The prognosis does not look good for the immediate future: too little is known about persisters to suggest ways of eradicating them. Knowing where to look for the cause of biofilm resistance, however, is a good place to start. Genes responsible for persistence can be identified (in addition to the examples discussed above), and these may serve as targets for drug discovery. Any inhibitor of a factor that causes persistence could then be combined with a conventional antibiotic such as a fluoroquinolone to eradicate a biofilm. Such a dual therapy is logistically similar to the currently used β-lactam–β-lactamase inhibitor combinations or MDR inhibitor-antibiotic combinations which are in development (37
). Both types of approaches are based on a combination of an antibiotic and a substance inhibiting the mechanism of resistance to this antibiotic.
Development of drugs disabling the persister phenotype is likely to provide an effective therapy for biofilm infections and other types of infections in which persisters are a problem, but this will take time. Meanwhile, a variety of approaches that can be used to fight or prevent biofilm infections are being tested.
The only specific antibiofilm therapy presently in use is based on the incorporation of antibiotics into the material of indwelling catheters (16
). The combination of rifampin and minocycline is especially effective. This approach decreases the probability of colonization and is in essence a prophylactic measure. This seems to be a straightforward and useful approach, although it has obvious limitations. Bacteria resistant to the impregnated antibiotic will colonize the indwelling device; the method is probably limited to relatively short-term catheters and will not be effective for artificial joints or heart valves, nor does it address the issue of biofilm infections unrelated to indwelling devices.
Two interesting physical approaches to the eradication of biofilms are being developed: the use of an electromagnetic field (38
) or ultrasound (55
), both in conjunction with antibiotic therapy. These promising methods are in preclinical stages of development.
Biofilm development is an area of intense research (see reference 47
for a recent comprehensive review), and the components involved in development have been considered possible targets for therapy. Random transposon insertion libraries were used for a generalized screen for “biofilm genes” by detecting the ability of mutant clones to adhere to the wells of microtiter plates. This approach was pioneered by Pierre Genevaux and coworkers (21
) and was originally applied to E. coli
. Similar studies were then performed with a number of other species: Pseudomonas fluorescens
), P. aeruginosa
), and Vibrio cholerae
). An independent study of E. coli
was done as well (52
). In all cases, biofilm formation was impaired in nonmotile mutants. Pili, although different kinds for each species, were found to facilitate initial adherence. Genes coding for the synthesis of exopolysaccharide were found to be necessary for biofilm formation in V. cholerae
. A detailed analysis of the dynamics of biofilm formation with some of these insertion mutants showed that motile cells are better at reaching the surface, accounting for the need for flagella. Pili are specialized attachment organelles and, not surprisingly, assist with biofilm formation. These findings confirmed previous observations that motility (30
) and pili (64
) are needed for biofilm formation. Some other surface adhesion factors as well as regulators of expression of surface compounds were found to be involved in biofilm formation (47
Is the presence, then, of flagella, pili, and exopolysaccharide sufficient to build a biofilm? Perhaps exopolysaccharide alone is sufficient in species that lack flagella or pili? Interestingly, exopolysaccharide synthesis defects were found to prevent biofilm formation only in V. cholerae
and not the other species studied. This might be due to the redundancy of different polysaccharides. For example, alginate is formed copiously in some strains of P. aeruginosa
and is believed to contribute to the pathology of cystic fibrosis. However, strains deficient in alginate production use other exopolysaccharides to form biofilms. It is also important that the ability of cells to adhere to a surface strongly depends on the nature of the surface. For example, the presence of pili was found to actually inhibit the attachment of P. aeruginosa
to contact lenses (20
). It is clear that both the surface of the cell and the surface of the substratum determine the effectiveness of adhesion in biofilm formation. Numerous surface adhesins of pathogens, of which the pilus is only one example, will facilitate binding to host cells (2
) and abiotic surfaces (27
). These adhesins might or might not play a role in biofilm formation on a particular artificial surface.
One limitation of the transposon insertion screening studies is that they test the mass of the cells making up the biofilm, which will not report defects in biofilm architecture. It was recently found that the quorum-sensing factor N
-homoserine lactone (HSL) is required for the formation of a biofilm with a complex “wild-type” architecture: rather loosely packed cell masses with a mushroom appearance with substantial amounts of exopolysaccharide and aqueous channels traversing the entire biofilm (17
). A P. aeruginosa lasI
mutant defective in HSL production formed thin, dense biofilms on a glass surface that were easily dislodged by sodium dodecyl sulfate (SDS), unlike the wild-type biofilms, which were not affected by SDS. We found that this mutant formed biofilms on polystyrene in the Calgary Biofilm Device that were not affected by SDS (11
). More importantly, the mutant biofilm showed the same level of resistance to ofloxacin as the wild type, suggesting that architecture or other properties of this defective biofilm do not affect its ability to produce persister cells and to resist killing by an antibiotic. The “mushroom” architecture of biofilms, with cell columns separated by water channels, evokes function. This would ease delivery of nutrients and release of metabolic products (17
). The sophisticated architecture in turn suggests that a dedicated program is in place to build a biofilm (47
). However, the usefulness of a well-structured biofilm compared to that of its flattened version has not been experimentally demonstrated.
