c d a journal, v o l 29, n º 5 Biofilms: sensing and signaling Elinor deLancey Pulcini abstra ct Biofilms are a community of surface-attached microorganisms that can have far-reaching effects. Biofilms are costly to industry and affect human health in a variety of ways. Research is only now beginning to discern the complexities of biofilm formation. author he problem of bacterial issues beyond the problem of dental contamination of dental waterline contamination. For example, elinor delancey Pulcini waterlines is an excellent biofilms in drinking water systems may is a Phd candidate at illustration of a basic precept act as a reservoir for potential pathogens.2 the Center for Biofilm Engineering at Montana in biofilm science: Biofilms In the human body, there is a direct State University. Prior tare the preferred mode of growth for relationship between the presence and to that, she was head of most bacte ria. Existence as a biofilm severity of dental plaque biofilm and an the Science Department provides bacteria with a protective increase in the potential of suffering a and a science instructor environment that effectively prevents heart attack.3 Despite the growing body of at Bigfork High School in Montana. attack by antimicrobials, biocides, and research into biofilm formation, relatively even immunologic factors. Biofilms little is known about the metabolism and are costly for industry due to their physiology of biofilm bacteria.4 biofouling potential, which can cause a Antony van Leeuwenhoek could be pressure drop or product degradation.1 considered one of the first biofilm The detachment of biofilms has been researchers when, in the late 1600s, he implicated in the contamination of scraped dental plaque from his mouth and food and household products during looked at it with his microscope. In 1943, manufacturing and processing. Biofilms ZoBell published a study of the affinity of are also associated with public health marine bacteria for attaching to surfaces. m a y 2001 351 c d a journal, v o l 29, n º 5 However, it was not until the 1970s that as a protective polymer for the cells cells. In contrast, ciprofloxacin was research in the formation of biofilms embedded within. As the biofilm grows shown to be able to diffuse through the really started. Two assumptions were and thickens, it begins to develop into a K. pneumoniae biofilm in as little as 20 pervasive in early biofilm research: that heterogeneous matrix interspersed with minutes. However, K. pneumoniae cells biofilm bacteria and planktonic or free- channels that allow nutrients and oxygen were resistant to ciprofloxacin at even 10 floating bacteria are the same and that to penetrate into the depths of even times its established minimal inhibitory biofilms were relatively simple systems of the thickest biofilms. Researchers have concentration.13 This suggests that the homogeneous slime. The more-traditional shown that the cells within the biofilm genetic changes the planktonic bacterium microbiological methodologies of plating matrix exhibit differences in physiology undergoes as it becomes a biofilm cell may and broth culturing of bacteria have, until depending on their location. This concept somehow also affect its susceptibility to recently, warped the view of how bacteria of spatial heterogeneity within a biofilm various antimicrobials. really live and survive in the environment. has been applied to oxygen limitations Ongoing research at the Center for Improvements in technology have allowed (from aerobic to anaerobic), pH, nutrients, Biofilm Engineering at Montana State biofilm scientists to prove otherwise.5 and rates of growth.8-10 Within a thick University in Bozeman has been working biofilm, there are various microniches that to delineate the changes that occur in current research allow for numerous types of metabolic P. aeruginosa during initial attachment. When a bacterial cell comes in contact processes to take place. Dental plaque is Proteomics involves the analysis of with a surface, it may or may not stick an excellent example of the complexity differentially expressed (induced or immediately. The confocal scanning of microorganisms that can exist within repressed) proteins and allows researchers laser microscope allows for the visual a biofilm with a range of metabolic to analyze the protein expression of an examination of biofilms in real time with capabilities.10 organism at a particular point in time minimal preparation. Using the confocal The development of a biofilm appears or under a particular condition. Results scanning laser microscope, individual cells to be a very effective survival strategy indicate that cells of P. aeruginosa of Pseudomonas aeruginosa containing for bacteria. The cells within the biofilm attaching to