online at dartmed.dartmouth.edu—Dartmouth Medicine 27Fall 201026 Dartmouth Medicine—online at dartmed.dartmouth.edu Fall 2010

Biofilms are everywhere. These colonies ofbacteria and other microorganisms can befound in mundane places, such as shower

curtains, sink drains, and toothbrushes, as well as inmore exotic locales, from chilly Arctic waters toscalding deep-sea vents.In some cases, biofilms are composed of a single

species. Elsewhere—such as on human teeth, wherethey form plaque—a biofilm may include hundredsof species. And in addition to bacteria, biofilms caninclude fungi, algae, protozoa, and other organisms.Some biofilms are large enough to be visible to thenaked eye, but most remain very small, perhaps 30to 50 micrometers across—roughly half the widthof a human hair. For many microorganisms, joininga biofilm is business as usual. Most bacterial cellsspend at least part of their life cycle in a biofilm,and for some bacteria this settled lifestyle makes upthe bulk of their existence.Biofilms’ ability to thrive wherever there is

moisture, food, and a surface for microbes to attachto has made them a subject of scientific curiosity.But it also makes them a treacherous threat to hu-man health. They are the culprits, for example, inmore than half of all hospital-acquired infections,and they form large colonies in the lungs of peoplewith cystic fibrosis, causing chronic infection and,eventually, premature death.The difficulty scientists and physicians have had

combating biofilms might seem surprising, giventhat one of the great medical advances of the 20thcentury was the development of antibiotics to fightoff bacterial infections. But the problem with bio -films, explains DMS microbiologist George O’Toole,

Biofilms can be as

mundane as slime on a

shower curtain, or as

menacing as antibiotic-

resistant bacterial colonies

on a medical device

or in a human lung.

But luckily, scientists

are making progress in

understanding these

mysterious aggregations

of microorganisms.

Amos Esty is the managing editor of Dartmouth Medicine.

Findings about biofilmsfrom George O’Toole’slab have graced thecovers of numerousjournals, includingthose pictured behind him here. Science

Slime

Text by Amos Esty • Photographs by Jon Gilbert Fox

Ph.D., is that although they are composed of organ-isms that can often be treated effectively in their in-dependent form, when those organisms gather as abiofilm they are suddenly much less vulnerable.Bacteria in bio films can be 1,000 times more resis-tant to antibiotics than free-swimming bacteria.This increased resistance is only one of the manyways bacteria in biofilms differ from bacteria outsidebiofilms. In some ways, biofilms are more like astand-alone organism than a community of indi-viduals. And the more scientists learn about bio -films, the more complicated, sophisticated, and im-pressive these microbial aggregations seem.Scientists have been interested in bacteria for

centuries—ever since an amateur Dutch scientistnamed Antonie Van Leeuwenhoek observed “lit-tle animals” through his homemade microscope inthe 1670s. But researchers only began to pay seri-ous attention to biofilms three centuries later, inthe 1970s, when they realized that bacteria inbiofilm form were causing medical problems, suchas dental plaque and hospital-acquired infections.They began their research by trying to tease apartthe mechanisms that allow biofilms to adhere todifferent surfaces, thinking that perhaps the bacte-ria were positively charged and the surface nega-tively charged, for example. “There wasn’t a lot offocus on the microbe,” O’Toole says.

O’Toole began reading about biofilms in thelate 1980s, when he was a graduate stu-dent at the University of Wisconsin.

What he learned convinced him to focus onbiofilms when he moved on to a postdoctoral fel-lowship at Harvard. At the time, the mechanicalapproach to biofilm research made sense, he says,

versus

An electronmicrograph of aPseudomonas aeruginosa biofilm

For a with links to more onbiofilms and some of O’Toole’s papers, seedartmed. dartmouth.edu/f10/we05.

