0 1995 by John Wiley & Sons, Ltd. Bacterial membranes may change when ..... Noorda WD, Purdell-Lewis DJ, de Koning W, van. Montfort AMAP, Weerkamp AH.
MICROBIAL ECOLOGY IN HEALTH AND DISEASE
VOL.
8: 3 13-3 16 (1995)
Metabolic Responses in Biofilms D. J. BRADSHAW Microbial Pathogenicity Department, CAMR, Salisbury, Wiltshire SP4 OJG, UK
INTRODUCTION The majority of bacteria in natural habitats exist in association with solid surfaces, such as microscopic particles in aquatic environments, clay or sand particles in soils, or on the surfaces of animal or plant tissues. Despite this, most in vitro studies have focused on growth of bacteria in liquid cultures, usually maintained in a homogeneous state. Recently, there has been an explosion in interest in bacterial associations with many types of surfaces, and the problems which they may pose in a wide variety of situations. Many authors have stated that metabolic responses of bacteria in biofilms differ markedly from their planktonic counterparts (reviewed by Costerton et al. 6 ) . The aim of this review is to consider the mechanisms which may lead to such differences, and to describe some of these ‘biofilm-specific’ responses; some of the difficulties which may be encountered in studying biofilm bacteria will also be discussed.
Bacterial membranes may change when in direct contact with a surface. Differences in LPS patterns of Pseudomonas aeruginosa have bten found between cells grown planktonically or as a biofilrn.l4 Some workers have also suggested that there may be changes in cell surface hydrophobicity, resultin from contact of the bacterium with the ~urface!~ Furthermore, specific genes may be ‘switched on’, or up-regulated by contact with a surface. When Streptococcus mutans was grown as a biofilm on hydroxylapatite (HA) surfaces, more lactate was produced, compared with planktonic cells, and this effect was not linked to any surface buffering effect of HA.3 More recently, the production of cloned glucosyltransferase (GTF)-B was found to be up-regulated in biofilm cells of S. mutans. l 7 Similarly, alginate production was increased in biofilm cells of P. aeruginosa compared with planktonic cells.7 However, many other ‘effects of growth at the surface’ may in reality be indirect.
Indirect eflects Indirect effects of the surface include those that affect the local environment of the cell, rather than having a direct influence on the bacteria per se. There may be effects on the local pH of the environment, for example, the presence of clay Direct eflects particles may stimulate growth, primarily through Direct effects can be defined as those which stem maintaining pH close to the surface at near from the effect of the proximity of the surface to optimal levels, and also by supplying inorganic the bacterium. Ellwood et al. l o reported that cells nutrition to the nearby b a ~ t e r i a . ~ ~ , ~ ~ ? ~ ’ Bacteria grown in association with an ionin a biofilm appeared to grow at a greater rate than planktonic equivalents, and postulated that as a exchange resin showed reduced yield and substrate result of contact with a substratum the diffusion of affinity, presumably due either to binding of essenprotons away from the cell may be hindered, tial substrateskofactors or limitations of mass increasing the efficiency of membrane H+- transport through the biofilm15 (also see below). ATPases, and creating a more energised bacterial Alternatively, binding of substrate to the surface membrane adjacent to the surface. Thus, the cells might promote growth, due to concentration of attached to the surface would have an energetic nutrients at the interface. This was suggested advantage. There has been little experimental by the classical experiments of Heukelekian & evidence to back up this hypothesis, and there are HellerI6 and Z0Be11~~ in ‘low-nutrient’ environments. More recently, however, these observed several theoretical objection^.^^
POSSIBLE MECHANISMS FOR DIFFERENCES IN METABOLIC RESPONSES BETWEEN BIOFILM BACTERIA AND THEIR PLANKTONIC COUNTERPARTS
