Battling the Superbugs

Science wages war on bacteria that are resisting modern drugs

This story has echoes of heroes tramping the Earth (or the galaxy) on a quest to defeat the forces of darkness. Along the way, the travelers encounter strange creatures with remarkable powers. They endure harrowing tests of mental strength and technological prowess. In the end, they prevail, bringing down the enemy and discovering a truth that saves civilization.

On a light box, an OSU researcher observes protein gels used in biochemical experiments. (Photo: Jan Sonnenmair)

It’s not a huge stretch to say a story like this is unfolding at Oregon State University. Against legions of bacteria and other microbes that cause TB, cholera, malaria and other infectious diseases, a cadre of OSU scientists has taken up arms. Their light sabers are machines called chromatographs and mass spectrometers. Their droids are “high-throughput” plate readers and underwater robots. Their curative elixirs derive from weird and remote organisms like gelatinous “sea squirts” from Africa’s Cape of Good Hope and giant tubeworms from the Axial Volcano a mile beneath the Pacific Ocean.

The story’s ending has yet to be written. But so far, the odds are with the germs. As Earth’s first inhabitants, bacteria have a 3-billion-year evolutionary jump on Homo sapiens. Superbly adept at adaptation, they’ve found genetic avenues into every ecological and biological niche, from polar icecaps to the human gut. They divide like crazy (some can double their population in nine minutes) and use an astounding array of strategies to make themselves at home. Many bacterial species do good things, like recycle waste. But other species, the ones scientists call pathogens, can invade and quickly overwhelm their host organisms, whether animal or plant. Miracle drugs like penicillin, once seen as impregnable shields against deadly infection, are losing their power as the bacteria regroup and recalibrate.

For all its brainpower and glittering technology, modern science is struggling to stay ahead of these microscopic shape shifters. The microbes have outmaneuvered just about every drug medical science has thrown at them.

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This is the story of OSU’s heroic battle to outwit these cunning adversaries. From running high-speed experi- ments in Corvallis, to surveying patients at Portland’s Oregon Health & Science University (OHSU), to combing health-care data- bases for trends, the researchers are attacking infection and prevention from every conceivable angle. They search vast international databases for promising compounds. They decipher mechanisms for disease-promoting phenomena like bacterial sliming and swarming behaviors. They ponder unlikely-seeming disease pathways, such as those between pigs, fish and humans.

The eight professors in the colleges of Pharmacy, Science and Veterinary Medicine you will meet in these pages gather biweekly to trade insights and design collaborative investigations, ranging from microbes’ molecular structures to hospitals’ dosing protocols to urbanites’ risks for drug-resistant infections. Here we look in on their journey, from lab bench to sickbed to public square.

Deep Ocean Dive

Kerry McPhail reaches into a Styrofoam cooler and lifts out a jagged black rock the size of a cantaloupe. “We collected this from an active volcano on the bottom of the Pacific, a mile deep,” she says, her voice accented with the musical tones of southern Africa. Under her office’s fluorescent lights, the rock’s sharp facets shine like obsidian.

In their natural-products lab, Kerry McPhail (left) and Taifo Mahmud examine a flask containing an antibiotic-producing red marine cyanobacterium growing in modified seawater. The cyanobacterium produces different toxins depending on the composition of the seawater. (Photo: Jan Sonnenmair)

Her pride in this solidified chunk of deep-sea lava might suggest that McPhail is an Earth scientist. Why else would she join a 2009 NOAA expedition exploring the Axial Seamount 250 miles beyond Oregon’s shore? So it’s curious to learn that McPhail is neither a geologist nor an oceanographer, but a medicinal chemist in the OSU College of Pharmacy. Curious, too, is the fact that her research endeavors aboard the voyage were funded in part by the National Institutes of Health (NIH), along with Oregon Sea Grant.

