Biofilms - Science is Fun in the Lab of Shakhashiri1... · Biofilms A new understanding of these niicrobial communities is driving a revolution that may transform the science of microbiology

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Biofilms

A new understanding of these niicrobial communities is drivinga revolution that may transform the science of microbiology

Joe J. Harrison, Raymond J. Turner, Lyriam L. R. Marques and Howard Ceri

When we think about bacteria, mostof us imagine a watery milieu,

with single-celled organisms swim-ming about. We might envision thesesolitary entities getting together withsome of their brethren now and then tocause some disease or spoil some food,but once the job is done they return totheir isolated existence. This image ofbacterial existence, it turns out, is notonly oversimplified but perhaps mis-leading as well. In nature, the majorityof microorganisms live together in largenumbers, attached to a surface. Ratherthan living as lonely hermits in tbe so-called planktonic form, most bacteriaspend much of their lives in tbe micro-bial equivalent of a gated community—a biofilm.

A mature biofilm is a fascinating con-struction: It can form layers, clumpsand ridges, or even more complex mi-crocolonies that are arranged into stalksor mushroom-like formations. The resi-dents of the biofilm may be a singlespecies or a diverse group of microor-ganisms distributed in various neigh-borhoods. Their common bond is a ma-trix made of polysaccbarides, DN A and

joe I Harrison is a doctoral candidate and Raymond

j . Turner an associate professor in the Departmciit

of Biological Sciences at the University of Calgary.

Lyriam L R. Marques was a fxistdoctoral fcllo'd'

with the university's Biofilm Research Group mid

now serves as associate research director at MSEC

BioProducts, Inc. Hoiuard Ceri is a professor of bio-

logical sciences at the iinivtrsitii and chairs tlie Bio-

filin Researcli Group, tie aha senvs on tlie Sigma

Xi Committee on Publications. Address far Ceri:

Deparinmd ofBioiogical Sciences, 2500 University

Drive N. W., Calgary. Alberta, Canada T2N J N4.

Internet: ceri@uca!gari/.ca

proteins, which together form an extra-cellular polymeric stibstiince—what manymicrobiologists just call slime.

It's becoming increasingly clear thatthe communal life offers a microor-ganism considerable advantages. Thephysical proximity of other cells favorssynergistic interactions, even betweenmembers of different species. Theseinclude the horizontal transfer of ge-netic material between microbes, thesharing of metabolic by-products, anincreased tolerance to antimicrobials,shelter from changes in the environ-ment and protection from the immunesystem of an infected host or fromgrazing predators. The formation of abiofilm has even been likened to theprogram by which cells within a mul-ticellular organism differentiate.

An appreciation of the significanceof biofilms is a relatively recent phe-nomenon. Only witbin the past 15 to 20years have biologists begun to exam-ine the physiology of these microbialcommunities. This is an extraordinarystate of affairs, given that the Dutch mi-croscopist Antonie van Leeuwenhoekfirst described biofilms in the late 1600s.Using acetic acid, he had tried to killa biofilm—the dental plaque on hisdentures—^but noted that only the free-swimming cells could be destroyed.Despite the early discovery of microbialcommunities, microbiology departedfrom tbese observations to focus pri-marily on planktoiiic bacteria.

To be sure, not everyone agrees thatbiofilms are the predominant form ofbacteria in nature. The vast majorit}' oflaboratory methods used today exam-ine cultured microorganisms in their

planktonic mode. But we believe thatmicrobiology is experiencing a shift inhow bacteria are conceptualized. Wepredict that this new perspective ofbow microorganisms live will have fun-damental consequences for medicine,industry, ecology and agriculture.

Biofilms Are EverywhereMost people are familiar with the slip-pery substance covering the rocks in ariver or a stream. This particular slimeis an aquatic biofilm made up of bacte-ria, fungi and algae. It begins to formafter bacteria colonize tbe rock's sur-face. These microbes produce the ex-tracellular polymeric substance, whichis electrostatically charged so that ittraps food particles and clay and otherminerals. The matter trapped in theslime forms microscopic niches, eachwith a distinct microenvironment, al-lowing microorganisms that have dif-ferent needs to come together to forma diverse microbial consortium.