a surface begin to express a genetic insert called green fluorescent exhibit an increased resistance to biocides changes in their protein profiles (when protein were followed as they attached to and antimicrobials in comparison to compared to planktonic cells) in as little a surface. The green fluorescent protein planktonic cells. A number of hypotheses as 10 minutes after inoculation. These genes, which come from jellyfish, cause have been put forth to attempt to explain changes in protein expression continue the cells to fluoresce, allowing for the this phenomenon. In some cases, there during the time of the experiments (three visualization of bacteria without the is a limitation to the penetration of the hours).14 These differences in protein use fixatives or stains that kill the cells. antimicrobials into the biofilm matrix. expression during initial adhesion indicate Results indicated that some bacteria will Since cells within the matrix are living at physiologic changes are taking place permanently attach to the surface while different physiologic states, the rate of within cells as they attach to a surface. others will attach briefly and then move uptake into the cell of the antimicrobial As the biofilm develops, bacterial cells on to another position.6 During this time can be affected. The exopolysaccharide within the matrix will release chemical of initial adhesion, there are a number of of the biofilm matrix may provide a signals. These signal molecules may changes taking place within the bacterial physical barrier to the penetration of enable the bacterial colonies to develop cell. Bacteria that are dividing at the rate antimicrobials.11 The differences in the characteristics of a more mature of minutes in culture will stop dividing for bacterial cell physiology within the biofilm biofilm. A number of bacterial species, hours when first attached to a surface.6 will reduce the susceptibility of cells to both gram-positive and gram-negative, During this time, there are numerous some antimicrobials such as growth- use these chemical signal molecules to changes occurring as that bacterial cell dependent antibiotics.12 However, coordinate activity.15 The action of these makes the transition from a planktonic diffusion and growth limitations alone signal molecules relies on a process called to a biofilm cell. Eventually, the biofilm may not account for the entire decrease quorum sensing. In quorum sensing, the bacterial cell will be metabolically and in susceptibility to antimicrobials seen ability of the molecule to cause an action physiologically very different from its in biofilm cells. A study of the effects of is dependent on its concentration within planktonic counterpart to the point that antibiotics on Klebsiella pneumoniae the environment. That concentration can there may even exist what is now termed biofilms grown on microporous increase only when there is a sufficient the biofilm phenotype.7 polycarbonate membranes showed that number of bacterial cells producing that Attached bacteria produce an ampicillin, unable to penetrate the biofilm particular signal. Probably some of the exopolysaccharide matrix that can act matrix, cannot kill K. pneumoniae biofilm best-known quorum sensing systems are 352 m a y 2001 c d a journal, v o l 29, n º 5 found in marine bacteria of the genus is obviously critical. To successfully r eferences Vibrio. Species of this bacterial genus minimize contamination, it is important 1. Costerton JW, Lewandowski Z, et al, Microbial biofilms. Annu Rev Microbiol 49:711-45, 1995. symbiotically colonize the light organs to understand the physiology and 2. Stickler D, Biofilms. Curr Opin Microbiol 2:270-5, 1999. of certain fish or squid and will emit metabolism of biofilm bacteria. That 3. Potera C, Biofilms invade microbiology. Science. 273:1795-7, luminosity only when the population bacteria do not usually live in the 1996. 4. Costerton JW, Stewart PS, and Greenberg EP, Bacterial density has reached sufficient quorum environment in suspensions of single cells biofilms: a common cause of persistent infections. Science density numbers.16 has significant ramifications both for the 284:1318-22, 1999. The cell-to-cell signaling systems relevance of how most bacterial species 5. Costerton JW, Nonculturable Microorganisms in the Environment, Colwell RR and Grimes DJ, eds. ASM Press, of P. aeruginosa have been extensively are studied and for the treatment options Washington DC, 2000, pp 131-45. studied as a model for quorum sensing utilized for biofilm control. 6. Rice A, Hamilton MA, and Camper AK, Apparent surface during biofilm development by gram- associated lag time in growth of primary biofilm cells. Microb Ecol 40:8-15, 2000. negative bacteria. Mutant strains of 7. Costerton JW