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Inside the complex world of bacteria: aplace where germs talk to each.orher;build cities, and execute precisely coordinated attacks.

BY WENDY ORENT

.,

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erhaps you notice it after a visit to the dentist. Youpass your tongue across the front of yowr teeth and they feel slick and squeaky-clean. Four hours later,althouqh you might not yet be able to tell the difference, the oeginning of a rough fuzz is growing. Theseare streptococci, the first bacterial settlers in the film that saliva deposits on your teeth. Another fourhours and the bridge germs, the fusobacteria, have climbed on board. They are the ones that make it pos-sible for the really bad actors, like Porphyromonas gingiva/is, to grab on and start building colonies.

By the next morning, if you still have not brushed your teeth, a definite fuzzy scum is startingto form. If you Gould look at that fuzz under a microscope without disturbing its structure, youwould see towers or entire communities of bacteria, each building upon others. Some of thosemicrobes are dangerous indeed. P. gingivatis not only grows in the pockets of your gums, helpingto loosen your teeth from your jaws, but also causes the release of inflammatory chemicals thatget into your circulation, complicating diabetes treatment and possibly increasing the risk of heartdisease. Traces of the germ have also been found in arterial plaque.

If you have ever been admitted to a hospital, it is very likely you have experienced another, related

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kind of scary bacterial growth-and inthis case you almost certainlydid not notice it. Hospitalized patients are routinely hooked up tourinary catheters that enable doctors and nurses to measure urineflow (not incidentally, the catheters also liberate health-care work-ers from having to take patients to the bathroom). Swiftly coatedby a conditioning film made of proteins in the urine, the cathetersare then inexorably covered by layers of interacting bacteria, whichalter the chemistry of their surface and can cause crystals to form.Within a week, an infection is growing on the catheters of 10 percentto 50 percent of catheterized patients. Within a month the infectionhas reached virtually everyone.

These slimy bacterial colonies, known as biofilms, add a remark-able new dimension to our understanding of the microbial world.Ever since Louis Pasteur first grew bacteria in flasks, biologistshave pictured bacteria as individual invaders floating or swimmingin a liquid sea, moving through our blood and lymph like a schoolof piranhas down the Amazon. But in recent years, scientists havecome to understand that much, and perhaps most, of bacteriallife is collective: 99 percent of bacteria live in biofilms. They varywidely in behavior. Sometimes these collectives are fixed, like acluster of barnacles on a ship's hull; other times they move, orswarm, like miniature slime molds. Bacteria may segregate intosingle-species biofilms, or they may, as in the case of dental bac-teria, join together in groups that function like miniature ecologicalcommunities, competing and cooperating with each other.

The unifying factor in all these biofilms-the thing that makes themso strange and wonderful and dangerous-is that their cooperationis, in a sense, verbal. Using streams of chemicals that they pump out-side their cell walls and membranes, they "talk" incessantly, amongtheir own clones and species and even to unrelated bacteria dwell-ing nearby. Understanding that chatter could be vital for gaining theupper hand in the endless battle against infectious disease.

B iofilms were first discovered in 1978 in the clear waters of afrigid mountain stream in British Columbia. MicrobiologistWilliam Costerton, now of Allegheny General Hospital inPittsburgh, and his team of scientists wondered why there

were so few bacteria in the water, while billions upon billions of thegerms nestled in the crevices of the streambed's rocks. "We werefinding 9 bacteria per milliliter in the water, but there must havebeen 100 million in a square centimeter when we took a rock outof a stream and brought it down to the lab," Costerton says.

The bacteria were not just sitting idly on the rocks, he found. Theywere forming complicated structures, cities of germs encased in aslippery substance the bacteria exude called an exopolysaccharidematrix. This slime protects them from grazing amoebas and pro-vides them with food that is excreted by bacteria within the biofilmor even bits of DNA released when other germs die.

When Costerton published his results, he coined the term bio-film and introduced a whole new understanding of how bacteriabehave. "We reasoned one stubborn fact," he recalls. "Bacteriahave no idea of where they are. They are just programmed to dotheir thing." In other words, they are always going to form biofilms-whether they are living on a rock or in the human body.

