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What drives bacteria to produce a bioﬁlm?
Kimberly K. Jeﬀerson
The Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115, USA Received 26 March 2004; received in revised form 1 June 2004; accepted 3 June 2004 First published online 15 June 2004
Abstract Nearly 40 years ago, Dr. R.J. Gibbons made the ﬁrst reports of the clinical relevance of what we now know as bacterial bioﬁlms when he published his observations of the role of polysaccharide glycocalyx formation on teeth by Streptococcus mutans [Sci. Am. 238 (1978) 86]. As the clinical relevance of bacterial bioﬁlm formation became increasingly apparent, interest in the phenomenon exploded. Studies are rapidly shedding light on the biomolecular pathways leading to this sessile mode of growth but many fundamental questions remain. The intent of this review is to consider the reasons why bacteria switch from a free-ﬂoating to a bioﬁlm mode of growth. The currently available wealth of data pertaining to the molecular genetics of bioﬁlm formation in commonly studied, clinically relevant, single-species bioﬁlms will be discussed in an eﬀort to decipher the motivation behind the transition from planktonic to sessile growth in the human body. Four potential incentives behind the formation of bioﬁlms by bacteria during infection are considered: (1) protection from harmful conditions in the host (defense), (2) sequestration to a nutrient-rich area (colonization), (3) utilization of cooperative beneﬁts (community), (4) bioﬁlms normally grow as bioﬁlms and planktonic cultures are an in vitro artifact (bioﬁlms as the default mode of growth). Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Bioﬁlm; Genetics; Microbial communities
1. Introduction Genetic adaptation is the cornerstone of ﬁtness and survival and can ensue from mutations and recombination within genes, acquisition of new genetic material, or from the regulated expression of existing genetic material. Flexibility in bacterial gene expression permits survival in environments with rapidly changing conditions, and bacteria, being particularly adaptable, have ﬂourished in nearly every environmental niche on our planet. Bacterial species that are able to colonize humans are especially creative in their regulatory processes. Many pathogenic and commensal bacteria are capable of transitioning between life in the environment and in the human host, and all must be able to adapt to sudden shifts in nutrient availability as well as to primary and secondary host immune defenses. One par*
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ticularly important and clinically relevant example of bacterial adaptation through systematized gene expression is the ability to grow as part of a sessile, exopolymer-enshrouded community referred to as a bioﬁlm. Scientiﬁc interest in the process of bacterial bioﬁlm formation has erupted in recent years and studies of the molecular genetics of bioﬁlm formation have begun to shed light on the driving forces behind the transition to the bioﬁlm mode of existence. It is now recognized that bioﬁlm formation is an important aspect of many, if not most bacterial diseases, including native valve endocarditis, osteomyelitis, dental caries, middle ear infections, medical device-related infections, ocular implant infections, and chronic lung infections in cystic ﬁbrosis patients . Established bioﬁlms can tolerate antimicrobial agents at concentrations of 10–1000-times that needed to kill genetically equivalent planktonic bacteria, and are also extraordinarily resistant to phagocytosis, making bioﬁlms extremely diﬃcult to eradicate from living hosts .
0378-1097/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.06.005
K.K. Jeﬀerson / FEMS Microbiology Letters 236 (2004) 163–173
Consequently, bioﬁlm-related infections that appear to respond to a therapeutic course of antibiotics may relapse weeks or even months later, making surgical removal and replacement of the infected tissue or medical device a frequent and unfortunate necessity. The serious and pervasive clinical impact of bacterial bioﬁlms has inspired many researchers to investigate the regulatory mechanisms behind their formation and dissolution, with the ultimate goal of pinpointing speciﬁc targets for chemotherapeutic agents. A number of target gene-directed as well as global proteomics- and genomics-based studies have lead to the identiﬁcation of a plethora of genes associated with bioﬁlm development. Sorting out the roles of all of these genes is however, an exceedingly complex task. For one thing, the regulatory processes of bioﬁlm elaboration are cyclical and dynamic. In other words, external conditions trigger alterations in the expression of a subset of genes re-
quired for bioﬁlm formation. The formation of a bioﬁlm in turn alters the microenvironment of its own inhabitants which then leads to additional alterations in gene expression and further maturation of the bioﬁlm, and so on. Complicating the study of gene expression in bioﬁlms even further, bioﬁlm inhabitants are heterogeneous. Disease-related bioﬁlms can be multi-species or even multi-kingdom, such as the bioﬁlms involved in tooth decay, or single-species such as those involved in endocarditis, but even bacteria within single-species bioﬁlms are heterogeneous with respect to gene expression. This is due to diﬀusion limitations imparted by the bioﬁlm, which result in local variations in pH, nutrient and oxygen availability, and concentrations of bacterial metabolites. On top of all of these complications, bioﬁlm studies are confounded by the intrinsic limitations of the in vitro bioﬁlm models and the techniques available to study the roles of these genes.
