Engineering Biofilms

Understanding how bacteria function in communities could lead to a host of new applications.

“Anywhere there’s a surface and water in the liquid state,” Tom Wood confirms, “you’re going to have biofilms.”

In riverbeds and showerheads. On the hulls of ships and inside pipelines. On contact lenses and joint prostheses and the gleaming white surfaces of your teeth. Biofilms, says Wood, professor of chemical engineering and biochemistry at Penn State, “are communities of bacteria that have the ability to cement themselves to a solid surface, and then—if you picture them in a river, say—rather than going with the flow they anchor down to a rock, and as the river goes by they get the nutrients they need and they’re able to thrive.”

“Communities” is the operative word. The biofilm that coats your teeth harbors more than 300 species of bacteria, working in concert. Most of these microbes either do no harm or are actually beneficial, but the few bad actors can saddle you with tooth decay and gum disease. 

Biofilms cause corrosion, a huge economic drain on industry and infrastructure, and are also increasingly recognized as a leading culprit in chronic disease, from childhood middle-ear infections to cystic fibrosis. Hospital infections are largely due to their ubiquitous presence.

These complex microbial communities, in short, cause a variety of problems, both inside the human body and out. But they also have the potential to do great good, from wastewater treatment to oil-spill clean-up to producing alternative fuels--if their biochemistry can be controlled. Wood believes that it can.

Complex microbial communities have the potential to resolve practical problems in ways that combine the laws of nature with engineering principles.

“The whole idea of my lab,” he says, “is that if we can understand the genetic basis of biofilm formation, then we can either get rid of a biofilm, or promote it to do whatever we want.”

Sleeper Cells

The Dutch scientist Anton van Leeuwenhoek first noticed biofilms back in 1683. When placing a scraping of plaque from his own teeth under one of his first-generation microscopes, he spotted a host of “very little living animalcules, very prettily a-moving.” For most of the next 300 years, however, biofilms were largely ignored, as microbiology focused on individual organisms in their free-floating, or planktonic, state.

“But bacteria do have this desire to hunker down and form an attachment to a solid surface,” Wood says.  “That’s the way they are in nature, primarily—living in communities.”

Living in communities, bacteria are much hardier than when floating around free. They’re far more resistant to antibiotics—up to a thousand times more resistant, according to common estimates. “They’re much harder to kill,” Wood acknowledges, “but they’re even trickier than that.” Standard antibiotic treatments, he notes, target bacteria that are growing, dividing, evolving. “But in a biofilm, up to 10 percent of the population is not actively metabolizing.”

Bacterial cells living in biofilm communities "talk" to each other through chemical signals.  As more and more cells aggregate, the concentration of signals increases. “The chemical will build up and up, and eventually you’ll reach a threshold,” Tom Wood says. "This is how the cell monitors what’s going on around it.”

Cell-Cell Communication

Bacterial cells living in biofilm communities "talk" to each other through chemical signals.  As more and more cells aggregate, the concentration of signals increases. “The chemical will build up and up, and eventually you’ll reach a threshold,” Tom Wood says. "This is how the cell monitors what’s going on around it.”

Image: Center for Biofilm Engineering, Montana State University

Under antibiotic attack, Wood explains, these bacteria in effect “put themselves to sleep” to avoid destruction. “If a cell is asleep, not dividing, the antibiotic has no effect,” he says. Then, when the coast is clear and the drug has run its course, these sleeper cells have the ability to wake themselves up and kick off a whole new infection.

Appropriately, they’re called persisters. Their discovery is fairly recent, and when and how they work are hot topics among researchers of infectious disease. “What’s really fascinating to me,” Wood says, “is that they don’t undergo genetic change at all. There’s no mutations, no change in the DNA. It’s the opposite of building up genetic resistance.”

Chemical Messages

Wood arrived at Penn State in January 2012, to fill the Endowed Biotechnology Chair in chemical engineering, with a joint appointment in biochemistry and molecular biology. “I’m a microbiologist in practice,” he likes to say, “but an engineer by training.” As such, he has always had an eye for down-the-road applications.

“At first, I was just interested in trying to clean up the world—engineering bacteria to get rid of toxic waste,” Wood recalls. Then he started thinking more broadly, about sustainable practices for other types of chemical manufacture. “We got to wondering, how could we use these bacteria we were creating to do remediation, and also to do green chemistry? And we figured it would have to be in biofilm reactors”—engineered systems for growing and exploiting bacterial communities. But in order to build successful reactors, Wood knew, he first had to get a better handle on how biofilms form.

Biofilms are, he suggests, “basically the beginning of a tissue--the beginning of us. What I mean is that as these bacterial cells join together and grow, they differentiate, like the cells of higher organisms. It’s not like one group of cells becomes a tooth and another group becomes an ear, as in a developing mouse or a human. But they differentiate themselves by turning on different genes at different times, according to what’s needed.”

Acting in the common interest requires communication, something bacteria achieve by cell-to-cell signaling. “Bacteria cells, whether free-floating or attached, are constantly secreting chemical signals,” Wood explains. As cells aggregate, however, held together by the slime that makes up the biofilm matrix, the concentration of signals increases. “The chemical will build up and up, and eventually you’ll reach a threshold,” he says. At that point, the signal crosses back into the cell and spurs it to act in some appropriate fashion. “It’s called quorum sensing. This is how the cell monitors what’s going on around it.”

