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Contamination of abiotic surfaces: what a colonizing bacterium sees and how to blur it
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Contamination of abiotic surfaces: what a colonizing bacterium sees and how to blur it Philippe Lejeune Unite´ de Microbiologie et Ge´ne´tique, Composante INSA de Lyon, CNRS UMR 5122, Baˆtiment Andre´ Lwoff, 10 rue Dubois, 69622 Villeurbanne, France
Many microbes are able to interfere with solid surfaces and trigger highly sophisticated colonization responses that include expression of specific properties such as increased resistances to antimicrobial agents. An anticontamination strategy might be to prevent adhesion by interfering with the surface-sensing processes and repelling the pioneering cells, to maintain the cellular sensitivity to antimicrobial agents. Recent studies have shown that differences in the physiological state of freefloating and attached bacteria, which could explain the increased levels of resistance, are triggered very early during attachment. Two-component-mediated signalling mechanisms are involved in these surface-sensing processes. Drugs and surface treatments able to interfere with the stimulation factors of these sensing systems (water activity and accumulation of proteins within the periplasm) could ‘blind’ the colonizing bacteria and delay surface contamination. In natural environments, every material that is exposed to the minimal conditions required for life (presence of water, or simply wet air, with a little dissolved gas, mineral salts and organic molecules) is generally contaminated by microbes. Depending on the availability of these substances, the absence of detrimental cells (such as protozoans or macrophages) and environmental parameters (such as temperature, pH and osmolarity), the initial contamination can evolve into a complex consortium: a highly organized layer of matrix-embedded microbial populations called a biofilm [1]. For human societies, the most detrimental property of surface-associated contaminants is probably the expression of specific characters, such as increased resistance to detergents, disinfectants, antibiotics and immunological defenses [1 – 3]. Many nosocomial infections are now considered to be consequences of surface contaminations of indwelling medical devices, or of equipment such as air conditioning and water-distribution networks [3]. In spite of expensive cleaning procedures, the survival and proliferation of Pseudomonas, Salmonella or Listeria at the surface of production equipment used in food-processing industries are also common causes of foodborne infections. Corresponding author: Philippe Lejeune ([email protected]).
In recent years, numerous studies have been devoted to the process of surface colonization and the physiology of mature biofilms [4 – 6]. Knowledge gained in these areas could now be exploited against detrimental biofilms. In my opinion, two different strategies need to be adopted: first, to prevent adhesion, by interacting with the surfacesensing processes to repel the pioneering cells and to maintain the cellular sensitivity to antimicrobial agents; and second, to destroy the organization of differentiated biofilms and then wipe them out by conventional cleaning procedures. In this Review, I will focus on the first strategy: even if, at present, it seems impossible to protect definitively the surface of biomaterials or equipment against microbial contamination, it is now conceivable to develop drugs and coatings that are able to delay colonization. As the development of infections on prostheses frequently results from bacteria introduced at the same time as the implanted device, any delay (even a few hours) in the colonization process could successfully increase the capacities of both the preventive antibiotic therapy and the immunological defenses to eradicate the infection. Likewise, simply delaying the contamination of catheters could constitute significant progress because, in most cases, the catheters are implanted for less than three or four days. In hospital and industrial settings, surface treatments able to retard adhesion could greatly enhance the efficiency of daily cleaning and disinfection procedures because free-floating microbes remain sensitive to detergents and biocides. The very first stages of surface colonization An individual bacterium can reach a surface by three different modes [7]: passive transport due to the flow of a gas or liquid; diffuse transport resulting from Brownian motion; and active movement involving flagella. Afterwards, what happens at the very moment the bacterium collides with the surface is ‘simply’ a question of physicochemical interactions between two bodies carrying, or not carrying, reactive domains. The present state of these two bodies is, of course, the result of past events, namely interactions of the surface with molecules of water and dissolved substances of the liquid phase, and the physiological modulation of particular components of the bacterial envelope by various parameters of the medium, such as temperature and nutrient availability. It seems
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obvious, therefore, that both the nature of the surface and the culture conditions can have a profound influence on the colonization kinetics in laboratory flask environments. And this is what is actually observed. For instance, Chavant et al. [8] have shown that the hydrophilic surface properties of Listeria monocytogenes cells are negatively correlated with growth temperature and that the colonization kinetics are significantly different on hydrophilic (stainless steel) and hydrophobic (Teflon) substrata. In real life, the situation is largely complicated by a pivotal feature of materials exposed to natural fluids: surface conditioning by macromolecules. It has been recognized for a long time that immersion of a clean substratum is immediately followed by fast and efficient adsorption of organic molecules onto the surface [9], forming so-called conditioning films, but the characterization of these structures by modern physicochemical techniques is only just beginning. Although it already seems clear from pioneer