Two years later Tom Marrie, a young doctor working in Halifax,Nova Scotia, 'examined a feverish homeless man who had wan-dered off the street and into his emergency room. The man had araging staph infection and, on his chest, a lump the size and shapeof a cigarette pack. It was an infected pacemaker, Marrie reasoned.

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Staphylococcusaureus (above) buildbiofilms everywherefrom hospitalcatheters toyour mouth andteeth.

For three weeks the man was given huge doses of antibiotics butdid not get better, so Marrie and his team decided to operate. Theyinvited Costerton to sit in. "If there were ever going to be a biofilminfection in a human being, it was going to be on the end of thatpacemaker," Costerton says. "We took out the pacemaker andthere was our first medical biofilm. It was a great big thick layer ofbacteria and slime, just caked on."

Biofilms on implants are now recognized as a serious and grow-ing health problem. Bacterial infections hit 2 percent to 4 percent ofall implants. Of the 2 million hip and knee replacements performedworldwide each year, 40,000 become infected. More than a third ofthese infections lead to amputation, and not with very successfulresults: Most of those people die. "Implant operations have a 98percent success rate, so people don't want to talk about the infec-tions," Costerton says. "They're a bit of a disgrace, really."

Biofilm infections are not limited to implants. They can be found inthe bodies of the young and the healthy. Many children suffer fromundiagnosed biofilm infections in their ears, which require monthsof oral antibiotic therapy while the underlying infection smoldersuntouched. Millions of others live with chronic biofilms: urinary tractinfections in women that last for years; prostatitis that no antibiot-ics permanently cure; bone infections (osteomyelitis) that crippleand immobilize people for the rest of their lives. Each year roughly500,000 people in the United States die of biofilm-associated infec-tions, nearly as many as those who die of cancer.

As Marrie's experience shows, biofilms repel antibiotics,although scientists do not fully understand how. Some drugs can-not fully penetrate the, biofilm's protective matrix. lri other cases,

molecules that produce more of themselves inside the cell) controlthe switch that turns the light genes off and on. Each bacteriumsecretes a bit of this light-evoking substance into the environ-ment. When a crowd of bacteria and their autoinducers becomedense enough, the lights in all the bacteria switch on at once. "Thiscounting of heads is called quorum sensing," Bassler explains.More broadly, this is how bacteria coordinate their actions in largegroups: When the local concentration of autoinducers gets highenough, the bacteria know a crowd is present, and they flip overfrom solitary mode to group behavior.

The autoinducer molecule that triggers bacterial glow is madeby a protein called Luxl, which has a very focused effect. "Themolecule that the Luxl protein makes is acylated homoserine lac-tone, or AHL," Bassler says. "Each Luxl protein and the molecule itproduces is species-specific. There are two kinds of bacteria, andeach talks in a different language. Gram-negative bacteria [whichhave a thin cell wall surrounded by an outer membrane] use the

Bacteriologists had it wrong for the past 300 years - bacteria don't livealone. They grow best when each one does its own thing ...together. -even though most of the germs die, enough remain alive to regroupand develop another biofilm. The matrix also keeps its residentgerms under cover, hiding the chemical receptors on the bacteriaso that drugs cannot latch onto them and kill the germs.

The study of this newly discovered behavior is rooted in the basicand ancient biology of bacteria. Geneticist Bonnie Basslerof Prince-ton University thinks group-living bacteria may give us a windowonto the origins of multicellular life. "Bacteria grow best when eachone does its own thing ... together," she says. "Bacteriologists had itwrong for the past 300 years-bacteria don't live alone."

As these social bacteria talk to each other, we can now listenin. Bassler and other scientists are learning how to eavesdrop onthe chemical language of bacteria, seeking ways to scramble orblock those messages. Disrupting the formation of films could bea powerful way to neutralize harmful infections.