A biofilm matrix is considered to bea fn/drogel, a complex polymer hydrat-ed with many times its dry weight inwater. The hydrogel characteristics ofthe slime confer fluid and elastic prop-erties that allow the biofilm to with-stand changes in fluid shear within itsenvironment. So biofilms often formstreamers—gooey assemblages of mi-crobes that are tethered to a surface.As running water passes over the bio-film, some pieces may break free and sospread the microbial community down-stream. It is believed that bacteria cancolonize the lungs of patients on ven-tilators in this way, causing often-fatalpneumonia in critically ill patients.

508 American Scientist, Volume 93

Figure 1. Yellowstone National Park is full of unusual microscopic life, including thermophilic algae and (at bottom nght) filamentous bacteria.The biofilms that these organisms often form may be obvious to the nature photographer's eye, but they are not well understood. Despite the dis-covery of microbial biofilms as far back as the 17th century, scientists have largely focused their aHentions on the solitary, or planktonic, forms ofmicrooi^anisms. In nature, however, most microorganisms live together in large communities attached to a surface, a lifestyle that profoundly af-fects their interaction with other organisms and their resilience as pathogens. New studies of biofilms may change the direction of microbiologicalresearch—with the promise of controlling infections by bacteria and other microorganisms. (Photographs courtesy of the National Park Service.)

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A microorganism's extraordinaryability to spread explains how biofilmsshow up in the unlikeliest of places.The steel hull of a ship at sea can becoated with biofilms that increase thedrag on the vessel and so compromiseits speed. Other biofilms wreak havocin the oil industry by facilitating themicroscopic corrosion of metals andlimiting the lifespan of pipelines. Somebiofilms, made up of the ancient lin-eage of prokaryotes (organisms lack-ing a nucleus) called ardinea, can evensurvive the hostile hydrothermal envi-ronments of hot springs and deep-seahydrothermal vents. The aptly namedarchaebacterium Pyrodictium thrivesat the bottom of the sea, growing in amoldlike layer on sulfur crystals in thedark, anaerobic environment of a hy-drothermal vent, where temperaturesmay exceed 110 degrees Celsius.

Perhaps one of the most extraordi-nary environments where one can finda biofilm is in the belly of a dairy cow.Biofilms are part of the normal com-plement of microbes in many healthyanimals, but the presence of these mi-crobiai communities in ruminants pro-

vides a rich example of the interactionswithin a biofilm.

We begin with the rumen, the largestcompartment of the bovine stomach,which can hold a liquid volume in ex-cess of 150 liters. It is filled with somany microbes that microbiologistsrefer to cows as mobile fermenters.Bacteria colonize the digestive tract ofa calf two days after it is born. Withitithree weeks the microorganisms havemodified the chemistry inside the ru-men, which soon becomes home to areported 30 species of bacteria, 40 spe-cies of protozoa and 5 species of yeast.The cells in this biofilm thrive in themucous layer of the stomach and growon the food ingested by the animal.Cows, of course, eat grass, which con-sists largely of cellulose, a complex car-bohydrate that caruiot be broken downby mammalian digestive enzymes. Butcellulose is a perfect fuel for the bac-teria in the biofilm, which convert itinto a microbial biomass that in turnsupplies the proteins, lipids and carbo-hydrates needed by the cow.

The heart of this process is a micro-scopic ecosystem that begins when a

pioneering planktonic bacterium in therumen, a species such as Ruminococ-cus flavefaciens, gains access to the innerparts of a leaf, perhaps one that mighthave been broken by tiie cow's chewing.These bacteria attach themselves to thecellulose in the inner layers of the leafand proliferate to form a rudimentarybiofiin. The microbes release cellulose-degrading enzymes, which produce sim-ple sugars and metabolic by-productsthat attract other bacteria^anaerobicfermenters such as the spiral-shapedTreponenia byrantii, which ingest the sug-ars and produce organic acids, includingacetic acid and lactic acid.

The acidic metabolites would nor-mally slow the growth of the bacteriaby a process of feedback inhibition, butit so happens that other microorganismsjoin the biofilm community and eat theorganic acids. These are the methano-gens, archaea whose actions acceleratethe growth of the bacterial communityand prevent the inhibitory feedback.As the name suggests, methanogensproduce methane—lots of it. Approxi-mately 15 to 25 percent of the globalemission of methane, which totals 7.5

extracellularpolymericsubstance"slime")

y ^ " - ' •'•growth and dfvision multispecies consortia

Figure 2. Formation of a biofilm is analogous to the development of a multicellular organism, with intercellular signals regulating growth anddifferentiation. A typical biofilm forms (follow arrows from upper left) when free-swimming planktonic bacteria adsorb to a biotic or inani-mate surface—an association that is initially reversible, but then irreversible. Adhesion triggers the first physiological changes on the path toa biofilm lifestyle. As the bacteria grow and divide, molecular signals passed between the cells provide information on cell density—a processcalled quorum sensing. In a maturing colony, the microbes produce an extracellular polymeric substance—a matrix of poly saccharides, DNAand proteins that encases the microcolony structure. Planktonic cells may leave the biofilm to establish new biofilm structures. Signals fromthe collective may also recruit new microbial species to join the consortium.