studies that, in artificial [10] and natural [11] conditions, the organic molecules never cover the whole surface, conditioning films are an important source of heterogeneity and surface property modification. Their inevitable occurrence in any particular situation in which biofilms are detrimental has therefore to be carefully considered when trying to develop surface treatments. An individual bacterium that approaches a solid surface has to overcome a possible repulsive force and interact with the solid phase or the conditioning film. Depending on the strength of the bonds that can be formed, bacterial motility and gliding properties are often of crucial importance in initiating efficient attachment. In different bacterial species, transposon insertions conferring the loss of the adherent phenotype have been found in genes involved in flagellar motility (Table 1). Accordingly, a nonadherent phenotype could be observed after transposon inactivation of two types of bacterial gliding movement: twitching motility relying on type IV pilus extension and Table 1. Appendages and envelope structures involved in abiotic surface colonization Species
Structure
Refs
Escherichia coli
Flagella Type I fimbriae Lipopolysaccharide Curli Conjugative fimbriae NplE lipoprotein Type III fimbriae Flagella Type IV fimbriae Putative organelle (CupA) Flagella Curli Flagella Lipopolysaccharide Bap adhesin Teichoic acids AtlE autolysin SSP1 adhesin Peptidoglycan Flagella Mannose-sensitive hemagglutinin fimbriae
[13] [13] [28] [18] [29] [23] [30] [31] [31] [32] [33] [34,35] [26] [26] [36] [37] [38] [39] [40] [41] [42]
Klebsiella pneumoniae Pseudomonas aeruginosa
Pseudomonas fluorescens Salmonella enterica
Staphylococcus aureus Staphylococcus epidermidis Streptococcus gordonii Vibrio cholerae
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retraction in Pseudomonas aeruginosa, and swarming due to overflagellation in Salmonella enterica (Table 1). The next step in early attachment events is an interaction sufficiently strong to prevent disruption by convective forces or Brownian motion. The first link between the bacterium and the material (or the conditioning film) is presumably a combination of weak chemical bonds, dipole interactions, and hydrophobic interactions [12]. Two situations are possible: direct interactions between constitutive components of the bacterial envelope and the surface, or bridging mediated by specialized bacterial structures of attachment, such as fimbriae or adhesins. Different genetic strategies have recently been used to identify these bacterial structures (Table 1). Interestingly, given that type I pilus-associated attachment of E. coli requires flagellar motility [13], whereas adhesion mediated by curli is independent of strain motility [14], the adhesion processes mediated by two types of fimbriae can be different, presumably because of differences in the strength of the chemical bonds that these structures are able to form. Surface sensing A major concept of biofilm study is the difference in the physiological state between free-floating and attached bacteria. During the past 70 years, changes in global functions, such as growth rate, respiration and assimilation, have been correlated with substratum-attached growth (for a review of the early literature on the influence of surfaces on microbial behaviour, see Refs [7,9]). Recently, the use of microscopy and reporter gene techniques has clearly established that precise changes in gene expression are triggered very early during the transition between the swimming and attached states. Using lacZ fusions, Davies et al. [15] examined the expression of algC, a gene required for alginate synthesis, within colonizing P. aeruginosa cells. As early as 15 minutes after the initial attachment, they observed induction of algC expression. In addition, time-lapse microscopic observations of P. aeruginosa adhesion showed that the organisms move along the surface before attachment, almost as if they are scanning for an appropriate location for initial contact [4]. These observations indicate that sensing systems are activated when individual bacteria come into contact with the surface. To gather information on these cognitive processes, Prigent-Combaret et al. [16,17] isolated a library of lacZ gene fusions by random insertion of a transposon (carrying the promoterless lacZ gene) in the genome of an E. coli K-12 mutant strain able to colonize hydrophobic and hydrophilic materials [18]. By using a simple screen, these authors showed that, among 446 Lacþ fusions, 98 are significantly more expressed in the attached cells (1.5- to 10-fold), and 73 are less expressed in these cells (2- to 10-fold), after 24 hours of colonization. The synthesis of flagella is one of the repressed functions, whereas production of colanic acid, a major matrix exopolysaccharide, is induced in attached bacteria. To identify the external parameters and bacterial structures that enable the bacteria to discriminate between the liquid phase and the interface, the same
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authors assumed that the presence of electric charges on the solid surface and on the molecules of the conditioning film could increase osmotic strength or, more precisely, decrease water activity (i.e. the proportion of water molecules acting as pure solvent), at the liquid– solid interface by interacting with ions and water molecules. Because it is well-established that, in E. coli, during the initial hours after an osmotic shift, there is a quasi-linear relationship between the osmolarity of the external medium and the intracellular concentration of potassium, Prigent-Combaret et al. compared the intracellular Kþ concentration of planktonic and adherent cell populations [16]. Ten hours after inoculation, the attached bacteria displayed a significantly higher concentration than the free-floating cells. This result is important because it strongly indicates that the water activity of the attached cell micro-environment, at an early stage of the colonization process, is lower than in the liquid phase. Furthermore, because the control of water activity inside cells is an essential function that requires permanent