Originally trained as a biochemist at Johns Hopkins University, 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 in 10 years. Bassler was riveted by what she heard. Silverman talked about how bacteria make light inside the inch-long luminescent 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 nutrients —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) 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 moonlight on the water. Both squid and bacteria benefit. “The host wants the light, the bacteria get fed,” Bassler says.

The glow of V. fischeri provides an instructive glimpse into the communal behavior of bacteria. Autoinducers (chemical signaling molecules that produce more of themselves inside the cell) control the switch that turns the light genes off and on. Each bacterium secretes a bit of this light-evoking substance into the environment. When a crowd of bacteria and their autoinducers become dense enough, the lights in all the bacteria switch on at once. “This counting of heads is called quorum sensing,” Bassler explains. More broadly, this is how bacteria coordinate their actions in large groups: When the local concentration of autoinducers gets high enough, the bacteria know a crowd is present, and they flip over from solitary mode to group behavior.

The autoinducer molecule that triggers bacterial glow is made by a protein called LuxI, which has a very focused effect. “The molecule that the LuxI protein makes is acylated homoserine lactone, or AHL,” Bassler says. “Each LuxI protein and the molecule it produces is species-specific. There are two kinds of bacteria, and each talks in a different language. Gram-negative bacteria [which have a thin cell wall surrounded by an outer membrane] use the AHLs as autoinducers, while gram-positives [which have a thick cell wall] use peptides. This is a very ancient split.” When the V. fischeri make enough AHL autoinducer—called AI-1 for short—the cells wink 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 a wide range of bacteria emit. In many species this chemical, called autoinducer 2 (AI-2) has properties of a waste product, says molecular biologist Stephen Winans of Cornell University. AI-2 is the by-product of a complex process of metabolism in these species. Not all bacteria create AI-2, however. According to Winans, eons ago one line of early bacteria began to break down waste products along a pathway leading to the excretion of AI-2; another line did not. The latter are the bacteria that eventually gave rise to eukaryotic organisms, including humans. “That’s why you don’t excrete AI-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 as another bacterial language, one that can carry messages between different kinds of germs. Most forms of quorum sensing, including V. fischeri’s luminescence circuit, act as a private language—that is, each germ speaks only to others of its own kind. But AI-2 is a kind of bacterial Esperanto, Bassler determined. After she and her team purified the small AI-2 molecule and its protein receptor, they were able to show that the two form a lock-and-key structure, the telltale sign of a chemical signaling mechanism.

The big question was, what are different germs saying when they talk to each other? Bassler says that in some instances—such as in dental biofilms, in which some 600 species may be growing at a time—AI-2 is necessary for collective or cooperative behavior. First, though, the bacteria must be right next to each other to receive the signal, especially in a dynamic system like the mouth, where saliva is constantly washing across the teeth. The earliest colonists on freshly cleaned teeth, the streptococci, produce only low levels of AI-2; the fusobacteria produce moderate levels. The appallingly destructive germs love a very high level of AI-2, which sends them into overdrive. “They grow like gangbusters,” says Paul Kolenbrander of the National Institute of Dental and Craniofacial Research of the National Institutes of Health.

Quorum-sensing molecules also play an important part in bacterial virulence, or deadliness. If a lethal germ released toxic chemicals immediately after entering the host’s body, the immune system would quickly sense the toxin and go after the invader. So it pays for bacteria to wait, stealthily multiplying until the unwitting host 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 are searching for ways to scramble the quorum-sensing signals of cholera germs. The researchers have demonstrated that in test tubes a particular chemical, called CAI-1, can induce deadly cholera cells to turn off their virulence genes.

Building on our understanding of how germs communicate, Naomi Balaban, a molecular biologist at Tufts University, has spent 17 years studying Staphylococcus aureus, a strain of bacterium that is the main cause of hospital-acquired infections. Antibiotic-resistant forms of S. aureus, known collectively as methicillin-resistant Staphylococcus aureus, or MRSA, have spread widely in hospitals throughout the world, forming long chains of infection. There are 19,000 MRSA-associated deaths in the United States alone each year.

Other forms of MRSA have begun to spread outside of hospitals; one strain, known as USA300, is especially deadly. It has infected and killed children and athletes, and no one knows where it came from or exactly how it spreads, though athletic locker rooms have been implicated in some cases. Like other forms of staph, USA300 can form invisible biofilms outside the body, making it almost impossible to eradicate. It is difficult to judge the actual prevalence of MRSA, since many staph infections do not get much more serious than a small pimple. Some cases do progress, though, and they may cause debilitating and almost untreatable soft-tissue infections like cellulitis and folliculitis, pneumonia, and often-fatal heart infections, or endocarditis. Another form of staph, Staphylococcus epidermidis, grows commonly in sheets of invisible biofilm on our skin, where it is normally benign. But if it is introduced into the body during a medical procedure—especially if a joint implant, catheter, or pacemaker is contaminated during insertion—both S. epidermidis and S. aureus can form dangerous biofilms that often cannot be treated without removal of the infected implant.

