Engineering Biofilms

“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.

“In joint replacement surgery,” Wood says, “if an infection takes hold, there’s no drug they can add to get rid of it. They have to go back in, take out the original prosthesis, and put another one in—and hope the same thing doesn’t happen all over again.” Over 65 percent of all microbial infections are attributable to biofilms, according to the National Institutes of Health.

intraorally developed biofilm
By Ronald Ordinola Zapata (Own work) [CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons

Bacterial communities just like these thrive on your gums and teeth (and everyone else's too).

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.

“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. Over the last couple of decades, scientists have started to look at that state, and there’s been an exponential increase in biofilm literature and studies. There’s now even a mouthwash that talks about including anti-biofilm compounds—so the public’s waking up to it, too.”

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.”

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.

“Right out of college,” he remembers, “I was working in industry, making things that kill bacteria.” At Rohm and Haas, the chemical manufacturing giant headquartered near Philadelphia, Wood developed cosmetics. “Then I just got interested in understanding more about how bacteria live,” he says simply. He made the decision to go to graduate school at North Carolina State, focusing on biotechnology.

“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.

explanatory graphic of the formation of bio-film over time, from a few minutes up to a few months
By Clemencedg (Own work) [CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0) or GFDL (http://www.gnu.org/copyleft/fdl.html)], via Wikimedia Commons

After landing on a suitable substrate in the presence of water, bacteria form communities, held together by an an extracellular polymer matrix (a.k.a., slime). Eventually, elements of the biofilm move on to colonize other substrates.

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.”

When first approaching the problem, Wood says, he figured the cell’s options under the circumstances are somewhat limited. “If you look at the classic steps of protein synthesis, you go from DNA to messenger RNA, and then the messenger RNA becomes protein,” he notes. If you’re a toxin, and you want to stop the cell from producing a protein, “You could eat up all of your DNA, but if you do that you’re never going to wake up, because you’ve lost your genetic blueprint. You could gobble up the protein itself—but the protein is changing all the time anyway, to adapt to conditions.

“That leaves the thing in the middle—RNA. What the toxin will do is eat that intermediate message, and thereby put the cell to sleep.” A series of experiments confirmed his hunch: “We found the specific enzymes that eat the RNA.”

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.

“We knew biofilms are going to form naturally on metal,” Wood remembers, “so we engineered the ‘good’ bacteria that were present to secrete small antimicrobial peptides that would inhibit the 'bad’ bacteria—in this case sulfate-reducing bacteria that grab electrons from the metal and make it corrode. That was the beginning—the first inkling that we could control biofilm reactions.”

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. Marine biologists had noted that this species doesn’t have a slimy feel to it like most seaweed does. Subsequent work showed that the seaweed secretes furanones to turn off signaling between bacteria and prevent a biofilm from forming on its surface and interfering with its process of photosynthesis.

“We figured out how it worked,” Wood says, “and we showed that it could decrease signaling in E. coli by tens of thousands of times.” 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 Nature Communications 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. As Wood explains, the researchers first laid down a biofilm of a certain bacterial strain; then they introduced a second biofilm of a different strain, programmed to release a signal that would cause the first biofilm to break up. When it had done so, they activated another chemical signal to make the second biofilm dissolve.

“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. “We’re trying to create biofilms with hundreds of different microenvironments,” he explains, “and different kinds of chemistries occurring at different positions. If we can pull that off, we will have gone a long way to show how biofilms could be used in a biorefinery.” Doing so would also bring Wood 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.”

Sidebar: 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.”

Thomas K. Wood, Ph.D., is Endowed Biotechnology Chair and Professor of Chemical Engineering and Professor of Biochemistry and Molecular Biology. He can be reached at twood@engr.psu.edu.

Last Updated November 29, 2012