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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 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 pre ferred mode of growth for relationship between the presence and to that, she was head of most . Existence as a biofilm severity of 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 , biocides, and research into biofilm formation, relatively even immunologic factors. Biofilms little is known about the and are costly for industry due to their of biofilm bacteria.4 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.

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However, it was not until the 1970s that as a protective 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 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 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) and allows researchers laser microscope allows for the visual a biofilm with a range of metabolic to analyze the 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 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 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 .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 . 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

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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 . 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 and Stewart PS, Biofilms and device-related P. aeruginosa deficient in one of the infections. In Persistent Bacterial Infection. ASM Press, quorum sensing systems (lasR) have Washington DC, 2000, pp 423-39. 8. Huang C-T, Xu KD, et al, Spatial patterns of alkaline been shown to produce biofilms that lack phosphatase expression within bacterial colonies and biofilms the towers and channels often seen in P. in response to phosphate starvation. Appl Environ Micriobiol aeruginosa biofilms. In addition, these 64:1526-31, 1998. 9. Xu KD, Stewart PS, et al, Spatial physiological heterogeneity mutant biofilms lack the resistance to in biofilm is determined by oxygen treatment by sodium dodecyl sulfate availability. Appl Environ Micriobiol 64:4035-9, 1998. seen in wild-type biofilms.17 Recently, 10. Whittaker CJ, Klier, CM, and Kolenbrander PE, Mechanisms of adhesion by oral bacteria. Annu Rev Microbiol 50:513-52, researchers have isolated quorum-sensing 1996. molecules produced by P. aeruginosa from 11. Stewart PS, Theoretical aspects of diffusion into the sputum of patients, microbial biofilms. Antimicrob Agents Chemother 40:2517-22, 1996. suggesting that this is a biofilm disease of 12. Brown MRW, Allison DG and Gilbert P, Resistance of the lungs.18 bacterial biofilms to antibiotics: a growth-rate related effect? Research into the cell-to-cell signaling J Antimicrob Chemother 22:777-83, 1988. 13. Anderl JN, Franklin MJ and Stewart PS, Role of antibiotic capabilities of gram-positive biofilm- penetration limitation in Klebsiella pneumoniae biofilm forming bacteria has also been ongoing. resistance to ampicillin and ciprofloxacin. Antimicrob Agents Mutants of Streptococcus gordonii, a Chemother 44:1818-24, 2000. 14. Pulcini E, manuscript in progress. 2001. gram-positive oral bacterium that initiates 15. Parsek MR and Greenberg EP, Acyl-homoserine lactone the formation of dental plaque, were quorum sensing in gram-negative bacteria: a signaling assayed for defective biofilm formation. mechanism involved in associations with higher organisms. Proc Natl Acad Sci USA 97:8789-93, 2000. In this particular study, nine mutants 16. Ruby EG, Lessons from a cooperative, bacterial-animal shown to have defects in genes of known association: the Vibrio fischeri-Euprymna scolopes light organ function could not form biofilms. One of symbiosis. Annu Rev Microbiol. 50:591-624, 1996. 17. Davies DG, Parsek MR, et al, The involvement of cell-to-cell the genes identified, ComD, is a known signals in the development of a bacterial biofilm. Science component of the cell-to-cell signaling 280:295-8, 1998. system in gram-positive bacteria.19 18. Singh PK, Schaefer AL, et al, Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature 407:762-4, 2000. conclusion 19. Loo CY, Corliss DA and Ganeshkumar N, Streptococcus The majority of bacteria in the gordonii biofilm formation: identification of genes that code for biofilm phenotypes. J Bact 182:1374-82, 2000. environment are found attached to To request a printed copy of this article, please contact/Elinor surfaces rather than as unicellular, freely deLancey Pulcini, Center for Biofilm Engineering, 366 EPS suspended planktonic cells. Biofilms are Building, Montana State University, Bozeman, MT 59717 or at [email protected]. found in almost every environmental system studied and in nearly every industrial and medical setting where microbial contamination is a problem. Dental water lines can provide just the environment conducive to biofilm growth. The quality of dental water

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