The Biofilm Mode of Life
Recent advances in studies of biofilm systems have generated a wealth of novel information on multicellular prokaryotic biology and have established models for the formation of biofilms and the biology of their lifecycles. As a prelude to the subsequent chapters in this volume, this introductory article is aimed at identifying the contextual scientific and experimental framework for contemporary biofilm research programs, and addresses the strengths and weaknesses of some of the current key biofilm models. We will discuss whether or not a unique biofilm specific gene expression underpins our observations on biofilm structure and biology. Further, we will highlight the limitations inherent to current genetic and physiological analyses of bacterial biofilms, including the strengths and weaknesses of the molecular toolbox and the biofilm assays commonly employed. Moreover, the extent by which multiple parallel pathways of biofilm formation exist will be addressed, with reference also to applications for novel control strategies based on contemporary advances in studies of bacterial biofilms. The chapter will conclude by discussing the relevance of a consensus view of bacterial biofilm formation and biology. Biofilm organization and differentiation Bacterial biofilms are multicellular consortia in which cells are embedded in an extracellular matrix at close proximity to one another. Such consortia are generally studied to assess particular properties of biofilms attached to solid surfaces, but they occur also as multicellular aggregates, flocs and granules suspended in the aqueous phase in many habitats. Biofilms of these kinds can be accommodated by single species, but in most cases in natural as well as artificial systems, they are mixed species consortia, ranging from phylogenetically highly diverse communities Juretschko et al. 2002; Lyautey et al. 2005; Taylor et al. 2004) to those in which one or a restricted number of species will be numerically and functionally dominant (Tujula et al. 2006) Intriguingly, whether biofilms consist of one or a mixture of a range of species, they display similarities with respect to architectural features and behavioral responses. For example, the microcolony structure observed in established mature biofilms is strikingly similar across monoand multispecies biofilms, across different habitats, as well as for different organismal levels (Lawrence et al. 1991; O'Toole et al. 2000; Sauer et al. 2002) The latter applies to the similarities found between the multicellular consortia of well studied caister.com/biofilmsbooks Kjelleberg and Givskov 6 model bacterial systems, those of highly differentiated multicellular prokaryotes such as streptomycetes and myxobacteria, and colonial or sessile marine invertebrates. Moreover, strong similarities in behavioral responses and functional features have been reported for monovs. multispecies biofilms, as well as across organismal levels on the evolutionary scale. For example, it is now appreciated that biofilms have unique differentiation events that generate subpopulations of cells with apparently defined tasks and hence sharing of labor in the biofilm population (Allesen Holm et al. 2006; Webb et al. 2003) These events include different cells that build the different parts of the mushroom like, or equivalent structures, of biofilms, and the generation of specialized dispersal cells or propagules for colonization of new surfaces (Caldwell et al. 2000; Labbate et al. 2004; Mai Prochnow et al. 2006) The advances recently made in our understanding of biofilms systems whether well defined or of more complexity, derive in the main from a series of seminal studies on a restricted set of monospecies biofilm (Ghannoum and O'Toole G, 2004) The experimental approaches have preferentially been focusing on high resolution genetic and microscopic techniques. These studies have laid the foundation for our current models of bacterial biofilms, their formation and life cycle, and they have subsequently been extrapolated and assessed for more complex systems (e.g. Auschill et al. 2001; Hope et al. 2002) Specifically, it is now appreciated that biofilm formation can be defined as a process that consists of defined stages. Commonly, we recognize that these stages include: reversible and irreversible attachment; surface motility and initiation of microcolony (or alternative structural arrangements) formation; maturation, ageing and differentiation of microcolonies; and, finally, biofilm dissolution and generation of specialized dispersal cells. It is further appreciated from the research reported on our currently best defined model systems that these stages and structural rearrangements include physiological characteristics and phenotypic responses apparently supportive of suggestions for the presence of a unique biofilm biology. Intuitively, the appearance of morphological arrangements uniquely observed for biofilm