510 American Scientist, Volume 93

cellulose-degradingbacteria «»

fermenter

cellulose-degradingbiofilm

Figure 3. A multispecies biofilm in a cow's rumen provides an example of the intricate relations between the cells in a microbial community,not to mention the roles biofilms play in the nutrition of ruminants and other animals. The colony begins with cellulose-degrading bacteria,which digest the grass eaten by the ruminant. (A cow's cud can be passed between its mouth and rumen several times before these productsare passed to its remaining stomachs and intestines.) The simple mono- and disaccharide sugars produced by these cellulolytic bacteriaattract fermenting microorganisms, which convert the sugars into organic acids. In turn, the organic acids attract methanogenic microbesthat join the biofilm. The organic acids not neutralized by the cow's saliva would normally inhibit further growth in the biofilm, but themethanogens convert these molecules into methane. The entire process produces a protein-rich microbial mass that can be digested by thecow, providing the bulk of the animal's nutrients.

billion kilograms per year, is attribut-able to the flatulence of ruminants. Be-cause methane traps heat in the atmo-sphere, the biofilm hidden away in acow's stomach may play a nontrivialrole in global climate change.

Animals aren't the only li\-ing thingsthat provide a home to biofilms. Micro-bia! colonies have been recognized ontropical plants and grocery-store pro-duce since the 1960s, but it wasn't untilthe past decade that the term biofilmwas used to describe bacterial growthon a plant's surface. In this domain, lifein a biofilm confers many advantagesto the individual cell, including protec-tion from a number of environmentalstresses—ultraviolet radiation, desic-cation, rainfall, temperature variations,wind and humidity. The biofilm alsoenhances a microorganism's resistanceto antimicrobial substances producedby competing microorganisms or thehost's defenses.

Relations between plants and bio-films can be quite varied. In some in-stances the plant merely serves as amechanical support, so the biofilm issimply a harmless epiphyte. In other

cases, the plant may provide some nu-trients for the microbes, such as the sap-rophytes that feed on decaying plantmatter; these too pose no danger to theplant. But there can be trouble whencertain epiphytic populations with thegenetic potential to initiate a pathogen-ic interaction with the host grow largeenough to overwhelm the host's de-fense mechanisms. Then the cells in thebiofilm coordinate the release of toxinsand enzymes to degrade the plant tis-sue. What began as an innocuous rela-tionship ends in disease.

Belowground, plants and biofiimsmay also engage in some fairly elabo-rate interactions. For example, Pseudo-monas fluorescens colonizes roots andprotects plants from pathogens by pro-ducing antibiotics that exclude fungiand other bacterial colonizers. But fun-gal biofilms can also be beneficial tothe plant. Certain mycorrhizal fungipenetrate a plant's root cells while alsoforming an extensive network in thesoil; thus they provide a drastic in-crease in the surface area that the plantcan use for the absorption of water andnutrients.

On the other hand, bacteria of thegenus Rhizobiutn fix nitrogen from theatmosphere by converting N-, gas intoammonia (NH^). This process can in-volve some intricate chemical signal-ing between the plant and the bacteriathat results in the formation of noduleswithin the root where the bacterial ag-gregates engage in nitrogen fixation.Perhaps the most intricate relationinvolves an interaction between therhizobia, the mycorrhizal fungi and aplant host. The bacteria form a biofiimon the surface of the fungus, whichin turn makes its connection with theplant, and so creates a tripartite symbi-otic system that relies on the formationof biofilms by two microorganisms.(Unless the soil is alkaline, the systemrequires another player, nitrifying bac-teria to oxidize the ammonia; they livenot in the nodule but in nearby soil.)

