Cell death in Pseudomonas aeruginosa biofilm development

Bacteria growing in biofilms often develop multicellular, three-dimensional structures known as microcolonies. Complex differentiation within biofilms of Pseudomonas aeruginosa occurs, leading to the creation of voids inside microcolonies and to the dispersal of cells from within these voids. However, key developmental processes regulating these events are poorly understood. A normal component of multicellular development is cell death. Here we report that a repeatable pattern of cell death and lysis occurs in biofilms of P. aeruginosa during the normal course of development. Cell death occurred with temporal and spatial organization within biofilms, inside microcolonies, when the biofilms were allowed to develop in continuous-culture flow cells. A subpopulation of viable cells was always observed in these regions. During the onset of biofilm killing and during biofilm development thereafter, a bacteriophage capable of superinfecting and lysing the P. aeruginosa parent strain was detected in the fluid effluent from the biofilm. The bacteriophage implicated in biofilm killing was closely related to the filamentous phage Pf1 and existed as a prophage within the genome of P. aeruginosa. We propose that prophage-mediated cell death is an important mechanism of differentiation inside microcolonies that facilitates dispersal of a subpopulation of surviving cells.     

黄色粘球菌基于复杂社会性细胞行为的生物被膜

黄色粘球菌具有多样化的细胞行为,具备典型多细胞水平的社会性特征。其形成的生物被膜是目前认知的最为复杂的单种群细菌生物被膜之一。黄色粘球菌的社会性细胞行为主导了其生物被膜形成过程中的关键环节,包括固体介质表面的细胞运动、群体细胞的捕食、亲缘细胞的识别、子实体的发育、粘孢子的分化以及细胞程序性死亡等行为过程。文中将介绍相关领域的研究进展。     

Prophage spontaneous activation promotes DNA release enhancing biofilm formation in Streptococcus pneumoniae

Streptococcus pneumoniae (pneumococcus) is able to form biofilms in vivo and previous studies propose that pneumococcal biofilms play a relevant role both in colonization and infection. Additionally, pneumococci recovered from human infections are characterized by a high prevalence of lysogenic bacteriophages (phages) residing quiescently in their host chromosome. We investigated a possible link between lysogeny and biofilm formation. Considering that extracellular DNA (eDNA) is a key factor in the biofilm matrix, we reasoned that prophage spontaneous activation with the consequent bacterial host lysis could provide a source of eDNA, enhancing pneumococcal biofilm development. Monitoring biofilm growth of lysogenic and non-lysogenic pneumococcal strains indicated that phage-infected bacteria are more proficient at forming biofilms, that is their biofilms are characterized by a higher biomass and cell viability. The presence of phage particles throughout the lysogenic strains biofilm development implicated prophage spontaneous induction in this effect. Analysis of lysogens deficient for phage lysin and the bacterial major autolysin revealed that the absence of either lytic activity impaired biofilm development and the addition of DNA restored the ability of mutant strains to form robust biofilms. These findings establish that limited phage-mediated host lysis of a fraction of the bacterial population, due to spontaneous phage induction, constitutes an important source of eDNA for the S. pneumoniae biofilm matrix and that this localized release of eDNA favors biofilm formation by the remaining bacterial population.     

Distinct roles of extracellular polymeric substances in Pseudomonas aeruginosa biofilm development

Bacteria form surface attached biofilm communities as one of the most important survival strategies in nature. Biofilms consist of water, bacterial cells and a wide range of self-generated extracellular polymeric substances (EPS). Biofilm formation is a dynamic self-assembly process and several distinguishable stages are observed during bacterial biofilm development. Biofilm formation is shown to be coordinated by EPS production, cell migration, subpopulation differentiation and interactions. However, the ways these different factors affect each other and contribute to community structural differentiation remain largely unknown. The distinct roles of different EPS have been addressed in the present report. Both Pel and Psl polysaccharides are required for type IV pilus-independent microcolony formation in the initial stages of biofilm formation by Pseudomonas aeruginosa PAO1. Both Pel and Psl polysaccharides are also essential for subpopulation interactions and macrocolony formation in the later stages of P. aeruginosa PAO1 biofilm formation. Pel and Psl polysaccharides have different impacts on Pseudomonas quinolone signal-mediated extracellular DNA release in P. aeruginosa PAO1 biofilms. Psl polysaccharide is more important than Pel polysaccharide in P. aeruginosa PAO1 biofilm formation and antibiotic resistance. Our study thus suggests that different EPS materials play distinct roles during bacterial biofilm formation.     

