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Volume 86 Issue 23 | pp. 15-23
Issue Date: June 9, 2008

Cover Stories

Communal Living

Scientists across academia and industry are making a concerted effort to understand and control bacteria that form biofilms
Department: Science & Technology
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Careful Growth
This clean room will be used to generate and study biofilms as part of the BASF Advanced Research Initiative at Harvard University.
Credit: Harvard University
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Careful Growth
This clean room will be used to generate and study biofilms as part of the BASF Advanced Research Initiative at Harvard University.
Credit: Harvard University

IT WAS LONG ASSUMED that bacteria were loners that floated through their single-celled existence without need of companionship. That simple, independent lifestyle made it pretty easy to study them in a test tube or grow them in a petri dish.

But it turns out that bacteria are actually social creatures. They congregate and chemically communicate, working together to stay alive. More often than not, they exist in complex communities called biofilms, started up when one cell sticks to a surface in an aqueous environment and somehow signals others to join in. Those recruits, along with descendents of that first cell, create a protective matrix through which nutrients are distributed. In the end, they are better off living as a colony than going it alone.

That safety in numbers poses a challenge for anyone trying to get rid of a biofilm, which can plant itself virtually anywhere. Bacteria are tricky enough to deal with on their own; they've spent billions of years learning to overcome whatever environmental threats come their way.

So imagine the power of bacteria banded together, living under something akin to a force field, and acting in unison in ways scientists have only recently begun to understand. In addition to being able to thrive in pretty much any environment, biofilms can be made up of a multitude of bacterial species, some of which may have never been studied in a lab. They make for a formidable adversary.

But researchers are starting to accumulate enough knowledge about how biofilms form to think seriously about how to control them. "In the past 10 or 15 years, we've seen the techniques and approaches of modern biology and genetic and molecular tools applied to biofilms," says Phil Stewart, director of the Center for Biofilm Engineering (CBE) at Montana State University. "That has helped change the biofilm from an amorphous primitive slab of slime into a biologically sophisticated, differentiated, and regulated community."

Indeed, academic researchers are intrigued and industry executives seem ready to devote money to real-world methods of manipulating biofilms. Everyone involved believes it is going to take a coordinated effort across the biological and physical sciences to better understand and harness these bacterial communities.

Kolter
Credit: BASF
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Kolter
Credit: BASF

Biofilms' ubiquity makes them a ripe subject for both academia and industry. If you have ever slipped on a rock while walking in a stream, you've encountered a biofilm. Run your tongue across the surface of your teeth; that plaque buildup you feel is actually a biofilm. When you're standing over your sink wondering why the water refuses to drain, blame a biofilm.

But biofilms can have a more costly impact. Bacteria like to freeload on the hulls of ships. Biofouling, or the accumulation of bacteria and other material on an underwater surface, exacts hefty fuel costs on the shipping industry. Antifouling paints can delay the process, but eventually bacteria will glom on. And removing the films takes a toll on the environment; those bacterial communities adhere to the surface so strongly that it takes chlorine and heavy-duty scraping to remove them.

Meanwhile, biofilms also have a major impact on human health. Hospital-acquired infections are now the fourth-leading cause of death in the U.S. behind heart disease, cancer, and stroke. The culprit behind most of those infections? Biofilms. Bacteria can thrive for months on hospital surfaces—floors, instruments, gloves—and be passed on to open wounds or to implanted devices such as catheters, heart valves, and artificial hips where biofilms can form.

YET BIOFILMS can also be useful. Industrial wastewater treatment centers have long exploited bacteria's propensity to suck in anything that looks tasty. Biofilms are incorporated into water treatment systems to filter out organic compounds and pathogens.

Despite the prevalence of biofilms, the discovery that bacteria like to form their own gated communities was discounted for years. Most scientists finally took notice of biofilms in the mid-1990s. Since then, the number of journal articles about the topic has risen exponentially, CBE's Stewart says.

Several underlying developments are spurring interest in biofilms. For one, ideas about bacteria are changing. "It used to be that we thought of microorganisms as being these little things that were all roughly the same clonal descendents of some maternal line who talked to one another, if at all, distantly," says George M. Whitesides, professor of chemistry at Harvard University and surface science expert.