What McPhail was seeking in those lightless depths, with the help of a diving robot named Jason II, was new sources of life-saving drugs. The black rock, grabbed by Jason II’s mechanical arm from inside the caldera, was coated with billions of protozoa that thrive on super-heated sulfurous waters at vent sites, where magma bubbles up from the subterranean. As unlikely as it seems, this bluish “microbial mat” may harbor healing chemical compounds never seen before. McPhail and her team have brought home other promising vent-dwelling extremophiles, as well, collected from giant tubeworms, “snow blowers” (white clouds of microorganisms spewing out in 200-degree plumes), orange-colored biofilms (slimy layers of swarming microbes). Some of her specimens were collected during the famous 2008 NOAA expedition when a never-before-witnessed eruption of the underwater volcano was caught on tape by OSU scientists.

“There’s an enormous diversity of microbes down there that people just had no idea about,” she says.

But the cold Pacific isn’t McPhail’s only odd-organism goldmine. Closer to Zimbabwe, where she spent her childhood on an agricultural research station and later in the capital city of Harare, she has been collecting and studying gelatinous creatures called tunicates. Known colloquially as “sea squirts,” these sac-like filter feeders are plentiful in the waters off South Africa. Having previously isolated potential anti-cancer compounds from tunicates, McPhail now has begun testing them for possible antibiotic properties. She also collaborates with OSU medicinal chemist Taifo Mahmud, who studies the curative powers of rare microbes living in the soils of “blackwater ecosystems” in the tangled jungles of Indonesia (see “Nature’s Medicine Chest,” Terra, Fall 2010).

Why are these strange and remote creatures so intriguing to McPhail? Why are deep-sea vent organisms significant enough for the NIH to award her one of its coveted “R21” grants for high-risk, exploratory projects? What makes tunicates worth the regular trips she makes to South Africa to collaborate with a fellow scientist at Rhodes University, where she got her Ph.D.?

It’s their very rarity that makes them promising in drug discovery.

“Unique organisms from unusual, diverse ecosystems have unusual chemistry,” says McPhail. “My lab is testing these organisms for unknown bioactive compounds — ones that target pathogens in unexpected ways. These compounds then can be used to design a new generation of drugs to fight infection.”

Tracing the chemical “fingerprints” of these novel compounds against known compounds catalogued in databases and examining microbial growth patterns in Petri dishes are the first steps in drug discovery. Next, McPhail will move on to studying disease progres- sion in animals. Once again, she’s eyeing a singular creature. This time, it’s the waxworm. Surprisingly, this caterpillar larva of the wax moth (a member of the “snout moth” family) has an immune system similar to that of mammals. That trait, along with being cheap and plentiful, makes the waxworm an excellent subject for drug discovery.

Nature of the Beast

In the global fight against infectious disease, new drugs are urgently needed. That’s because bacteria and other pathogens are evolving day-by-day, minute-by-minute, to withstand the onslaught of existing drugs. They survive by creating mutant versions of themselves or by swapping whole chunks of DNA with other microbes. Not only have pathogens learned to foil single drugs, they’re now fending off multiple drugs simultaneously. These multidrug-resistant germs have been dubbed “superbugs” in recognition of their ninja-like powers of intracellular infiltration and assassination.

A lab technician separates and analyzes DNA fragments isolated from a bacterium in an experiment to find natural-product biosynthetic genes. (Photo: Jan Sonnenmair)

A lab technician separates and analyzes DNA fragments isolated from a bacterium in an experiment to find natural-product biosynthetic genes. (Photo: Jan Sonnenmair)

Worldwide, nearly a half-million cases of multidrug-resistant tuberculosis take hold in human lungs each year, causing 150,000 deaths, according to the World Health Organization. (Learn about the TB research of OSU microbiologist Luiz Bermudez in “Targeting an Old Foe,”Terra, Winter 2009.) Drugs also are failing to cure malaria in countries ravaged by the mosquito-borne protozoan, which annually kills 650,000 people, mostly African children. Other killers include cholera, typhoid and pneumonia. Dangerous staph infections resistant to an older antibiotic called methicillin, as well as many current antibiotics, are rampant in hospitals and making forays into the community at large (See sidebar).

“For every antibiotic that’s ever been used, resistance has developed,” says OSU researcher David Bearden.

“It’s a hard game to play, because the truth is, the more you use it, the less well it’s going to work. That’s just the nature of the beast.”

It’s that beast’s nature — elusive, mutable — that captures Bearden’s imagination as a clinician. Infectious agents, he says, are a lot like those moving targets in the carnival booth. By the time you get one in your sights, a new one has taken its place.

