CBE News Update February, 2004 Volume 7, Issue 2   A Digest: Biofilms 2003 Themes     by P.S. Stewart February 16, 2004 ________________________________________________    Click to enlarge image & caption        The American Society for Microbiology meeting “Biofilms 2003,” held in Victoria, British Columbia in early November 2003, was the largest biofilm meeting ever held. Seventy-six oral presentations and 369 posters were presented over five days. This selective summary of the meeting (which is derived from a presentation made at the Center for Biofilm Engineering Technical Advisory Conference on February 5, 2004), focuses on five themes that especially stood out: 1) the expanding world of biofilm research and technology, 2) advances in characterizing microbial ecology in biofilms, 3) the occurrence of phenotypic variants in biofilms, 4) dispersal (detachment) from biofilms, and 5) interactions between microbial biofilms and higher organisms.   The expanding world of biofilm research and technology      Over the past few years, the biofilm world has expanded in both the human and microbial dimensions.  Presenters and attendees appeared at Biofilms 2003 who had not previously been involved in studying biofilms. Posters and talks discussed biofilm formation by microorganisms that have not been previously investigated in the context of biofilms. The expansion of biofilm research activity is also reflected in the increasing numbers of publications indexing keywords “biofilm” or “biofilms” in electronic databases such as PubMed and the Institute of Scientific Information (ISI) Web of Science. Biofilm-related publications have expanded by approximately 14 percent annually according the ISI data over the past seven years.   Advances in characterizing microbial ecology in biofilms      Molecular approaches for studying microbial ecology, in particular those based on 16s ribosomal RNA sequences, have been around for years. Now these techniques are being applied to biofilms with spectacular results. Two techniques are aiding researchers in characterizing the microbial species composition of biofilms. The first technique is Fluorescence In-Situ Hybridization or FISH. This method uses a fluorescently-tagged piece of DNA that hybridizes with high specificity to certain species or groups of species. FISH is used to locate, by fluorescence microscopy, individual cells of a certain species or group. Beautiful examples of FISH in natural biofilms ranging from wastewater (Michael Wagner and Satoshi Okabe) to subgingival plaque (Annette Moter) were presented. The second technique enables researchers to identify all the microbial members of a biofilm community. Molecular methods including denaturing gradient gel electrophoresis, PCR, and cloning are involved. The fruit of these methods is commonly presented in the form of a phylogenetic tree that identifies individual species and their relationships to one another. Dave Stahl and Dave Ward showed the power of these methods to characterize complex natural biofilms. Both FISH and the community analysis techniques mentioned above are culture-independent, which is important because many, or even most, of the microorganisms in natural biofilms are not readily cultured in the laboratory. The take-home message is that molecular techniques are now capable of providing detailed information about the composition and spatial organization of species in real-world biofilms.   The occurrence of phenotypic variants in biofilms      When biofilms of Pseudomonas aeruginosa are grown for several days, then dispersed and plated, unusual colony morphologies can be found. The term “phenotypic variant” is being used to describe these special strains. These are not contaminants, but are P. aeruginosa sub-strains that display distinct phenotypes. Some variants appear to be stable, while others readily revert to the wild type. The concept of phenotypic variants in biofilms took Biofilms 2003 by storm, as several investigators described the isolation of variants from P. aeruginosa biofilms. For example, Mary Jo Kirisits described a “sticky type” variant that occupies a stable fraction of the biofilm population. When grown by itself, the sticky type forms a robust biofilm with an architecture that is distinct from that exhibited by the parent strain. Pradeep Singh described a similar variant, which he labeled “wrinkly,” that forms robust biofilms that resist killing by high concentrations of chlorine. Eliana Drenkard presented data suggesting a connection between phenotypic variation and antibiotic resistance. Considerable discussion of the hypothetical persister cell – a protected cell state – begs the question of whether persisters might also be phenotypic variants. Could phenotypic variants represent a primitive process of differentiation in biofilms, in which variants within a particular species occupy separate niches or perform specialized functions? Will a better understanding of phenotypic variants allow us to defeat the antimicrobial tolerance mounted by microorganisms in biofilms?   