Now, scientists are beginning to understand that bacteria have a complex communication system, the properties of which are dictated, at least in part, by biofilms. In a film, there's no circulation or blood flow, no convective mass transport, Whitesides notes. "Concentrations of a chemical can build up to higher levels; there's an efficiency of chemical communication that occurs in a biofilm that would not occur otherwise."

Biofilm topography
Weibel's lab uses interferometry to create two-dimensional arrays of Pseudomonas biofilms.
Credit: Courtesy of Douglas Weibel
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Biofilm topography
Weibel's lab uses interferometry to create two-dimensional arrays of Pseudomonas biofilms.
Credit: Courtesy of Douglas Weibel

And importantly, more people—be they scientists or members of the public—understand that "biofilms actually matter," Stewart says. "When you go out into nature, or in engineered systems, and increasingly in the medical context, the microorganisms really are banded together in aggregate."

The human health threat, in particular, has attracted attention to the problem. Although ships have long dragged and pipes have forever clogged, those problems never seemed urgent, notes Roberto Kolter, professor of microbiology and molecular genetics at Harvard Medical School. But the proliferation of implants and other medical devices means more and more pernicious infections caused by the biofilms that love to call the devices home. Antibiotics are perplexingly ineffective against bacteria contained within a biofilm.

Once a few geneticists started pointing out that bacteria like to spend most of their time on surfaces, "interest really exploded," Kolter says. Advanced microscopy and other scientific tools have aided both in convincing people that bacteria like the communal lifestyle and in facilitating researchers' ability to study and understand how biofilms form.

One sure sign that biofilms are ripe for exploration is industry involvement. After all, the ultimate goal is learning to control the films in the real world. Last fall, BASF formed the BASF Advanced Research Initiative at Harvard University, a partnership that brings together researchers from both organizations to tackle scientific challenges. BASF put up $20 million to fund the five-year program, and biofilms are the first project on tap.

"Biofilms on surfaces cause multi-billion-dollar losses each year," Franz Brandstetter, head of BASF's Polymer Research Competence Center, told reporters at a symposium on biofilms held in April to mark the launch of the partnership. Montana State's CBE also works closely with industry on biofilms. It has 32 member companies and ran projects aimed at solving biofilm-related problems for 43 companies last year.

Because biofilms are magnificently complex systems, it will take a concerted effort by academia and industry to unravel the details of their formation and function.

SCIENTISTS ARE also weighing a chicken-or-egg question: Should they first develop a better understanding of biofilm formation and makeup, or is it possible to head straight to products and methods to control bacteria and then work backward to glean knowledge about how the systems work?

Whitesides
Credit: BASF
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Whitesides
Credit: BASF

CBE's Stewart wants to charge ahead. "If we try to wait until we have every gene in the circuit figured out, we're not going to get there," he says. "We need to do some fundamental science and build the information base, but this is the right time to begin actually exploring technologies."

Greenberg
Credit: BASF
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Greenberg
Credit: BASF

Biofilms' complexity is daunting, but it also lends itself to myriad methods of attack. Scientists can imagine many ways to prevent or control a biofilm. One route is to create surfaces that don't let bacteria stick at all. There are also multiple opportunities for intercession during the formation of a biofilm or after it has matured; researchers are considering everything from how the film feeds itself to how its composition changes over time. Scientists are even considering how they might encourage a benign biofilm to grow on a surface, thereby preventing a more pernicious one from attaching itself.

Many scientists are working to find the overarching principles to which, despite their diversity, all biofilms subscribe. Finding those commonalities could accelerate the search for broad means of control. Thus, the hunt is on for general themes, such as how surface properties affect biofilm formation and how a change in the environment affects the destiny of a colony.

The varying perspectives of the chemists, biologists, medical doctors, and physicists involved in the field means a wide range of scientific approaches. Harvard's Kolter, for example, is trying to understand the cellular changes that occur during biofilm formation and how different cell types organize themselves within the film matrix. By monitoring the gene expression of three important cell types—those responsible for motility, matrix production, and sporulation within a Bacillus subtilis biofilm—scientists in Kolter's lab found that the population of each type changed over time and was distributed based on the architecture of the colony.

