Microbial biofilms: from ecology to molecular genetics.

Biofilms are complex communities of microorganisms attached to surfaces or associated with interfaces. Despite the focus of modern microbiology research on pure culture, planktonic (free-swimming) bacteria, it is now widely recognized that most bacteria found in natural, clinical, and industrial settings persist in association with surfaces. Furthermore, these microbial communities are often composed of multiple species that interact with each other and their environment. The determination of biofilm architecture, particularly the spatial arrangement of microcolonies (clusters of cells) relative to one another, has profound implications for the function of these complex communities. Numerous new experimental approaches and methodologies have been developed in order to explore metabolic interactions, phylogenetic groupings, and competition among members of the biofilm. To complement this broad view of biofilm ecology, individual organisms have been studied using molecular genetics in order to identify the genes required for biofilm development and to dissect the regulatory pathways that control the plankton-to-biofilm transition. These molecular genetic studies have led to the emergence of the concept of biofilm formation as a novel system for the study of bacterial development. The recent explosion in the field of biofilm research has led to exciting progress in the development of new technologies for studying these communities, advanced our understanding of the ecological significance of surface-attached bacteria, and provided new insights into the molecular genetic basis of biofilm development.     

Communication among Oral Bacteria

Human oral bacteria interact with their environment by attaching to surfaces and establishing mixed-species communities. As each bacterial cell attaches, it forms a new surface to which other cells can adhere. Adherence and community development are spatiotemporal; such order requires communication. The discovery of soluble signals, such as autoinducer-2, that may be exchanged within multispecies communities to convey information between organisms has emerged as a new research direction. Direct-contact signals, such as adhesins and receptors, that elicit changes in gene expression after cell-cell contact and biofilm growth are also an active research area. Considering that the majority of oral bacteria are organized in dense three-dimensional biofilms on teeth, confocal microscopy and fluorescently labeled probes provide valuable approaches for investigating the architecture of these organized communities in situ. Oral biofilms are readily accessible to microbiologists and are excellent model systems for studies of microbial communication. One attractive model system is a saliva-coated flowcell with oral bacterial biofilms growing on saliva as the sole nutrient source; an intergeneric mutualism is discussed. Several oral bacterial species are amenable to genetic manipulation for molecular characterization of communication both among bacteria and between bacteria and the host. A successful search for genes critical for mixed-species community organization will be accomplished only when it is conducted with mixed-species communities.     

Communication among oral bacteria.

Human oral bacteria interact with their environment by attaching to surfaces and establishing mixed-species communities. As each bacterial cell attaches, it forms a new surface to which other cells can adhere. Adherence and community development are spatiotemporal; such order requires communication. The discovery of soluble signals, such as autoinducer-2, that may be exchanged within multispecies communities to convey information between organisms has emerged as a new research direction. Direct-contact signals, such as adhesins and receptors, that elicit changes in gene expression after cell-cell contact and biofilm growth are also an active research area. Considering that the majority of oral bacteria are organized in dense three-dimensional biofilms on teeth, confocal microscopy and fluorescently labeled probes provide valuable approaches for investigating the architecture of these organized communities in situ. Oral biofilms are readily accessible to microbiologists and are excellent model systems for studies of microbial communication. One attractive model system is a saliva-coated flowcell with oral bacterial biofilms growing on saliva as the sole nutrient source; an intergeneric mutualism is discussed. Several oral bacterial species are amenable to genetic manipulation for molecular characterization of communication both among bacteria and between bacteria and the host. A successful search for genes critical for mixed-species community organization will be accomplished only when it is conducted with mixed-species communities.     