~ Originally trained as a biochemist at Johns Hopkins University,~5 the blue-eyed, athletic Bassler walked into a lecture hall on a whim~ in the late 1980s to listen to a talk by geneticist Michael Silverman~ of the Agouron Institute in La Jolla, California. It was one of only a! handful of talks that the notoriously reserved Silverman had given'I' in 10 years. Bassler was riveted by what she heard. Silverman~ talked about how bacteria make light inside the inch-long lumines-~ cent squid that live in the shallow waters off the Hawaiian coast~ (see "Aliens of the Sea," page 76). Infant squid cannot glow until~ they excrete a mucuslike net to entrap the ubiquitous luminescent~ bacteria floating in the water. The squid draw captured bacteria~ into their "light pouches," where the bacteria are bathed in nutrientst~ -a diet richer than what they can find outside in the sea. In return,~ the bacteria (Vibrio fischeri, a close relative of the cholera germ).i produce a dim blue-green light that is directed downward through- small reflective organs in the squid to shine into the water below.~~ When the squid swim at the ocean surface at night, hunting for~~ shrimp, they are invisible to predators below because they look~ like moonligh on the water. Both squid and bacteria benefit. "The~ host wants the ligh, the bacteria get fed," Bassler says.[3 The glow of V. fischeri provides an instructive glimpse into the~ communal behavior of bacteria. Autoinducers (chemical signaling

AHLs as autoinducers, while gram-positives [which have a thick cellwall] use peptides. This is a very ancient split." When the V. fischerimake enough AHL autoinducer-called AI-1 for short-the cellswink on. But that is far from the only autoinducer.

Working with a related bacterium, Vibrio harveyi, in the early'1990s, Bassler discovered another kind of chemical signal that awide range of bacteria emit. In many species this chemical, calledautoinducer 2 (A1-2)has properties of a waste product, says molec-ular biologist Stephen Winans of Cornell University. AI-2 is the by-product of a complex process of metabolism in these species. Notall bacteria create AI-2, however. According to Winans, eons agoone line of early bacteria began to break down waste productsalong a pathway leading to the excretion of AI-2; another line didnot. The latter are the bacteria that eventually gave rise to eukary-otic organisms, including humans. "That's why you don't excreteAI-2," Winans says.

But Bassler found that AI-2 is much more than a waste product."This little leftover molecule," she says, got pressed into service asanother bacterial language, one that can carry messages betweendifferent kinds of germs. Most forms of quorum sensing, includingV. fischeri's luminescence circuit, act as a private language-thatis, each germ speaks only to others of its own kind. But AI-2 is akind of bacterial Esperanto, Bassler determined. After she and herteam purified the small AI-2 molecule and its protein receptor, theywere able to show that the two form a lock-and-key structure, thetelltale sign of a chemical signaling mechanism.

The big question was, what are different germs saying whenthey talk to each other? Bassler says that in some instances-suchas in dental biofilms, in which some 600 species may be growingat a time-AI-2 is necessary for collective or cooperative behav-ior. First, though, the bacteria must be right next to each other toreceive the signal, especially in a dynamic system like the mouth,where saliva is constantly washing across the teeth. The earliestcolonists on freshly cleaned teeth, the streptococci, produce onlylow levels of AI-2; the fusobacteria produce moderate levels. Theappallingly destructive germs love a very high level of AI-2, whichsends them into overdrive. "They grow like gangbusters," says

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Paul Kolenbrander of the National Institute of Dental and Cranio-facial Research of the National Institutes of Health.

Ouorurn-senslnq molecules also play an important part inbacterial virulence, or deadliness. If a lethal germ released toxicchemicalsirnrnediatety after entering the host's body, the immunesystem would quickly sense the toxin and go after the invader. Soit pays for bacteria to wait, stealthily multiplying until the unwittinghost is full of them. Then they can release their toxins all at once,overwhelming immunity and sickening or killing the host.

In their more recent work, Bassler and her colleagues aresearching for ways to scramble the quorum-sensing signals ofcholera germs. The researchers have demonstrated that in testtubes a particular chemical, called CAI-1 , can induce deadly chol-era cells to turn off their virulence genes.

