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Rediscovering Biology: Molecular to Global Perspectives

Microbial Diversity Expert Interview Transcripts: Phil Stewart, PhD

Professor of Chemical Engineering
Stewart is the Deputy Director and Research Coordinator a professor of Chemical Engineering at the Center for Biofilm Engineering at Montana State University. He studies biofilm control with antimicrobial agents, transport phenomena in biofilms, biofilm modeling, and biofilm detachment.

Interview Transcript

Interview with Phil Stewart, Ph.D., is the Deputy Director and Research Coordinator a professor of Chemical Engineering at the Center for Biofilm Engineering at Montana State University. He studies biofilm control with antimicrobial agents, transport phenomena in biofilms, biofilm modeling, and biofilm detachment.

Can you talk about biofilms?
We used to think of bacteria as these very primitive, single-cell organisms with a limited capacity to differentiate, communicate, and cooperate. I think one of the most exciting ideas in the biofilm field right now is that when these organisms get together in a biofilm, they act like a multi-cellular organism. They exhibit behaviors that we associate with higher organisms. They communicate with each other. They differentiate so cells of the same species kind of spin off into different cells states, in what could be imagined as a division of labor. And they cooperate with each other, metabolically and in other ways.

Has there been a change in thinking about bacteria?
I think there is a paradigm shift that is going to change the way that we do things, the way that we control biofilming problems and think about useful technologies for cleaning up wastewater or bioremediation. When we start to understand the multi-cellular nature of life in a biofilm, it’s going to lead us into whole new technologies that we didn’t imagine before.

Are there different kinds of biofilms?
Biofilms form on virtually any wetted surface, so you can find them in drinking water pipes and on your teeth as dental plaque, or the scum on the side of the flower vase.

We now recognize a growing number of medical settings where the infection really is rooted in a biofilm and is a very persistent infection when that happens. The big category are infections associated with implanted devices, so anytime that we put some foreign material in a human body, an artificial joint or a catheter or even a contact lens, there’s a chance that bacteria will adhere to the surface and form a biofilm and cause an infection.

When that happens, the infection is very difficult to treat, does not respond well to antibiotics, does not respond well to that person’s own defenses that are usually very effective at dispatching microbial interlopers. So any implanted device has a chance of an infection and with the huge explosion in the use of these kinds of devices in medicine, just in the last 50 years, I think the problem has become more apparent.

There are also some infections that don’t involve any kind of device, that involve damaged or poorly defended tissue or, sites that are compromised and these would be examples like the lungs in people with cystic fibrosis. This genetic defect reduces the barrier to infection in the lung just enough that biofilms can get established.

There’s some evidence now that certain types of ear infections are really a biofilm disease and that’s why they’re so hard to resolve.

What is the first step in forming biofilms?
Biofilms start with free-floating organisms that are swimming around and alight on a surface and stick to that surface, so that’s the very first step is that initial attachment.

Very shortly after bacteria hit a surface, they begin to respond to that and change so new genes are turned on, others are turned off, and we can see this process of differentiation happening very quickly.

How do the bacteria decide to glob onto a surface?
They may not know where the surface is but once they hit a surface, there must be some way of sensing that they hit a surface. We don’t really know what that is right now.

One idea is that there’s reduced motility, so bacteria that are naturally motile if they stick to a surface they don’t move as well. Even if they are trying to get the motors running-it may be that this is a signal to them that they’re attached. But that’s a good question and obviously it would be really exciting to discover that because that’s a really fundamental switch in lifestyles from the free-floating lifestyle to the biofilm lifestyle. We don’t know what it is.

So after attachment, the bacteria stay?
There’s a period of a kind of loose attachment or association, but one of the types of genes that gets turned on in that early process of making a biofilm are genes that produce adhesions that help them stick more firmly to the surface. These are materials like polysaccharides that actually help cement and glue the bacteria to the surface and to each other.

So that period of twitching and uncertainty is perhaps the time when they’re just turning on the glue and the matrix that’s going to hold the whole thing together. Once that kicks in they do become a little bit more cemented.