consortia, and the development of traits allowing for remarkable resilience against antimicrobials (Fux et al. 2004; Hall Stoodley et al. 2004) or removal of biofilm cells by the predatory actions of protozoans and phagocytes (Bjarnsholt et al. 2005; Matz et al. 2005; Queck et al. 2006) to name a few characteristics commonly observed for biofilms, would support the notion of a biology different from that of planktonically living bacteria. In further support of this, recent reports on transcriptomic and proteomic analysis of biofilm vs. free living cells of different monospecies systems appear to lay a strong foundation for the existence of genes and proteins uniquely expressed by biofilm cells (Hume et al. 2004; Sauer, 2003) For example, swarming, or coordinated population migration on surfaces, involves a defined differentiation event with distinct temporal stages in certain species (Eberl et al. 1999; Hume et al. 2004; Shapiro, 1988; Shapiro, 1998) Following the observation of apparent program like responses in this and other specific multicellular surface dependent responses, it is logical to anticipate the involvement of a development program of which biofilm specific genes are expressed in a defined order reflective of a biofilm life cycle. In the following sections we will address whether a unique program, and hence biofilm specific gene expression, underpins the structural and functional features discussed above, caister.com/biofilmsbooks The Biofilm Mode of Life 7 and whether the interpretations made from contemporary molecular analyses of biofilm genetics and physiology accurately reflect the biology of these communities. The implications of our analysis of currently available data sets on biofilm gene expression and seemingly similar behavioral responses of biofilms or multicelluar consortia of different organisms will also be explored. In addition, we will discuss whether multiple, parallel, pathways of biofilm formation and development of specific traits exists not only for different taxa, but also within a species. The implications of such parallel pathways for biofilm research and biofilm control measures will also be assessed. Finally, we will address strengths and limitations with current biofilm assays and conclude by discussing whether or not there should be a consensus view on how bacterial biofilms form and function. Biofilm program and specific gene expression Recent years have witnessed detailed mechanistic studies of biofilm formation in a range of species, including E. coli, Vibrio cholera, Staphylococcus aureus, and Serratia marcescens (Davey and O'Toole, 2000; Ghannoum and O'Toole G, 2004; Labbate et al. 2004) For in depth studies, one of the preferred model organisms is Pseudomonas aeruginosa. There are several good reasons for the many reports on biofilm biology in this organism. First, a number of molecular tools are available for this organism including commercial DNA arrays for the study of genome wide expression. Second, the genus Pseudomonas has simple growth requirements and tolerance to a wide range of temperatures (4degC to 42degC) significantly facilitating experimental efforts. Third, the organism represents a highly diverse and ecologically significant group of bacteria. Fourth, P. aeruginosa is also the most common bacterium found in nosocomial and life threatening infections of immune compromised patients for which the development of effective treatments are desirable. The bacterium causes a variety of acute and chronic infections (as listed by Van Delden and Iglewski, 1998) and it is particularly known for its involvement in chronic infections of the respiratory pathways including, diffuse panbronchiolitis, bronchiectasia and cystic fibrosis (CF) (Frederiksen et al. 1997; Hoiby, 1974; Kobayashi, 2005; Koch and Hoiby, 1993) Much of what we know about P. aeruginosa biofilm formation derives from studies of monospecies biofilms established in flow cells. A number of specific features are required for the type of biofilm development we see in these flow cells: (1) Attachment to the surface involving specific adhesive proteins, (2) cell to cell binding involving proteins, extra cellular DNA and polysaccharides in order for the cells to resist the hydrodynamic forces, and (3) cell motility to enable the cells to crawl on the surface (Ghannoum and O'Toole G, 2004) When the biofilm is fully developed, the bacteria are less likely to be displaced by physical processes including shear forces. Moreover, they are better able to withstand competition by invading organisms and they are more resilient to predation challenges (in nature or by host immune responses) In fact, biofilm cells show a remarkable tolerance to a variety of antimicrobial measures as compared to their, planktonic, growing, counterparts (Bjarnsholt et al. 2005; Drenkard and Ausubel, 2002; Fux et al. 2004; Hentzer et al. 2003; Lewis, 2001) Most standard antibiotics will kill only minor parts of the populati