“It’s a foreign-invader scenario,“ explains Bearden, who chairs the Department of Pharmacy Practice. “You’re giving drugs to the person to kill the living organisms that are attacking them from inside. And all the while, the thing you’re fighting is changing.”

Just outside the window of his 12th-floor Portland office, the aerial tramcars connecting OHSU’s South Waterfront to Pill Hill creep up and down the forested slope like silver beetles. On the adjacent lot below, workers in hardhats are running cranes and positioning girders for the Collaborative Life Sciences Complex, a joint project of OHSU and the Oregon University System. Bearden and another dozen pharmacy researchers who work at the OSU Center for Health and Healing will join scientists and clinicians of OHSU and Portland State University in the new complex when it opens in 2014.

While McPhail and Mahmud are rummaging in some of nature’s most peculiar ecosystems for new drugs, Bearden is looking for better ways to use the drugs already available. Resistance gets a boost when too many people take too many antibiotics, he points out. Patients suffering from colds and flu may request — even demand — antibiotics from their doctors. But because those common ailments are caused not by bacteria but by viruses, taking antibacterial drugs is an exercise in futility. Adding to the problem, many patients take their prescriptions inappropriately or stockpile antibiotics for future use. Remnant germs may lurk in the organs or tissues of their human host, building strength for another assault.

To combat the misuse and overuse of antibiotics, Bearden is looking into optimizing dosages and calibrating them for special groups, such as the obese. “Substandard dosing — concentrations that fail to inhibit or kill all of the bacteria — can induce or enrich resistance,” says Bearden. “Say you have a population of 1 million bacteria, and maybe 1,000 of them are very resistant. If you kill off 999,000 of them, the rest of them have this nice, free niche to become the dominant population.”

From her office a few strides from Bearden’s, OSU epidemiologist Jessina McGregor elaborates: “Optimizing the choice of drug, the dosage, the duration of therapy and the route of therapy — whether oral, topical or intravenous — are the next steps in prudent antibiotic use after first cutting down on overuse. That’s the focus of a lot of pharmaceutical research on infectious disease.”

Bio-Bargain Hunter

When Aleksandra Sikora gets excited about a buy-one-get-one-free deal, it has nothing to do with the half- yearly sale at the mall. Rather, she does her bargain hunting in scientific supply catalogs, such as the dog- eared booklet from Greiner Bio-One that sits on her desk. By stretching dollars, the OSU microbiologist can run more experiments with the startup grants that currently fund her research on cholera (which sickens more than 300,000 yearly) and gonorrhea (the most prevalent infectious disease in the United States).

Aleksandra Sikora, along with her husband and research partner Ryszard Zielke, is investigating ways to combat the germs that cause cholera and gonorrhea. (Photo: Jan Sonnenmair)

Aleksandra Sikora, along with her husband and research partner Ryszard Zielke, is investigating ways to combat the germs that cause cholera and gonorrhea. (Photo: Jan Sonnenmair)

Opening a cupboard in her level-2 bio-safety lab in OSU’s Pharmacy Building, Sikora takes out a crisp new package of clear-plastic trays, each about the size of a slice of bread.

“We use 20 to 30 of these sterile plates each time we run a screening for bioactivity,” says Sikora, who grew up in Gdansk, Poland, and has a Ph.D. from the University of Gdansk. “They’re expensive, about $3 to $4 each. If you buy them from Greiner Bio-One, you get one free for every two you order.”

What she’s after in those screenings are “hits” — that is, signs of bioactivity. A bioactive agent or compound is one that affects a living organism such as the gonorrhea-causing bacterium Neisseria gonorrhoaeae or the cholera-causing bacterium Vibrio cholerae, Sikora’s current subjects of study. A hit can show up as a faint glow (“bioluminescence”), which some microbes emit as they send chemical signals back and forth. It can also show up in patterns or rates of bacterial growth. Lack of growth, too, can tell a story.