Table 1 Genes required for bioﬁlm formation Gene Adhesion abpA sspA/B gbpA tarC icaADBC hla clfA dltA atlE aap bopABCD esp agn43 Quorum sensing comX comABCDE luxS? lasI Cell wall PBP2B PBP5 glmM bacA brpA Metabolism ccpA crc Stress response dgk rB ? purR rpoS? mutT Plasmids tra Protein/function Amylase binding Human salivary protein and collagen binding Polysaccharide formation Regulator of glucosyltransferase S and glucan binding protein Intercellular adhesin synthesis Hemolytic toxin Clumping factor A, ﬁbrinogen binding protein D -alanine esteriﬁcation of teichoic acids Autolysin/adhesin Accumulation associated protein Bioﬁlm on plastic surfaces operon Enterococcal surface protein Antigen protein involved aggregation Competence Competence Quorum sensing Synthesis of 3OC12-HSL quorum-sensing signal Peptidoglycan synthesis Peptidoglycan synthesis Peptidoglycan synthesis Peptidoglycan synthesis Possible regulator of autolysis Carbon catabolite control protein Global carbon metabolism regulator Stress response regulator, lantibiotic regulator Alternate sigma factor-stress response Regulator of purine synthesis, metabolism Regulator involved in slow growth DNA mismatch repair Conjugative pilus of F plasmid Species S. gordonii S. gordonii S. mutans S. mutans S. aureus, S. epidermidis S. aureus S. aureus S. aureus S. epidermidis S. epidermidis Enterococcus faecalis E. faecalis E. coli S. gordonii S. mutans S. mutans P. aeruginosa S. S. S. S. S. gordonii gordonii gordonii gordonii mutans References                          [13,14]  [15,18]  
S. mutans P. aeruginosa S. mutans S. aureus, S. epidermidis S. epidermidis E. coli S. gordonii E. coli
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Despite the complications associated with the study of bioﬁlms, comparative and comprehensive analysis of all of the existing data can enable one to elucidate congruencies and subsequently, globally signiﬁcant
Table 2 Gene expression UP or DOWN in bioﬁlms with respect to planktonic cells Gene Adhesion algC wcaB csgA clfA scaA abpA rggD int/CoA ﬂpA has Protein/function Alginate synthesis Colanic acid synthesis Curli Clumping factor/ﬁbrinogen binding Co-aggregation Amylase binding Glucosyltransferase inducer Intrageneric co-aggregation-relevant adhesin Fibronectin-binding Streptococcal hemagglutinin
truths become unveiled. Tables 1 and 2 present a compilation of genes from various clinically relevant bacteria that have been implicated in the bioﬁlm mode of growth. Table 1 lists genes that appear to be required for
Regulation UP UP UP DOWN DOWN DOWN UP DOWN DOWN DOWN DOWN UP UP DOWN DOWN UP DOWN UP UP UP DOWN DOWN DOWN DOWN DOWN UP UP UP UP UP UP DOWN UP DOWN DOWN DOWN DOWN DOWN DOWN DOWN UP UP UP
Species P. aeruginosa E. coli E. coli S. aureus S. gordonii S. gordonii S. gordonii S. gordonii S. gordonii S. gordonii P. aeruginosa S. mutans P. aeruginosa S. gordonii S. gordonii P. aeruginosa P. aeruginosa E. coli S. mutans S. mutans S. mutans S. gordonii S. mutans S. mutans S. mutans S. gordonii E. coli E. coli E. coli S. mutans S. mutans S. mutans E. coli P. P. P. P. P. P. E. aeruginosa aeruginosa aeruginosa aeruginosa aeruginosa aeruginosa coli
References                                           
Quorum sensing pA4296 Probable two-component response regulator comD,E Competence factors Cell wall mreC dltA ddl Cell morphology
D -Alanine-D -Alanyl
carrier D -Alanine: D -Alanine ligase
Stress response rpoH Stress/stationary phase s factor rpoS Heat shock s factor proU Transport-osmotic adaptation DnaK: Protein folding(heat shock) Grpe: folding(heat shock) 60 kDa chaperonin: heat shock htgX Putative heat shock protein Carbohydrate metabolism Fructose bisphosphate aldolase Pyruvate kinase 6-Phospho-B-galactosidase pbg Phospho-b-glucosidase P02925 D -ribose-binding periplasmic protein P02927 D -galactose-binding protein Q6150 Malate dehydrogenase Division minicell associated protein Div IVA: cell division FTSZ: septum formation ATP-dependent DNA helicase RECG: DNA replication H-NS: DNA binding Motility PA2128 pilA ﬂgD PA1092 ﬂiD ﬂgE ﬂiC Phage Coat protein of bacteriophage Pf1 Helix destabilizing protein of bacteriophage Pf1 Probable coat protein of bacteriophage Pf1 Probable ﬁmbrial protein Pilin protein Flagellar basal-body rod modiﬁcation protein Flagellin type B Flagellar capping protein Flagellar hook protein Flagellar synthesis
P. aeruginosa P. aeruginosa P. aeruginosa
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bioﬁlm formation (the cause genes) and Table 2 lists the genes that are diﬀerentially regulated in an established bioﬁlm (the eﬀect genes). Answers from many questions can be extricated from trends apparent in these tables. For example, the conspicuous presence of surface adhesions in Table 1 addresses the question ‘‘How do bioﬁlms adhere to solid surfaces?’’. Answering such mechanistic questions is, inarguably, critical to our understanding of the bioﬁlm mode of growth. However, excellent reviews dedicated to understanding the ‘‘How?’’ of bioﬁlm formation have already been published [2,4], and I would like to venture instead, to address the fundamental, albeit somewhat philosophical question ‘‘Why do bacteria form bioﬁlms in the human host?’’.
