Chemist Helen Blackwell is shown next to one of the deadly microbes she’s aiming to scuttle: antibiotic-resistant MRSA bacteria, enveloped in insidious biofilm slime, growing on the inner wall of a catheter.

Bacteria, Interrupted:
Moving Beyond Antibiotics

By Barbara Ellis

Microbes and mankind have coexisted in good ways and bad for millennia, but by the latter half of the 20th century, humans seemed to have gained a decisive edge when penicillin and a host of other antibiotics began to be used with astonishing success against a wide range of bacterial infections. In recent years, however, bacteria, in textbook Darwinian fashion, have staged a comeback, evolving new defenses against antibiotics and passing their resistant genes on to their progeny. Today’s medical journals are full of reports about childhood ear and throat infections that no longer respond to standard antibiotic therapies, garden-variety cuts and scrapes that erupt into body-ravaging infections, and hospitals that have become breeding grounds for new and ever deadlier strains of bacteria. In response, university scientists have begun to ramp up their research into new antibacterial therapies. But bacteria have been around much longer than humans. How easy will it be to mount a new offensive against an opponent that has had a head start of more than 2 billion years?

In a lab at the University of Wisconsin–Madison, assistant professor of chemistry Helen Blackwell, PhD ’99, is one of a small but highly motivated group of academic scientists looking into novel approaches to combat bacterial infection. Earlier this year, her research group reported in Chemistry & Biology that they had discovered four new chemicals that, in the Petri dish at least, disabled several types of pathogenic bacteria as effectively as the most powerful antibiotics in current use. The compounds worked especially well against one of the most worrisome bacterial pathogens yet to emerge, methicillin-resistant Staphylococcus aureus, otherwise known as MRSA, some strains of which are now resistant to all antibiotics. One strain even became resistant to a new antibiotic less than a year after the drug won FDA approval.

According to U.S. health experts, MRSA is responsible for an estimated 90,000 deaths a year. Although Blackwell’s findings are highly preliminary and have yet to be tested on animals, let alone humans, her team’s results are being hailed as a promising breakthrough in what is shaping up to be a long and complicated fight against antibiotic-resistant bacteria.

“There’s an urgent need for new antibacterial therapies,” says Blackwell. “It’s a problem that unfortunately is not being addressed by most big pharmaceutical companies right now. Bacterial infections are not chronic diseases whose treatment generates a lot of profit. Further, the bar for the development of new antibacterials is extremely high.” But several pharmaceutical companies are following her work with interest, and two have already given her seed funding for her research.

“One of the best things about the four new compounds we’ve found is that they’re not related to antibiotics or any other antibacterial compounds used so far, so the bacteria haven’t developed any resistance to them,” Blackwell says. “We’re not precisely sure how they work, but we’re delighted they do.” Finding out what their mechanism of action is, and improving on it, is shaping up as a focus for her future research.

Blackwell’s team made their discovery while testing out their latest small-molecule macroarray, a small-molecule screening apparatus that has considerably shortened the time it takes to engineer and evaluate novel compounds. In the trial reported in the journal, just under 200 new molecules were screened on a variety of bacteria in less than two days. If the researchers were able to identify four promising new molecules (which they’ve already patented) in such a short space of time, says Blackwell, conceivably they will find dozens more in the space of a year.

As well as looking for potential new drugs that kill bacteria, Blackwell’s multi-disciplinary group of 11 graduate students and three undergraduates is also pioneering research into molecules that are able to interfere with the way that many bacteria, including MRSA, organize themselves into disciplined and formidable collectives known as biofilms. For most of us, who recall gazing at a magnified bacterium or two under the microscope in biology class, it may come as a surprise that for much of their lives, many bacteria aren’t autonomous, free-living individuals at all, but live in structured communities. Sheltered by a sticky protective coating, the bacteria in biofilms take on different roles and coordinate their activities using a language of simple chemical signals. In this, they are not unlike a colony of ants or a multi-cellular organism.

The above illustration charts a biofilm’s development from a single bacterium to a small aggregate, to a collective known as a quorum, which triggers the formation of the biofilm.

“Like humans with words, bacteria use a language of simple small molecules to communicate,” Blackwell explains. “They use these molecules as signals to sense their local population density, and when they reach a sufficiently high density, which we call a quorum, bacteria can change their mode of growth and behave as a group. This is when many bacteria form these nasty biofilms.” This mechanism, known as quorum sensing, is a relatively new concept in bacteriology.

Many biofilms are harmless, and some—like those that clean sewage water in treatment plants—are beneficial, but the ones that harbor pathogenic bacteria are a serious health problem. It has been estimated that biofilms are responsible for 80 percent of bacterial infections in humans. Dental plaque, the first biofilm to be identified, is, like many others, a mixed community of bacterial species, some good and some bad. The latter cause gum disease and tooth decay, but the good ones may counteract this to some extent. Biofilms formed by Pseudomonas aeruginosa in the lungs are a leading cause of death for cystic fibrosis sufferers, AIDS patients, and burn victims. Other biofilms are responsible for chronic ailments such as cystitis, inner ear infections, and bacterial endocarditis, a potentially deadly infection of the heart valves. As tenacious as they are ubiquitous, these microbial colonies are almost impossible to remove from the surfaces to which they’ve attached themselves. In hospitals, a major breeding ground for antibiotic resistance, biofilms on the insides of reusable medical equipment such as catheters, ventilators, and breathing tubes are a huge problem because they survive sterilization procedures and pose the risk of infecting each new patient who uses the equipment.