Experiments have shown that quorum sensing figures in a remarkable range of coordinated behaviors. As Wood notes, “Some cells in the disease state will hide until they reach critical numbers and realize they can overwhelm the immune system. Then they attack.” Other biofilms will “agree” to a division of labor: one group of cells will remove oxygen and another group will secrete building blocks for the community.

In still other cases, a cell may be programmed to attack itself. As Wood explains it, bacterial cells contain enzymes called toxins, and, typically, corresponding antitoxins that—under normal circumstances—hold the toxins in check. In E. coli, the most-studied of all bacteria, researchers have so far identified 37 of these toxin/antitoxin pairings.

Under certain stresses, however, the antitoxins can be eliminated, freeing the toxin to damage the cell. Intriguingly, in the case of persister cells this is not a calamity but a survival choice. “The cell is not trying to kill itself,” Wood explains, “but just to slow down its growth rate, or make itself go to sleep.” Figuring out the nuts and bolts of how a persister actually achieves this feat and then manages to ‘wake up’ again when the time is right, he adds, “has been one of the thrusts of my lab.”

Harnessing the Potential

In 1999, at the University of Connecticut, Wood became one of the first researchers to engineer a biofilm for a real-world application, putting a protective film on a mild steel to prevent corrosion. “That was the beginning,” he remembers—“the first inkling that we could control biofilm reactions.”

An electron micrograph shows round Staphylococcus aureus bacteria, bound by a biofilm, on the surface of an indwelling catheter. Biofilms shield bacteria from antibiotic attacks and are responsible for most hospital infections.

Bacteria Bound by a Biofilm

An electron micrograph shows round Staphylococcus aureus bacteria, bound by a biofilm, on the surface of an indwelling catheter. Biofilms shield bacteria from antibiotic attacks and are responsible for most hospital infections.

Image: Janice Haney Carr/CDC

A decade later, he and colleagues discovered that fluorouracil, well known as an anticancer compound, could also be deployed to prevent quorum sensing. “There’s a company now in Canada that is using it to coat hospital catheters,” he says. The protective layer is intended to slow the inevitable formation of biofilm on the catheter surface, lessening the risk of infection.

Wood has studied another family of cell-signaling disruptors, called furanones, in a species of seaweed that lives off the coast of Australia. Furanones are now being considered as a possible alternative to standard antibiotics in the aquaculture industry, where antibiotic resistance is a serious problem.

In a paper published in January 2012, Wood and Arul Jayaraman of Texas A&M reported still another important advance. After characterizing a previously unknown signaling protein called BdcA, they engineered it to make biofilms disperse on command. “What this experiment shows,” Wood says, “is that we can control bacteria in consortia—more than one at a time. That means we can hope to control biofilm formation for more complicated applications.”

The next hurdle is to be able to dictate the positions of individual bacteria within a given biofilm. “If we can pull that off,” Wood explains, “we will have gone a long way to show how biofilms could be used in a biorefinery.” Doing so would also bring him closer to his old graduate-school dream of a sustainable chemistry.

“In my profession,” he says, “we need to manufacture chemicals. With conventional chemistry, that often requires harsh, polluting processes and solvents, and results in lots of waste.

“But what’s becoming clear is that just about anything you can make by conventional means, you can also make with a bacterium, with enzymes, and you can do it all in water. At the end of the day, then, everything is biodegradable. In essence, you can make the same chemicals for the same price without hurting the environment.

“That’s green chemistry. And that’s the kind of thing that I envision.”

 

Microbial Cunning

“The most basic part of our research,” Tom Wood says, “is the toxin/antitoxin systems that are key to persistence with antibiotics. Twenty years ago, nobody knew why they were there. Why would the bacteria cell incorporate something that could hurt it?”

Our research has since been able to link these systems to stress resistance, to biofilm formation, even to a kind of molecular altruism, Wood says. “Under viral attack, some cells will actually kill themselves to save the cells around them.” Amazingly, bacteria have apparently pilfered the machinery of toxin/antitoxin from their arch enemy—viruses.

“There’s always been this war between viruses and bacteria,” he explains. “It’s been going on for at least two-and-a-half billion years—and the viruses are winning.”

When a virus invades a bacterial cell, it incorporates its DNA into the cell’s DNA, so that whenever the cell divides, a copy of the virus will be made. “That’s its whole reason for being: to reproduce itself,” Wood says. “If the cell stops dividing, the virus will jump out, kills its host—it’s not a very polite guest—and go off in search of other healthy cells to invade.

“Meanwhile, though, spontaneous mutations are always happening in the cell’s DNA—that’s part of the way the bacteria evolves. Well, every once in a while a mutation will occur within the region of the embedded virus, before that virus has a chance to jump out.” With its genetic instructions scrambled and thus disabled, the virus in effect is captured. Frozen in time.

“So,” Wood continues, “you have some viruses that are inside the bacterial chromosome that have been stuck there unchanged for 50 million years—we call them viral fossils. And when we look at the genes of these fossils, we find they code for toxin/antitoxins.

“This is the kind of thing that continues to fascinate me. The cell is clever. It takes and adapts the tools of its enemy in order to control its own metabolism.”