monitoring of external water activity, all unicellular organisms are equipped with efficient sensors for this parameter. It can, therefore, be assumed that local water activity is a universal parameter that enables contaminating microbes to identify a solid– liquid interface and evaluate its physicochemical state. The role of sensor-regulator systems in surface sensing Another indication of the involvement of water activity in surface recognition is the importance of the signal transduction system EnvZ/OmpR in the curli-mediated adherence of E. coli strains. Vidal et al. [18] described a mutant allele of the ompR gene able to confer an adherent phenotype when transduced into many K-12 strains. A single point mutation resulting in the replacement of a leucine by an arginine residue is responsible for this property. The production of curli and the expression of the curlin-encoding gene are increased in the presence of this allele. The authors explained their observation by a possible increased efficiency of interaction between the mutant OmpR protein and the RNA polymerase at the regulatory sites of the target gene. On the other hand, the transduction of knockout mutations, in either ompR or the curlin gene, results in a loss of the adhesion properties of several biofilm-forming strains, including two clinical strains isolated from patients with catheterrelated infections [18]. EnvZ and OmpR are members of a large family of homologous proteins that are collectively referred to as sensor-regulator systems. These two-component systems enable bacteria to respond to various environmental parameters by means of the phosphorylation of a cytoplasmic response regulator by an inner membrane sensor. EnvZ/OmpR has been described as an osmosensing system that responds to external osmolarity by regulating the transcription of the ompF and ompC porin-encoding genes (for a review, see Ref. [19]). Given that EnvZ/OmpRmediated osmotic induction of curli and colanic acid syntheses has been demonstrated [16,18,20], the implication of this two-component system in surface sensing is highly probable. http://timi.trends.com
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CpxA/CpxR is an E. coli two-component system that responds to signals caused by disturbances in the cell envelope and activates genes encoding periplasmic protein folding and degrading factors [21]. The Cpx pathway negatively affects the expression of the operon coding for curli synthesis [22], and specific binding of CpxR to promoter regulatory sequences has been demonstrated [20]. Recently, Otto and Silhavy [23] showed that this signal transduction system is activated after adhesion of E. coli to hydrophobic surfaces. The expression of Cpx-regulated genes (cpxR, cpxP, spy, dsbA, degP) is induced during the first hour of the adhesion process. Inactivation of either the cpxR or the cpxA gene abolishes the adhesion-induced expression of one of the target genes, which demonstrates that the increased transcriptional activity of Cpx-dependent genes during adhesion follows the known molecular mechanisms of signal transduction by means of the CpxA/CpxR system. Moreover, the contact-dependent induction of the signal transduction pathway requires the outer-membrane lipoprotein NplE, which could be responsible for hydrophobic interactions with the surface. The forces created might cause a major perturbation in the envelope, resulting in monomer and misfolded protein accumulation within the periplasm, consequently activating the Cpx pathway. Another link between two-component systems and biofilm formation has been recently identified by Drenkard and Ausubel [24]. They found that antibiotic-resistant variants of P. aeruginosa with enhanced ability to form biofilms occur very frequently both in vitro and in the lungs of cystic fibrosis patients. There is a class of resistant colonies that are smaller than wild-type and display a rough phenotype. When cultivated on antibiotic-free medium, wild-type revertants, characterized by a large colony size, smooth appearance, wild-type levels of susceptibility to antibiotics and reduced biofilm-forming ability, arise at high frequency, indicating that the phenotypic changes observed in the resistant variants are the result of a phase variation mechanism. To identify putative P. aeruginosa factors involved in the switching between the two phenotypic forms, the authors transferred a cosmid library of P. aeruginosa chromosomal DNA to a resistant clone. One of the transconjugant clones is able to greatly enhance the rate of reversion: 100% of the cosmid-carrying cells form wild-type colonies after overnight incubation. Subcloning experiments identified a single open reading frame (ORF) responsible for this high reversion rate. The ORF shows sequence similarities to the response regulator elements of two-component systems, including VieA of Vibrio cholerae and a probable two-component regulator identified in the P. aeruginosa strain PAO1. These last results indicate that overexpression of the regulator component restores the wild-type pattern of gene expression and they strongly suggest that, in wildtype P. aeruginosa cells, a two-component system is able to switch on and off several functions, such as biofilmforming capacity and antibiotic resistance, in response to unknown environmental parameters.
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Table 2. Signalling systems involved in surface colonization Species
Sensing system
Stimulation factor
Refs
Bacillus subtilis Escherichia coli
PkrC Ser/Thr protein kinase EnvZ/OmpR two-component system CpxA/CpxR two-component system Unknown CsrA/CsrB carbon storage regulator BarA/UvrY two-component system Crc catabolite repression control protein GacA/GacS two-component system LasI production of homoserine lactone ComD histidine kinase ComD/ComE two-component system
Unknown Water activity, pH Disturbances in the cell envelope Cellular density Nutrient availability Hydrogen peroxide Nutrient availability Unknown Cellular density Cellular density Cellular density
[43] [18,20] [22,23] [16] [44,45] [45] [46] [47] [48] [40] [49]
Pseudomonas aeruginosa
Streptococcus gordonii Streptococcus mutans