The spatial architecture of Bacillus subtilis biofilms deciphered using a surface-associated model and in situ imaging

The formation of multicellular communities known as biofilms is the part of bacterial life cycle in which bacteria display cooperative behaviour and differentiated phenotypes leading to specific functions. Bacillus subtilis is a Gram-positive bacterium that has served for a decade as a model to study the molecular pathways that control biofilm formation. Most of the data on B. subtilis biofilms have come from studies on the formation of pellicles at the air-liquid interface, or on the complex macrocolonies that develop on semi-solid nutritive agar. Here, using confocal laser scanning microcopy, we show that B. subtilis strains of different origins are capable of forming biofilms on immersed surfaces with dramatically protruding "beanstalk-like" structures with certain strains. Indeed, these structures can reach a height of more than 300 μm with one undomesticated strain from a medical environment. Using 14 GFP-labeled mutants previously described as affecting pellicle or complex colony formation, we have identified four genes whose inactivation significantly impeded immersed biofilm development, and one mutation triggering hyperbiofilm formation. We also identified mutations causing the three-dimensional architecture of the biofilm to be altered. Taken together, our results reveal that B. subtilis is able to form specific biofilm features on immersed surfaces, and that the development of these multicellular surface-associated communities involves regulation pathways that are common to those governing the formation of pellicle and/or complex colonies, and also some specific mechanisms. Finally, we propose the submerged surface-associated biofilm as another relevant model for the study of B. subtilis multicellular communities.     

Role of a putative polysaccharide locus in Bordetella biofilm development

Bordetellae are gram-negative bacteria that colonize the respiratory tracts of animals and humans. We and others have recently shown that these bacteria are capable of living as sessile communities known as biofilms on a number of abiotic surfaces. During the biofilm mode of existence, bacteria produce one or more extracellular polymeric substances that function, in part, to hold the cells together and to a surface. There is little information on either the constituents of the biofilm matrix or the genetic basis of biofilm development by Bordetella spp. By utilizing immunoblot assays and by enzymatic hydrolysis using dispersin B (DspB), a glycosyl hydrolase that specifically cleaves the polysaccharide poly-beta-1,6-N-acetyl-D-glucosamine (poly-beta-1,6-GlcNAc), we provide evidence for the production of poly-beta-1,6-GlcNAc by various Bordetella species (Bordetella bronchiseptica, B. pertussis, and B. parapertussis) and its role in their biofilm development. We have investigated the role of a Bordetella locus, here designated bpsABCD, in biofilm formation. The bps (Bordetella polysaccharide) locus is homologous to several bacterial loci that are required for the production of poly-beta-1,6-GlcNAc and have been implicated in bacterial biofilm formation. By utilizing multiple microscopic techniques to analyze biofilm formation under both static and hydrodynamic conditions, we demonstrate that the bps locus, although not essential at the initial stages of biofilm formation, contributes to the stability and the maintenance of the complex architecture of Bordetella biofilms.     

细菌生物膜研究技术

细菌生物膜是细菌生长过程中为适应生存环境而在固体表面上生长的一种与游走态细胞相对应的存在形式。只要条件允许,绝大多数细菌都可以形成生物膜。一旦形成了生物膜细菌就具有极强的耐药性,在医疗、食品、工业、军事等诸多领域给人类社会带来了严重的危害,造成巨大的经济损失。因此,细菌生物膜已成为全球关注的重大难题,也是目前科学界研究的前沿和热点。本文结合细菌生物膜研究技术的最新进展,重点介绍了几种常用生物膜发生装置及检测量化技术,并对其原理及优缺点进行了讨论。     

Rhamnolipid surfactant production affects biofilm architecture in Pseudomonas aeruginosa PAO1