Balaban has discovered that all forms of staph, whether in a free-floating state or in a biofilm, have a complex form of chemical communication that can activate the agr (accessory gene regulator) system, producing a number of toxins. Somewhat controversially, Balaban also claims to have discovered another system that controls the agr system. The second system involves two proteins known as RNAIII activating protein (RAP) and TRAP, which Balaban calls “the most beautiful protein in the world.” TRAP is RAP’s target protein, Balaban says. It is found both on and within the staph cell. S. aureus secretes RAP into the environment, where the chemical collects and binds to the TRAP molecules on the cells. When enough RAP molecules adhere to enough target molecules, staph bacteria switch on their cell-to-cell communication and stress-response systems and begin producing the toxin that makes them so lethal. S. aureus bacteria, depending on their strain, can produce 40 or more different toxins. The toxins break down the cells in the host—which could very well be you—in order to release nutrients to the germs. That is why staph infections can be so destructive. When there are enough staph germs present, the host’s immune system is overwhelmed, and tissues are destroyed at a frightening rate, leading sometimes to shock and death.

Balaban reasoned that if she could find a way to block RAP from reaching its target molecule, she could break down the signaling system that allows the release of staph’s devastating toxins. She discovered a chemical she calls RIP (RNAIII inhibiting peptide), which blocks RAP from linking to its target. It is as if an outfielder were standing ready to catch a fly ball heading his way, but he already has a grapefruit in his mitt, preventing the ball from going in. If RAP does not reach its target molecule, the whole communication process breaks down, toxins do not get made, and human immune cells converge on the now-helpless staph germs, ready to mop them up. Balaban claims that RIP can have this effect on free-floating and biofilm-embedded staph alike.

Some researchers remain unpersuaded by Balaban’s work, however. Richard Novick of the NYU Langone Medical Center, a well-respected staph expert who was also Balaban’s postdoctoral adviser, insists that the TRAP protein does not have any known role in staph biology. In a series of letters to the journal The Scientist, he argues that only one quorum-sensing system has been discovered in Staphylococcus: the agr system. Neither Novick nor any 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. But whatever it’s doing, it’s not inhibiting agr,” he says. “I would guess it could work by interfering with assembly of a biofilm. It should not have any effect on planktonic Staphylococcus. If it did, I would have to revise my view.”

Despite these questions, RIP—which Balaban discovered in Novick’s laboratory—is in the first stages of preclinical testing as a new kind of antibiotic. It costs millions of dollars to develop drugs and get them tested in animals before they can ever be used in clinical trials for safety and efficacy in humans. Fortunately, Balaban has found a naturally occurring chemical equivalent to RIP: hamamelitannin, an extract of witch hazel bark. She has shown that this old-fashioned household remedy, long used by Native Americans, also serves to knock the ball from the outfielder’s mitt. In her tests, hamamelitannin has the same chemical effect as synthetically produced RIP.

In 2003 Balaban’s work caught the attention of Randall Wolcott, a doctor who runs the Southwest Regional Wound Care Center in Lubbock, Texas. Wolcott was developing new antibiofilm treatments, so he contacted the Tufts scientist to discuss both RIP and hamamelitannin. The result: Wolcott broke new ground by adding hamamelitannin to the xylitol and lactoferrin—other natural compounds—he was already using to fight infections. Xylitol is a plant-sugar alcohol that has powerful antibiofilm properties. “I learned about it from a local general practitioner,” Wolcott says. Lactoferrin, a protein found in milk and saliva, kills bacteria by sequestering the iron that they need to function normally.

Since biofilms in chronic wounds are built up by multiple species of bacteria, it is important to attack as many as possible at the same time, Wolcott says. He starts by scrubbing a wound to physically break up the biofilm. Then he hits it with a whole cocktail of antibiofilm agents to attack the colony defenses instead of the bacteria 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 to his 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 chronic wounds on one of his legs. Diabetic wounds typically start as an innocent cut or scratch, and they often go unnoticed because the disease damages nerves so the patient does not feel it. The cut may grow into an ugly, oozing ulcer with areas of yellow, black, or a terrifying green. It consumes more and more tissue until the only recourse is amputation. Several years ago, Porter limped into Wolcott’s medical center. Explaining that he took care of a wife who was completely disabled with multiple sclerosis, he said that he could not manage as an amputee. “Do anything you want, doctor,” he said. “But don’t take my leg.”

Had Porter gone nearly anywhere else, he would most likely have had an amputation, as thousands of Americans do every year. “First we take one leg, then we take the other leg, and then they die,” Wolcott says. “A diabetic amputee has a worse five-year prognosis than anyone with anything except the worst forms of cancer. They die piece by piece, and their suffering is terrible.” Under Wolcott’s care, though, the horrible colors faded from Porter’s foot, and clean pink tissue grew over the wound sites. It took Wolcott’s team nine months to heal the wounds, but Porter walked out of the medical center, leaning on a cane, on his own two legs. Wolcott claims that his methods could save tens of thousands of lives a year nationwide.

In 2006 Wolcott’s work in chronic wound healing, done in collaboration with the Montana State University Center for Biofilm Engineering (once directed by William Costerton), earned the research center a four-year, $2.9 million grant from the National Institutes of Health. Wolcott has found that more than three-quarters of patients with serious diabetic wounds keep their limbs with his 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 use them at the same time, not sequentially. Quorum-sensing inhibitors are the long-term solution.”