Building on our understanding of how germs communicate,Naomi Balaban, a molecular biologist at Tufts University,hasspent 17 years studying Staphylococcus aureus, a strain ofbacterium that is the main cause of hospital-acquired infec-

tlons, Antibiotic-resistant forms of S. aureus, known collectivelyas methicillin-resistant Staphylococcus aureus, or MRSA, havespread widely in hospitals throughout the world, forming longchains of infection. There are 19,000 MRSA-associated deaths inthe United States alone each year.

Other forms of MRSA have begun to spread outside of hos-pitals; one strain, known as USA300, is especially deadly. It has

.infected and killed children and athletes, and no one knows whereit came from or exactly how it spreads, though athletic lockerrooms have been implicated in some cases. Like other forms ofstaph, USA300 can form invisible biofilms outside the body, mak-.ing it almost impossible to eradicate. It is difficult to judge theactual prevalence of MRSA, since many staph infections do notget much more serious than a small pimple. Some cases do prog-ress, though, and they may cause debilitating and almost untreat-able soft-tissue infections like cellulitis and folliculitis, pneumonia,and often-fatal heart infections, or endocarditis. Another form ofstaph, StaphylococcUs epidermidis, grows commonly in sheetsof invisible biofilm on our skin, where it is normally benign. But ifit is introduced into the body during a medical procedure-espe-cially if a joint implant, catheter, or pacemaker is contaminatedduring insertion-both S. epidermidis and S. aureus can formdangerous biofilms that often cannot be treated without removalof the infected implant.

Balaban has discovered that all forms of staph, whether in afree-floatinq state or in a biofilm, have a complex form of chemicalcommunication that can activate the aqr (accessory gene regula-tor) system, producing a number of toxins. Somewhat controver-sially, Balaban also claims to have discovered another system thatcontrols the agr system. The second system involves two proteinsknown as RNAIIIactivating protein (RAP)and TRAp,which Balabancalls "the most beautiful protein in the world." TRAP is RAP'stargetprotein, Balaban says. It is found both on and within the staph cell.S. aureus secretes RAP into the environment, where the chemi-cal collects and binds to the TRAP molecules on the cells. When

"fenough RAP molecules adhere to enough target molecules, staphbacteria switch on their cell-to-cell communication and stress-response systems and begin producing the toxin that makes themso lethal. S. aureus bacteria, depending on their strain, can pro-duce 40 or more different toxins. The toxins break down the cells

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in the host-which could very well be you-in order to releasenutrients to the germs. That is 'why staph infections can be sodestructive. When there are enough staph germs present, thehost's immune system is overwhelmed, and tissues are destroyedat a frightening rate, leading sometimes to shock and death.

Balaban reasoned that if she could find a way to block RAPfromreaching its target molecule, she could break down the signalingsystem that allows the release of staph's devastating toxins. Shediscovered a chemical she calls RIP (RNAIII inhibiting peptide),which blocks RAP from linking to its target. It is as ltan.outnelderwere standing ready to catch a fly ball heading his way, but healready has a grapefruit in his mitt, preventing the ball from goingin. If RAP does not reach its target molecule, the whole cornrnunl-cation process breaks down, toxins do not get made, and humanimmune cells converge on the now-helpless staph germs, readyto mop them up. Balaban claims that RIP can have this effect onfree-floating and biofilrn-ernbedded staph alike.

Some researchers remain unpersuaded by Balaban's work,however. Richard Novick of the NYU Langone Medical Center, awell-respected staph expert who was also Balaban's postdoctoraladviser, insists that the TRAP protein does not have any knownrole in staph biology. In a series of letters to the journal The Sci-entist, he argues that only one quorum-sensing system has beendiscovered in Staphylococcus: the agr system. Neither Novick norany other scientist has been able to reproduce Balaban's RAP/TRAP experiments in the laboratory. Novick does acknowledge,though, that RIP works. "I don't question that it has activity. Butwhatever it's doi~g, it's not inhibiting agr," he says. "I would guessit could work by interfering with assembly of a biofilm. It shouldnot have any effect on planktonic Staphylococcus. If it did, I wouldhave to revise my view."