What is the role of cell signaling in this process?
We can’t tell you exactly when it happens, but we know relatively early on when there’s a few bacteria present in small clusters that signaling’s likely to be important. [We know] that they’re talking to each other and that that communication may be part of the kind of switch into the biofilm state and the decision to make the matrix and change their phenotype.The bacteria are making small diffusible signal molecules that can escape from one cell and accumulate in the local environment. When enough bacteria are releasing these signals, they accumulate to a level in a particular spot that’s high enough to be sensed and causes them then to make new genetic moves.

What is quorum sensing?
Quorum sensing is just another name for this process of cell-cell communication. Quorum sensing conveys a little bit more the idea that this is a way for bacteria to sense their neighbors and how many of them are banded together.

When do they form polysaccharides?
Quorum sensing may be involved in helping them turn on the synthesis of polysaccharides in proteins that glue the biofilm together. Then the bacteria grow just like they do in conventional cultures. They reproduce and they build structures that lift off the surface.

When those structures reach a certain size, they’re characterized by gradients in nutrient availability. For example, if the bacteria require oxygen, oxygen may be available at the outer surface of that cell cluster but it may be increasingly diminished as it goes into the interior of that cluster. The core of the cluster might be completely lacking in oxygen.

Whereas in the traditional planktonic culture, the traditional test tube culture is generally well mixed and the bacteria are experiencing a very homogenous population environment. One of the characteristics of biofilms is that there’s a lot of microscale heterogeneity from one spot to another, even just tens of microns apart, the environment can be very different.

And that microscale chemical variation allows for the coexistence of diverse species, so biofilms can be incredibly diverse communities. In a very small space, we can find bacteria that are completely different metabolically. Back to the oxygen example: You might have aerobic bacteria on the surface of the cluster and in the core, we can find bacteria that are strictly anaerobic, that actually can’t live in the presence of oxygen. They’re coexisting because the aerobes are scavenging the oxygen, actually creating the niche for these other bacteria, the anaerobes, to thrive.

There are not very many engineers looking at the antibiotic or antimicrobial questions, so I’m a pioneer in the engineering side of that particular question.

How long is it before the genes are turned on?
We see signs of genes being turned on and off within 15 minutes of a bacterium associating with a surface, so it’s quick. I think the old model, even if it was unspoken, was that this was just a passive clomping to a surface and bacteria happen to stick. And the stuck bacteria were just like a free-floating bacteria. But what we can understand from the genetic evidence is that they’re not the same. There are all kinds of regulation that happens. There’s a lot of biology taking place within 15 minutes of that association with the surface and then throughout the lifecycle of the biofilm.

What gets turned on in the first 15 minutes might get turned off on the second day and something else gets turned on so there’s a whole sequence that we’re just beginning to get a glimpse of right now.

How do you know when the genes get turned on?
The technologies for probing the cell that has appeared in just the last five years have allowed us to do this so there are a couple of different approaches. One is DNA chips that actually allow you to take the contents (the RNA) of the cell and read on a little chip in which genes are turned on and off directly from the genetic code.

Another way to do it is by taking the proteins out of a cell-these are the gene products-and ask what proteins are present in the cell, i.e., proteomics. So both of those have been applied to biofilms.

Can you talk about the biofilm structures?
The structures are highly variable. One thing that I get concerned about these days is that we’ve propagated a certain baroque structure as the structure of a biofilm and if it doesn’t have that structure it’s not a biofilm. In fact, there is a lot of variation in what a biofilm looks like, from very thin, sparsely attached bacteria to structures that lift off the surface and look like a field of mushrooms or a city full of skyscrapers. In those more complex structures, there can be flow in-between the mushrooms of the clusters.

What is the purpose of these flow channels?
These water channels are one way to deliver nutrients into the biofilm and they are also a way to remove waste products. Maybe there are other functions too. That’s an interesting question. Why does the system have this interesting complex architecture? What is it doing? We don’t have all the answers for that but there is a nice analogy to the idea of a city full of skyscrapers with diverse occupants and also with occupants that are mobile.

One of the things that we now understand about biofilms is that they’re very dynamic. The old snapshot under the microscope tends to convey the idea that these are static structures. When we make movies under flow conditions we find that these structures can move and wiggle in the flow and that there’s a lot of detachment. There are chunks and individual bacteria that are released from the biofilm. Even within a cluster of bacteria we sometimes find motion.