Biofilms as a Mode of Existence of Bacteria in External Environment and Host Body: The Phenomenon,Genetic Control,and Regulation Systems of Development

Studies of the last decade have shown that most bacteria exist in natural ecosystems as specifically organized, attached to substrates biofilms rather than as freely floating plankton cells. The formation of these biofilms is a complex and highly regulated process. The development of biofilm communities is a primary strategy of bacterial survival not only in the external environment but also in the bodies of infected macroorganisms. In these organisms, bacteria are joined by complicated cell–cell associations, which makes them functionally similar to multicellular organisms. In the present review, we consider the structural organization of biofilms, factors affecting initiation of the biofilm formation, differential expression of bacterial genes at various stages of the biofilm development and their regulation. The significance of studies in this field for medicine, in particular, for prevention and protection against pathogenic bacteria, is discussed.     

The Matrix Reloaded: How Sensing the Extracellular Matrix Synchronizes Bacterial Communities

In response to chemical communication, bacterial cells often organize themselves into complex multicellular communities that carry out specialized tasks. These communities are frequently referred to as biofilms, which involve the collective behavior of different cell types. Like cells of multicellular eukaryotes, the biofilm cells are surrounded by self-produced polymers that constitute the extracellular matrix (ECM), which binds them to each other and to the surface. In multicellular eukaryotes, it has been evident for decades that cell-ECM interactions control multiple cellular processes during development. While cells both in biofilms and in multicellular eukaryotes are surrounded by ECM and activate various genetic programs, until recently it has been unclear whether cell-ECM interactions are recruited in bacterial communicative behaviors. In this review, we describe the examples reported thus far for ECM involvement in control of cell behavior throughout the different stages of biofilm formation. The studies presented in this review have provided a newly emerging perspective of the bacterial ECM as an active player in regulation of biofilm development.     

A new colorimetric microtitre model for the detection of Staphylococcus aureus biofilms

Aims: Research on biofilms requires validated quantitative models that focus both on matrix and viable bacterial mass. In this study, a new microplate model for the detection of Staphylococcus aureus biofilms was developed. Methods and Results: Dimethyl methylene blue (DMMB) dye was used to quantify biofilm matrix colorimetrically. Initially developed for the detection of glycosaminoglycans, the DMMB protocol was optimized for S. aureus biofilm research. In addition, the redox indicator resazurin was used to determine the viable bacterial biofilm burden. Conclusion: A new, simple and reproducible microplate test system based on DMMB and resazurin, offering a reliable differentiation between biofilm matrix and cellular activity, was developed and validated for the detection of S. aureus biofilms. Significance and Impact of the Study: The DMMB–resazurin microtitre plate model is a valuable tool for high capacity screening of biocides and for the development of synergistic mixtures of biocides, destroying both biofilm matrix and bacteria.     

Modeling biocide action against biofilms

A phenomenological model of biocide action against microbial biofilms was derived. Processes incorporated in the model include bulk flow in and out of a well-mixed reactor, transport of dissolved species into the biofilm, substrate consumption by bacterial metabolism, bacterial growth, advection of cell mass within the biofilm, cell detachment from the biofilm, cell death, and biocide concentration-dependent disinfection. Simulations were performed to analyze the general behavior of the model and to perform preliminary sensitivity analysis to identify key input parameters. The model captured several general features of antimicrobial agent action against biofilms that have been observed widely by experimenters and practitioners. These included (1) rapid disinfection followed by biofilm regrowth, (2) slower detachment than disinfection, and (3) reduced susceptibility of microorganisms in biofilms. The results support the plausibility of a mechanism of biofilm resistance in which the biocide is neutralized by reaction with biofilm constituents, leading to a reduction in the bulk biocide concentration and, more significantly, biocide concentration gradients within the biofilm. Sensitivity experiments and analyses identified which input parameters influence key response variables. Each of three response variables was sensitive to each of the five input parameters, but they were most sensitive to the initial biofilm thickness and next most sensitive to the biocide disinfection rate coefficient. Statistical regression modeling produced simple equations for approximating the response variables for situations within the range of conditions covered by the sensitivity experiment. The model should be useful as a tool for studying alternative biocide control strategies. For example, the simulations suggested that a good interval between pulses of biocide is the time to minimum thickness. (c) 1996 John Wiley & Sons, Inc.     

