CBE Biofilm Control Research Area:

Mechanisms of Biofilm Resistance to Antimicrobial Agents

Biofilms evade antimicrobial challenges by multiple mechanisms. These we have grouped into three broad categories:
    1) reduction of the antimicrobial concentration in the bulk fluid surrounding the biofilm;
    2) failure of the antimicrobial agent to penetrate the biofilm; and
    3) adoption of a resistant physiological state or phenotype by at least a fraction of the cells in a biofilm.

In the first scenario, the antimicrobial agent is depleted to ineffectual levels before it gets to the biofilm. In the second scenario, the antimicrobial agent is delivered to the surface of the biofilm, but is not effectively transported into the depth of the biofilm. In the third scenario, the antimicrobial agent permeates the biofilm, but is unable to kill microorganisms because they exist in a phenotypic state that confers reduced susceptibility. We further distinguish two versions of the last scenario. In the first, a nutrient limitation leads to slow-growing or starved regions in the biofilm. In the second, an intrinsic phenotypic switch occurs in the biofilm that does not depend on nutrient limitation. These mechanisms of biofilm protection are not mutually exclusive. Indeed, it seems likely that combinations of these three general types of resistance may work in concert.

The reduced susceptibility of biofilms has not been attributed to the usual mechanisms— mutation or acquisition of genetic elements coding for specific resistance genes—that account for conventional antibiotic resistance. For these mechanisms to explain biofilm resistance, the genetic modifications would have to be present in the biofilm but absent in the planktonic state. This does not appear to be the case. Even clearly susceptible microorganisms acquire marked resistance in the biofilm mode of growth. When dispersed from a biofilm, however, these microorganisms rapidly return to a susceptible state.

1.  Antimicrobial Depletion in the Bulk Fluid

If a biofilm exerts a chemical demand for the antimicrobial agent with which it is being challenged, then it is possible for these neutralizing reactions to reduce the bulk fluid concentration of the agent. Consider, for example, an antimicrobial susceptibility test performed first against a dilute suspension of planktonic cells and then against a heavily fouled biofilm specimen. It is not difficult to imagine that the antimicrobial concentration could be maintained during the planktonic test but significantly decreased during the course of the biofilm test. One could argue that this phenomenon is not a true resistance mechanism, but simply an unfair comparison. The biofilm is not being subjected to the same antimicrobial concentration as the planktonic reference test. However, it seems that this straightforward mechanism has been often overlooked. It would pay to keep antimicrobial depletion in the bulk fluid in mind as a possible explanation for poor antimicrobial performance against a biofilm. What this mechanism lacks in glamour it may recoup in practical importance.

Bulk fluid antimicrobial depletion can be diagnosed experimentally by measuring antimicrobial residual concentrations during both planktonic and biofilm tests. In some experimental systems, biofilm and planktonic disinfection can be measured in the same fluid, an approach that elegantly eliminates the possibility of unequal treatment concentrations.
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2.  Transport Limitation of Antimicrobial Penetration

Failure of an antimicrobial agent to rapidly or completely penetrate a biofilm is perhaps the most intuitively appealing explanation for biofilm resistance. It is this mechanism that Antonie van Leeuwenhoek invoked over three centuries ago in his seminal studies of dental plaque (which he called "scurf"):

"From whence I conclude, that the Vinegar with which I washt my Teeth, kill’d only those Animals which were on the outside of the scurf, but did not pass thro the whole substance of it."

There are two versions of the transport limitation resistance mechanism that we would like to distinguish. The first postulates that the biofilm matrix constitutes a barrier to the inherent mobility of antimicrobial agents. According to this hypothesis, the matrix physically excludes antimicrobial compounds from the biofilm. While powerful and generic in its ability to explain antimicrobial resistance, this mechanism poses the paradox of how such a matrix barrier allows nutrients to pass while excluding biocidal molecules of similar size. Measurements of effective diffusion coefficients in biofilms (Stewart, 1998) indicate that, while diffusion is somewhat retarded in biofilms, diffusive transport nevertheless proceeds at rates that are of the same order of magnitude as those in pure water. Even molecules of the size of oligonucleotide probes, lectins, and others readily penetrate intact biofilms as evidenced by their ability to stain the interior regions of biofilm specimens. The direct experimental demonstration of permeation of certain antimicrobial agents through biofilm also argues against a generic barrier to antimicrobial agent access.

The second and more plausible version of antimicrobial transport limitation in biofilms requires an interaction between the antimicrobial agent and the biofilm that neutralizes antimicrobial activity. The barrier to penetration in this case is reactive rather than physical: the rate of deactivation of the antimicrobial exceeds the rate of diffusive penetration. This mechanism is supported by experimental evidence in the case of hypochlorite (de Beer et al, 1994; Chen and Stewart, 1996; Xu et al, 1996). It is likely to be important for other highly reactive oxidants, such as ozone and hydrogen peroxide, and may be a factor for some non-oxidizing biocides as well (Stewart et al, 1998). It is also realistic for certain antibiotics, such as the beta-lactams, that are subject to rapid enzymatic degradation.

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3. Physiological Limitation of Antimicrobial Efficacy

It is clear that there are many instances in which a biofilm is too thin or is insufficiently reactive with the antimicrobial agent to manifest either of the preceding resistance mechanisms. In these cases, biological explanations for the reduced susceptibility of biofilm microorganisms are called for. We wish to distinguish two general types of biological limitations to biofilm susceptibility. The first type of biological limitation to biofilm susceptibility requires that at least some of the cells in a biofilm experience nutrient limitation and therefore exist in a slow growing or starved state. Such slow- or non-growing cells are hypothesized (or have been shown experimentally) to be less susceptible to many antimicrobial agents. The second type of biological limitation of biofilm susceptibility invokes the existence of a distinct, and relatively resistant, biofilm phenotype. This phenotype is not the result of a nutrient limitation. The hypothesized biofilm phenotype is adopted by a subset of the microbial population in a biofilm as a result of some other stimulus, for example, contact with a solid surface or attainment of a threshold cell density.

The spatial heterogeneity of physiological status within a biofilm may be a crucial issue in determining susceptibility to antimicrobial agents. To illustrate this point, suppose that the action of an antimicrobial agent is known to be growth rate or nutrient status dependent. Consider the two distinct scenarios regarding microbial growth rate profiles within the biofilm depicted below.