In response to certain environmental signals, bacteria will differentiate from an independent free-living mode of growth and take up an interdependent surface-attached existence. These surface-attached microbial communities are known as biofilms. In flowing systems where nutrients are available, biofilms can develop into elaborate three-dimensional structures. The development of biofilm architecture, particularly the spatial arrangement of colonies within the matrix and the open areas surrounding the colonies, is thought to be fundamental to the function of these complex communities. Here we report a new role for rhamnolipid surfactants produced by the opportunistic pathogen Pseudomonas aeruginosa in the maintenance of biofilm architecture. Biofilms produced by mutants deficient in rhamnolipid synthesis do not maintain the noncolonized channels surrounding macrocolonies. We provide evidence that surfactants may be able to maintain open channels by affecting cell-cell interactions and the attachment of bacterial cells to surfaces. The induced synthesis of rhamnolipids during the later stages of biofilm development (when cell density is high) implies an active mechanism whereby the bacteria exploit intercellular interaction and communication to actively maintain these channels. We propose that the maintenance of biofilm architecture represents a previously unrecognized step in the development of these microbial communities.     

Quorum sensing in streptococcal biofilm formation

Bacteria in their natural ecosystems preferentially grow as polysaccharide-encased biofilms attached to surfaces. Although quorum-sensing (QS) systems directing the 'biofilm phenotype' have been extensively described in Gram-negative bacteria, there is little understanding of the importance of these systems in Gram-positive biofilm formation. Streptococci are a diverse group of Gram-positive bacteria that colonize epithelial, mucosal and tooth surfaces of humans. In several streptococci, competence-stimulating peptide (CSP)-mediated QS has been connected with competence development for genetic transformation. Recent work, especially with bacteria that inhabit the biofilm of dental plaque, has linked CSP stimuli to other cell-density adaptations, such as biofilm formation.     

Biofilm: set-up and organization of a bacterial community

Bacterial attachment on various surfaces mostly takes place in the form of specialised bacterial communities, referred to as biofilm. The biofilm is formed through series of interactions between cells and adherence to surface, resulting in an organised structure. In this review we have been using Pseudomonas aeruginosa as a model microorganism to describe the series of events that occurred during this developmental process. P. aeruginosa is an opportunistic pathogen that has a wide variety of hosts and infectious sites. In addition to biofilm formation in certain tissues, inert surfaces, such as catheters, are also target for bacterial biofilm development. The use of convenient genetic screens has made possible the identification of numerous biofilm-defective mutants, which have been characterised further. These studies have allowed the proposal for a global model, in which key events are described for the different stages of biofilm formation. Briefly, flagellar mobility is crucial for approaching the surface, whereas type IV pili motility is preponderant for surface colonisation and microcolonies formation. These microcolonies are finally packed together and buried in an exopolysaccharide matrix to form the differentiated bio-film. It is obvious that the different stages of biofilm formation also involved perception of environmental stimuli. These stimuli, and their associated complex regulatory networks, have still to be fully characterised to understand the bacterial strategy, which initiates biofilm formation. One such regulatory system, called Quorum sensing, is one of the key player in the initial differentiation of biofilm. Finally, a better understanding, at the molecular level, of biofilm establishment and persistence should help for the design of antimicrobials that prevent bacterial infections.     

Staphylococcal biofilm disassembly

Staphylococcus aureus and Staphylococcus epidermidis are a frequent cause of biofilm-associated infections that are a tremendous burden on our healthcare system. Staphylococcal biofilms exhibit extraordinary resistance to antimicrobial killing, limiting the efficacy of antibiotic therapy, and surgical intervention is often required to remove infected tissues or implanted devices. Recent work has provided new insight into the molecular basis of biofilm development in these opportunistic pathogens. Extracellular bacterial products, environmental conditions, and polymicrobial interactions have all been shown to influence profoundly the ability of these bacteria to colonize and disperse from clinically relevant surfaces. We review new developments in staphylococcal biofilm disassembly and set them in the context of potential strategies to control biofilm infections.     

Is there a role for quorum sensing signals in bacterial biofilms?