A model for surface sensing As shown in Table 2, other sensing systems have recently been related to biofilm formation. Although there are many fascinating open questions that require further
investigation, all these data are consistent and a model of surface sensing can be proposed (Fig. 1). The very first step of the sensing process occurs when a swimming or free-floating bacterium perceives the presence
(a)
Surf
Appendage monomer
Organic molecule of the conditioning film Secreted organic molecule
OM Per IM Cyt
(b)
Surf
OM Per IM Cyt
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Fig. 1. A model of surface sensing. When swimming (a) convective forces (symbolized by two arrows) break bacterial appendages and disperse secreted molecules. Immobilization (b) reduces dispersion and appendage breaks. A new micro-environment is created between the solid phase and the surface of the cell. The electric charges of the solid surface, the organic molecules of the conditioning film, the appendages, the microbial surface, and the secreted organic molecules decrease water activity. Because of the ‘traffic jam’ appendage, monomers and other molecules accumulate within the periplasm, and also decrease water activity. Activation of sensing mechanisms, such as two-component systems, occurs and various metabolic changes are triggered. Surf, abiotic surface; OM, outer membrane (with two trimers of porin); Per, periplasm; IM, inner membrane (with two sensors, red points: phosphate groups); Cyt, cytoplasm (with two regulators). http://timi.trends.com
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of a solid phase. For sure, every microbe is equipped with sensor structures that constitutively measure water activity, pH and nutrient concentrations. These structures have generally been studied in laboratory conditions as ‘stress’ response components. However, in reality, one of their functions is probably to allow the cell to adopt instantaneous changes in behaviour, such as positive and negative chemotaxis. Kolter’s group has reported the importance of surface scanning and chemotaxis mechanisms for biofilm formation [4,13] and it seems, therefore, to be established that flagellated bacteria can be attracted to or repelled by the surface. This is obviously of primary importance for the design of anti-biofilm coatings. A pivotal question that remains to be answered concerns the behaviour of non-flagellated bacteria, such as Staphylococcus aureus and Staphylococcus epidermidis. Are these bacteria attracted by the surface as iron by a magnet, or are they able to make a choice and ‘refuse’ the contact ? The second step is the initial attachment. At this moment, three fundamental changes occur: first, because of immobilization, the convective forces that are able to break bacterial appendages and rapidly remove secreted molecules are weakened; second, a new micro-environment is created between the solid phase and the cell, and its composition will be modified by the cellular metabolism; and third, part of the envelope is exposed to short-distance interactions determined by the physicochemical state of the solid phase. In addition, the assembly of certain fimbriae, for instance curli, is an extracellular process. It has been reported that E. coli excretes copious amounts of monomers to synthesize its curli [25]. When a bacterium swims, it is conceivable that a large part of the monomers could be wasted. However, following immobilization, fimbriae synthesis could be much more efficient and significantly modify binding, water activity around the cell, and monomer concentration within the periplasm. All these changes constitute signals that can be transduced to the genome by two-component systems, such as EnvZ/OmpR and CpxA/CpxR. The future: how to blind pioneering bacteria In my opinion, there are three possible pharmacological targets that could be used to prevent microbial adhesion: the sensor systems involved in chemotaxis; the twocomponent regulatory systems; and the cellular structures responsible for interaction with the solid phase. To target the sensor systems involved in chemotaxis, diffusible repellent molecules could be incorporated into a gel smeared on implanted medical devices, such as prostheses. These molecules would divert the passage of motile bacteria and keep them in the liquid phase, where they would remain sensitive to antibiotics and to immune defense mechanisms. The two-component regulatory systems are now considered to be promising therapeutic targets for general antibiotics [26] but, in addition, it can be assumed that particular compounds, old or new, that have been rejected during classical screening procedures for reasons of toxicity, secondary effects or weak activity in vivo could http://timi.trends.com
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be very efficient when applied locally via diffusion from a gel covering the biomaterial. Lastly, the third target named are the cellular structures responsible for interaction with the solid phase. When grafted or smeared on the surface, surfactants [27] or polymers carrying electric charges could modify the physicochemical properties of the interface and decrease the binding ability of the colonizing organisms. To find molecules able to weaken, and consequently delay, adhesion is a difficult but fascinating task. I think that we have to be aware that, in nature, most microbes live within highly organized multispecies consortia. During consortium development, bacteria carry out precisely, in terms of time and place, certain functions necessary for the construction of the whole. Within these very stable communities, the microbes are constantly giving out messages to their partners and also to eventual enemies, of which antibiotics are an example. It would seem to me very worthwhile to look for those compounds that, at low doses, are able to act on the functions essential to the structuring of the microbial ecosystems and, afterwards, to look at their anticontamination, and even antimicrobial, capacities at high doses. Acknowledgements Research in the author’s laboratory was funded by grants from the French Defense Ministry (96/048 DRET) and the Centre National de la Recherche Scientifique (Re´seau Infections Nosocomiales).