Kolter also uncovered evidence of a developmental pathway by which the motile cells transition to matrix production and then to sporulation. Furthermore, he found checkpoints during biofilm formation when interrupting the development of a cell will change its fate. "If you arrest normal development by having a mutation that does not make the matrix, the bacteria don't progress to the next stage; they somehow get stuck," Kolter says.

These checkpoints are something scientists previously wouldn't have associated with bacterial communities, Kolter says, and could eventually provide the means to control or mediate them.

Whitesides' lab, meanwhile, is bringing its expertise in shaping and defining the mechanical properties of gels—a good proxy for slimy biofilms—to help elucidate the interface between a biofilm and a surface. The goal is to learn which properties of the biofilm are molecular and which are mechanical. "We don't know whether the characteristics of the biofilm are intimately connected to the properties of the molecules that are there or primarily a result of the gel and its influence on mass transport," Whitesides says.

Biofilm formation occurs in two stages, Whitesides points out. The first is when a bacterial cell has to stick to a surface—be it steel, Teflon, or bone—and form a gel-like matrix. The second is when subsequent parts of the biofilm stick to that matrix. Whitesides is interested in building gels to understand those two interfaces—one between a surface and a microbial gel and one between two gels.

Researchers in Whitesides' lab are taking several tacks to explore those interactions. For example, they are using self-assembled monolayers (SAMs) to build structured molecular interfaces that have both adsorbing and nonadsorbing surfaces and are then studying how well compounds attach to them. One goal is to understand how biofilm formation correlates with hydrophilicity, hydrophobicity, and other surface properties.

By screening for nonfouling surfaces, his group has been successful in finding a handful of SAMs that resist protein adsorption. Whitesides is also trying to build three-dimensional microfluidic devices—systems of micrometer-scale channels and wells that mimic the chemistry and physics of biological systems. Those 3-D approaches could help scientists build layered biofilm systems that contain different organisms and closely replicate what is going on in nature.

Rustem F. Ismagilov, a chemist at the University of Chicago, is using microfluidics to understand biofilms' spatial structure, which dictates the flow of nutrients, environmental signals, and how cells within the film chemically talk to each other. The structure also influences metabolic interactions within films, including the breakdown of molecules.

"We can't understand biofilms until we understand the spatial structure," Ismagilov says. This means determining the main bacterial players, how those players function depending on their location, and how that function changes as the biofilm matures and the external environment varies.

THE GOAL is to better understand how the highly organized structure enables cells within biofilms to act in concert. Such an understanding could provide new ideas about how to break the structure down or recreate it in synthetic systems that mimic some of the functions performed by biofilms.

Douglas B. Weibel, a biochemist at the University of Wisconsin, Madison, is studying the point in biofilm development when that first bacterial cell adheres to a surface. His lab is trying to understand how a cell senses it is on a surface—a seemingly basic step that is well understood in mammalian cells but is a mystery in bacterial cells.

Weibel first wants to figure out the environmental trigger that tells the cell it is in contact with a surface and next to determine the transcriptional change that tells it to abandon its solitary lifestyle. Although scientists have yet to prove whether that change is chemical or physical, Weibel, like Whitesides, is looking at a host of physical phenomena including surface stiffness, surface tension, hydrophobicity, hydrophilicity, viscosity, and osmolality. The idea is that, despite differences between bacteria, commonalities based on physical phenomena could provide starting points for controlling bacterial behavior.

Others are diving directly into real-world problems by tapping general knowledge about bacteria and human biology and then extrapolating to how biofilms distribute nutrients. For example, microbiology professors E. Peter Greenberg and Pradeep Singh, both at the University of Washington, Seattle, are exploiting basic information about bacteria's dependence on iron, which is required for biofilm development, to develop drugs that treat biofilm-related infections.