Bacteria form multicellular biofilm communities on most surfaces. Genetic analysis of biofilm formation has led to the proposal that extracellular signals and quorum-sensing regulatory systems are essential for differentiated biofilms. Although such a model fits the concept of density-driven cell-cell communication and appear to describe biofilm development in several bacterial species and conditions, biofilm formation is multifactorial and complex. Hydrodynamics, nutrient load and intracellular carbon flux have major impacts, presumably by altering the expression of cellular traits essential for bacterial adaptation during the different stages of biofilm formation. Hence, differentiated biofilms may also be the net result of many independent interactions, rather than being determined by a particular global quorum sensing system.     

Mapping of Bacterial Biofilm Local Mechanics by Magnetic Microparticle Actuation

Most bacteria live in the form of adherent communities forming three-dimensional material anchored to artificial or biological surfaces, with profound impact on many human activities. Biofilms are recognized as complex systems but their physical properties have been mainly studied from a macroscopic perspective. To determine biofilm local mechanical properties, reveal their potential heterogeneity, and investigate their relation to molecular traits, we have developed a seemingly new microrheology approach based on magnetic particle infiltration in growing biofilms. Using magnetic tweezers, we achieved what was, to our knowledge, the first three-dimensional mapping of the viscoelastic parameters on biofilms formed by the bacterium Escherichia coli. We demonstrate that its mechanical profile may exhibit elastic compliance values spread over three orders of magnitude in a given biofilm. We also prove that heterogeneity strongly depends on external conditions such as growth shear stress. Using strains genetically engineered to produce well-characterized cell surface adhesins, we show that the mechanical profile of biofilm is exquisitely sensitive to the expression of different surface appendages such as F pilus or curli. These results provide a quantitative view of local mechanical properties within intact biofilms and open up an additional avenue for elucidating the emergence and fate of the different microenvironments within these living materials.     

Biofilm control with natural and genetically-modified phages

Bacteriophages, as the most dominant and diverse entities in the universe, have the potential to be one of the most promising therapeutic agents. The emergence of multidrug-resistant bacteria and the antibiotic crisis in the last few decades have resulted in a renewed interest in phage therapy. Furthermore, bacteriophages, with the capacity to rapidly infect and overcome bacterial resistance, have demonstrated a sustainable approach against bacterial pathogens-particularly in biofilm. Biofilm, as complex microbial communities located at interphases embedded in a matrix of bacterial extracellular polysaccharide substances (EPS), is involved in health issues such as infections associated with the use of biomaterials and chronic infections by multidrug resistant bacteria, as well as industrial issues such as biofilm formation on stainless steel surfaces in food industry and membrane biofouling in water and wastewater treatment processes. In this paper, the most recent studies on the potential of phage therapy using natural and genetically-modified lytic phages and their associated enzymes in fighting biofilm development in various fields including engineering, industry, and medical applications are reviewed. Phage-mediated prevention approaches as an indirect phage therapy strategy are also explored in this review. In addition, the limitations of these approaches and suggestions to overcome these constraints are discussed to enhance the efficiency of phage therapy process. Finally, future perspectives and directions for further research towards a better understanding of phage therapy to control biofilm are recommended.     

The interconnection between biofilm formation and horizontal gene transfer

Recent research has revealed that horizontal gene transfer and biofilm formation are connected processes. Although published research investigating this interconnectedness is still limited, we will review this subject in order to highlight the potential of these observations because of their believed importance in the understanding of the adaptation and subsequent evolution of social traits in bacteria. Here, we discuss current evidence for such interconnectedness centred on plasmids. Horizontal transfer rates are typically higher in biofilm communities compared with those in planktonic states. Biofilms, furthermore, promote plasmid stability and may enhance the host range of mobile genetic elements that are transferred horizontally. Plasmids, on the other hand, are very well suited to promote the evolution of social traits such as biofilm formation. This, essentially, transpires because plasmids are independent replicons that enhance their own success by promoting inter-bacterial interactions. They typically also carry genes that heighten their hosts' direct fitness. Furthermore, current research shows that the so-called mafia traits encoded on mobile genetic elements can enforce bacteria to maintain stable social interactions. It also indicates that horizontal gene transfer ultimately enhances the relatedness of bacteria carrying the mobile genetic elements of the same origin. The perspective of this review extends to an overall interconnectedness between horizontal gene transfer, mobile genetic elements and social evolution of bacteria.     