Dispersal (detachment) from biofilms      One of the most exciting developments in biofilm research is the progress being made in elucidating mechanisms of detachment and dispersal. The release of cells from a biofilm to the surrounding fluid is poorly understood and has been understudied. Now many groups are describing observations of biofilm cell cluster hollowing and detachment. Clues about mechanisms are also beginning to emerge. Staffan Kjelleberg showed how a bacteriophage causes cell death and lysis in the center of Pseudomonas aeruginosa biofilms cell clusters once they reach a certain stage of maturity. Pradeep Singh described a phenotypic variant of P. aeruginosa that appears to detach prematurely due to its overproduction of a rhamnolipid biosurfactant. Alfred Spormann presented evidence that detachment of Shewanella oneidensis biofilms is triggered by oxygen starvation. The theme of detachment resulting from nutrient starvation was reinforced by 3D cellular automata simulations generated by Steve Hunt showing dynamic growth and hollowing of cell clusters predicted by this mechanism. Jeff Kaplan described the isolation of mutants of Actinobacillus actinomycetemcomitans that are defective in the usual dispersal process that is observed with this organism in vitro. A very interesting enzyme discovered in this way appears to be involved in the degradation of extracellular polymeric matrix material. Small amounts of the purified enzyme cause Actinobacillus or Staphylococcus biofilms to detach. From these presentations one comes away with the idea that there must be diverse, parallel pathways for biofilm dispersal and detachment. It is also natural to imagine that this research will someday lead to new strategies for controlling the formation of unwanted biofilms by breaking biofilms apart rather than just killing cells.   Interactions between microbial biofilms and higher organisms      An interesting session shed light on the interaction between bacterial biofilms and higher organisms. Interactions with plants, protozoa, fibroblasts, leukocytes, epithelial cells, and marine fouling organisms were described. What emerges from these presentations is that the interaction between the bacteria in a biofilm and a neighboring eukaryote is complex and involves two-way communication. Here is just one example. It comes from Mike Hadfield at the University of Hawaii, who studies marine fouling of surface by creatures such as the tubeworm Hydroides elegans. The larvae of fouling eukaryotes settle on submerged surfaces in response to signals from the bacterial biofilm that forms on the surface. Some bacteria repel larvae, but others actually call them in. The interaction would appear to be specific between a particular bacterial species and a particular eukaryote. Once a larva has attached, the progression of fouling depends on the metamorphosis of the larva. Metamorphosis also appears to be directed by cues from the bacterial biofilms. Certain species of bacteria trigger metamorphosis of most of the settling larvae of H. elegans, while biofilms of other bacteria shut the process down entirely. An understanding of these complex interactions could eventually lead to new strategies, more subtle than applying biocides or antibiotics, for controlling the fouling or infection process by steering it in a favorable direction. To quote Bill Costerton, “Biofilms are hot!” Start preparing now for Biofilms 2006.   Contact information for this article:   Dr. Phil Stewart Deputy Director and Research Coordinator Center for Biofilm Engineering Montana State University—Bozeman Bozeman, MT, USA phil_s@erc.montana.edu   Affiliations of scientists named in this article:   Michael Wagner Technische Universitat Lehrstuhl fur Microbiologie Freising, Germany Satoshi Okabe Hokkaido University Dept. of Urban & Environmental Engineering Sapporo, Japan Annette Moter Inst fur Mikro Und Hygiene Berlin, Germany David Stahl University of Washington Civil & Environmental Engineering Seattle, WA, USA David Ward Montana State University—Bozeman Dept. of Land Resources and Environmental Sciences Bozeman, MT, USA Mary Kirisits Northwestern University Evanston, IL, USA Pradeep Singh University Iowa Hospitals & Clinics Internal Medicine Iowa City, IA, USA Eliana Drenkard Dept. of Molecular Biology Massachusetts General Hospital Boston, MA, USA Staffan Kjelleberg School of Biotechnology & Biomolecular Science University of New South Wales Australia Alfred Spormann Stanford University Terman Engineering Center Stanford, CA, USA Stephen Hunt Montana State University—Bozeman Center for Biofilm Engineering Bozeman, MT, USA Jeffrey Kaplan New Jersey Dental School Newark, NJ, USA Michael Hadfield University of Hawaii, Manoa Kewalo Marine Laboratory Honolulu, HI, USA