SUMMARY 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.

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.Although bacteria are commonly viewed as solitary life forms, these organisms are more typically colonial creatures. In their natural settings, bacteria persist within microbial communities, where they exploit elaborate systems of intercellular interaction and communication to adjust to changing environmental parameters. Moreover, biofilm formation has also been linked to the emergence of a variety of opportunistic human pathogens (5). For example, organisms such as Staphylococcus epidermidis and Pseudomonas aeruginosa form biofilms on implants and dead or living tissue, thereby contributing to a variety of persistent infections.Thinking about bacterial populations as connected organisms capable of concerted multicellular activities has provided researchers with novel insights into microbial biology. The key to such multicellular behavior lies in the ability of each individual cell to sense and respond to information from nearby cells, and this behavior requires a certain population size or quorum of cells. The development of biofilms is a process that involves both a quorum of cells and multicellular behavior (6). Single-species biofilms are of particular interest due to their clinical importance and the monospecies biofilms formed by P. aeruginosa has become a prominent model for studying this aspect of microbial biology.The complex structure of microbial biofilms has only recently been determined. Detailed analysis by scanning confocal laser microscopy has shown that biofilms of P. aeruginosa formed on solid surfaces and exposed to a continuous flow of fresh nutrients are open, highly hydrate...

Genomic DNA was isolated from the active layer of tundra soil collected from the Kolyma lowland, Northeast Eurasia, near the Arctic Ocean coast. The SSU (small subunit) rRNA genes were amplified with eubacterial primers from the bulk genomic community DNA and cloned into plasmid vectors. Forty-three SSU rDNA clones were obtained, and all of them had different RFLP patterns. Phylogenetic analysis based on partial sequences (about 300 bp) established with the maximum likelihood method revealed the presence of three major and several minor groups that fell into 11 of the established lines of bacteria, and one sequence that could not be assigned to any of the described groups. Most of the clones belonged to the alpha (20.9%) and delta (25.6%) subdivisions of the Proteobacteria, with lesser proportions in the beta (93 O/ O) and gamma (47 %) subdivisions, groups typically isolated from soil by culture methods. Fewer than 12 O/ O of the clones belonged to Gram-positive bacteria, and 16% of the clones were related to fibrobacter. The majority of the clones (70%) had sequences that were 5-15% different from those in the current databases, and 7% of the clones had sequences that differed by more than 20% from those in the database. The results suggest that these tundra-derived clones are very diverse in phylogeny, and that many probably reflect new genera or families. Hence, most of the tundra soil bacterial community has never been isolated and thus the physiology and function of its dominant members appears to be unknown.

Periodontitis is a biofilm-mediated disease. Porphyromonas gingivalis is an obligate anaerobe consistently associated with severe manifestations of this disease. As an opportunistic pathogen, the ability to proliferate within and disseminate from subgingival biofilm (plaque) is central to its virulence. Here, we report the isolation of a P. gingivalis transposon insertion mutant altered in biofilm development and the reconstruction and characterization of this mutation in three different wild-type strains. The mutation responsible for the altered biofilm phenotype was in a gene with high sequence similarity (ϳ61%) to a glycosyltransferase gene. The gene is located in a region of the chromosome that includes up to 16 genes predicted to be involved in the synthesis and transport of capsular polysaccharide. The phenotype of the reconstructed mutation in all three wild-type backgrounds is that of enhanced biofilm formation. In addition, in strain W83, a strain that is encapsulated, the glycosyltransferase mutation resulted in a loss of capsule. Further experiments showed that the W83 mutant strain was more hydrophobic and exhibited increased autoaggregation. Our results indicate that we have identified a gene involved in capsularpolysaccharide synthesis in P. gingivalis and that the production of capsule prevented attachment and the initiation of in vitro biofilm formation on polystyrene microtiter plates.The clinical importance of dental plaque and its accessibility for in vivo research makes it one of most studied and bestunderstood biofilm communities. It is now well documented that gram-negative anaerobic bacteria play a significant role in the development of periodontitis, with Porphyromonas gingivalis being implicated as one of the major players in the progression of this chronic disease (10,19,30,61). A number of factors are associated with the virulence of the organism, including a variety of proteases, endotoxins, and collegenases and the production of surface structures, such as fimbriae and capsular polysaccharide (38). This opportunistic pathogen also attaches to and invades human epithelial cells (18,53,56,57), connective tissue, and endothelial cells (15,17,55), and invasion has been shown to be mediated by the expression of fimbriae and a variety of surface adhesins. It is evident that the growth of P. gingivalis within the subgingival plaque is central to the disease process; however, although there have been numerous studies of the pathogenicity of the organism and its interactions with other organisms within the biofilm community, little is known about the molecular-genetic basis of biofilm formation in P. gingivalis.Research on oral biofilms has focused on determining the spatial organization and complex development of plaque by examining the succession of organisms in the growing biofilm. By monitoring the development of the microbial community on the tooth surface after professional cleaning, studies have demonstrated that the early colonizers are primarily grampositive organisms and the late, o...