Copper affects biofilm inductiveness to larval settlement of the serpulid polychaete Hydroides elegans (Haswell)

Copper (Cu) contamination is a potential threat to the marine environment due to the use of Cu-based antifouling paints. Cu stress on larval settlement of the polychaete Hydroides elegans was investigated, and this was linked to Cu stress on biofilms and on the biofilm development process. The inductiveness of young biofilms was more easily altered by Cu stress than that of old biofilms, indicating the relative vulnerability of young biofilms. This might result from changes in bacterial survival, the bacterial community composition and the chemical profiles of young biofilms. Cu also affected biofilm development and the chemical high performance liquid chromatograph fingerprint profile. The results indicate that Cu affected larval settlement mainly through its effect on the process of biofilm development in the marine environment, and the chemical profile was crucial to biofilm inductiveness. It is strongly recommended that the effects of environmentally toxic substances on biofilms are evaluated in ecotoxicity bioassays using larval settlement of invertebrates as the end point.     

植物提取物及其活性成分抑制细菌生物被膜的研究进展北大核心CSCD

大量研究报道生物被膜细菌对抗生素的耐药性是浮游菌的10–1 000倍,据报道细菌生物被膜是80%以上细菌感染的罪魁祸首,对医疗保健领域构成了严峻的挑战。植物提取物及其活性成分对细菌生物被膜有明显的抑制作用,包括减少生物被膜量、生物被膜活菌数以及清除已经成熟的生物被膜等。该文对这些有效的植物提取物及其活性成分进行了总结,并分析了其抗细菌生物被膜的作用机制。旨在为防治细菌生物被膜感染的植物类药物的开发提供参考。     

Extracellular DNA is essential for maintaining Bordetella biofilm integrity on abiotic surfaces and in the upper respiratory tract of mice

Bacteria form complex and highly elaborate surface adherent communities known as biofilms which are held together by a self-produced extracellular matrix. We have previously shown that by adopting a biofilm mode of existence in vivo, the gram negative bacterial pathogens Bordetella bronchiseptica and Bordetella pertussis are able to efficiently colonize and persist in the mammalian respiratory tract. In general, the bacterial biofilm matrix includes polysaccharides, proteins and extracellular DNA (eDNA). In this report, we investigated the function of DNA in Bordetella biofilm development. We show that DNA is a significant component of Bordetella biofilm matrix. Addition of DNase I at the initiation of biofilm growth inhibited biofilm formation. Treatment of pre-established mature biofilms formed under both static and flow conditions with DNase I led to a disruption of the biofilm biomass. We next investigated whether eDNA played a role in biofilms formed in the mouse respiratory tract. DNase I treatment of nasal biofilms caused considerable dissolution of the biofilm biomass. In conclusion, these results suggest that eDNA is a crucial structural matrix component of both in vitro and in vivo formed Bordetella biofilms. This is the first evidence for the ability of DNase I to disrupt bacterial biofilms formed on host organs.     

Biofilm dispersion in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

In recent decades, many researchers have written numerous articles about microbial biofilms. Biofilm is a complex community of microorganisms and an example of bacterial group behavior. Biofilm is usually considered a sessile mode of life derived from the attached growth of microbes to surfaces, and most biofilms are embedded in self-produced extracellular matrix composed of extracellular polymeric substances (EPSs), such as polysaccharides, extracellular DNAs (eDNA), and proteins. Dispersal, a mode of biofilm detachment indicates active mechanisms that cause individual cells to separate from the biofilm and return to planktonic life. Since biofilm cells are cemented and surrounded by EPSs, dispersal is not simple to do and many researchers are now paying more attention to this active detachment process. Unlike other modes of biofilm detachment such as erosion or sloughing, which are generally considered passive processes, dispersal occurs as a result of complex spatial differentiation and molecular events in biofilm cells in response to various environmental cues, and there are many biological reasons that force bacterial cells to disperse from the biofilms. In this review, we mainly focus on the spatial differentiation of biofilm that is a prerequisite for dispersal, as well as environmental cues and molecular events related to the biofilm dispersal. More specifically, we discuss the dispersal-related phenomena and mechanisms observed in Pseudomonas aeruginosa, an important opportunistic human pathogen and representative model organism for biofilm study.     

