Antibiotics have become a regular feature in our medication. They are now recommended for a number of treatments of various diseases. But this over dependence on antibiotics has led to a number of problems, mostly notably diseases have become resistant to their effect.
A number of mechanisms have been explained in this regard. In recent years biofilms have come forward as a bacterial pathogenic tool.
Biofilms are a natural phenomenon of some pathogenic bacteria by which they gain protection against the host’s defence mechanisms. They are communities of slime-enclosed microorganisms that colonize ‘surfaces’. An example of a biofilm is the fuzzy coating we all feel on our teeth before brushing in the morning. It is the film that develops and can cause corneal infection on contact lenses that are not properly cleaned. It is the slippery substance on the bottom of a swimming pool that, unless removed by chemical applications, can transmit illnesses.
Biofilms are of two types: one is in which bacterial cells are embedded more randomly in a polysaccharide matrix or slime layer which often mediates adherence of cells to surfaces and second in which the bacteria produces slime materials to attach themselves to a surface or substrate.
Bacteria can attach to the surface, produce slime, divide and produce micro-colomes within the slime layer, and construct a biofilm which becomes an enriched and protected environment for themselves and other bacteria. Many bacteria have this ability. They can attach to solid surfaces and form biofilms that are defined as matrix-enclosed microbial populations adherent to each other and to surfaces or interfaces.
A number of forces are involved in the attachment of bacteria to the surface. The precise nature of the interaction is still a matter of intense debate. The final stage in the irreversible adhesion of a cell to an environmental surface, is its association with the production of extracellular polymer substances or EPS.
Most biofilm EPS contains sugars such as glucose, galactose, mannose, fructose, rhamnose, N-acetylglucosamine and others. This slime layer of EPS and bacteria entraps particulate of a variety of material.
Although biofilm production is associated with all wetted surfaces, living or non-living, yet in a medical context its formation itself may be considered a significant virulence factor that enables the infection to persist or spread in the host. Biofilm microorganisms commonly produce an extracellular polymeric slime matrix that appears to play both protective (increasing resistance to anti-microbials and desiccation) and mechanical (attachment of the biofilm to the surface and maintenance of mechanical stability) roles. Biofilms may provide a mechanism for the survival of clinically relevant microorganisms in many patient populations. Biofilms are responsible for more than 60 per cent of all bacterial infections. Its role in antibiotic resistance and pathogenicity is well established.
Biofilms are responsible for diseases such as otitis media, the most common acute ear infection in children in the US. Other diseases in which biofilms play a role include bacterial endocarditis (infection of the inner-surface of the heart and its valves), cystic fibrosis (a chronic disorder resulting in increased susceptibility to serious lung infections) and Legionnaire’s disease (an acute respiratory infection resulting from the aspiration of clumps of Legionnella biofilms detached from air and water heating/cooling and distribution systems). Biofilms may be responsible for a wide variety of “nosocomial” (hospital-acquired) infections.
Sources of biofilm-related infections can include the surfaces of catheters, medical implants, wound dressings or other types of medical devices. Bacteria growing in biofilms cannot be eradicated by antibiotics and biofilms resist the immunological and non-specific defence mechanisms of the body. Consequently, very high and/or long-term doses are often required to eradicate biofilm-related infections. The chronic P. aeruginosa infection in cystic fibrosis patients is an endobronchiolitis caused by bacteria producing a biofilm which makes them resistant to antibiotics and to the defence mechanisms of the body.
The resistance against antibiotics (e.g. aminoglycosides, beta-lactam antibiotics, fluoroquinolones) of biofilm growing bacteria can be due to several factors such as slow growth, penetration barrier and the presence of some enzymes from the bacteria, which cleaves and/or traps beta lactams antibiotics. The increased resistance of the biofilm growing bacteria means that antibacterial therapy usually fails with respect to eradication of the bacteria in the biofilm, although the susceptibility tests in the laboratory demonstrate sensitivity to the antibiotics used. Therefore, the ordinary techniques used in the laboratory to determine antibiotic susceptibility of planktonic growing bacteria cannot predict the possibility of eradication of the bacteria growing in biofilms. Biofilms evade antimicrobial challenges by multiple mechanisms. These we have grouped into three broad categories:
* Reduction of the antimicrobial concentration in the bulk fluid surrounding the biofilm.
* Failure of antimicrobial agent to penetrate the biofilm.
* 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 the 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.
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.
If the antimicrobial agent is reactively neutralized within the biofilm, it may fail to penetrate the full depth of the biofilm. This resistance mechanism depends on the relative rates of antimicrobial agent reaction and diffusion within the biofilm. 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 indicate that, while diffusion is somewhat retarded in biofilms, diffusive transport nevertheless proceeds at rates that are of the same order of magnitude of 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, 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. It is also realistic for certain antibiotics, such as the betalactams, that are subject to rapid enzymatic degradation.
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.
Biofilm development is an area of intense research, and the components involved in development have been considered possible targets for therapy. The exopolymer 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.
Antibiotics have become a regular feature in our medication. They are now recommended for a number of treatments of various diseases. But this over dependence on antibiotics has led to a number of problems, mostly notably diseases have become resistant to their effect.