WEB EXTRA

online at dartmed.dartmouth.edu—Dartmouth Medicine 29Fall 2010

downstream, eventually attaching to a new part ofthe surface or a different surface altogether to cre-ate another biofilm.Once part of a biofilm, individual bacterial cells

can change their identity dramatically. They maybecome larger or smaller, change their shape, or ac-quire significant differences in their gene expres-sion. In P. aeruginosa, for example, the expression ofmore than 70% of a cell’s genes can be affected bywhether it’s free-swimming or part of a biofilm.

When O’Toole began studying biofilms asa postdoc, he didn’t assume that the at-tachment of a bacterial cell to a surface

was merely a mechanical process. He looked at themicrobe itself, trying to figure out what was goingon inside the cell that might allow it to take thefirst step in biofilm formation. To do that, he decid-ed to examine whether there was a genetic compo-nent to the attachment.O’Toole created thousands of strains of P. aerug-

inosa that had various genetic mutations, lookingfor those that could not form biofilms. He foundtwo different strains lacking the ability to formcolonies, and when he looked closer he realized thatthere was a different explanation in each case. P.aeruginosa has two methods of moving around, andboth are required for a cell to attach to a surface toform a biofilm. One type of movement involves theuse of the cell’s flagellum, a sort of tail that extendsoutward from one end of the cylindrical cell. “Theflagellum is essentially a bacterial propeller,” O’Toole says. When a cell decides to attach to asurface, the flagellum helps it form the initial at-tachment. Once it’s attached, the cell can use theflagellum to move across the surface.The second kind of movement is called twitch-

ing motility, and it involves the use of pili, whichare like little hairs on the surface of the bacteria.“You can almost think of it as throwing out a grap-pling hook and then pulling themselves along,” O’Toole explains. “They can extend their pili, thetip of the pili attaches, and then they retract the piliand pull themselves sort of hand over hand, or piliover pili.”After a bacterial cell uses its flagellum to estab-

lish the initial attachment, it uses its pili to form astronger attachment to the surface. The mutantsO’Toole identified in those early studies lacked ei-ther flagellar motility or twitching motility, pre-venting biofilm formation.“For us, it was exciting to see because it suggests

different genes playing a role at different steps inthe process,” O’Toole says. “That was some of thefirst evidence that maybe there was a regulated se-ries of steps.” In other words, the attachment of a

bacterial cell to a surface is not merely the result ofmechanical forces, as is the case in the attachmentof a magnet to the door of a refrigerator. Instead, thebacteria have to “decide” to attach to a surface.

I n recent years, O’Toole and the other re-searchers in his lab have continued to breakdown the steps involved in that decision, iden-

tifying more genes and proteins involved in theprocess of biofilm formation. “The unfolding pic-ture of biofilm formation has certainly becomequite complex,” says Sherry Kuchma, Ph.D., a re-searcher in O’Toole’s lab. “These organisms that wethink of as being so simple actually can engage invery complex behaviors.” The more scientists learnabout attachment and the more steps they identi-fy, the more targets there may be to try to preventbiofilm formation in situations where that can beharmful.While O’Toole has shown how complicated

biofilms are, he has also helped unify the field byproviding a framework to study them. In 2000, hewrote an article proposing what he called the de -velopmental model of biofilm formation. His idea,in short, was to compare the growth of bio filmsto the development of multicellular organisms.

Alicia Ballok, a graduate student in microbiology, has beena member of O’Toole’s lab for the past two years. She isone of five graduate students currently based in his lab.

“That was some of the

first evidence that

maybe there was a

regulated series of

steps,” explains O’Toole.

In other words, the

attachment of a bacterial

cell to a surface is not

merely the result of

mechanical forces, as

in the attachment of a

magnet to the door of a

refrigerator. Instead, the

bacteria have to “decide”

to attach to a surface.

Fall 2010

because the study of biofilms really started as an en-gineering problem. It had long been realized thatcolonies of microorganisms would form on the hullsof ships, slowing their progress through the water.That led to attempts to figure out what the organ-isms were and how they were able to stick so effec-tively to ships and other surfaces—as well as to at-tempts to develop surfaces that were resistant tothese microbial communities.These studies, however, often overlooked the

complexity of the organisms that made up thecolonies. “A lot of the time the microbes were treat-ed as inanimate spheres,” O’Toole says.