For the last five years, Blackwell’s group has been studying the dynamics of these pathogenic biofilms and analyzing their quorum-sensing pathways. “We’re looking at efficient ways to synthesize the types of molecules that the bacteria use for quorum sensing” she says, “and then we’ll make subtle changes to them.” Like code breakers in time of war, Blackwell and her colleagues are looking to decipher and hijack the language bacteria use to talk to one another, and then turn it against them.

Biofilms form when the population density of free-swimming bacteria reaches a quorum, and a quorum-sensing signal summons them to come together and settle down. The clustering bacteria attach themselves to a surface, preferably a nice damp one with a regular supply of nutrients, and secrete a sticky polymer coating that glues them to their base and to one another. The hardened outer part of this polymer also acts as a strong, watertight, protective sheath. “It gives them a nice place in which to live,” Blackwell says, “but because it acts physically like a shell, it’s an enormous problem when it comes to dealing with pathogenic bacteria,” because antibiotics have a hard time penetrating it. “And it’s a double whammy,” she adds. “Not only are the bacteria in the biofilm resistant to treatment, but their mutation rates are accelerated.” The crowding in the colony seems to stimulate certain bacteria such as MRSA to rapidly evolve and swap antibiotic-resistant genes.

Surprisingly, pathogenic bacteria in a biofilm are harmless until they reach another, higher, quorum density that triggers the activation of virulence genes. Then they turn ugly and surge out of the biofilm en masse to attack their host. “It makes sense that they should wait until there are enough of them before invading our cells, because the immune system has a harder time when it’s attacked by a mob,” Blackwell says. It’s when the immune system is overwhelmed that we get ill.

“We’ve known about quorum sensing for the past 30 years, but it’s only in the last 15 or so that people have started looking for the compounds that control it—it’s still not a heavily researched area.” So it’s a wide-open field for her group as they work on identifying the chemical signals that the bacteria use to count themselves. Once they’ve found these, they can make small changes to the chemical structure of the signals to develop what Blackwell calls quorum-sensing modulators to make the microbial arithmetic go awry. One type of modulator might trick the bacteria into thinking (so to speak) that their numbers are insufficient to start up a biofilm, while another type might convince the pathogens that their numbers are greater than they actually are, in which case they’ll prematurely turn virulent, and go on the attack, only to be picked off by the immune system. A big advantage of using these modulators is that “the compounds we’re developing to control quorum sensing won’t kill the bacteria, they’ll just keep them from talking, if you will,” Blackwell says. “There’s a hope that if they’re merely inhibited, there’s less chance they’ll develop resistance to the chemicals.” Antibiotics will still be used to mop up the loose germs swimming around.

A native of Cleveland, Blackwell attended Oberlin College, also in Ohio, for her undergraduate degree, and found her calling. “When I took chemistry in college, it clicked, especially the research. I realized then that it was what I wanted to do,” she recalls. Her father, an engineering professor at Case Western Reserve University, may have also been an influence, although Blackwell says that he never pressured her to follow in his footsteps. In 1994, she took the advice of Beckman Professor of Biology Harry Gray, “who came to Oberlin when I was a senior, and told me to apply to Caltech, because it had such great research opportunities in chemistry.” As her particular interest at that time was polymer chemistry, she joined the group of Atkins Professor of Chemistry Robert Grubbs. “But after I arrived, I did almost no polymer chemistry. I worked on a synthetic methodology project, which was biologically inspired. Quite a jump from polymer chemistry!”

It was an exciting time to be in the Grubbs group, she recalls, working on the reactions that would lead, a few years later, to Grubb’s 2005 Nobel Prize in Chemistry, “though, while we knew that the work was amazingly powerful, we really had no idea at the time that he would get it.” A seasoned outdoorsman and rock climber, Grubbs likes to take his research group on camping trips to such venues as Yosemite and Sequoia national parks. Blackwell enjoyed the camping, but left the rock climbing to others, one of whom—fellow grad student Dave Lynn—she married in 2002.

Grubbs encouraged his students to try things on their own, Blackwell recalls. “We had to teach ourselves. You could flounder with so much freedom, but it taught me self-reliance.” Nearly a decade later, she has come to have fond memories of the Institute. “I didn’t appreciate as much at the time as I do now that Caltech is a very special place, open and supportive, full of brilliant people, with a wonderful culture of excitement and possibilities.”