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17 Prigent-Combaret, C. and Lejeune, P. (1999) Monitoring gene expression in biofilms. Methods Enzymol. 310, 56 – 79 18 Vidal, O. et al. (1998) Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression. J. Bacteriol. 180, 2442 – 2449 19 Pratt, L.A. and Silhavy, T.J. (1995) Porin regulon of Escherichia coli. In Two-Component Signal Transduction (Hoch, J.A. and Silhavy, T.J., eds) American Society for Microbiology 20 Prigent-Combaret, C. et al. (2001) Complex regulatory network controls initial adhesion and biofilm formation in Escherichia coli via regulation of the csgD gene. J. Bacteriol. 183, 7213 – 7223 21 Danese, P.N. and Silhavy, T.J. (1997) The sE and the Cpx signal transduction systems control the synthesis of periplasmic proteinfolding enzymes in Escherichia coli. Genes Dev. 11, 1183 – 1193 22 Dorel, C. et al. (1999) Involvement of the Cpx signal transduction pathway of E. coli in biofilm formation. FEMS Microbiol. Lett. 178, 169 – 175 23 Otto, K. and Silhavy, T.J. (2002) Surface sensing and adhesion of Escherichia coli controlled by the Cpx-signalling pathway. Proc. Natl. Acad. Sci. U. S. A. 99, 2287 – 2292 24 Drenkard, E. and Ausubel, F.M. (2002) Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation. Nature 416, 740 – 743 25 Bian, Z. and Normark, S. (1997) Nucleator function of CsgB for the assembly of adhesive surface organelles in Escherichia coli. EMBO J. 16, 5827 – 5836 26 Stephenson, K. and Hoch, J.A. (2002) Two-component and phosphorelay signal-transduction systems as therapeutic targets. Curr. Opin. Pharmacol. 2, 507 – 512 27 Mireles, J.R. II et al. (2001) Salmonella enterica Serovar Typhimurium swarming mutants with altered biofilm-forming abilities: surfactin inhibits biofilm formation. J. Bacteriol. 183, 5848– 5854 28 Genevaux, P. et al. (1999) Identification of Tn10 insertions in the rfaG, rfaP, and galU genes involved in lipopolysaccharide core biosynthesis that affect Escherichia coli adhesion. Arch. Microbiol. 172, 1 – 8 29 Ghigo, J.M. (2001) Natural conjugative plasmids induce bacterial biofilm development. Nature 412, 442 – 445 30 Di Martino, P. et al. Klebsiella pneumoniae type 3 pili facilitate adherence and biofilm formation on abiotic surfaces. Res. Microbiol. 154, 9 – 16 31 O’Toole, G.A. and Kolter, R. (1998) Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol. Microbiol. 30, 295 – 304 32 Vallet, I. et al. (2001) The chaperone/usher pathways of Pseudomonas aeruginosa: identification of fimbrial gene clusters (cup) and their involvement in biofilm formation. Proc. Natl. Acad. Sci. U. S. A. 98, 6911 – 6916
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Contamination of abiotic surfaces: what a colonizing bacterium sees and how to blur it Philippe Lejeune Unite´ de Microbiologie et Ge´ne´tique, Composante INSA de Lyon, CNRS UMR 5122, Baˆtiment Andre´ Lwoff, 10 rue Dubois, 69622 Villeurbanne, France
Many microbes are able to interfere with solid surfaces and trigger highly sophisticated colonization responses that include expression of specific properties such as increased resistances to antimicrobial agents. An anticontamination strategy might be to prevent adhesion by interfering with the surface-sensing processes and repelling the pioneering cells, to maintain the cellular sensitivity to antimicrobial agents. Recent studies have shown that differences in the physiological state of freefloating and attached bacteria, which could explain the increased levels of resistance, are triggered very early during attachment. Two-component-mediated signalling mechanisms are involved in these surface-sensing processes. Drugs and surface treatments able to interfere with the stimulation factors of these sensing systems (water activity and accumulation of proteins within the periplasm) could ‘blind’ the colonizing bacteria and delay surface contamination. In natural environments, every material that is exposed to the minimal conditions required for life (presence of water, or simply wet air, with a little dissolved gas, mineral salts and organic molecules) is generally contaminated by microbes. Depending on the availability of these substances, the absence of detrimental cells (such as protozoans or macrophages) and environmental parameters (such as temperature, pH and osmolarity), the initial contamination can evolve into a complex consortium: a highly organized layer of matrix-embedded microbial populations called a biofilm [1]. For human societies, the most detrimental property of surface-associated contaminants is probably the expression of specific characters, such as increased resistance to detergents, disinfectants, antibiotics and immunological defenses [1 – 3]. Many nosocomial infections are now considered to be consequences of surface contaminations of indwelling medical devices, or of equipment such as air conditioning and water-distribution networks [3]. In spite of expensive cleaning procedures, the survival and proliferation of Pseudomonas, Salmonella or Listeria at the surface of production equipment used in food-processing industries are also common causes of foodborne infections. Corresponding author: Philippe Lejeune ([email protected]).
In recent years, numerous studies have been devoted to the process of surface colonization and the physiology of mature biofilms [4 – 6]. Knowledge gained in these areas could now be exploited against detrimental biofilms. In my opinion, two different strategies need to be adopted: first, to prevent adhesion, by interacting with the surfacesensing processes to repel the pioneering cells and to maintain the cellular sensitivity to antimicrobial agents; and second, to destroy the organization of differentiated biofilms and then wipe them out by conventional cleaning procedures. In this Review, I will focus on the first strategy: even if, at present, it seems impossible to protect definitively the surface of biomaterials or equipment against microbial contamination, it is now conceivable to develop drugs and coatings that are able to delay colonization. As the development of infections on prostheses frequently results from bacteria introduced at the same time as the implanted device, any delay (even a few hours) in the colonization process could successfully increase the capacities of both the preventive antibiotic therapy and the immunological defenses to eradicate the infection. Likewise, simply delaying the contamination of catheters could constitute significant progress because, in most cases, the catheters are implanted for less than three or four days. In hospital and industrial settings, surface treatments able to retard adhesion could greatly enhance the efficiency of daily cleaning and disinfection procedures because free-floating microbes remain sensitive to detergents and biocides. The very first stages of surface colonization An individual bacterium can reach a surface by three different modes [7]: passive transport due to the flow of a gas or liquid; diffuse transport resulting from Brownian motion; and active movement involving flagella. Afterwards, what happens at the very moment the bacterium collides with the surface is ‘simply’ a question of physicochemical interactions between two bodies carrying, or not carrying, reactive domains. The present state of these two bodies is, of course, the result of past events, namely interactions of the surface with molecules of water and dissolved substances of the liquid phase, and the physiological modulation of particular components of the bacterial envelope by various parameters of the medium, such as temperature and nutrient availability. It seems