Finally, let us consider the patho-genic interactions of biofilms withinthe plant's vasculature. Unfortunately,vascular diseases are currently un-treatable and tend to be devastating tomany economically important crops.A few pathogenic biofilms have been

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health disease

epiphyticbiofilm(protects bacteria)

fungal biofilms(nutrientuptake)

antitungaisantibiotics

bacteriocinskill or intiibit pathogenic bacteria

Figure 4. Relations between plants and bio-films run the gamut from healthy (above, leftside) to pathogenic (right side). Many biofilmsare harmless: Saprophytes merely digest deadleaves, whereas epiphytes often simply usethe plant for mechanical support. Some inter-actions may even be valuable: Bacteria-fillednodules below ground enable a plant to fixnitrogen, and certain fungal biofilms give theplant's roots a greater surface area for the ab-sorption of water and nutrients. Some com-mensal bacteria release substances that killpotential pathogens. Unfortunately, biofilmsmay overwhelm the plant's defense mecha-nisms, causing disease processes that attackthe plant from below the ground or even fromthe vasculature within. Xyletia fastidiosa bio-films (right, a 25-inicrometer-zi'ide segment) area problem for grape and citrus growers andothers. (Micrograph courtesy of the authors.)

described in the water-carrying xylemof plants, but here we'll merely ad-dress Xylella fastidiosa. This pathogencauses Pierce's disease in grapevinesand citrus variegated chlorosis insweet oranges—diseases that have hada major in^pact on the wine industryin California and the citrus industry inBrazil, with economic losses exceeding$14 billion in the past decade. Pierce'sdisease also limits the development ofa wine industry in Florida because thebacterium is endemic in that region.

X. fastidiosa is transmitted by xylem-feeding insects, called sharpshooters,tbat acquire the bacteria while feed-ing from infected plants. The bacteriaform a rudimentary biofilm inside theinsect's gut, and this allows them tobe sloughed off indefinitely in aggre-gates sufficient to infect another plantwben the insect feeds again. In turn,the biofilms clog the plant's xylemand cause symptoms related to waterstress. So the biofilm plays a key rolein the colonization of the plant vessels,the propagation of tbe disease and itspatbogenicity.

The appreciation of biofilms' im-portance in plant disease has only justbegun, and it will prtjbably take sometime for the idea to be applied in plantmicrobiology. However, tbe benefitscould be significant. A better under-standing of the associations betweenplants and biofilms may lead to moreefficacious and environmentally friend-ly treatments for disease. It may alsolead to the development of commer-cial applications that could improve thebeneficial interactions between plantsand microorganisms. Indeed, variousrhizobia are now being used on com-mercial farms as a biotic fertilizer.

United We StandThe Centers for Disease Control andPrevention estimates tbat up to 70percent of tbe human bacterial infec-tions in the Western world are causedby biofilms. Tbis includes diseasessuch as prostatitis and kidney infec-tions, as well as illnesses associatedwith implanted medical devices suchas artificial joints and catheters andthe dental diseases—both tooth de-cay and gum disease—that arise fromdental plaque, a biofilm. In the lungsof cvstic fibrosis patients, Psciidoinonasaeruginosa frequently forms biofilmsthat cause potentially lethal pneumo-nias. There is a long list of biofilm-related ailments, and many scientists

512 American Scientist, Volume 93

believe the list will continue to growas we learn more about the functionof these microbial structures.

In almost all instances, the biofilmplays a central role m helping microbessurvive or spread within the host.That's because the slimy matrix acts asa shield, protecting pathogenic bacteriafrom antibodies and white blood cells,the sentinels of the immune system.Biofilms are also notorious for theirability to withstand extraordinarilyhigh concentrations of antibiotics thatare otherwise lethal in smaller doses totheir planktonic counterparts. In fact, abiofilm can be 10 to 1,000 times less sus-ceptible to an antimicrobial substancethan the same organism in suspension.

This challenge, with its grave im-plications for the fight against patho-gens, has been the focus of our researchgroup's investigations. We have devel-oped and licensed to a Canadian start-up company a technology (the CalgaryBiofilm Device, now called the MBECAssay) that can be used to rapidlyscreen biofilms for their sensitivity toantimicrobials. A pharmaceutical labo-ratory testing a potential drug to fightpneumonia or catheter-related infectioncan now find out whether a drug thatis effective against free-floating patho-gens will be successful in eradicatingthe same organisms in a biofilm.

During the development of this tech-nology, we have learned some remark-able things about biofilms. We havemoved on to exploring some patho-genic "co-biofilms" of unrelated spe-cies living together, along with specificmechanisms that may be important indrug development. For example, bio-films' resistance to high metal concen-trations makes them useful in remov-ing toxic metals from the environment.But a detailed understanding of howthe films handle metal toxicity mayalso open the door to antimicrobialtreatments targeted at biofilms.