Grubbs remembers that “when Helen arrived, she decided to go in a new direction for herself and our group, taking us in a more biological direction. She developed techniques for modifying peptides by using the catalysts we were developing, and had the personality and style to recruit collaborators so we could move into this new area and make progress. She was also a leader in the social, interactive scene. At Madison, Helen’s found a new area for herself which is extremely important, and she brings to it a unique background—the ability to do biological research and the ability to make molecules.”

After graduating in 1999, Blackwell went to Harvard as a postdoc. She “got the bug,” as she expresses it, for biological research when she joined a group headed by pioneering chemical biologist Stuart Schreiber. The team was working to develop small organic molecules for use as probes in biology and medicine, and, toward the end of her stay, Blackwell initiated some novel experiments on plants, evaluating whether nonnative compounds—that is, substances to which plants aren’t usually exposed—could interfere with their development as seedlings. The experience was a useful prelude to her current research on organisms that use a simpler form of chemical communication, but about which much less is known.

Currently, Blackwell’s Wisconsin lab is one of approximately seven university labs worldwide that focus on the chemical aspects of bacterial quorum sensing. “What makes my lab’s approach unique,” she says, “is that we are applying novel combinatorial chemistry approaches to the discovery of nonnative quorum-sensing modulators.” A technique pioneered by chemists, combinatorial chemistry enables scientists to put together hundreds of different molecular combinations quickly and cheaply by combining chemical “building blocks” in a variety of ways, then trying them out one after another on an organism to see what happens. “It’s really effective for our type of research, as it accelerates the discovery process,” Blackwell says.

When she embarked on her quorum-sensing work, it wasn’t clear which compounds would have the ability to intercept and disrupt the appropriate bacterial signals, so Blackwell and her students designed an initial collection, called a library, of 100 or so molecules that she thought might have the desired properties, and tested them on selected bacteria to see what effect they had. After identifying a handful of molecules that showed some effect, they made another batch of compounds with similar molecular structures, and tested those. Through repeated fine-tunings, the team gradually honed in on compounds that actively interfered with bacterial quorum counting.

Newly minted molecules effective against MRSA leave white circles of dead bacteria among the living, red-stained ones. One of these molecules was subsequently pipetted over the colony in the form of a W in tribute to the University of Wisconsin.

All this testing sounds like a lot of work, but Blackwell’s group has pioneered ways to automate and speed up the process. The state-of-the-art small-molecule macroarray that they used to identify the four MRSA antagonists can assemble and screen a 100-molecule library in less than 24 hours. In this system, the compounds are built up ingredient by ingredient in an array of small dots on a planar polymeric substrate (oftentimes made from cellulose, or simple filter paper), which, if needed, can be warmed in a microwave oven to “bake” the organic reactions. The dots of molecules are then punched out with a hole punch and placed on top of bacteria growing in a Petri dish. After a few hours of incubation to let the chemicals soak into the bacteria, the dishes are rinsed with a “live-dead” stain that colors living bacteria red and dead ones white so that it’s really easy to see which molecules have worked—they’re the ones that leave white dots on the dish. The bigger the dot, the more toxic the chemical.

Using these screening methods and others, Blackwell and her colleagues have already found, and patented, several synthetic signaling molecules that effectively disrupt or promote cell-to-cell communication in a variety of bacteria. Interestingly, many of these molecules seem to be highly specific to a single bacterial strain, and minor changes to their chemical structures can make them effective against a different one. Each strain seems to have its own signaling words—which raises the possibility that quorum-sensing drugs will one day be able to target only the bad bacteria in a mixed biofilm community, leaving the beneficial ones untouched. That’s something antibiotics have never been able to do, which is why many people cannot take them without experiencing unpleasant side effects.

While Blackwell carries out her antibacterial investigations, her husband is working with biomedical polymers and devices in the department of chemical and biological engineering on the UW campus. “We’re lucky in that we’ve managed to move on to the same places together,” she says. Blackwell also has her teaching duties at UW, and last year she received the Chancellor’s Award for Distinguished Teaching, the university’s highest teaching honor. “It was a big thrill to win this,” she says, “because teaching is something I really enjoy doing.” She’s also gained a slew of other awards to help fund her research projects and, in 2005, MIT’s Technology Review named her a TR35 Young Innovator, “one of 35 up-and-coming scientists under the age of 35.”

Blackwell expects to spend the next few years making more libraries of molecules, finding out how they work against pathogenic bacteria, and honing them to be more effective. The rapid screening methods she’s developed should identify many new designer compounds, although she reckons it’ll be a decade or more before any of them move toward the clinic. She stresses that no “magic bullet” in the form of a super antibacterial agent is going to solve the problem of bacterial resistance. With billions of years of experience to rely on, microorganisms are simply too good at learning how to outsmart any chemicals used against them. New therapies will need to be developed all the time, simply to keep up with bacteria’s ability to mutate into resistant strains. But Blackwell’s upbeat about the future: “Bacteria are smart little beasts, but with continued research efforts toward new anti-infective targets—like quorum sensing and others—humans might someday get the upper hand. It will take a lot of work, but the payoff for humankind will be enormous.”