If she gets a hit, she can see it almost instantly on a computer screen. Thanks to her pricey plastic trays and an even-pricier BioTek plate reader she ordered for her lab soon after arriving at OSU last fall after her post-doctoral training at the University of Michigan Medical School, the whole process is automated. She can run nearly 3,000 tests in just minutes using the high-speed robotic machine, which purrs with perfect precision. That’s because each plate contains 96 “wells” — little troughs that hold samples of bacteria inoculated with whatever compounds are being tested — and the plate reader holds up to 30 plates. Scientists call this type of ultra-fast screening “high-throughput” — in other words, putting samples through chemical and biological analyses at accelerated rates (compared to the old days, when researchers had to run them manually, one at a time).

The gleaming stainless-steel gear gracing Sikora’s lab will let OSU’s team of infectious-disease researchers create their own electronic “compound library.” To that end, Sikora is testing bioactive compounds from McPhail’s vent organisms and sea squirts and Mahmud’s blackwater bacteria, along with her own experiments.

Sikora’s target in the infectious-disease battle is the microbe’s cell wall — the point of contact between the pathogen and the host. It’s where virulence gets a toehold. Instead of targeting the whole cell with a drug designed to kill it outright, Sikora and Ryszard Zielke, her husband and research partner, hope to block “virulence factors,” the actions of bacteria that cause disease. Toxins and other proteins secreted from the cell wall, as well as the composition of the wall itself, are of particular interest.

Chatty Bacteria

In the pantheon of weird sea creatures, the Hawaiian bobtail squid ranks near the top. This 2-inch “stealth bomber of the ocean,” as Natural History magazine calls it, is worthy of Dr. Seuss’s imaginary bestiary. This tiny nocturnal squid even has a biological buddy, a bacterium called Vibrio fischeri that dwells inside a sort of built-in lampshade on the belly of the eight-legged cephalopod. The squid nourishes the dense bacterial populace, which repay the favor by glowing just enough to cancel out the squid’s silhouette as it swims, rendering it invisible to predators. “Interestingly,” OSU microbiologist Martin Schuster notes, “the bacteria don’t glow when they’re out in the open ocean by themselves.”

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How do these one-celled wonders accomplish this stunning feat of variable bioluminescence? As biologists discovered in the 1970s, they do it by sending chemical signals back and forth. These bacteria essentially talk to each other in a process called “quorum sensing,” a stunning discovery that led to a paradigm-shift in microbiology: the realization that microbes aren’t loners but rather social creatures that communicate and cooperate with each other.

“Microbes talk,” says Schuster. “And we’re listening in.”

The discovery of quorum sensing in Vibrio fischeri, a microbe beneficial to its host, set off a flurry of new findings. Cell-to-cell communication, it turns out, is common in disease-causing bacteria, too. This includes Pseudomonas aeruginosa, the primary organism under study in Schuster’s laboratory. This notoriously antibiotic-resistant bacterium causes serious hospital-acquired infections and is the main cause of death among people suffering from cystic fibrosis.

Schuster is studying how germ-to-germ dialog fosters disease-causing actions among bacteria, such as secreting harmful toxins or enzymes that break down host tissue. Biofilm formation, a gabfest among millions of microbes, is another. These “cities of microbes” can be up to 1,000-fold more resistant to antibiotic treatment than free-floating bacteria and are the source of many chronic infections. They build a slimy coating that shields the germs deepest within the biofilm. They draw strength from their surrounding compatriots.

“Biofilms and nasty toxins that harm the host are produced by bacteria as a group,” Schuster says. “It’s a concerted effort.”

What Schuster hears as he eavesdrops on these secret chemical conversations constitutes a novel approach in antibiotic design: disarming rather than killing the pathogen with so-called anti-virulence drugs. “With traditional antibiotics, you basically wipe everybody out,” says Schuster. “Only the resistant clone remains and then just explodes.”

Scientists generally assume that if bacteria aren’t threatened with annihilation, they won’t work so hard to create new versions of themselves. Minus this “selective pressure” — evolution’s relentless push for genetic adaptation to environmental threats — resistance won’t develop. This assumption had never been tested experimentally, until now.