Bacterial biofilms as a natural form of existence of bacteria in the environment and host organism

Advances in microscopic analysis and molecular genetics research methods promoted the acquisition of evidence that natural bacteria populations exist predominately as substrate attached biofilms. Bacteria in biofilms are able to exchange signals and display coordinated activity that is inherent to multicellular organisms. Formation of biofilm communities turned out to be one of the main survival strategies of bacteria in their ecological niche. Bacteria in attached condition in biofilm are protected from the environmental damaging factors and effects of antibacterial substances in the environment and host organism during infection. According to contemporary conception, biofilm is a continuous layer of bacterial cells that are attached to a surface and each other, and contained in a biopolymer matrix. Such bacterial communities may be composed of bacteria of one or several species, and composed of actively functioning cells as well as latent and uncultured forms. Particular attention has recently been paid to the role of biofilms in the environment and host organism. Microorganisms form biofilm on any biotic and abiotic surfaces which creates serious problems in medicine and various areas of economic activity. Currently, it is established that biofilms are one of the pathogenetic factors of chronic inflection process formation. The review presents data on ubiquity of bacteria existence as biofilms, contemporary methods of microbial community analysis, structural-functional features of bacterial biofilms. Particular attention is paid to the role of biofilm in chronic infection process formation, heightened resistance to antibiotics of bacteria in biofilms and possible mechanisms of resistance. Screening approaches for agents against biofilms in chronic infections are discussed.     

Composition and Metabolic Activities of Bacterial Biofilms Colonizing Food Residues in the Human Gut

Bacteria growing in the human large intestine live in intimate association with the host and play an important role in host digestive processes, gut physiology, and metabolism. Fecal bacteria have been investigated extensively, but few studies have been done on biofilms that form on digestive wastes in the large bowel. The aims of this investigation were to investigate the composition and metabolic activities of bacterial communities that colonize the surfaces of food residues in fecal material, with respect to their role in the fermentation of complex carbohydrates. Fresh stools were obtained from 15 healthy donors, and food residues were separated by filtration. Adherent bacteria were removed by surfactant treatment for microbiological analysis and fermentation studies. Scanning electron microscopy and fluorescent in situ hybridization in conjunction with confocal laser scanning microscopy (CLSM) were used to visualize intact biofilms. Results showed that bacterial populations strongly adhering to particulate matter were phenotypically similar in composition to unattached communities, with bacteroides and bifidobacteria predominating. Biofilms comprised a mixture of living and dead bacteria, and CLSM showed that bifidobacteria in the biofilms occurred as isolated dispersed cells and in microcolonies near the interface with the substratum. Fermentation experiments with a variety of complex carbohydrates demonstrated that biofilm populations were more efficient in digesting polysaccharides, while nonadhering communities fermented oligosaccharides most rapidly. Acetate was the principal fermentation product formed by biofilm bacteria, whereas higher levels of butyrate were produced by nonadherent populations, showing that the two communities were metabolically distinct.     

Composition and metabolic activities of bacterial biofilms colonizing food residues in the human gut