Oral squamous cell carcinomas are a major cause of morbidity and mortality, and tobacco usage, alcohol consumption, and poor oral hygiene are established risk factors. To date, no large-scale case-control studies have considered the effects of these risk factors on the composition of the oral microbiome, nor microbial community associations with oral cancer. We compared the composition, diversity, and function of the oral microbiomes of 121 oral cancer patients to 242 age- and gender-matched controls using a metagenomic multivariate analysis pipeline. Significant shifts in composition and function of the oral microbiome were observed with poor oral hygiene, tobacco smoking, and oral cancer. Specifically, we observed dramatically altered community composition and function after tooth loss, with smaller alterations in current tobacco smokers, increased production of antioxidants in individuals with periodontitis, and significantly decreased glutamate metabolism metal transport in oral cancer patients. Although the alterations in the oral microbiome of oral cancer patients were significant, they were of substantially lower effect size relative to microbiome shifts after tooth loss. Alterations following tooth loss, itself a major risk factor for oral cancer, are likely a result of severe ecological disruption due to habitat loss but may also contribute to the development of the disease.

No abstract

Our understanding of how oral microbiota adapt in response to changes in their surroundings remains limited. This is particularly true of the slow-growing anaerobes that persist below the gum line. Here, we report that the oral anaerobe Porphyromonas gingivalis strain 381 can surface translocate when sandwiched between two surfaces. We show that during movement, this bacterium alters its metabolism, specifically side products of arginine utilization including citrulline and ornithine accumulated in the translocating cells; while arginine, N-acetyl-arginine, and the polyamine putrescine, which is produced from arginine were consumed. In addition, our results indicate that movement requires modification of the surrounding environment via proteolysis, cell dispersion, cell-on-cell rolling, and sub-diffusive cell-driven motility. We also show that production of fimbriae and fimbriae-associated proteins; as well as the regulation of contact-dependent growth inhibition genes, which are known to be involved in self-nonself discrimination, and the type IX secretion system are central to surface translocation. These studies provide a first glimpse into P. gingivalis motility and its relationship to ecological variables.

The primary goal of the human microbiome initiative has been to increase our understanding of the structure and function of our indigenous microbiota and their effects on human health and predisposition to disease. Because of its clinical importance and accessibility for in vivo study, the oral biofilm is one of the best-understood microbial communities associated with the human body. Studies have shown that there is a succession of select microbial interactions that directs the maturation of a defined community structure, generating the formation of dental plaque. Although the initiating factors that lead to disease development are not clearly defined, in many individuals there is a fundamental shift from a health-associated biofilm community to one that is pathogenic in nature and a central player in the pathogenic potential of this community is the presence of Porphyromonas gingivalis. This anaerobic bacterium is a natural member of the oral microbiome, yet it can become highly destructive (termed pathobiont) and proliferate to high cell numbers in periodontal lesions, which is attributed to its arsenal of specialized virulence factors. Hence, this organism is regarded as a primary etiologic agent of periodontal disease progression. In this review, we summarize some of the latest information regarding what is known about its role in periodontitis, including pathogenic potential as well as ecological and nutritional parameters that may shift this commensal to a virulent state. We also discuss parallels between the development of pathogenic biofilms and the human cellular communities that lead to cancer, specifically we frame our viewpoint in the context of ‘wounds that fail to heal’.