Despite these questions, RIP-which Balaban discovered inNovick's laboratory-is in the first stages of preclinical testing asa new kind of antibiotic. It costs millions of dollars to develop drugsand get them tested in animals before they can ever be used inclinical trials for safety and efficacy in humans. Fortunately, Bala-ban has found a naturally occurring chemical equivalent to RIP:hamamelitannin, an extract of witch hazel bark. She has shownthat this old-fashioned household remedy, long used by NativeAmericans, also serves to knock the ball from the outfielder's mitt.

beft: §. chest ;ay shows a pacemaker, the frequent target ofbiot'ilms. Above: lnal.Ower.;ity of Minnesota lab, biofilms are beingstudied. Dental plaque IJioIWnson a used toothbrush bristle.

In her tests, hamamelitannin has the same chemical effect as syn-thetically produced RIP.

In 2003 Balaban's work caught the attention of Randall Wolcbtt,a doctor who runs the Southwest Regional Wound Care Centetin Lubbock, Texas. Wolcott was developing new antibiofilm treat-ments, so he contacted the Tufts scientist to discuss both RIPand hamamelitannin. The result: Wolcott broke new ground byadding hamamelitannin to the xylitol and lactoferrin-other naturalcompounds-he was already using to fight infections. Xylitol isa plant-sugar alcohol that has powerful antibiofilm properties. "Ilearned about it from a local general practitioner," Wolcott says.Lactoferrin, a protein found in milk and saliva, kills bacteria bysequestering the iron that they need to function normally.

Since biofilms in chroriic wounds are built up by multiple spe-cies of bacteria, it is important to attack as many as possible atthe sametime, Wolcott says. He starts by scrubbing a wound tophysically break up the biofilm. Then he hits it with a whole cocktailof antibiofilm agents to attack the colony defenses instead of thebacteria themselves-a departure from usual antibiotic therapy,where one drug, when it fails to resolve the infection, is followed-by another, and another. Wolcott's therapy has,had good results.Patients are doing strikingly better with hamamelitannin added tohis chemical cocktail than without it.

Typical of Wolcott's patients is James Porter (not his real name),who, like many late-stage diabetics, suffered for years with chronicwounds on one of his legs. Diabetic wounds typically start as aninnocent cut or scratch, and they often go unnoticed because thedisease damages nerves so the patient does not feel it. The' cutmay grow into an ugly, oozing ulcer with areas of yellow, black,or a terrifyi'ng green. It consumes more and more tissue until theonly recourse is amputation. Several years ago, Porter limped intoWolcott's medical center, Explaining that he took care of a wifewho was completely disabled with multiple sclerosis, he said thathe could not manage as an amputee. "Do anything you want, doc-tor," he said. "But don't take my leg."

Had Porter gone nearly anywhere else, he would most likelyhave had an amputation, as thousands of Americans do everyyear, "First we take one leg, then we take the other leg, and thenthey die," Wolcott says. "A diabetic amputee has a worse five-yearprognosis than anyone with anything except the worst forms ofcancer.'They die piece by piece, and their suffering is terrible."Under Wolcott's care, though, the horrible colors faded from Por-ter's foot, and clean pink tissue grew over the wound sites, It tookWolcott's team nine months to heal the wounds, but Porter walkedout of the medical center, leaning on a cane, on his own two legs.Wolcott claims that his methods could save tens of thousands oflives a year nationwide.

In 2006 Wolcott's work in chronic wound healing, done in col-laboration with the Montana State University Center for BiofilmEngineering (once directed by William Costerton), earned theresearch center a tour-year.Sz.s million grant from the NationalInstitutes of Health. Wolcott .hasfound that more than three-quar-ters of patients with serious diabetic wounds keep their limbs withhis treatments, The secret is remembering that bacteria are social,not solitary. "Biofilms are not what we learned in microbiology,"Wolcott says. "To treat them in the short term, we need biocides[xylitol, lactoferrin, antibiotics] in combination. You have to usethem at the same time, not sequentially, Quorum-sensing inhibi-tors are the long-term solution,:'.

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