Bacteria growing in the human large intestine live in intimate association with the host and play an important role in host digestive processes, gut physiology, and metabolism. Fecal bacteria have been investigated extensively, but few studies have been done on biofilms that form on digestive wastes in the large bowel. The aims of this investigation were to investigate the composition and metabolic activities of bacterial communities that colonize the surfaces of food residues in fecal material, with respect to their role in the fermentation of complex carbohydrates. Fresh stools were obtained from 15 healthy donors, and food residues were separated by filtration. Adherent bacteria were removed by surfactant treatment for microbiological analysis and fermentation studies. Scanning electron microscopy and fluorescent in situ hybridization in conjunction with confocal laser scanning microscopy (CLSM) were used to visualize intact biofilms. Results showed that bacterial populations strongly adhering to particulate matter were phenotypically similar in composition to unattached communities, with bacteroides and bifidobacteria predominating. Biofilms comprised a mixture of living and dead bacteria, and CLSM showed that bifidobacteria in the biofilms occurred as isolated dispersed cells and in microcolonies near the interface with the substratum. Fermentation experiments with a variety of complex carbohydrates demonstrated that biofilm populations were more efficient in digesting polysaccharides, while nonadhering communities fermented oligosaccharides most rapidly. Acetate was the principal fermentation product formed by biofilm bacteria, whereas higher levels of butyrate were produced by nonadherent populations, showing that the two communities were metabolically distinct.     

A novel planar flow cell for studies of biofilm heterogeneity and flow-biofilm interactions

Biofilms are microbial communities growing on surfaces, and are ubiquitous in nature, in bioreactors, and in human infection. Coupling between physical, chemical, and biological processes is known to regulate the development of biofilms; however, current experimental systems do not provide sufficient control of environmental conditions to enable detailed investigations of these complex interactions. We developed a novel planar flow cell that supports biofilm growth under complex two-dimensional fluid flow conditions. This device provides precise control of flow conditions and can be used to create well-defined physical and chemical gradients that significantly affect biofilm heterogeneity. Moreover, the top and bottom of the flow chamber are transparent, so biofilm growth and flow conditions are fully observable using non-invasive confocal microscopy and high-resolution video imaging. To demonstrate the capability of the device, we observed the growth of Pseudomonas aeruginosa biofilms under imposed flow gradients. We found a positive relationship between patterns of fluid velocity and biofilm biomass due to faster microbial growth under conditions of greater local nutrient influx, but this relationship eventually reversed because high hydrodynamic shear leads to the detachment of cells from the surface. These results reveal that flow gradients play a critical role in the development of biofilm communities. By providing new capability for observing biofilm growth, solute and particle transport, and net chemical transformations under user-specified environmental gradients, this new planar flow cell system has broad utility for studies of environmental biotechnology and basic biofilm microbiology, as well as applications in bioreactor design, environmental engineering, biogeochemistry, geomicrobiology, and biomedical research.     

Development of a multispecies oral bacterial community in a saliva-conditioned flow cell

Microbial communities within the human oral cavity are dynamic associations of more than 500 bacterial species that form biofilms on the soft and hard tissues of the mouth. Understanding the development and spatial organization of oral biofilms has been facilitated by the use of in vitro models. We used a saliva-conditioned flow cell, with saliva as the sole nutritional source, as a model to examine the development of multispecies biofilm communities from an inoculum containing the coaggregation partners Streptococcus gordonii, Actinomyces naeslundii, Veillonella atypica, and Fusobacterium nucleatum. Biofilms inoculated with individual species in a sequential order were compared with biofilms inoculated with coaggregates of the four species. Our results indicated that flow cells inoculated sequentially produced biofilms with larger biovolumes compared to those biofilms inoculated with coaggregates. Individual-species biovolumes within the four-species communities also differed between the two modes of inoculation. Fluorescence in situ hybridization with genus- and species-specific probes revealed that the majority of cells in both sequentially and coaggregate-inoculated biofilms were S. gordonii, regardless of the inoculation order. However, the representation of A. naeslundii and V. atypica was significantly higher in biofilms inoculated with coaggregates compared to sequentially inoculated biofilms. Thus, these results indicate that the development of multispecies biofilm communities is influenced by coaggregations preformed in planktonic phase. Coaggregating bacteria such as certain streptococci are especially adapted to primary colonization of saliva-conditioned surfaces independent of the mode of inoculation and order of addition in the multispecies inoculum. Preformed coaggregations favor other bacterial strains and may facilitate symbiotic relationships.