There’s a lot of seething activity there, more like a beehive or an ant colony. If you study an individual ant you learn something about ants but you miss the amazing things that groups of ants do together, the kind of communal behaviors they have. I think that that’s the idea that we’re coming to in biofilms that like an ant colony, a cluster of bacteria probably has a few bacteria in this cell state and another one in a different cell state and they’re doing different things that further the community as a whole.

Would you say that the bacteria have different jobs?
That’s a hypothesis. We haven’t really tested it yet but there’s been some interesting work here showing that bacteria that land on a certain degradable chiton surface, some of them will turn on enzymes to begin degrading that chiton and neighbors very nearby won’t turn those enzymes on but will profit from the ones that are degrading with the enzyme. They’ll actually feed off the chewed up substrate so that’s one place where we invoke the idea the division of labor.

The other is in the resistance to antibiotics and biocides that we run up against all the time in biofilms. And here, one attractive mechanism is that there is some small fraction of the population in the biofilm that is shunted into a protected state, almost a sprawling state, and those bacteria might not be able to grow, but in the event of a catastrophic challenge they can survive and reseed the biofilm.

Their neighbors that are pending nutrient availability, growing, reproducing, making the matrix are probably relatively vulnerable, but they’re propagating the genome and building a structure and so that would be an example of a division of labor. No single cell can have it both ways. You can’t be growing and building a biofilm and shut down in a protected state at the same time but in a population, you can do that.

Why does a biofilm break up?
The last stage is the detachment or dispersion of bacteria from the biofilm back into the fluid bathing it. That’s a natural process that’s happening all the time in a biofilm, even as it’s growing there is a certain number of cells that are being shed. That detachment is in most cases the primary process balancing growth. If nothing ever detached, we would just get this progressive accumulation and the bacteria would have no way of moving from this spot to some other spot.

Detachments are a really important process. We know almost nothing about it because it can’t be studied in a test tube or a shake flask. It doesn’t exist in a planktonic culture. It’s a purely biofilm phenomenon. There are probably lots of different modes of detachment.

So these release cells then complete the cycle. They can move on and find a new surface and start a new biofilm. And when you think about the lifecycle of a biofilm that way, the free floaters, the ones that we’ve been focused on as a community of microbial scientists and engineers for a hundred years are really just the seeds that let this organism move from one spot to another. The interesting really robust morpha type of organism is the biofilm where it’s an inherently multi-cellular organism.

Do genes get turned on that help bacteria break away from biofilm?
There is some evidence that bacteria when they attach and start forming the biofilm lose the flagellum, lose their motility. And when there’s a mature cluster it can actually have some of the bacteria begin to become motile again, so maybe they expressed the flagellum again and they break out and swim off. There almost surely are genes being turned on to mediate that release.

And that’s one of the exciting ways that we might be able to control a biofilm. If we could learn how to push the detachment button and have the whole thing dissolve, that would be a potentially beautiful alternative to poisons and different toxic chemicals that we use to try and kill them.

Or what about before the bacteria start to form biofilms?
You could abort the whole process potentially and I think that’s the power of this hypothesis that it’s a multi-cellular organism is that now we have whole bunch of different ideas about how to control or manipulate the process one way or another. Whether we want more biofilm or less or this species or that species.

We can try to influence the process of attachment. We can try to influence the signaling or quorum sensing. We can try to attack the synthesis of the matrix. All the antimicrobials that we have right now for trying to control unwanted bacteria target the cell itself–we try to kill the cell.

In the biofilm, if we could learn how to stop the matrix from being made or chop it up, this would be a very effective way of eliminating the formation of the biofilm or dispersing it without having to actually kill bacteria. You can go through that whole sequence of stages in the development of biofilm through the detachment stage and at each one there’s at least a concept for a newer way to control a bacterial infection or to manipulate a biofilm to do something a little bit different.

So this would be a new approach to fighting illness?
I think it gives us hope. If we think about alternative approaches for dealing with infections, not that antibiotics will necessarily be obsolete-they won’t-but we can lay in these alternative strategies in combination with existing drugs to defeat infections that right now are pretty troublesome and difficult to deal with.