2. Why do bacteria form bioﬁlms? According to Darwin’s theory of evolution, the only true driving force behind the course of action of any organism is reproductive ﬁtness. Any action that increases proliferation will endure within a species. Therefore, when we talk about the driving force behind bioﬁlm formation we are asking the question ‘‘How does the bioﬁlm mode of growth promote survival and propagation of the cell?’’ It almost seems counter-intuitive that the bioﬁlm mode of growth could confer a reproductive ﬁtness advantage when one considers that bioﬁlm bacteria have a reduced rate of growth relative to bacteria growing planktonically in broth culture. Outside of the laboratory, however, bacteria rarely, if ever, ﬁnd themselves in an environment as nutrient rich as culture media, and in these less-than-ideal conditions, there are a number of ﬁtness advantages imparted by the bioﬁlm mode of growth. This review proposes four advantages which are illustrated in Fig. 1. The more we learn about the genetic regulation of bioﬁlm formation, the more we understand about the relative roles of these beneﬁts and about the forces that drive the switch to the bioﬁlm mode of growth. 2.1. Defense: bioﬁlm formation as a stress response Bioﬁlms are resistant to physical forces such as the shear forces produced by blood ﬂow and the washing action of saliva. Organisms within bioﬁlms can withstand nutrient deprivation, pH changes, oxygen radicals, disinfectants, and antibiotics better than planktonic organisms. Bioﬁlms are also resistant to phagocytosis, and the phagocytes that attempt an assault on the bioﬁlm may actually do more harm to surrounding tissues than to the bioﬁlm itself. The chronic nature of certain infections is inarguably due to the development of a resilient bioﬁlm. The invulnerability of bioﬁlms is not completely understood but is likely dependent upon a
Fig. 1. Dr. Sean D. Taverna’s artistic interpretation of the four driving forces behind bacterial bioﬁlm formation that are discussed in this review.
number of bioﬁlm-speciﬁc characteristics including slow growth and physiologic heterogeneity of the inhabitants. Another important trait that fortiﬁes bioﬁlm resistance is the sticky matrix which may contain DNA and other polymers but in general, is predominantly composed of exopolysaccharides. The important role of exopolysaccharide (EPS) in both the early and late stages of bioﬁlm formation is exempliﬁed by the conspicuous presence of genes involved in polysaccharide synthesis in Tables 1 and 2. In Escherichia coli, csgA, which encodes a protein involved in the synthesis of colanic acid, is involved in aggregation and algC, the gene required for alginate synthesis plays a role in Pseudomonas aeruginosa bioﬁlms [4–6]. EPS synthesis is important in the development of grampositive bioﬁlms as well. Glucan binding protein GbpA is a glucosyltransferase that has been implicated in sucrose-dependent polysaccharide production and bioﬁlm formation in S. mutans . In addition, the intercellular adhesin locus (icaADBC) in Staphylococcus aureus and Staphylococcus epidermidis encodes the gene products responsible for the synthesis of a b-1-6-linked poly-Nacetylglucosamine polymer called PNAG or PIA (polysaccharide intercellular adhesin) . Fig. 2 illustrates the important role for the exopolysaccharide PNAG in the morphology of S. aureus bioﬁlms. A weak PNAG-producing strain retains the simple morphology of a young bioﬁlm whereas a strong PNAG-producing strain forms tight mushroom-like microcolonies separated by wide channels. In addition to its roles in aggregation and bioﬁlm structure, EPS plays a part in defense, enabling bioﬁlms to resist shear forces and
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Fig. 2. Confocal microscopic imaging demonstrates the inﬂuence of exopolysaccharide elaboration on the structure of S. aureus bioﬁlms. S. aureus clinical isolate strain MN8, and the isogenic, constitutive PNAG over-producing derivative strain MN8m were allowed to form bioﬁlms on collagencoated glass coverslips for 48 h under ﬂow conditions. The bioﬁlms were stained using the BacLight Live/Dead kit which stains live bacteria green and dead bacteria red. These confocal images demonstrate that the level of PNAG synthesis plays a critical role in bioﬁlm structure. MN8 formed a somewhat unstructured bioﬁlm, whereas the PNAG-overproducing strain MN8m formed a highly structured bioﬁlm with dense mushroom-shaped microcolonies separated by large channels.
