Research Update
TRENDS in Microbiology Vol.9 No.1 January 2001
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Meeting Report
Biofilms adhere to stay Howard F. Jenkinson and Hilary M. Lappin-Scott The 147th Ordinary Meeting of the Society for General Microbiology was held at the University of Exeter, Exeter, UK, 12–15 September 2000.
In their natural environments, bacteria do not exist as isolated cells but grow and survive in organized communities. These can form flocculents in suspension, liquidsurface pellicles, or mats, but the microbial communities that develop at phase interfaces, such as solid–liquid or air–liquid interfaces, are termed biofilms. Biofilms have multiple impacts on the Earth’s resources: they can be essential (in sewerage processing); useful (for food digestion); inconvenient (on slippery steps); unpleasant (as dental plaque); destructive (within water conduits); or can even prove to be fatal (on medical prostheses). The biofilm concept, which was coined in 1978 by Bill Costerton (Montana State University, Bozeman, MT, USA), is now widely embraced by microbiologists, engineers and computer scientists, but sadly is still poorly represented within many teaching curricula. The 147th Ordinary Meeting of the Society for General Microbiology featured two symposia entitled ‘Community Structure and Cooperation in Biofilms’ and ‘Medical Implications of Biofilms’, and provided novel insights into how microorganisms organize and behave in communities, the collective strengths of which are inevitably greater than the sums of the individual components. Indeed, the biofilm could be considered as an evolutionary unit. It is from this microbial metropolis that pioneers emerge to found rural settlements upon which new microbial civilizations can arise. Poetry in motion
The biofilm icon is of interacting, organized, 3-D structures of organisms enveloped by extracellular polymeric substances (EPS), with networks of intervening water channels and multiple layers of cells1. However, the physical and biological structures of biofilms are subject to multiple intrinsic and environmental influences. Many physical properties of
biofilms can be attributed to the EPS matrix. Hans-Curt Flemming (University of Duisburg, Germany) described how EPS can behave as a gel or as a viscoelastic fluid, with proteins, Ca2+ ions, the alignment of the polysaccharide chains and the water content all influencing biofilm stability. Under turbulent flow, biofilms composed of complex communities ripple and creep across surfaces (Paul Stoodley, Montana State University, Bozeman, MT, USA), which is consistent with EPS behaving as a viscoelastic fluid. It is this structural fluidity, along with a biological fluidity of microbial cells continually entering into and departing from associations, that makes the biofilm a dynamic entity. From the initial adhesion of cells to a surface, their growth and accumulation – through diversification and reorganization – to dynamic maturity is surely poetry in motion (Fig. 1). In the life and times of a biofilm, the initial adhesion of the bacterial cell to a conditioned surface is considered a random event. If cells adhere then divide and accumulate, a linking film is produced (Henk Busscher, University of Groningen, Groningen, The Netherlands), onto which further attachment and accumulation of cells can occur (Fig. 1). During the development of polymicrobial populations such as dental plaque, every new organism that binds to the linking film presents a new surface (Paul Kolenbrander, National Institutes of Health, Bethesda, MD, USA) and therefore forms a basis for the accretion of defined organism groupings. The linking film also provides a means for stabilizing communities that are continuously subject to physical shear forces. So how do bacteria know when they are on a surface? The recognition signals are still a mystery, but it is clear that surface growth is associated with massive transcriptional upheavals. Thus, it has been reported that the transcription of 38% of Escherichia coli genes is affected following cell attachment2. These bacterial cells send signals to each other, but evidence for the Gram-negative bacterial communication molecules, acyl homoserine
lactones (AHLs), playing a major role in biofilm formation is surprisingly not compelling (David Davies, Binghampton University, New York, NY, USA). Although it does seem likely that AHLs and cell-density-dependent regulation play roles in EPS matrix deposition and in the dispersal of bacteria from biofilms3, precisely how the genetic switches and cascades are coordinated during surface growth still awaits discovery. Resilience and resistance
Aside from the fundamental interest in how these microbial communities form, it is their unique characteristics that are under scrutiny. The increased resistance properties of biofilm cells to external influences such as antibiotics, host defences, antiseptics and shear forces, concerns medicine and industry, and the resilience of the communities fascinates microbiologists (for a review on this subject see pp. 34–39). Resilience happens because microorganisms help each other. Phil Marsh (CAMR, Porton Down, Salisbury, UK) and colleagues have demonstrated that, within dental plaque communities, anaerobic bacteria survive and grow in close physical interconnections (coaggregations) with specific aerobic organisms that generate an oxygen-depleted environment. Paradoxically, bacterial species that are in direct competition for a primary nutrient source also work out ways to survive in biofilm communities, as demonstrated by Soeren Molin (Technical University of Denmark, Lyngby, Denmark) for Pseudomonas putida and Acinetobacter sp. degrading toluene. Using green or red fluorescent protein reporter–promoter fusions, Molin and colleagues have shown that P. putida cells quickly cluster around Acinetobacter cells in a nutritional interrelationship that temporally strangles the Acinetobacter before the biofilm remodels. But why are biofilm bacteria less susceptible to antimicrobial agents? It is now clear that EPS do not necessarily provide a diffusion barrier to inhibitory compounds (David Allison and Peter Gilbert, University of Manchester,
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Research Update
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TRENDS in Microbiology Vol.9 No.1 January 2001
Life and times of a biofilm Surface energy of substratum
Physicochemical factors Nutritional factors
Propinquity
Emergence Commensalism Mutualism
Hydrodynamic shear Grazing
Phenotypic changes
Flow
Linking film
Adhesion
Colonization
Accumulation
Climax community
Dispersal
TRENDS in Microbiology
Fig. 1. The life and times of a biofilm, depicted as a developmental cycle, the various stages of which are determined by a range of physical, biological and environmental factors, as indicated at the top of the diagram. Initial adhesion to a surface is a random event influenced by surface free energy and propinquity (nearness) of bacterial cells. Adhered bacteria then undergo cell division, colonize the surface, and thus provide for further cell adhesion and accumulation to generate a ‘linking film’ upon which a climax community is generated. The biofilm is a dynamic entity: cells continually enter or leave the community, promoting diversification or dispersal, and protozoal grazing and shear forces in flow systems lead to remodelling of the biofilm structure.