Why does our immune system have trouble fighting biofilms?
What we find across the board is that microorganisms in a biofilm are protected from all types of antimicrobial agents, everything from brute force, oxidizing agents like chlorine bleach, to antibiotics that have exquisitely specific cellular targets.

And that protection is also apparent for our own bodies’ natural antimicrobials, so one of the ways that we defend against bacteria is through a host of antimicrobial compounds. Just like other biocides and antibiotics they don’t work as well in a biofilm as they do on free-floating cells.

The other defense are the white blood cells, the leukocytes that attack and engulf bacterial invaders. We’re finding that they just don’t seem to be able to get their teeth around a biofilm and do the job. When the bacteria group together, it may be just a physical problem–literally the aggregate of bacteria is just physically too big for the white blood cell to engulf. Or it may be that there are more sophisticated interactions going on.

In the meantime, doesn’t the immune system blast the biofilm with enzymes?
That’s often the worst consequence of a biofilm infection: the body’s own defenses are chewing away slowly, attempting to battle the biofilm bacteria with proteoletic enzymes and with oxidative molecules that do collateral damage to the healthy tissue and that’s a real concern.

Can you explain why antibiotics don’t work on biofilms?
All the antibiotics we have and probably all the antimicrobials we have, have really been discovered and optimized based on culture techniques that use free-floating cells. And right away it’s not surprising that they don’t work as well as we’d like them to on a biofilm cell.

Bacteria in biofilms turn on genes that help them resist antibiotics?
There’s almost surely multiple defensive mechanisms here. It’s not just one mechanism and there’s not an easy answer. The antimicrobial may fail to penetrate all the way through the biofilm. It maybe that the biofilm has regions that are nutrient-starved and the bacteria in those regions will be either slow-growing or not growing at all. And just that difference in growth state can have a big effect on antibiotic sensitivity.

Non-growing bacteria are really sometimes impervious to antibiotics. Penicillin antibiotics won’t work on a cell that’s not growing, for example. So there’s variation in growth state, and then there is also the idea that perhaps bacteria in a biofilm are turning on protective genes and we’re just seeing some evidence of that coming out right now.

Efflux pumps are a really intuitively appealing idea. These are pumps that spit the antibiotic back out of the cell and a lot of people have jumped into that expecting that they’d find the pump turned on in the biofilm.

And for the most part, we haven’t seen that, so the efflux pumps explain a lot of conventional antibiotic resistance, but they’re not showing up as a big part of what’s happening in a biofilm. It may be that we’re looking at the wrong pumps. There may be completely different pumps that have not been very well characterized because they’re not important in free-floating cells, but they are important in the biofilm.

But it may be that the mechanisms in the biofilm are actually really completely different from what we’ve discovered in free-floating cells and really have to start back at the square one to discover what they are.

Planktonic vs. biofilm?
It depends a little bit on how that protection works. If it’s protection that’s afforded to a single cell like an efflux pump, then it doesn’t really matter what the neighbors are doing. If it’s protection that can have a kind of communal effect then it does have that synergistic power.

For example, one of the defenses against penicillin antibiotics are enzymes called “beta-lactamases” that cleave membrane that’s at the core of penicillin antibiotics and completely deactivate them. If the bacteria in a biofilm are expressing beta-lactamase even at a low level collectively, they have a lot of power to degrade penicillin antibiotics before they can make it into the interior of the biofilm.

It seems that that is a very specific defense.
I think it works both ways. There may be defenses that are aggregate defenses, multi-cellular defenses where the combined ability of bacteria to defend against an antimicrobial is important and there are others where it’s not.

If you imagine a lone cell out in a sea of water trying to defend against an antimicrobial by synthesizing some protective molecule it’s a losing battle. But when you’ve got a group of bacteria all making that protective molecule and they’re bunched together as a group, they can create a cloud of protection that allows them to just withstand an antimicrobial challenge that would devastate a lone cell.

