‐dependent selection maintains coexisting genotypes during fluctuating selection

Natural environments are rarely static; rather selection can fluctuate on timescales ranging from hours to centuries. However, it is unclear how adaptation to fluctuating environments differs from adaptation to constant environments at the genetic level. For bacteria, one key axis of environmental variation is selection for planktonic or biofilm modes of growth. We conducted an evolution experiment with Burkholderia cenocepacia, comparing the evolutionary dynamics of populations evolving under constant selection for either biofilm formation or planktonic growth with populations in which selection fluctuated between the two environments on a weekly basis. Populations evolved in the fluctuating environment shared many of the same genetic targets of selection as those evolved in constant biofilm selection, but were genetically distinct from the constant planktonic populations. In the fluctuating environment, mutations in the biofilm‐regulating genes wspA and rpfR rose to high frequency in all replicate populations. A mutation in wspA first rose rapidly and nearly fixed during the initial biofilm phase but was subsequently displaced by a collection of rpfR mutants upon the shift to the planktonic phase. The wspA and rpfR genotypes coexisted via negative frequency‐dependent selection around an equilibrium frequency that shifted between the environments. The maintenance of coexisting genotypes in the fluctuating environment was unexpected. Under temporally fluctuating environments, coexistence of two genotypes is only predicted under a narrow range of conditions, but the frequency‐dependent interactions we observed provide a mechanism that can increase the likelihood of coexistence in fluctuating environments.