Manchester, UK), but that biofilm bacteria are inherently more resistant. For example, small-colony variants of Staphylococcus aureus, generated within biofilm communities on medical devices, have diminished metabolic rates that make them less susceptible to antibiotics (Roger Bayston, University of Nottingham, Nottingham, UK). In mixed biofilms, one β-lactamase-producing bacterial species can protect cells of another species from penicillin. Julia Douglas (University of Glasgow, Glasgow, UK) described an intriguing protective effect of coadhering oral Streptococcus gordonii on the susceptibility of Candida albicans biofilms to antifungals. One of the many challenges is to devise effective means to prevent biofilm infections of indwelling medical
devices that are otherwise traumatic to the patient and costly to rectify. Impregnation of biomaterials with slow-release antimicrobials was one approach, but especially interesting was the notion from Simon Pickering (Derby Royal Infirmary, Derby, UK), that increased antibiotic sensitivity of biofilm staphylococci can be augmented electromagnetically. Spreading the word
Arguably, the most important issue to come out of this meeting is that the description of biofilm structure, morphology and metabolism should be tempered with the knowledge that biofilms are spatially and temporally heterogeneous (Julian Wimpenny, University of Cardiff, Cardiff, UK). The
Key conference outcomes • • •
•
The heterogeneity of biofilms has been underestimated and is beginning to be revealed by using more sophisticated molecular and metabolic probes. The dynamic properties of biofilms are now realized and new models developed with the application of time-lapse imaging techniques. Defined physical interactions between biofilm organisms, as well as nutrient supply and hydrodynamic flow, all influence the development of structured microbial communities. EPS might determine mechanical strength of biofilms as well as the positioning of cellular populations, but do not provide a diffusion barrier.
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concept of a single ‘biofilm mode of growth’ is not really tenable: biofilm cells demonstrate a spectrum of differentiated states, so any analysis that does not take this into account merely generates a synthesis of data. Photon-excitation microscopy, coupled with fluorescence lifetime imaging, clearly reveals a mosaic of pH microzones within oral biofilms (David Bradshaw, CAMR, Porton Down, Salisbury, UK), reflecting species distribution and metabolic fluxes. Fluorescent reporter systems reveal that cells within biofilm microcolonies metabolize at vastly different rates (Soeren Molin). We will see an explosion in the use of these reporter systems, which are compatible with confocal microscopy, to monitor expression of genes, signals and environments within biofilms. There will be a movement away from general descriptions of biofilm structure and function, and a deeper exploration into the activities and properties of individual cells, groupings and microcosms. Now that the word has spread and the biofilm concept has been universally embraced, there is no doubt that biofilms are here to stay. Acknowledgements We are grateful to our lab groups for input into this report, and to all the conference speakers for making the meeting such a stimulating and rewarding experience. The proceedings of this meeting are published as chapters in a book entitled Community Structure and Co-operation in Biofilms, Cambridge University Press (ISBN 0521 793025) as part of the Society for General Microbiology Symposium Series, Volume 59 (2000).
References 1 Costerton, J.W. et al. (1999) Bacterial biofilms: a common cause of persistent infections. Science 284, 1318–1322 2 Prigent-Combaret, C. et al. (1999) Abiotic surface sensing and biofilm-dependent regulation of gene expression in Escherichia coli. J. Bacteriol. 181, 5993–6002 3 Stoodley, P. et al. (1999) The role of hydrodynamics and AHL signalling molecules as determinants of the structure of Pseudomonas aeruginosa biofilms. In Biofilms: The Good, the Bad and the Ugly (Wimpenny, J. et al., eds), pp. 223–230, BioLine, Cardiff, UK
Howard F. Jenkinson* University of Bristol Dental School, Lower Maudlin Street, Bristol, UK BS1 2LY. *e-mail:
[email protected] Hilary M. Lappin-Scott School of Biological Sciences, University of Exeter, Hatherly Laboratories, Prince of Wales Road, Exeter, UK EX4 4PS.