Singh found that lactoferrin, an iron-binding antimicrobial factor present in human tears, sweat, mucus, and other bodily fluids, sends a signal to bacteria that iron in the area is low, prompting them to move on rather than squat and form a biofilm. Ehud Banin, of Israel's Bar Ilan University, and Greenberg showed that normal biofilm development needs a threshold level of cellular iron. Putting those concepts together led to the idea that the efficacy of antibiotics, which are notoriously bad at killing bacteria within a biofilm, could be increased by somehow limiting iron in the cell.

Singh decided to take a Trojan horse strategy and gain access to the cell using the ironlike element gallium, which interferes with bacterial iron metabolism. Banin and Greenberg combined efforts with Mottie Chevion, a chemist at Hebrew University of Jerusalem, to make gallium-containing compounds that look like a source of iron to bacteria. In the lab, several compounds seem to enhance the efficacy of antibiotics, and early experiments in animals are validating the gallium approach.

IN ADDITION to potentially yielding new drugs, the research by Greenberg and Singh could explain why antibiotics are unable to kill bacteria living in a biofilm. They have shown that the combination of a gallium-containing compound with the antibiotic gentamicin is more effective against biofilms than either treatment on its own. Greenberg hypothesizes that the antibiotic is killing the bacteria on the surface of the biofilm, which is well fed and therefore susceptible to treatment, while the gallium compound is killing the bacteria in the middle of the biofilm, which is starved and scavenging for iron or ironlike compounds.

Despite the wide range of approaches to unraveling the complexity of biofilms and inventing ways of controlling them, there are some basic housekeeping issues that need to be resolved before any product can make it to market.

Wisconsin's Weibel points to the issue of reproducibility. At present, the techniques for generating biofilms in the lab are not very precise, to the detriment of methodical study. For example, researchers would like to be able to make a whole slew of identical biofilms and then study each one under different conditions to decide which conditions are important and which are not.

Surface Study
Ye Jin (Jenna) Eun, a graduate student in Weibel's lab, works on a biofilm experiment.
Credit: Courtesy of Douglas Weibel
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Surface Study
Ye Jin (Jenna) Eun, a graduate student in Weibel's lab, works on a biofilm experiment.
Credit: Courtesy of Douglas Weibel

"This is one area where I think chemists can have a big impact," Weibel says. "We know how to control surface chemistry, which means we know how to control where cells get seeded on a surface. We can take those concepts and extend them into techniques to make reproducible biofilms."

Being able to generate reproducible films could also ease the registration of antibiofilm products, mainly disinfectants, with the Environmental Protection Agency. "The test EPA uses to register disinfectants is from the 1950s," CBE's Stewart says. CBE is working with EPA, the Food & Drug Administration, and a number of companies to develop standardized methods for testing biofilms.

The lack of agreed-upon standards for making biofilms also extends into the medical arena. The first drug to combat a biofilm "is going to be a hard one," University of Washington's Greenberg says. "Whoever does this is going to have to work closely with FDA."

Meanwhile, the Harvard and BASF researchers have ambitious goals for their biofilms collaboration. Andreas Kreimeyer, executive director for research at BASF, figures it will take about three years to parse through the general understanding of biofilms to decide which product opportunities are most viable. The approaches for tackling biofilms are vast, and the scientists at BASF and Harvard are likely to first consider how to develop controlled surfaces, adds David Weitz, a physicist at Harvard.

REAL-LIFE SOLUTIONS—new medical materials or ship coatings that can resist biofilms—are still years away. While work in the lab has largely focused on simple systems combining one surface and one strain of bacteria, the real world is never so simple. Most biofilm systems comprise multiple species of bacteria, including many organisms that have never been characterized, Kolter cautions. It's a big leap to get from studying the basic mechanisms of a single cell to understanding what's happening on a ship's hull, he says.

Scientists need to recognize the enormous diversity contained in a biofilm and consider that there may not be a single solution or even a set of them, Kolter says. Still, researchers are optimistic that a concerted push across many disciplines will accelerate efforts to control biofilms. "This is an area where we think the problem is both complicated enough to be interesting, but simple enough that well-defined approaches will lead to good results," Harvard's Whitesides says.

 
 
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