phagocytosis by inﬂammatory cells . Some evidence suggests that EPS may also be involved in tolerance of bioﬁlms to antimicrobial agents but this is still a matter of debate . However, if the sole force driving EPS production and bioﬁlm formation is resistance to dangers encountered in the body, then what are the environmental cues that warn bacteria that they need to bolster their defenses? Certain bacterial species may have evolved to switch on transcription of genes required for EPS synthesis in response to certain environmental stimuli that are encountered upon host entry, before the immune system mounts a speciﬁc attack . For example, certain stimuli that mirror the physiology of the human body, such as iron deprivation and osmotic stress, induce the expression of genes encoding proteins that synthesize EPS in staphylococci and enterococci [10,11]. The involvement of stress response regulators in bioﬁlm formation would support the hypothesis that the motivation behind bioﬁlm formation is defense, but the contribution of these regulators is somewhat unclear. As indicated in Table 1, studies have implicated the stress response regulators Dgk, rB , and RpoS, of S. mutans, S. aureus, and E. coli (respectively) in bioﬁlm formation [12–15]. Other studies however, dispute the roles of rB and RpoS in bioﬁlm formation [16–18]. In P. aeruginosa RpoS expression is down in bioﬁlms but expression of RpoH, a sigma factor that has been linked to the stationary phase and stress, is elevated . The role of the stress response regulators may depend upon the conditions under which bioﬁlm formation is triggered or upon the genetic background of the bacterial cell, again suggesting that what we categorize as a bioﬁlm actually represents a collection of diﬀerent growth states. Bio-
ﬁlms are inarguably resilient, but if defense is the major driving force behind the bacterial mode of growth in the human body, then why would the bacteria form a sessile community in such an inhospitable place? In sum, it is apparent that the stress is not the only trigger for this mode of growth. 2.2. Colonization: bioﬁlm formation as a mechanism to remain in a favorable niche Humans and other animals have developed intricate immune systems for one critical reason: microorganisms are continually trying to inhabit their bodies. The body, or at least parts of it, is nutrient rich and relatively stable with respect to water content, oxygen availability, and temperature. Consequently, there is a never-ending race between the development of the host immune system and the progression of bacterial strategies to evade it. In some cases, a compromise has been made, and as such, the body is inhabited by a large number of commensals, many of which exist as bioﬁlms. As the body is obviously an appealing place for bacteria to live, it may be that the primary motivation for switching to the bioﬁlm mode of growth is to remain ﬁxed. Bacteria have a number of strategies to ensure that they remain ﬁxed in the human body. Bacterial surface proteins that bind to host extracellular matrix proteins such as ﬁbronectin, ﬁbrinogen, vitronectin, and elastin are referred to as MSCRAMMs (microbial surface component recognizing adhesive matrix molecules) and often play a key role in initial adherence of bacteria to solid surfaces within the host . S. aureus is particularly notable for the abundance of MSCRAMMs that
K.K. Jeﬀerson / FEMS Microbiology Letters 236 (2004) 163–173
it can produce, including clumping factors A and B (ClfA/B), ﬁbronectin binding factors A and B (FnBA/ B), and a collagen binding protein (Cna). S. epidermidis produces at least two autolysin–adhesins that bind to ﬁbronectin and the ﬁbrinogen binding protein Fbe . Streptococcus pyogenes contains genes for ﬁbronectin (prtF) and ﬁbrinogen (emm) binding proteins . The oral streptococci have evolved to bind to the pellicle or conditioning ﬁlm on tooth surfaces which is composed of salivary glycoproteins and lipids. The salivary agglutinin glycoprotein binding proteins (SspA and SspB in Streptococcus gordonii and SpaP in S. mutans), and salivary amylase binding proteins of various oral streptococci aid in binding to the pellicle on the surface of teeth [7,21]. Flagella, pili, and ﬁmbriae have also been implicated in adherence of Vibrio cholerae, E. coli, P. aeruginosa, and Salmonella enterica . Interestingly, Table 2 indicates that once the bioﬁlm is established, expression of a number of the adhesins and motility factors is suppressed. This suggests that the main role of adhesins, pili, and ﬂagella is in initial attachment, and that once the development of the bioﬁlm has passed this stage, the proteins are no longer needed and their expression is inhibited. Overall, bacteria produce an impressive array of adhesins that appear to have evolved as a means to inhabit the human body. Also in support of the hypothesis that bioﬁlm formation is a mechanism for organisms to stay put in the favorable environment of the human host, is the ﬁnding that carbon catabolite induced gene regulation plays a critical role in bioﬁlm formation (Table 1). Exopolysaccharide expression and bioﬁlm elaboration are markedly enhanced in certain bacteria, including the pseudomonads, V. cholerae, and E. coli, the staphylococci and the streptococci, when glucose or another readily utilizable carbon source is abundant [4,23–25]. When nutrient sources are depleted, the bacteria detach and become planktonic, suggesting that nutrient deprivation is a trigger to move on, in search of a better habitat. Glucose-induced exopolysaccharide production may be multi-functional. It is possible that glucose simply serves as a substrate in the EPS synthesis pathway but studies with S. aureus in our laboratory suggest that, at least for this organism, this is not the case. Glucose appears to augment EPS elaboration at the level of transcriptional regulation rather than at the level of EPS synthesis . A second possibility, which supports the bioﬁlm as a mode of defense is that bacteria may have evolved to interpret elevated glucose levels as a cue that it is in the bloodstream, and that it needs to form a bioﬁlm to remove itself from circulation and protect itself from the immune system. Alternatively, polysaccharide production may function as a mechanism of glucose storage during times of plenty, and/or as a mechanism to augment the accumulation