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obvious, therefore, that both the nature of the surface and the culture conditions can have a profound influence on the colonization kinetics in laboratory flask environments. And this is what is actually observed. For instance, Chavant et al. [8] have shown that the hydrophilic surface properties of Listeria monocytogenes cells are negatively correlated with growth temperature and that the colonization kinetics are significantly different on hydrophilic (stainless steel) and hydrophobic (Teflon) substrata. In real life, the situation is largely complicated by a pivotal feature of materials exposed to natural fluids: surface conditioning by macromolecules. It has been recognized for a long time that immersion of a clean substratum is immediately followed by fast and efficient adsorption of organic molecules onto the surface [9], forming so-called conditioning films, but the characterization of these structures by modern physicochemical techniques is only just beginning. Although it already seems clear from pioneer studies that, in artificial [10] and natural [11] conditions, the organic molecules never cover the whole surface, conditioning films are an important source of heterogeneity and surface property modification. Their inevitable occurrence in any particular situation in which biofilms are detrimental has therefore to be carefully considered when trying to develop surface treatments. An individual bacterium that approaches a solid surface has to overcome a possible repulsive force and interact with the solid phase or the conditioning film. Depending on the strength of the bonds that can be formed, bacterial motility and gliding properties are often of crucial importance in initiating efficient attachment. In different bacterial species, transposon insertions conferring the loss of the adherent phenotype have been found in genes involved in flagellar motility (Table 1). Accordingly, a nonadherent phenotype could be observed after transposon inactivation of two types of bacterial gliding movement: twitching motility relying on type IV pilus extension and Table 1. Appendages and envelope structures involved in abiotic surface colonization Species
Structure
Refs
Escherichia coli
Flagella Type I fimbriae Lipopolysaccharide Curli Conjugative fimbriae NplE lipoprotein Type III fimbriae Flagella Type IV fimbriae Putative organelle (CupA) Flagella Curli Flagella Lipopolysaccharide Bap adhesin Teichoic acids AtlE autolysin SSP1 adhesin Peptidoglycan Flagella Mannose-sensitive hemagglutinin fimbriae
[13] [13] [28] [18] [29] [23] [30] [31] [31] [32] [33] [34,35] [26] [26] [36] [37] [38] [39] [40] [41] [42]
Klebsiella pneumoniae Pseudomonas aeruginosa
Pseudomonas fluorescens Salmonella enterica
Staphylococcus aureus Staphylococcus epidermidis Streptococcus gordonii Vibrio cholerae
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retraction in Pseudomonas aeruginosa, and swarming due to overflagellation in Salmonella enterica (Table 1). The next step in early attachment events is an interaction sufficiently strong to prevent disruption by convective forces or Brownian motion. The first link between the bacterium and the material (or the conditioning film) is presumably a combination of weak chemical bonds, dipole interactions, and hydrophobic interactions [12]. Two situations are possible: direct interactions between constitutive components of the bacterial envelope and the surface, or bridging mediated by specialized bacterial structures of attachment, such as fimbriae or adhesins. Different genetic strategies have recently been used to identify these bacterial structures (Table 1). Interestingly, given that type I pilus-associated attachment of E. coli requires flagellar motility [13], whereas adhesion mediated by curli is independent of strain motility [14], the adhesion processes mediated by two types of fimbriae can be different, presumably because of differences in the strength of the chemical bonds that these structures are able to form. Surface sensing A major concept of biofilm study is the difference in the physiological state between free-floating and attached bacteria. During the past 70 years, changes in global functions, such as growth rate, respiration and assimilation, have been correlated with substratum-attached growth (for a review of the early literature on the influence of surfaces on microbial behaviour, see Refs [7,9]). Recently, the use of microscopy and reporter gene techniques has clearly established that precise changes in gene expression are triggered very early during the transition between the swimming and attached states. Using lacZ fusions, Davies et al. [15] examined the expression of algC, a gene required for alginate synthesis, within colonizing P. aeruginosa cells. As early as 15 minutes after the initial attachment, they observed induction of algC expression. In addition, time-lapse microscopic observations of P. aeruginosa adhesion showed that the organisms move along the surface before attachment, almost as if they are scanning for an appropriate location for initial contact [4]. These observations indicate that sensing systems are activated when individual bacteria come into contact with the surface. To gather information on these cognitive processes, Prigent-Combaret et al. [16,17] isolated a library of lacZ gene fusions by random insertion of a transposon (carrying the promoterless lacZ gene) in the genome of an E. coli K-12 mutant strain able to colonize hydrophobic and hydrophilic materials [18]. By using a simple screen, these authors showed that, among 446 Lacþ fusions, 98 are significantly more expressed in the attached cells (1.5- to 10-fold), and 73 are less expressed in these cells (2- to 10-fold), after 24 hours of colonization. The synthesis of flagella is one of the repressed functions, whereas production of colanic acid, a major matrix exopolysaccharide, is induced in attached bacteria. To identify the external parameters and bacterial structures that enable the bacteria to discriminate between the liquid phase and the interface, the same