Why can’t antibiotics enter biofilms?
There are a couple of issues there. One is the channels. While those channels deliver in some cases nutrients right down to the attachment surface, they don’t guarantee access of nutrients into the interior of cell clusters. So this convoluted geometry can have flow through it, but for the interior cell clusters, the access of a nutrient is still controlled by diffusion. Diffusion is a slow process and so there are gradients in the nutrient availability and there are zones of nutrient limitation in a biofilm, even when there are water channels present. The same thing could happen with an antimicrobial. It may not penetrate into the interior cell clusters.

What we’re actually finding is that most antibiotics do penetrate adequately, so that at least for antibiotics inadequate penetration’s probably not the main protective mechanism. It may add a little bit, in some cases but probably not the main one. Inadequate penetration is important for reactive oxidants like hydrogen peroxide or bleach. Those agents are neutralized out of the surface of the biofilm cluster faster than they can diffuse in, and that can very profoundly retard their access to all parts of the biofilm.

Why is it difficult to treat a biofilm with one antibiotic?
Our understanding of biofilms now is that the microbial population is very diverse in terms of metabolic state physiological activity and the antibiotics that we have probably only work on a certain slice of that physiology. Most of the antibiotics we have probably will only work on actively growing metabolically active cells and don’t work nearly as well on those cells that are shut down and not active.

The fact that we’ve got this whole spectrum of physiological states in a biofilm means that they’re really hard to treat with a single agent and that right now we just don’t have the right agents. All the agents we have are only going to hit one end of the spectrum. Of course, that means there’s an opportunity, too, to discover whole new agents that target the biofilm state specifically and I think we’re going to see a lot of work in that area in the next decade.

What is the focus of your research?
I’m not so much in the business of hunting for compounds as I am hunting for specific biological or chemical mechanisms that confer resistance. If we understood these physiological states a little better, where they are, how they’re determined, then we can get ideas about how to design or screen for compounds that might work against that those states.

Another scheme that a collaborator and I are working on is to screen for genes that are important in antibiotic resistance in biofilms. At the genetic level, we can devise ways to discover which genes are important in antibiotic resistance in a biofilm and that’s very powerful because once you have the gene, you often know what that gene is likely to be doing and you have a concrete target for interfering with development of antibiotic resistance in the biofilm.

What about trying to disarm the bacteria’s ability to signal to one another?
If cell-cell communication, if quorum sensing turns out to be an important part of the development of a biofilm, then we ought to be able to cripple the biofilm or prevent it from forming simply by jamming that communication, disrupting the natural crosstalk that bacteria use as part of the regulation of forming a biofilm.

So there’s a lot of interest in finding molecules, chemical compounds, that will jam that communication process that might go in and block the receptor for the natural signaling molecule so that it doesn’t do its job anymore or interfere with it in some other way. Conceptually, it is a very powerful strategy and there are examples of compounds that do this.

Is this research into biofilms changing the mindset of treating infections?
Absolutely, we have to start using biofilm methods, techniques, and concepts. The test tube planktonic culture won’t take us very much further in fighting biofilm diseases. There’s a huge opportunity here for making new discovery when we embrace the idea that an organism that we may be well acquainted with in planktonic culture is really a different creature, a multi-cellular creature, when it grows as a biofilm. And we take that idea and apply biofilm methods to it and there will be a lot of opportunity for new discovery of alternative ways to fight these infections.

What are the ramifications for patients?
I certainly think that there’s hope in it. There’s lots of great work being done in cystic fibrosis that has nothing to do with biofilms. But there’s a lot of hope that out of the biofilm research we could potentially see therapies for dealing with the infections that plague the lungs with cystic fibrosis and slowing the deterioration of the lung.

I think there’s a lot of hope in the possibility that we can find that there are good ideas for dealing with biofilms in the labs. And what that would do for somebody with cystic fibrosis is reduce the bad side effects of the infections that they have in their lungs, reduce the deterioration of the lung and help them breathe a little easier.

What are still the driving questions in this research?
I would like to understand why biofilms are so hard to kill with antibiotics and biocides and disinfectants that work pretty well on our conventional cultures. Again, I think there are multiple overlapping explanations that are different for different agents. But I think that insight that we get from understanding those fundamental protective mechanisms will allow us to learn more about the basic biology of biofilm and devise better ways of controlling detrimental biofilms.

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