Biofilm development and cell death in the marine bacterium Pseudoalteromonas tunicata

The newly described green-pigmented bacterium Pseudoalteromonas tunicata (D2) produces target-specific inhibitory compounds against bacteria, algae, fungi, and invertebrate larvae and is frequently found in association with living surfaces in the marine environment. As part of our studies on the ecology of P. tunicata and its interaction with marine surfaces, we examined the ability of P. tunicata to form biofilms under continuous culture conditions within the laboratory. P. tunicata biofilms exhibited a characteristic architecture consisting of differentiated microcolonies surrounded by water channels. Remarkably, we observed a repeatable pattern of cell death during biofilm development of P. tunicata, similar to that recently reported for biofilms of Pseudomonas aeruginosa (J. S. Webb et al., J. Bacteriol. 185:4585-4595, 2003). Killing and lysis occurred inside microcolonies, apparently resulting in the formation of voids within these structures. A subpopulation of viable cells was always observed within the regions of killing in the biofilm. Moreover, extensive killing in mature biofilms appeared to result in detachment of the biofilm from the substratum. A novel 190-kDa autotoxic protein produced by P. tunicata, designated AlpP, was found to be involved in this biofilm killing and detachment. A Delta alpP mutant derivative of P. tunicata was generated, and this mutant did not show cell death during biofilm development. We propose that AlpP-mediated cell death plays an important role in the multicellular biofilm development of P. tunicata and subsequent dispersal of surviving cells within the marine environment.     

The biological role of death and lysis in biofilm development

Recent studies have revealed that the regulated death of bacterial cells is important for biofilm development. Following cell death, a sub-population of the dead bacteria lyse and release genomic DNA, which then has an essential role in intercellular adhesion and biofilm stability. This Opinion focuses on the role of regulated cell death and lysis in biofilm development and provides a functional comparison between bacterial programmed cell death and apoptosis. The hypothesis that the differential regulation of these processes during biofilm development contributes to the antibiotic tolerance of biofilm cells is also explored.     

Chemical defenses of seaweeds against microbial colonization

Any living or non-living surface immersed in seawaterrapidly acquires a bacterial biofilm. For living marineorganisms, biofilm formation can result in the death ofthe host, and thus there is strong evolutionary pressure formarine eukaryotes to evolve mechanisms which inhibit orcontrol the development of biofilms on their surfaces.Some marine eukaryotes are indeed successful incontrolling biofilms on their surfaces, and in manyinstances this control is achieved by the production ofinhibitory chemicals which act at or near the surface ofthe organism. In some cases these natural inhibitors aresimply toxic to bacteria. However, increasingly it appearsthat at least some of these compounds act by interferingspecifically with bacterial characteristics which effect theability of bacteria to colonize their hosts, such asattachment, surface spreading, or the production ofextracellular macromolecules. As an example, theAustralian seaweed Delisea pulchra appears tocontrol bacterial colonization by interfering with abacterial regulatory system (the acylated homoserinelactone system) that regulates several colonizationrelevant bacterial traits. Understanding how marineorganisms control specific bacterial colonization traitsshould provide us with insights into new technologies forthe control of biofilms on artificial surfaces.     

Bacterial biofilms and surface fouling

Bacteria are attracted to surfaces. Their surface adhesion, with subsequent binary fission and exopolymer production, leads to the formation of biofilms. Such biofilms consist of bacterial cells in a matrix of their own exopolysaccharide glycocalyces. In addition to the bulk fluid and the surface, biofilms constitute a third physical phase. The close proximity of the bacterial cells in the biofilm matrices assists the formation of metabolically dependent consortia. The chemical and physical activities of these microbial communities produces a heterogeneous system at the colonised surface. Metabolites, produced at specific points on the surface, can lead to the development of effective anodes and cathodes at adjoining locations on the surface. In this way the fouling of a surface by bacterial biofilm development facilitates focal attack on that surface. This pit formation is characteristic of bacterial surface activities as diverse as dental decay and metal corrosion. In this review, we examine bacterial adhesion, biofilm formation and several instances of focal bacterial attack on colonised surfaces. However, pathogenic biofilms and the fouling of biological surfaces, with the exception of caries formation, is outside the scope of this paper.     

Aggregation of ammonia-oxidizing bacteria in microbial biofilm on oyster shell surface

Summary Formation and activity of bacterial nitrifying biofilms play an important role in the closed seawater systems for shrimp cultivation. The structure of microbial biofilm on empty oyster shells, used as a biofilm carrier in biofiltration of aquacultural water, was studied using fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy. FISH was performed with specific oligonucleotide probes for Bacteria and ammonia-oxidizing Nitrosomonas spp. The bacterial cells were arranged within the biofilm as a layer of vertically elongated aggregates. Aggregates of ammonia-oxidizing bacteria were embedded within the matrix formed by other bacteria. Vertically elongated cell aggregates may be ecologically important in bacterial biofilms because they have a higher surface-to-volume ratio than that of laminated biofilms.