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authors assumed that the presence of electric charges on the solid surface and on the molecules of the conditioning film could increase osmotic strength or, more precisely, decrease water activity (i.e. the proportion of water molecules acting as pure solvent), at the liquid– solid interface by interacting with ions and water molecules. Because it is well-established that, in E. coli, during the initial hours after an osmotic shift, there is a quasi-linear relationship between the osmolarity of the external medium and the intracellular concentration of potassium, Prigent-Combaret et al. compared the intracellular Kþ concentration of planktonic and adherent cell populations [16]. Ten hours after inoculation, the attached bacteria displayed a significantly higher concentration than the free-floating cells. This result is important because it strongly indicates that the water activity of the attached cell micro-environment, at an early stage of the colonization process, is lower than in the liquid phase. Furthermore, because the control of water activity inside cells is an essential function that requires permanent monitoring of external water activity, all unicellular organisms are equipped with efficient sensors for this parameter. It can, therefore, be assumed that local water activity is a universal parameter that enables contaminating microbes to identify a solid– liquid interface and evaluate its physicochemical state. The role of sensor-regulator systems in surface sensing Another indication of the involvement of water activity in surface recognition is the importance of the signal transduction system EnvZ/OmpR in the curli-mediated adherence of E. coli strains. Vidal et al. [18] described a mutant allele of the ompR gene able to confer an adherent phenotype when transduced into many K-12 strains. A single point mutation resulting in the replacement of a leucine by an arginine residue is responsible for this property. The production of curli and the expression of the curlin-encoding gene are increased in the presence of this allele. The authors explained their observation by a possible increased efficiency of interaction between the mutant OmpR protein and the RNA polymerase at the regulatory sites of the target gene. On the other hand, the transduction of knockout mutations, in either ompR or the curlin gene, results in a loss of the adhesion properties of several biofilm-forming strains, including two clinical strains isolated from patients with catheterrelated infections [18]. EnvZ and OmpR are members of a large family of homologous proteins that are collectively referred to as sensor-regulator systems. These two-component systems enable bacteria to respond to various environmental parameters by means of the phosphorylation of a cytoplasmic response regulator by an inner membrane sensor. EnvZ/OmpR has been described as an osmosensing system that responds to external osmolarity by regulating the transcription of the ompF and ompC porin-encoding genes (for a review, see Ref. [19]). Given that EnvZ/OmpRmediated osmotic induction of curli and colanic acid syntheses has been demonstrated [16,18,20], the implication of this two-component system in surface sensing is highly probable. http://timi.trends.com
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CpxA/CpxR is an E. coli two-component system that responds to signals caused by disturbances in the cell envelope and activates genes encoding periplasmic protein folding and degrading factors [21]. The Cpx pathway negatively affects the expression of the operon coding for curli synthesis [22], and specific binding of CpxR to promoter regulatory sequences has been demonstrated [20]. Recently, Otto and Silhavy [23] showed that this signal transduction system is activated after adhesion of E. coli to hydrophobic surfaces. The expression of Cpx-regulated genes (cpxR, cpxP, spy, dsbA, degP) is induced during the first hour of the adhesion process. Inactivation of either the cpxR or the cpxA gene abolishes the adhesion-induced expression of one of the target genes, which demonstrates that the increased transcriptional activity of Cpx-dependent genes during adhesion follows the known molecular mechanisms of signal transduction by means of the CpxA/CpxR system. Moreover, the contact-dependent induction of the signal transduction pathway requires the outer-membrane lipoprotein NplE, which could be responsible for hydrophobic interactions with the surface. The forces created might cause a major perturbation in the envelope, resulting in monomer and misfolded protein accumulation within the periplasm, consequently activating the Cpx pathway. Another link between two-component systems and biofilm formation has been recently identified by Drenkard and Ausubel [24]. They found that antibiotic-resistant variants of P. aeruginosa with enhanced ability to form biofilms occur very frequently both in vitro and in the lungs of cystic fibrosis patients. There is a class of resistant colonies that are smaller than wild-type and display a rough phenotype. When cultivated on antibiotic-free medium, wild-type revertants, characterized by a large colony size, smooth appearance, wild-type levels of susceptibility to antibiotics and reduced biofilm-forming ability, arise at high frequency, indicating that the phenotypic changes observed in the resistant variants are the result of a phase variation mechanism. To identify putative P. aeruginosa factors involved in the switching between the two phenotypic forms, the authors transferred a cosmid library of P. aeruginosa chromosomal DNA to a resistant clone. One of the transconjugant clones is able to greatly enhance the rate of reversion: 100% of the cosmid-carrying cells form wild-type colonies after overnight incubation. Subcloning experiments identified a single open reading frame (ORF) responsible for this high reversion rate. The ORF shows sequence similarities to the response regulator elements of two-component systems, including VieA of Vibrio cholerae and a probable two-component regulator identified in the P. aeruginosa strain PAO1. These last results indicate that overexpression of the regulator component restores the wild-type pattern of gene expression and they strongly suggest that, in wildtype P. aeruginosa cells, a two-component system is able to switch on and off several functions, such as biofilmforming capacity and antibiotic resistance, in response to unknown environmental parameters.