At Harvard, O’Toole started to study thebacterium Pseudomonas aeruginosa. Thespecies was already widely used for re-

search because it is possible to manipulate it genet-ically to decipher which genes are involved inwhich processes; this made it a good model organ-ism for examining biofilms. Since then, O’Toolehas contributed to the growing understanding oftwo important aspects of biofilms: the moleculardetails of the attachment process and why bacteriain biofilm form are so resistant to antibiotics. Overthe course of his career, he has helped to show thatbiofilms are more complex than anyone imaginedback when he first learned about them.Depending on the species of microorganisms in-

volved, the process of biofilm formation can be trig-

gered by a number of factors. “It’s clear that bacte-ria actually regulate whether or not they want toattach to a surface,” O’Toole says. In other words,these tiny, single-celled organisms take an activerole in determining whether to live on their own orsettle down and join a biofilm.If certain environmental conditions are met, an

individual bacterial cell may decide to attach to asurface, usually in a moist environment with a goodsupply of nutrients. The colony grows as the cell di-vides and other bacteria also attach to the surface.As the bacteria attach, they secrete a mix of mole-cules that together form what even scientists oftenrefer to as “slime”—or, in more technical terms, anextracellular matrix. This slime is essential for theformation of a healthy biofilm. It surrounds thecolony, giving it shape as it grows, helping to pro-tect it from threats such as antibiotics and immunecells, and facilitating the collection of nutrients tofeed the bacteria.Bacteria within a biofilm can communicate with

each other through a process called quorum sens-ing. They release molecules that act as signals, ba-sically creating a group discussion that helps thebacteria decide when to form a biofilm, how to re-act to a threat, or when to move on to a new spotif the nutrient level begins to drop.A successful biofilm doesn’t just grow in one

place but can also colonize new areas. Some of thebacteria within a biofilm may detach and float

28 Dartmouth Medicine—online at dartmed.dartmouth.edu

This illustration shows the life cycle of a biofilm. In panel 1, free-swimming (or planktonic) bacteria attach to a surface; those attached (or sessile) cells then createmicrocolonies. In panel 2, the colony grows and matures as the cells divide and other cells attach to it; EPS stands for extracellular polymeric substances, also calledthe extracellular matrix. Eventually, in panel 3, some bacteria split off, either individually (seeding) or in clusters, and attach to another surface to start a new colony.

An individual bacterial

cell may decide to attach

to a surface, usually in

a moist environment

with a good supply of

nutrients. The colony

grows as the cell divides

and other bacteria also

attach to the surface.

As the bacteria attach,

they secrete a mix of

molecules that forms

what even scientists

often refer to as “slime.”

Attachment1

Growth2

Detachment3

Planktonic Cells Mature BiofilmDetachment of Clusters

SeedingDispersalEPS

SessileCells Microcolonies

An electronmicrograph of aPseudomonas aeruginosa biofilm

MSU CENTER FOR BIOFILM ENGINEERING / PEG DIRCKX

online at dartmed.dartmouth.edu—Dartmouth Medicine 31Fall 2010

thing, DMS has a strong cystic fibrosis researchgroup that fosters collaborations among physiciansand scientists in a number of disciplines.

P. aeruginosa is found in many places, O’Toolepoints out. “Chances are you’ve inhaled a bunchevery time you take a shower. But a normal, func-tioning lung is able to clear those infections.”