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effects have been attributed to experimental limi- system.27 There is an obvious potential for tations.” Many surfaces also have the potential to metabolic interaction between these species.23 act as electron acceptors or donors, which might Various studies have shown that DNA stability facilitate is increased in association with various surPotential substrates or inhibitors may leach f a c e ~ , ’ and ~ , ~ ~this appears to result in higher from the surface itself, with consequent effects on transformation rates of Bacillus subtilis in associabacterial activity and/or growth rate. Many plastic tion with sand particles.20 In addition, plasmid materials used in piping systems may, for example, transfer rates may be higher in biofilms, partially leach carbon sources into the medium, which may due to greater DNA stability, and ossibly due to promote g r ~ w t h .Fluoride ~ can be freed from juxtaposition of cells in a biofilm. 2 f728 fluorapatite/calcium fluoride in dental tissues, parA further possibility is that differences observed ticularly at low pH values, and is believed to be a in biofilm cells compared to planktonic are due to major factor in the cariostatic action of f l ~ o r i d e . ’ ~growth-rate related effects. Changes in the immediate environment of the cell, resulting from the formation of the biofilm, may lead to changes in PHYSICO-CHEMICAL EFFECTS O F growth rate. It has been well-documented over BIOFILM FORMATION many years that changes in growth rate affect all ~~ these During the development of biofilms, limitations aspects of microbial p h y ~ i o l o g y .Among on diffusion may allow gradients to develop. Al- effects are changes in antimicrobial sensitivity and though some workers have questioned the extent toxic product formation. Another ‘biological’ effect in biofilms relates to of such diffusion barriers,26 it seems clear that metabolism in biofilms can lead to the develop- the metabolic diversity of the population in the ment of gradients, and these may cause a variety of biofilm (dependent on the species diversity). Once effects, including: lack of penetration of antimicro- again, because of the close proximity of cells to bial agents (discussed in more detail below), or one another, there is the possibility of complex gradients of pH, oxygen, redox or metabolites. For metabolic interactions taking place, which might example, deep layers of the biofilm may be rela- not be possible in the more diffuse liquid phase. tively reduced, whereas the planktonic phase may When mixed cultures of nine or ten oral bacteria be relatively oxidised. In addition, the biofilin may were grown in a two-stage chemostat biofilm provide physical shielding for the organisms from model, in which the second stage was aerated, shear forces, engulfment by protozoa or host the presence of an oxygen-consuming organism, defences, or attack by viruses. As the matrix of Neisseriu subfuva, allowed greater numbers of the biofilm builds up, so the local chemistry anaerobes to grow in the biofilm compared to the may change, for example, the buffering capacity planktonic phase.4 However, in the absence of N. of dental plaque decreases as the extracellular subfuvu, the numbers of anaerobes were reduced, polysaccharide (EPS) content relative to bacterial both in the planktonic and the biofilm phase, and mass increases8 The significance of this is empha- particularly in younger biofilms. This work indisised by the fact that maximal enamel deminer- cates that, as expected, oxygen has an important alisation occurred when the EPS:bacteria ratio effect on cultures, but also that in biofilms, the presence of an aerobe can protect obligate was ~ 5 . ’ ~ anaerobes from the inhibitory effects of oxygen. ‘BIOLOGICAL’ EFFECTS OCCURRING IN BIOFILMS
ANTIMICROBIAL EFFECTS IN BIOFILMS
‘Biological’ effects comprise a number of specific interactions or effects which do not fit readily in the other categories of effects described, but have a biological basis. These include specific interspecies coaggregations” which may also involve close metabolic interactions. S. mutans and Veillonellu ulcalescens grown together gave greater demineralisation than S. mutans alone, when grown on HA surfaces in an artificial plaque
It is widely accepted that bacteria in biofilms show greater resistance to antimicrobial agents than equivalent planktonic cells.6,26The question which remains to be answered is whether cells in biofilms are inherently more resistant to antimicrobial treatment, compared to planktonic cells, or whether the observed reductions in sensitivity are merely the result of shielding by the biofilm matrix, or other indirect effects. Costerton and colleagues6 have