We and other investigators havelearned that part of the extraordinaryresilience of bacteria arises from the re-markable heterogeneity inside the bio-film. Microbes closest to the fluid thatsurrounds the biofilm have greater ac-cess to nutrients and oxygen comparedwith those in the center of the matrixor near the substratum. As a result, thebacteria in the outer layers of the com-munity grow more quickly than thoseon the inside. This comes into play asa defense mechanism because manyantibiotics are effective only against

Figure 5. Many biofilms can cause disease and discomfort in human beings. The fungus Asper-githis ftimigatus (top left) causes potentially lethal lung infections. The opportunistic pathogenPseiidomojiiis aertiginosa (bottom left) can be fata! to patients with cystic fibrosis. Bacterial bio-films growing on contact lenses (top right) or catheters (bottom right) can cause serious infections,(Micrographs courtesy of the authors. Merle Olson and Liz Middlemiss, University of Calgary.Image-area widths range from 14 [contact lens film] to 66 micrometers [A. fiimigatus].)

negativelycharged matrix

/

CH)

CH)

cell-to-cell Asignals A

X A A

positively chargedantimicrobial

(binds to negativelycharged slime)

change inphysiology

CH)

nutrient gradient.fewnutrients

oxygen gradient

CH)\ fast growers

slowgrowers

geneticdiversity

CH)

Figure 6. Biofilms derive their extraordinary tolerance to antimicrobial compounds from severalfactors. Bacteria near the center of a microcolony grow very slowly because they are exposed tolower concentrations of oxygen and nutrients f J>. They are thus spared the effects of antibioticdrugs, which are much more effective against fast-growing cells. Intercellular signals (.2) can alterthe physiology of the biofilm, causing members to produce molecular pumps that expel antibioticsfrom the cells and allow the community to grow even in the presence of a drug. The biofilm matrixis negatively charged (3) and so binds to positively charged antimicrobials, preventing them fromreaching the cells within the colony. Specialized populations of persister cells ii) do not grow inthe presence of an antibiotic, but neither do they die. When the drug is removed, the persisters cangive rise to a normal bacterial colony. This mechanism is believed to be responsible for recurrentinfections in hospital settings. Finally, population diversity (5J, genetic as well as physiological,acts as an "insurance policy," improving the chance that some cells will survive any challenge.

www.americansdentist.cirg 2005 No\-ember-December 513

A New Way to Look at Microorganisms

The conventional way to grow bacteria is to inoculate a flask that containsa broth of nutrients. If you stir the broth constantly, the cells will have

plenty of oxygen and a homogeneous distribution of food. Under these opti-mal growth conditions, you'll get a nice batch of planktonic bacteria floatingin the solution.

Of course, nature rarely provides such a perfectly uniform environment.Bacteria in a biofilm grow in a matrix of heterogeneous microenvironmentsthat vary in oxygen content, nutrient distribution and countless other chemicalvagaries. The bacteria that stick to the sides of the laboratory flask form maturebiofilms. Ironically, until recently these were largely ignored or destroyed.

Several new technologies have been explicitly developed to grow andexamine biofilms in the laboratory. One method uses a rotating disk insidean inoculated broth. The shear force caused hy the rotation encourages theformation of a biofilm on the disk. Our laboratory group has also recentlydeveloped a biofilm-based assay for examining the effectiveness of antimi-crobials in a high-throughput fashion—that is, the device allows us to create96 statistically equivalent biofilms, and it can also be used to test various dilu-tions of antimicrobial compounds with a standard microtiter plate, the MBECassay. We are currently using this fool to discover new substances that may beeffective against biofilms.

Another device, called a flow cell, consists of a chamber and an opticallytransparent surface, such as a glass coverslip. A growth medium is pumpedthrough the chamber, promoting the formation of a thick biofilm on theglass surface. This method allows scientists fo examine microbial communi-ties in a confocal laser-scanning microscope (CLSM). Specialized computersoftware can be used to assemble images captured by CLSM to create athree-dimensional view of a biofilm.

CLSM might be considered as a complement to scanning electron mi-croscopy (SEM). SEM can achieve magnifications tbat are 10 times greaterthan CLSM and so can be used to examine the shape and arrangement ofsingle cells, whereas CLSM provides an overview of the hiofilm's structure.SEM also kills the microbial community, whereas CLSM is not as invasive.Sequences of images can be compiled into movies that show how microor-ganisms live and die in a biofilm.