phase so that when the organism ﬁnds itself in an environment rich in nutrients it can occupy that niche. With all of the complex mechanisms that pathogenic and commensal bacteria have evolved to survive in the human body, it is clear that the beneﬁts that we aﬀord them outweigh the hurdles imparted by our immune systems.
3. Community: bioﬁlms and communal behavior 3.1. Are bioﬁlms multicellular organisms? The idea that bacteria often exist within nature as bioﬁlms rather than individual, free-ﬂoating cells was brought into wide acceptance by the ideas and intuition of Dr. J. William Costerton . Following the excitement generated by his trailblazing revolution of longstanding scientiﬁc dogma, other revolutionary hypotheses about the nature of bacterial behavior began to emerge. One such hypothesis is that bioﬁlms should be regarded as multicellular organisms and that bioﬁlm bacteria exhibit cooperative, unselﬁsh behavior . The behavior of bacteria within bioﬁlms has even sparked skepticism about the Darwinian theories of evolution . Hypothetical challenges to well-accepted theories are entertaining but without scientiﬁc evidence to back them up they remain purely philosophical, and there has not yet been an eﬀort to scientiﬁcally validate the challenge to our current ideas about evolution. Recently however, mathematical modeling of bioﬁlm systems and scientiﬁc experiments are being designed to test whether cooperative, altruistic behavior in bacteria is compatible with the mainstream theory of evolution . There are indeed similarities between bioﬁlm bacteria and multicellular organisms. For instance, bacteria (including planktonic bacteria) can sense their surroundings, and this enables them to adjust their metabolic processes to maximize the use of available substrates and to protect themselves from detrimental conditions. When bacteria are growing within a bioﬁlm, these changes in gene expression result in phenotypic heterogeneity within the bioﬁlm which can be interpreted as specialization or division of labor similar to cellular diﬀerentiation seen in multicellular organisms. In addition, bacteria secrete substances referred to as autoinducing signals, which inﬂuence gene expression and may be a means by which cells communicate with one another. There is even a growing body of evidence that bacteria exhibit altruistic behavior and can undergo a process similar to programmed cell death, again suggestive of multicellularity . However, there are fundamental distinctions between bacteria and multicellular organisms. For example, while bacterial cells can react and adapt to their environmental surroundings, they do
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not permanently diﬀerentiate. For instance, one can isolate colonic epithelial cells from a human and grow them in tissue culture medium, and even though the cells are suddenly faced with radical changes in their surrounding milieu, they continue to grow as colonic epithelial cells and can even form a polarized monolayer similar to colonic epithelium. Scientists have devoted much eﬀort to developing methods to ‘‘undiﬀerentiate’’ diﬀerentiated cells, but the process of cellular diﬀerentiation, even in simple multicellular organisms, is not easily reversed by any natural means. This is because their genetic regulatory patterns have been permanently altered. If however, you remove bacterial cells from a bioﬁlm and radically change their environmental conditions then they will quickly adapt to their new environmental surroundings and exhibit phenotypic changes. Depending on the conditions in which they are grown, they will even convert back to the planktonic mode of growth. Bacterial cells do not diﬀerentiate, rather they respond to their environmental surroundings by adapting their gene expression to suit their own needs for survival. For this reason, it is more accurate to refer to bioﬁlms as interactive communities rather than comparing them to multicellular organisms. Nonetheless, the community lifestyle is likely an important motivation for bioﬁlm formation and provides its members with a number of beneﬁts. In addition to the advantage of resistance to environmental changes, which is discussed in the defense section, the bioﬁlm may beneﬁt from a number of properties of a communal existence including division of the metabolic burden, gene transfer, and selﬂess behavior. 3.1.1. Division of the metabolic burden Diﬀusion limitations imparted by the bioﬁlm structure result in local variations in nutrient availability, pH, and oxygen tension. Therefore, the bacteria within bioﬁlms are inevitably heterogeneous with respect to gene expression. Many bioﬁlms are made up of a variety of bacterial species and some even contain mixtures of bacteria and fungi. The members of these mixed bioﬁlms have diﬀerent requirements and perform diﬀerent metabolic functions making commensalism a widespread phenomenon in bioﬁlms . For example, whereas early colonizers of the oral cavity are aerobic or facultatively anaerobic, limited oxygen diﬀusion through the bioﬁlm provides an environmental niche allowing for later colonization by obligate anaerobes . A study in which promoter activity was monitored as a function of the expression of a ﬂuorophore indicated that heterogeneity in the gene expression proﬁles of the individual cells exists even within single-species bioﬁlms . It is likely that this heterogeneity translates into specialized functions of cells within a bioﬁlm . Fruiting body formation by Myxobacteria is frequently cited as an example of cell specialization in bacteria and it is an