The genetic mutation that causes cystic fi-brosis creates a buildup of mucus in thelungs, which provides a welcoming envi-

ronment for P. aeruginosa. The bacteria form bio -films, creating a chronic infection. “There’s reason-ably good clinical data that the longer you can pre-vent chronic Pseudomonas infection, the better it isfor patients,” O’Toole says. “So there’s a real driveto figure out ways to prevent infection of Pseudo -monas, and, if you can’t prevent it, to eliminate thecolonization once it occurs.”So far, antibiotics alone have not proven effec-

tive. But if scientists could figure out what makesbiofilms so resistant to antibiotics, they might beable to overcome that resistance. For years, scien-tists thought that the extracellular matrix—the lay-er of slime—simply prevented antibiotics from dif-fusing all the way through a biofilm, protecting atleast some of the cells.But in 2003, O’Toole identified an entirely dif-

ferent mechanism that protects bacteria in biofilms.His lab created mutant strains of P. aeruginosa thatwere able to form biofilms but were not as resistantto antibiotics as normal strains. O’Toole reasonedthat if the extracellular matrix surrounding a bio -film was perfectly fine but the biofilm still couldnot resist an antibiotic, then there must be otherfactors involved in protecting biofilms.His lab found that a strain of P. aeruginosa lack-

ing the gene ndvB was able to form normal biofilmsbut was less resistant than it should have been to to-bramycin, an antibiotic often used against the bac-terium in people with CF. Looking closer, the re-searchers realized that ndvB causes the productionof molecules called glucans that trap antibiotics be-fore they can reach their target.A glucan, O’Toole explains, is a cylindrical sug-

ar molecule. “You can think of it almost as a barrel,and it can hold molecules inside of that barrel,” hesays. Tobramycin works by targeting the ribosomesof bacterial cells, which are found in the cytoplasm.O’Toole discovered that glucans bind tobramycinwhile it is still in the periplasm—which is outsidethe cytoplasm—thus preventing the drugs fromreaching the ribosomes.This finding led to a more accurate picture of

how antibiotic resistance works but also made itclear that biofilms are more devious than had been

previously thought. They have mechanisms otherthan simply a layer of slime to protect themselvesfrom drugs and other threats.

Dartmouth has turned out to be a good placefor O’Toole to try to figure out ways aroundthat resistance. He is now the associate di-

rector of the CF Research Development Programat DMS, one of only a few sites in the U.S. fundedby the Cystic Fibrosis Foundation to carry out CFresearch. When he arrived at DMS 12 years ago,notes O’Toole, perhaps 10 people would show up atmeetings of the CF group. Now, he says, it’s closerto 35 or 40 people, including cell biologists, immu-nologists, microbiologists, and physicians.Over the past several years, O’Toole has worked

closely with Bruce Stanton, Ph.D., a physiologistat DMS. Together, they and other researchers intheir labs have developed a model of the cells foundin the lungs of people with CF. There is no good an-imal model that can be used to explore treatmentsfor CF, so the cellular model developed by Stantonand O’Toole filled a major gap and is making it pos-sible to get a more realistic look at CF’s effects.But one of the first findings from this model was

daunting. The researchers found that bio films

Jack Hammond, a technician in O’Toole’s lab for the pastthree years, is entering DMS’s Ph.D. program this fall.

In 2003, O’Toole

identified an entirely

different mechanism

that protects bacteria

in biofilms. This finding

led to a better picture

of how antibiotic

resistance works but

also made it clear that

biofilms are more

devious than had been

thought. They have

mechanisms other than

a layer of slime to

protect themselves from

drugs and other threats.

Fall 2010

“It turns out that bacteria actually perform manyof the behaviors that multicellular organisms can,”O’Toole says. “They can talk to each other. Theycan discriminate self from nonself. They can evenspecialize.” This last point is important, he adds,because it is part of the definition of multicellularorganisms. In humans, for example, the cells thatmake up the heart, the lungs, and other organs allhave their own characteristics and their own func-tions. But they also all grow out of the same fewstem cells that are created after fertilization.