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found that biofilm cells are more resistant, and REFERENCES proposed that this is due to diffusion limitation. 1. Allison DG, Evans DJ, Brown MR, Gilbert P. However, other workers have suggested that diffu(1990). Possible involvement of the division cycle in sion limitations may not be sufficient to account dispersal of Escherichia coli from biofilms. Journal for differences in susceptibility to antibiotics.26 of Bacteriology 172, 1667-1669. Anwar et a1.’ found that ‘old’ biofilms are much 2. Anwar H, Strap JL, Costerton JW. (1992). Estabmore resistant to a variety of antimicrobial agents lishment of aging biofilms: possible mechanism of bacterial resistance to antimicrobial therapy. Antithan ‘young’ biofilms. A similar result was obmicrobial Agents and Chemotherapy 36, 1347-135 1. tained for biofilms of Streptococcus sanguis treated 3. Berry CW, Henry CA. (1977). The effect of adsorpwith ~ h l o r h e x i d i n eThese . ~ ~ results may be due to tion on the acid production of caries and differences between growth rates in the older and noncaries-producing streptococci. Journal of younger biofilms. Dental Research 56, 1193-1200. Gilbert and colleagues, in a number of studies 4. Bradshaw DJ, Marsh PD, Watson GK, Schilling using novel techniques to grow constant growth K. (1994). Effect of oxygen and inoculum comporate-biofilms,’ ‘,12,13 have found that most effects sition on development of mixed culture biofilm. purported to be due to the biofilm mode of growth Journal of Dental Research 73, 849 (abstract 504). are actually related to differences in growth rate. 5. Colbourne JS. (1985). Materials usage and When bacterial growth rate in a biofilm is controltheir effects on the microbiological quality of led, there are fewer differences between biofilm and water supplies. Journal of Applied Bacteriology Symposium Supplement, 47s-59s. planktonic cells. However, re-suspended biofilms 6. Costerton JW, Cheng KJ, Geesey GG, Ladd TI, are more susceptible than intact biofilm, suggestNickel JC, Dasgupta M, Marrie TJ. (1987). Bacing some effect on antimicrobial sensitivity of the terial biofilms in nature and disease. Annual ‘glycocalyx’ matrix of biofilm,’ and there is also Reviews in Microbiology 41, 435464. some evidence of cell-cycle dependency.’ 7. Davies DG, Chakrabarty AM, Geesey GG. (1993). Exopolysaccharide production in biofilms: substratum activation of alginate gene expression by SUMMARY Pseudomonas aeruginosa. Applied and EnvironIn conclusion, it is clear that the metabolic remental Microbiology 59, 1181-1 186. sponses of bacteria in biofilms d o differ from their 8. Dibdin GH, Shellis RP. (1988). Physical and bioplanktonic counterparts. However, there are a chemical studies of Streptococcus mutans sediments suggest new factors linking the cariogenicity of number of important points which must be borne plaque with its extracellular polysaccharide conin mind when attempting to identify biofilmtent. Journal of Dental Research 67, 890-895. specific responses. Firstly, many effects are 9. Duguid IG, Evans E, Brown MR, Gilbert P. indirect-resulting from the effect of the surface on (1992). Effect of biofilm culture upon the susceptithe immediate environment of the cell, rather than bility of Staphylococcus epidermidis to tobramycin. directly on the microorganism p e r se. Experiments Journal of Antimicrobial Chemotherapy 30, 803are difficult to control-for example, growth rates 810. in biofilms may be highly variable, and if these 10. Ellwood DC, Keevil CW, Marsh PD, Brown CM, effects cannot be controlled (or measured) then Wardell JN. (1982). Surface-associated growth. results may be difficult to interpret. Methods for Philosophical Transactions of the Royal Society of studying biofilms are far from standardised, London Series B 297, 517-532. and therefore direct comparisons between studies 11. Evans DJ, Allison DG, Brown MR, Gilbert P. (1990). Effect of growth-rate on resistance of from different groups are often difficult. Finally, Gram-negative biofilms to cetrimide. Journal of artifacts may be difficult to detect; for example, is Antimicrobial Chemotherapy 26, 473478. it possible that biofilm microorganisms are simply Gilbert P, Allison DG, Evans DJ, Handley PS, 12. sub-populations of the planktonic population, so Brown MRW. (1989). Growth rate control of that changes attributed to the biofilm mode of adherent bacterial populations. Applied and growth are merely a reflection of the different Environmental Microbiology 55, 1308-1 311. properties of these sub-populations? 13. Gilbert P, Collier PJ, Brown MR. (1990). Influence The study of biofilms in the future will require, of growth rate on susceptibility to antimicrobial therefore, the use of carefully designed experimenagents: biofilms, cell cycle, dormancy, and stringent tal systems, with appropriate controls; there is also response. Antimicrobial Agents and Chemotherapy a clear need for the development of novel methods. 34, 1865-1868.
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