attractive idea that this phenomenon occurs in other bacteria as well . Although it has not been deﬁnitively proven, the heterogeneity within bioﬁlms may indeed result in a ‘‘division of labor’’ of sorts and certainly increases the metabolic eﬃciency of the population as a whole. A popular notion is that such division of labor is coordinately regulated within bioﬁlms through intercellular communication. Autoinducing signals are small molecules, generally homoserine lactones in gram-negatives and peptides in gram-positives, that are constitutively released by bacteria and which, when present at a critical concentration, will induce the expression of certain genes. Autoinducing signals are frequently referred to as quorum-sensing signals because when a bacterial population reaches a high enough density, the local concentrations reach threshold levels and alter gene expression. However, it has not been shown conclusively that bacteria actually respond to the accumulation of a quorum, and it has been suggested that the more biologically signiﬁcant role of the autoinducing signals is to relay information to the bacterial cell about local diﬀusion rates . In her provocative review, Dr. Rosemary Redﬁeld suggests that expression of secreted proteins are induced in the presence of elevated levels of autoinducing signals not because the bacteria have evolved to work cooperatively, but because the beneﬁts of secreted proteins are realized by an individual bacterial cell when local conditions limit diﬀusion and mixing . One example used is the secretion of a protease which is required to degrade exogenous proteins so that the bacteria can assimilate amino acids. Under conditions of reduced diﬀusion or mixing, secreted proteases and the proteins degraded by them would remain in the vicinity of, and beneﬁt the cell. It is therefore logical that bacteria would restrict expression of secreted proteins under conditions of high mixing and diﬀusion. There are also examples of autoinducer eﬀects that do not readily ﬁt this model. The diﬀusion sensing model suggests that a bacterial cell responds to it’s own secreted signals, but recognition of and response to signals secreted by heterologous species has been welldocumented . It is likely that both diﬀusion-sensing and quorum-sensing are aspects of autoinducing signals but a more precise answer incorporating roles for both of these phenomena awaits further investigation. While the primary function of autoinducing signals remains unclear, their role in bioﬁlm development is even more ambiguous. As is indicated in Table 1, one study found a role for the LuxS system in S. mutans but two additional studies indicated that LuxS was not required for bioﬁlm formation [12,33,34]. Equally confounding results were obtained when diﬀerent investigators studied the role of the lasRlasI quorumsensing system in P. aeruginosa . Furthermore, there is evidence that the accessory gene regulator (Agr) which
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is involved in quorum-sensing in the staphylococci actually inhibits bioﬁlm formation . A more recent study suggests that the Agr eﬀect is dependent upon the ﬂow strength over the bioﬁlm and that under static conditions, Agr reduces bioﬁlm formation, under low to moderate ﬂow it does not aﬀect bioﬁlm formation, and under very strong ﬂow it increases bioﬁlm formation . These results may support the diﬀusion-sensing theory if one considers that under rapid ﬂow, the autoinducing signals may rapidly diﬀuse out of the bioﬁlm. Overall, a bioﬁlm represents both a quorum and imparts restrictions on diﬀusion, so regardless of whether one accepts the diﬀusion-sensing or the quorumsensing hypothesis, it would seem, at least superﬁcially, that auto-inducing signals inﬂuence its development. In parts of the human body however, especially in places such as heart valves and teeth, bioﬁlms are subjected to strong shear forces which may keep autoinducing signal levels low. A recent report indicates that in the human lung, the acyl-homoserine lactone quorum-sensing signal of P. aeruginosa is inactivated by some unidentiﬁed host cell-associated factor . Does this indicate the dispensability of quorum-sensing and mean that P. aeruginosa can establish a bioﬁlm in the lung despite immune-mediated inhibition of quorum-sensing, or is the ﬁnding that the immune system targets this signal suggestive of its importance in bioﬁlm formation? The answer is unclear and the role of quorum-sensing in bioﬁlm formation remains elusive. 3.1.2. Gene transfer Nucleic acid is the basis for evolution and consequently its one true purpose is to replicate and perpetuate its own speciﬁc code. A reproductive ﬁtness advantage will perpetuate the genetic material of an individual. The same rules apply to obligate infectious agents such as viruses and plasmids, and as these agents can not replicate their own genetic material, they would cease to exist without a means to spread from one organism to another. Bacteriophage and plasmids have therefore both evolved mechanisms to promote their maintenance within a bacterium and their spread to other bacteria. For example, bacteriophage slow down their replicative machinery and integrate into the bacterial chromosome so that their genome is replicated as the cell divides. Plasmid replication is an expensive process for a bacterial cell and plasmids are quickly lost unless there are required or beneﬁcial. Plasmids have therefore evolved a rather elaborate method for survival. They carry toxin–antitoxin gene pairs which make their maintenance necessary for bacterial survival . The plasmid-encoded toxin is a stable protein and the antitoxin is a labile protein. When a bacterial cell divides the daughter cell inherits both toxin and antitoxin. If the cell fails to replicate the plasmid as it divides or if the daughter cell fails to inherit the plasmid, then the supply