Finally, new methods in proteomics and transcriptomics allow scientiststo examine the distribution and patterns of proteins and gene expression inbiofilms. The development of these techniques has opened the door to a newview of bow microorganisms live.

Images assembled from "slices" created by confocal laser-scanning microscopy can providea detailed look at a microbial biofilm's structure. This is a biofilm of Esclwrichta coli that hasbeen grown in thelaboratoiy and made visible by splicing a gene fora fluorescent protein intoits DNA. A close-up appears on the magazine's cover. (Image courtesy of EDM Studio.)

fast-growing cells, so the slow growerswithin the biofilm tend to be spared.Moreover, the cells in the center of thecommunity are further protected fromthe environment because the biofilmmatrix is negatively charged. This re-stricts the entry of positively chargedsubstances, such as metal ions and cer-tain antibiotics.

One of tbe most intriguing defensemechanisms enabled by the formationof a biofilm involves a kind of inter-cellular signaling called quorum sens-ing. Some bacteria release a signalingmolecule, or inducer. As cell densitygrows, the concentration of these mol-ecules increases. The inducers interactwith specific receptors in each cell toturn on "quorum sensing" genes andinitiate a cascade of events, trigger-ing the expression or repression of anumber of other genes on the bacterialchromosome. Some bacterial strainsseem to rely on quorum sensing morethan others, but anywhere from 1 to 10percent of a microbe's genes may bedirectly regulated by this process.

Quorum sensing is known to affectthe production of en^^ymes involved incellular repair and defense. For exam-ple, the enzymes superoxide dismutaseand catalase are both regulated byquorum sensing in P. aerugiiiosn, whichforms mucoidal clusters of bacterialcells embedded in cellular debris fromthe airway epithelial layer in the cysticfibrosis patient's ltmg. The first enzymepromotes fhe destruction of the harmfulsuperoxide radical (O.,"), whereas thesecond converts the equally toxic hy-drogen peroxide molecule (H-,0-,) intowater and molecular oxygen, these en-zymes help fhe biofilm survive assaultsnot only from disinfectants, but alsofrom the cells of a host's immune sys-tem that typically kill bacteria by un-leashing antimicrobial agents, includingreactive oxygen species.

Quorum sensing may also be in-\ olved in the defense against antibioticdrugs. Here the mechanism increasesthe production of molecular pumpsthat expel compounds from the cell.These so-called miiltidrug efflux pumpsreduce the accumulation of the anti-biotics within the bacteriiun and evenallow the microbe to grow in the pres-ence of the dnigs.

There is also heterogeneity amongthe cell types in the biofilm that contrib-utes to antimicrobial tolerance. Special-ized survivor cells, called "persisters,"are slow-growing variants that exist in

Figure 7, Candida tropicalis, a yeast that causes vagtnitis, thrush and cardiac infections, formsbiofilms that are highly resistant to antifungal and antimicrobial treatments. The image wascreated using a confocal laser-scanning microscope, a new device thai provides a snapshotof microbial microcoionies, which make up a biofilm (see discussion on facing page). (Imagecourtesy of the authors.)

every bacterial population. They are ge-netically programmed to survive envi-ronmental stress, including exposure toantibiotics. Although persisters do notgrow in the presence of an antibiotic,they also do not die. Persisters are notmutants; even in a genetically uniformpopulation of cells a small portion un-dergo a spontaneous switch to the per-sistent form. This past year Kim Lewisof Northeastern University demonstrat-ed that persisters generate a toxin, RelE,that drives the bacterial cell into a dor-mant state. Once antibiotic therapy hasceased, the persisters give rise to a newbacterial population, resulting in a re-lapse of the biofilm infection.

The use of persister cells as a defensemechanism may have evolved early inthe history of life. In this post-genomicsera, scientists have learned that manyrelated genes are present in a varietyof distantly related bacteria, suggestingthat similar genes were present in theprimeval common ancestors. Yet thereduced growth rate of the persistersposes a paradox because slowed celldivision decreases the fitness of a popu-lation. Edo Kussell and his colleaguesat Rockefeller University recently pro-posed that bacterial persistence mayhave evolved as an "insurance policy"against rare antibiotic encounters. If so,in attempting to overcome bacterial an-tibiotic tolerance, scientists are battlingan ancient mechanism that may havebeen refining itself for billions of years.If we are ever to succeed in controlling

bacterial infection, more research effortsneed to be focused on biofilms rath-er than the comparatively vulnerableplanktonic form.

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