S imilarly, O’Toole explains, different cellswithin a biofilm carry out different tasks,grow at different rates, and live in very dif-

ferent environments within a biofilm. Yet the cellsare also individual organisms, even though they co-ordinate their behavior in a way that benefits thewhole community and that may mean some indi-vidual bacteria get less food or face more risk thanother bacteria within the biofilm. “It is pretty amaz-ing,” O’Toole says.“I think it has been a very useful framework or

hypothesis to test,” says DMS microbiologist Deb-orah Hogan, Ph.D., who also works on biofilms(and is married to O’Toole). She adds that this de-

velopmental model helped scientists explore andrecognize the many ways in which planktonic bac-terial cells are very different from the bacteria inbiofilms.One of the most important of those differences

is that biofilms are far more resistant to antibiotics.The question is why, and there is still not a com-plete answer. “From a medical standpoint, trying toincrease the efficacy of antibiotics toward biofilmcells is probably the most important problem thatneeds to be tackled,” Hogan says.When O’Toole arrived at DMS in 1999, he

knew that biofilms cause a number of medical prob-lems. But neither he nor anyone else knew for cer-tain how important biofilms are in making cystic fi-brosis (CF) such a deadly disease. It is only in thepast decade that scientists have learned that bacte-ria form biofilms in the lungs of people with CF,making treatment difficult. As a result, CF patientslive on average, even in the United States, onlyinto their late 30s.O’Toole has turned much of his attention to CF

research because of two factors, he says. For onething, P. aeruginosa is the primary culprit in thedeath of most people with CF and is found in thelungs of about 80% of adults with CF. For another

30 Dartmouth Medicine—online at dartmed.dartmouth.edu

Grad student Dae Gon Ha, left, explains the results of a recent experiment to O’Toole; in the background is another of the grad students in O’Toole‘s lab, Kyle Cady.

In biofilms, “bacteria

actually perform many

of the behaviors that

multicellular organisms

can,” O’Toole says. “They

can talk to each other.

They can discriminate

self from nonself. They

can even specialize.”

continued on page 62

An electronmicrograph of aPseudomonas aeruginosa biofilm

62 Dartmouth Medicine—online at dartmed.dartmouth.edu Fall 2010

Science versus Slime

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continued from page 31grown on human airway cells were evenmore resistant to antibiotics than were bio -films grown on the plastic surfaces often usedin experiments with bacteria. So the prob-lem they were trying to solve was even hard-er than they’d figured.

But there has been good news as well.The failure of individual antibiotics againstbiofilms led the DMS researchers to try var-ious combinations of therapies, hoping to si-multaneously address some of the multipleways that biofilms are able to escape the ef-fects of individual antibiotics.

In 2009, O’Toole, Stanton, and postdoc-toral researcher Sophie Moreau-Marquis,Ph.D., reported that they had been able toeliminate 90% of the bacteria in P. aerugi-nosa biofilms using a combination of tobra -mycin and iron chelators—chemicals thatbind to iron and prevent it from being usedby cells. And happily, the two iron chelatorsthey tested in the study had already been ap-proved by the Food and Drug Administra-tion for use in humans, making the resultsvery clinically relevant.

S ince then, O’Toole says, they havebeen testing different combinations ofantibiotics to find even more effective

treatments, and they hope soon to be able tostart clinical trials using a combination ofiron chelators and antibiotics.

“We don’t understand why iron is impor-tant and why it seems to be more importantparticularly to the cells in the center,” saysO’Toole. “But the bacteria die really quick-ly, so it’s likely not a growth issue. There’ssomething critical about that iron, and whenyou bind it up, the cells become hypersensi-tive to treatment with antibiotics.”

O’Toole didn’t anticipate at the begin-ning of his career that he would focus on CF,but he’s pleased that his work could have im-plications for the treatment of this difficultdisease. “I think we have the possibility ofcontributing to improving patient care,” hesays. “A lot of that has . . . been influencedby the colleagues that are here and their will-ingness to collaborate.”

It’s a good thing O’Toole is still energizedby the scientific challenge. For despite hisprogress, much remains to be learned aboutthe mysterious lives of biofilms.