of labile antitoxin rapidly declines whereas the toxin lingers and destroys the plasmid-free daughter cell. These cunning maintenance methods ensure vertical transfer of phages and plasmids but in order to be successful, a phage or infectious plasmid must also utilize horizontal transfer. A bioﬁlm is the ideal environment for horizontal exchange of genetic material . The close proximity fosters rapid spread of phage as well as conjugation and uptake of plasmid DNA by competent bacteria. Plasmids and phage have consequently developed methods to induce the transition to the bioﬁlm mode of growth in their host so that they can spread to uninfected bacteria and sometimes even to cross the species barrier . Dr. Jean-Marc Ghigo found that the pili encoded in a number of conjugative plasmids adhere nonspeciﬁcally to solid surfaces and to other bacteria leading to a dramatic increase in bioﬁlm formation of E. coli and other Gram-negative bacteria . By inducing the transition to the bioﬁlm mode of growth, the plasmid is likely increasing its chances for horizontal transmission. In addition, a number of phage genes, including phage coat protein genes, are activated in P. aeruginosa bioﬁlms, supporting the idea that an effective strategy for horizontal phage transmission is to re-enter the infectious cycle during the bioﬁlm mode of growth . In eﬀect, we may need to consider that bioﬁlm formation beneﬁts not only bacterial ﬁtness but the proliferative potential of bacteriophage and plasmids as well. Horizontal gene transfer within bioﬁlms may also directly beneﬁt the bacteria through the exchange of antibiotic resistance determinants. In S. gordonii, the expression of competence factors has been implicated as both a cause and an eﬀect of bioﬁlm formation supporting a role for exchange of genetic material in bioﬁlms [39,42]. Whether the primary function of competence factors is to assimilate external DNA as a means to increase their genetic diversity or simply to use it as a nutrient source is controversial, but the end result is that the bioﬁlm is an ideal environment for the exchange of genetic material. The motivation of bacteria themselves as well as plasmids and bacteriophage to exchange genetic material may all play an important role in the process of bioﬁlm development. 3.1.3. Selﬂess behavior Overall, bacteria enjoy a number of beneﬁts due to their community mode of growth but are bacteria truly cooperative and capable of exhibiting unselﬁsh or even altruistic behavior? Indeed, experimental evidence from mathematical modeling supports the concept that bacteria can exhibit altruistic behavior . While this may initially appear to defy the rules of survival of the ﬁttest, the models indicate that unselﬁsh behavior in bioﬁlm inhabitants can increase the overall growth yield. Therefore, altruistic bacteria, despite a sacriﬁce in
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growth rate are, under certain conditions, actually more ﬁt. Results from these studies also suggest that altruism is more eﬃcient when the bacteria exist in small, clonal clusters, perhaps explaining the microcolony formation characteristic of bioﬁlms. Fitness need not entail aggression, beneﬁcial symbiotic relationships can increase yield, and Darwin’s theories about evolution through competition and ﬁtness can be aptly applied to unselﬁsh behavior. There is mounting evidence that a process similar to apoptosis may actually occur in bacteria. Homologues of pro-apoptotic genes such as caspases are widespread among bacteria . Additionally, several examples of toxin–antitoxin cassettes, similar to those that ensure plasmid maintenance, have been found within the chromosomes of gram-negative and gram-positive bacteria, and it has been suggested that programmed cell death may occur within the bioﬁlm community . One rationalization for altruistic behavior in bacteria is that it reduces the metabolic load and increases nutrient availability to the survivors. This sort of unselﬁsh behavior could be explained in a clonal population of bacteria where death of some still results in perpetuation of the genetic code. This type of phenomenon is rationalized by the kin selection model . However, bacterial bioﬁlms are rarely clonal, and surviving bacteria would quickly out-compete sacriﬁcial bacteria. A more plausible hypothesis regarding the chromosomal toxin– antitoxin cassettes, has recently been proposed by Dr. Kim Lewis. He suggests that the cassettes induce, in a fraction of the bacterial population, not death, but a quiescent, persister state which enables survival in the event of a sudden unfavorable environmental change . Overall, the idea that bacterial cells can undergo programmed cell death, while appealing, does not make immediate sense with respect to the laws of evolution, but only further experimentation will resolve this question.
