Biofilms

A new understanding of these microbial communities is driving a revolution that may transform the science of microbiology

Joe Harrison, Raymond Turner, Lyrium Marques, Howard Ceri

United We Stand

Figure 5. Many biofilms can cause disease...

The Centers for Disease Control and Prevention estimates that up to 70 percent of the human bacterial infections in the Western world are caused by biofilms. This includes diseases such as prostatitis and kidney infections, as well as illnesses associated with implanted medical devices such as artificial joints and catheters and the dental diseases—both tooth decay and gum disease—that arise from dental plaque, a biofilm. In the lungs of cystic fibrosis patients, Pseudomonas aeruginosa frequently forms biofilms that cause potentially lethal pneumonias. There is a long list of biofilm-related ailments, and many scientists believe the list will continue to grow as we learn more about the function of these microbial structures.

In almost all instances, the biofilm plays a central role in helping microbes survive or spread within the host. That's because the slimy matrix acts as a shield, protecting pathogenic bacteria from antibodies and white blood cells, the sentinels of the immune system. Biofilms are also notorious for their ability to withstand extraordinarily high concentrations of antibiotics that are otherwise lethal in smaller doses to their planktonic counterparts. In fact, a biofilm can be 10 to 1,000 times less susceptible to an antimicrobial substance than the same organism in suspension.

This challenge, with its grave implications for the fight against pathogens, has been the focus of our research group's investigations. We have developed and licensed to a Canadian startup company a technology (the Calgary Biofilm Device, now called the MBEC Assay) that can be used to rapidly screen biofilms for their sensitivity to antimicrobials. A pharmaceutical laboratory testing a potential drug to fight pneumonia or catheter-related infection can now find out whether a drug that is effective against free-floating pathogens will be successful in eradicating the same organisms in a biofilm.

During the development of this technology, we have learned some remarkable things about biofilms. We have moved on to exploring some pathogenic "co-biofilms" of unrelated species living together, along with specific mechanisms that may be important in drug development. For example, biofilms' resistance to high metal concentrations makes them useful in removing toxic metals from the environment. But a detailed understanding of how the films handle metal toxicity may also open the door to antimicrobial treatments targeted at biofilms.

Figure 6. Biofilms derive their extraordinary tolerance...

We and other investigators have learned that part of the extraordinary resilience of bacteria arises from the remarkable heterogeneity inside the biofilm. Microbes closest to the fluid that surrounds the biofilm have greater access to nutrients and oxygen compared with those in the center of the matrix or near the substratum. As a result, the bacteria in the outer layers of the community grow more quickly than those on the inside. This comes into play as a defense mechanism because many antibiotics are effective only against fast-growing cells, so the slow growers within the biofilm tend to be spared. Moreover, the cells in the center of the community are further protected from the environment because the biofilm matrix is negatively charged. This restricts the entry of positively charged substances, such as metal ions and certain antibiotics.

One of the most intriguing defense mechanisms enabled by the formation of a biofilm involves a kind of intercellular signaling called quorum sensing. Some bacteria release a signaling molecule, or inducer. As cell density grows, the concentration of these molecules increases. The inducers interact with specific receptors in each cell to turn on "quorum sensing" genes and initiate a cascade of events, triggering the expression or repression of a number of other genes on the bacterial chromosome. Some bacterial strains seem to rely on quorum sensing more than others, but anywhere from 1 to 10 percent of a microbe's genes may be directly regulated by this process.

Quorum sensing is known to affect the production of enzymes involved in cellular repair and defense. For example, the enzymes superoxide dismutase and catalase are both regulated by quorum sensing in P. aeruginosa, which forms mucoidal clusters of bacterial cells embedded in cellular debris from the airway epithelial layer in the cystic fibrosis patient's lung. The first enzyme promotes the destruction of the harmful superoxide radical (O₂ ^-), whereas the second converts the equally toxic hydrogen peroxide molecule (H₂O₂) into water and molecular oxygen. These enzymes help the biofilm survive assaults not only from disinfectants, but also from the cells of a host's immune system that typically kill bacteria by unleashing antimicrobial agents, including reactive oxygen species.

Quorum sensing may also be involved in the defense against antibiotic drugs. Here the mechanism increases the production of molecular pumps that expel compounds from the cell. These so-called multidrug efflux pumps reduce the accumulation of the antibiotics within the bacterium and even allow the microbe to grow in the presence of the drugs.

There is also heterogeneity among the cell types in the biofilm that contributes to antimicrobial tolerance. Specialized survivor cells, called "persisters," are slow-growing variants that exist in every bacterial population. They are genetically programmed to survive environmental stress, including exposure to antibiotics. Although persisters do not grow in the presence of an antibiotic, they also do not die. Persisters are not mutants; even in a genetically uniform population of cells a small portion undergo a spontaneous switch to the persistent form. This past year Kim Lewis of Northeastern University demonstrated that persisters generate a toxin, RelE, that drives the bacterial cell into a dormant state. Once antibiotic therapy has ceased, the persisters give rise to a new bacterial population, resulting in a relapse of the biofilm infection.

The use of persister cells as a defense mechanism may have evolved early in the history of life. In this post-genomics era, scientists have learned that many related genes are present in a variety of distantly related bacteria, suggesting that similar genes were present in the primeval common ancestors. Yet the reduced growth rate of the persisters poses a paradox because slowed cell division decreases the fitness of a population. Edo Kussell and his colleagues at Rockefeller University recently proposed that bacterial persistence may have evolved as an "insurance policy" against rare antibiotic encounters. If so, in attempting to overcome bacterial antibiotic tolerance, scientists are battling an ancient mechanism that may have been refining itself for billions of years. If we are ever to succeed in controlling bacterial infection, more research efforts need to be focused on biofilms rather than the comparatively vulnerable planktonic form.

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