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Table 2. Signalling systems involved in surface colonization Species
Sensing system
Stimulation factor
Refs
Bacillus subtilis Escherichia coli
PkrC Ser/Thr protein kinase EnvZ/OmpR two-component system CpxA/CpxR two-component system Unknown CsrA/CsrB carbon storage regulator BarA/UvrY two-component system Crc catabolite repression control protein GacA/GacS two-component system LasI production of homoserine lactone ComD histidine kinase ComD/ComE two-component system
Unknown Water activity, pH Disturbances in the cell envelope Cellular density Nutrient availability Hydrogen peroxide Nutrient availability Unknown Cellular density Cellular density Cellular density
[43] [18,20] [22,23] [16] [44,45] [45] [46] [47] [48] [40] [49]
Pseudomonas aeruginosa
Streptococcus gordonii Streptococcus mutans
A model for surface sensing As shown in Table 2, other sensing systems have recently been related to biofilm formation. Although there are many fascinating open questions that require further
investigation, all these data are consistent and a model of surface sensing can be proposed (Fig. 1). The very first step of the sensing process occurs when a swimming or free-floating bacterium perceives the presence
(a)
Surf
Appendage monomer
Organic molecule of the conditioning film Secreted organic molecule
OM Per IM Cyt
(b)
Surf
OM Per IM Cyt
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Fig. 1. A model of surface sensing. When swimming (a) convective forces (symbolized by two arrows) break bacterial appendages and disperse secreted molecules. Immobilization (b) reduces dispersion and appendage breaks. A new micro-environment is created between the solid phase and the surface of the cell. The electric charges of the solid surface, the organic molecules of the conditioning film, the appendages, the microbial surface, and the secreted organic molecules decrease water activity. Because of the ‘traffic jam’ appendage, monomers and other molecules accumulate within the periplasm, and also decrease water activity. Activation of sensing mechanisms, such as two-component systems, occurs and various metabolic changes are triggered. Surf, abiotic surface; OM, outer membrane (with two trimers of porin); Per, periplasm; IM, inner membrane (with two sensors, red points: phosphate groups); Cyt, cytoplasm (with two regulators). http://timi.trends.com
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of a solid phase. For sure, every microbe is equipped with sensor structures that constitutively measure water activity, pH and nutrient concentrations. These structures have generally been studied in laboratory conditions as ‘stress’ response components. However, in reality, one of their functions is probably to allow the cell to adopt instantaneous changes in behaviour, such as positive and negative chemotaxis. Kolter’s group has reported the importance of surface scanning and chemotaxis mechanisms for biofilm formation [4,13] and it seems, therefore, to be established that flagellated bacteria can be attracted to or repelled by the surface. This is obviously of primary importance for the design of anti-biofilm coatings. A pivotal question that remains to be answered concerns the behaviour of non-flagellated bacteria, such as Staphylococcus aureus and Staphylococcus epidermidis. Are these bacteria attracted by the surface as iron by a magnet, or are they able to make a choice and ‘refuse’ the contact ? The second step is the initial attachment. At this moment, three fundamental changes occur: first, because of immobilization, the convective forces that are able to break bacterial appendages and rapidly remove secreted molecules are weakened; second, a new micro-environment is created between the solid phase and the cell, and its composition will be modified by the cellular metabolism; and third, part of the envelope is exposed to short-distance interactions determined by the physicochemical state of the solid phase. In addition, the assembly of certain fimbriae, for instance curli, is an extracellular process. It has been reported that E. coli excretes copious amounts of monomers to synthesize its curli [25]. When a bacterium swims, it is conceivable that a large part of the monomers could be wasted. However, following immobilization, fimbriae synthesis could be much more efficient and significantly modify binding, water activity around the cell, and monomer concentration within the periplasm. All these changes constitute signals that can be transduced to the genome by two-component systems, such as EnvZ/OmpR and CpxA/CpxR. The future: how to blind pioneering bacteria In my opinion, there are three possible pharmacological targets that could be used to prevent microbial adhesion: the sensor systems involved in chemotaxis; the twocomponent regulatory systems; and the cellular structures responsible for interaction with the solid phase. To target the sensor systems involved in chemotaxis, diffusible repellent molecules could be incorporated into a gel smeared on implanted medical devices, such as prostheses. These molecules would divert the passage of motile bacteria and keep them in the liquid phase, where they would remain sensitive to antibiotics and to immune defense mechanisms. The two-component regulatory systems are now considered to be promising therapeutic targets for general antibiotics [26] but, in addition, it can be assumed that particular compounds, old or new, that have been rejected during classical screening procedures for reasons of toxicity, secondary effects or weak activity in vivo could http://timi.trends.com
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be very efficient when applied locally via diffusion from a gel covering the biomaterial. Lastly, the third target named are the cellular structures responsible for interaction with the solid phase. When grafted or smeared on the surface, surfactants [27] or polymers carrying electric charges could modify the physicochemical properties of the interface and decrease the binding ability of the colonizing organisms. To find molecules able to weaken, and consequently delay, adhesion is a difficult but fascinating task. I think that we have to be aware that, in nature, most microbes live within highly organized multispecies consortia. During consortium development, bacteria carry out precisely, in terms of time and place, certain functions necessary for the construction of the whole. Within these very stable communities, the microbes are constantly giving out messages to their partners and also to eventual enemies, of which antibiotics are an example. It would seem to me very worthwhile to look for those compounds that, at low doses, are able to act on the functions essential to the structuring of the microbial ecosystems and, afterwards, to look at their anticontamination, and even antimicrobial, capacities at high doses. Acknowledgements Research in the author’s laboratory was funded by grants from the French Defense Ministry (96/048 DRET) and the Centre National de la Recherche Scientifique (Re´seau Infections Nosocomiales).
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