a bioﬁlm. And yet they are often grown planktonically in the laboratory. It is possible that the presence of a suitable substrate for attachment is all that is required to trigger bioﬁlm formation. There is mounting evidence that immediately subsequent to the initial adherence of bacterial to a solid surface, changes in gene regulation begin to occur [4,44]. This suggests that cells actually sense the solid surface to which they are attached and that this sensing system triggers a signaling cascade that may lead to some of the early gene expression patterns necessary for bioﬁlm formation. For example, in P. aeruginosa, expression of algC, a gene required for alginate synthesis, is increased within minutes of attachment, and when S. epidermidis makes contact with a solid surface, the normally spherical cell forms a leg-like appendage [45,46]. These ﬁndings suggest that, similar to eukaryotic cells, bacterial cells possess surface-sensing systems that induce intracellular signals powerful enough to result in transcriptional and morphologic changes. The sensing mechanisms utilized by bacteria to detect adherence are not well understood. Changes in the perceived osmolarity caused by charges on solid surfaces may be an important cue for bacteria to recognize surfaces . The EnvZ–OmpR two-component system which is involved in sensing environmental osmolarity, has also been shown to regulate expression of curli and colanic acid [47,48]. The ﬁbrillar surface structure curli plays a role in adherence and colanic acid is an exopolysaccharide involved in aggregation. The role of osmolarity in bioﬁlm regulation has also been noted in staphylococci  and Pseudomonas ﬂuorescens . Overall, the bioﬁlm mode of growth may be the default mode of growth for at least some bacterial species suggesting that we should be questioning what triggers the planktonic mode of growth rather than what motivates the bioﬁlm mode of growth.
5. Conclusions 4. The bioﬁlm as the default mode of growth In the laboratory, bacteria are generally grown planktonically, but the utopian microcosms created in culture vessels are designed to maximize bacterial growth rates, not to replicate natural growth conditions of the bacteria. In fact, some bacterial species appear to constitutively utilize the bioﬁlm mode outside of the lab. The oral streptococci are very highly adapted to sessile growth on the surface of teeth. Most of the oral bacterial species lack an environmental niche and are found almost exclusively within the mouth . For these bacteria, planktonic growth would cause them to be quickly washed away by saliva, swallowed and destroyed within the acidic juices of the stomach. These bacteria likely spend the majority of their natural existence growing as Scientiﬁc interest in bioﬁlms has exploded in the past decade. This fascination with bioﬁlms is due in large part to their presumed clinical relevance. But it is also, undeniably, due to the appeal of projecting traits of higher organisms on these life forms that were once thought to be so simple and autonomous. The ability of prokaryotes to adapt to their surroundings is indeed remarkable, but whether they actually communicate, coordinate, and specialize within bioﬁlms for the beneﬁt of the community, as opposed to simply reacting to their environments in order to selﬁshly promote their own survival, has not yet been suﬃciently established. Unfortunately, we often make the erroneous conclusion that certain questions are purely philosophical and can not be tested scientiﬁcally. In fact, such questions can
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often, if not always, be objectively examined, and scientists have recently begun to develop mathematical models that predict the relative impacts of altruistic vs. selﬁsh behavior on the survival and propagation of bacteria . In conclusion, it is evident that bacteria reap a number of beneﬁts from the bioﬁlm mode of growth and it is likely that diﬀerent forces motivate bacteria to transition to one of a variety of bioﬁlm states depending on the genetic makeup of the organism and its environment. The alternative motives for bioﬁlm formation presented in this review are by no means exhaustive or mutually exclusive and because it is such a complex process, they may all have a role.
Acknowledgement I thank Dr. Sean Taverna (Rockefeller University) for his artistic contribution. I would also like to acknowledge that there are a number of excellent publications on the molecular genetics of bioﬁlm formation which could not be referenced here due to space limitations.
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