This analysis brings me to an important question: are development proteins viable targets for antibiofilm drug discovery? Are antagonists of HSL good candidates for drug development? One problem with components like pili or flagella is that targeting of developmental components means that the therapy will provide a prophylaxis rather than a cure for a biofilm infection. The exopolysaccharide synthesis genes seem like a better potential choice as targets since these components are probably required for the maintenance of biofilm formation and not only for the initial steps of biofilm formation. However, redundancy of polysaccharides and the differences between the biosynthesis genes in various species is a serious limitation for possible drug development by use of this pathway as a target. Similarly, quorum-sensing factors vary among different species, and HSL does not appear to be required for the formation of a biofilm resistant to killing.
This analysis of options suggests that the development of a universal antibiofilm therapy, possibly on the basis of targeting of persister proteins, is a long-term project, yet a possible simple solution to biofilm infection follows directly from the dynamics of in vitro biofilm eradication. The rationale is to administer a cidal antibiotic, then withdraw it, and then add it again. The first application of antibiotic will eradicate the bulk of biofilm cells, leaving persisters. In a realistic example, ofloxacin decreases the size of a P. aeruginosa
biofilm from 108
cells to 105
). Withdrawal of the antibiotic will allow this persister population to start growing. Assume that after two divisions the persistence phenotype is lost. At this point, the new population of 4 × 105
cells will produce 40 persisters. A second application of antibiotic should then completely eradicate the biofilm. This type of a simple cyclical antibiotic regimen was proposed previously by Bigger (7
) for eradication of staphylococcal persisters. This approach might work in topical applications, in which the delivery of antibiotics can be well controlled. For example, biofilm infections are common in urinary catheters, into which a desired solution can be instilled. P. aeruginosa
biofilm infections of cystic fibrosis patients provide another example in which this approach might work well. Antibiotics can be delivered topically to cystic fibrosis patients as aerosols. The popular medication Tobra (PathoGenesis/Chiron) is a tobramycin aerosol. This antibiotic is very effective in eradicating planktonic cells, which explains the clinical usefulness of the preparation. However, as discussed above, biofilms are resistant to tobramycin. In a cyclical application, one would deliver an aerosol of a fluoroquinolone antibiotic like ciprofloxacin, which would penetrate the biofilm and kill the cells. A second antibiotic application after a minimal period of time that would be necessary for survivors to start growing and loose their persister phenotype could then eradicate the biofilm. The feasibility of a cyclical biofilm eradication approach will depend on the rate with which persisters lose resistance to killing and regenerate new persisters and on the ability to manipulate the antibiotic concentration. Development of resistance in a situation in which the antibiotic concentration is allowed to drop is a concern, but cycling of two different antibiotics could largely eliminate this problem. If this approach works for topical applications, it will encourage an inquiry into the possible use of cyclical treatment of systemic biofilms as well. It is entirely possible that successful cases of antimicrobial therapy of biofilm infections result from a fortuitous optimal cycling of an antibiotic concentration that eliminated first the bulk of the biofilm and then the progeny of the persisters that began to divide.
Another interesting possibility for biofilm elimination comes from the observations of biofilm self-destruction. P. fluorescens
readily forms a biofilm in a well-oxygenated environment, such as near the liquid surface on a glass slide inserted vertically in a beaker. As the oxygen gets depleted by the growing biofilm mass, a specific exopolysaccharide lyase is induced and digests the biofilm matrix, liberating the cells (1
). The result is a striking, almost complete disappearance of the biofilm. The authors suggested that the degradation of the matrix serves two functions: it provides nutrients for the starving biofilm and liberates cells, allowing them to seek greener pastures. The nutrient limitation in this experiment comes from oxygen deficiency rather than carbon deficiency, and it remains unclear whether a biofilm will self-destruct in response to any type of energy (or essential nutrient) limitation. It seems reasonable to expect that this dramatic and so obviously useful (to humans as well) ability of a biofilm to self-destruct is not limited to oxygen deficiency. Disassembly of the biofilm could be exploited to treat infections. One approach would be to emulate energy deprivation by providing inhibitors of oxidative phosphorylation. Such substances are usually toxic, but a number of topical antimicrobials are membrane-acting agents. The quaternary ammonium compound benzalkonium chloride or the cationic base chlorhexidine are pertinent examples. Salicylate, widely used in food preservation, is an uncoupler. It might very well appear that some of the topical antimicrobials are causing biofilm self-destruction to a certain extent. However, it must be pointed out that the aim of conventional antimicrobial therapy is to deliver and maintain the drug at the maximally achievable and safe level. A high concentration of an antiseptic like chlorhexidine will simply kill the majority of cells, will probably leave the persisters largely intact, and will not cause biofilm self-destruction. Synthesis and export of lyase are required for biofilm degradation, but these will happen only under conditions that decrease the energy level and that do not completely inhibit protein synthesis. The same logic would apply to industrial biofilm eradication (cooling towers, pipes, etc.): it might appear that an optimal low level of a biocide will be more effective than a high dose for the treatment of biofilms. Another and possibly more productive approach would be to develop specific drugs that interact directly with the components of the biofilm self-destruction pathway. In an experiment that could serve as a model for this approach, expression of alginate lyase from a controllable promoter increased sloughing of cells from a colony of mucoid P. aeruginosa
cells that overproduced alginate (10
). Genes controlling biofilm self-destruction might appear to be of more use than genes involved in biofilm formation.