What would happen, Schuster wondered, if he shut off the bacterial chatter? Could he halt the behaviors that bolster the disease process? Could he slow the evolution of resistance, the looming problem with traditional antibiotics? The answer to both turned out to be yes. Graduate student Brett Mellbye ran a number of “evolution-in-a-test-tube” experiments, mingling drug-resistant bacteria with non-resistant bacteria. The non-resistant bacteria, Mellbye discovered, got ahead by “cheating” — that is, by exploiting the nutrients and other resources supplied by the resistant bacteria. The cheating put the brakes on resistance.

“The suppression of resistance is contingent on the targeting of cooperative behaviors,” Schuster cautions. The next step in verifying these findings is to move the experiments from test tubes to animal models.

Fishy Germ Swap

How could an Atlantic whitefish caught off Boston pass a germ to a pig on an Iowa hog farm that winds up infecting a teenager in Seattle? It hasn’t happened yet, as far as we know. The pathway from fish to hog to human is not a straight line; it zigs and it zags. But OSU veterinary microbiologist Dan Rockey says it’s just a matter of time before all the dots connect.

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The reason: Antibiotic overuse isn’t limited to hospitals and doctors’ offices. Factory farms and feedlots, which routinely give antibiotics to healthy livestock to promote growth and prevent disease, can become breeding grounds for resistant germs. As a rule, pigs and cows don’t pass those dangerous germs to people. That’s because bacterial species rarely jump from animals to humans, or vice versa. For instance, chlamydia, the disease Rockey studies, is common across the animal kingdom, yet each animal has its own version of the germ. “A single chlamydia species generally dials in to a single animal species,” he explains. “The organisms that infect a koala bear or a horse or a lizard or a frog — those don’t cross over.”

But as we know, bacteria everywhere are hardwired to adapt — even on an Iowa pig farm. That’s where Rockey and some colleagues from Iowa State and the University of Washington recently discovered a strain of bacteria resistant to tetracycline, the most common antibiotic used to treat humans suffering from chlamydia (a range of diseases affecting eyes and sexual organs). The researchers traced the microbe to the pigs’ diet: fishmeal. It appears that a complex DNA switcheroo among fish pathogens created a genetic perfect storm for tetracycline resistance.

“One fish pathogen had all the right genes coded in the right sequence,” he says. “Lo and behold, crazy but true, this other fish pathogen, unrelated to the first pathogen, has a complimentary set of genes. Somehow, the two fish pathogens got together and then got into the chlamydia that infects pigs, which often are fed fish waste, especially in the Midwest. This story tells you how complicated it is for antibiotic resistance to spread between humans and animals.”

So far, chlamydia in humans remains treatable with tetracycline and other antibiotics. But Rockey’s research suggests that a day will come when the human chlamydia germs C. trachomatis and C. pneumoniae join the ranks of resistant germs. In his lab, Rockey was able to engineer a tetracycline-resistant human chlamydia strain using DNA from the resistant pig strain. If science can do it in the lab, nature can do it in the wild — and sooner or later, it will.

“If tet-resistance were to get into the human strain, it could spread around pretty quickly,” Rockey says. “Tetracycline is a primary drug of choice against chlamydia infections. This could really be a problem, especially in developing countries.”

Evidence is mounting that animal-human crossover can and does occur. Rockey cites a significant paper by the Phoenix-based nonprofit TGen (Translational Genomics Research Institute) describing how the creation of methicillin-resistant Staphylococcus aureus (MRSA) likely was generated by contacts among humans, pigs, bacteria and antibiotics. Here’s what the study found: Pigs acquired a strain of S. aureus from humans, a strain that initially was treatable by antibiotics. But because pig farms are awash in antibiotics, the strain quickly developed resistance inside the pigs. Today’s MRSA problem may well have originated with the next step in that chain of infection: the bacterium’s jump back to humans, this time in its resistant form.

Lance Price, the TGen study’s lead author, sounded a warning. “Our findings underscore the potential public health risks of widespread antibiotic use in food animal production,” he said in announcing the study results in February. “Staph thrives in crowded and unsanitary conditions. Add antibiotics to that, and you’re going to create a public health problem.”

Skin-to-Skin Contact

Factory farms and feedlots aren’t the only “crowded and unsanitary conditions” that promote staph infections. Gyms, dorms, barracks, playgrounds, day-care centers — close quarters where people have skin-to-skin contact — are a growing worry in health-care circles. In most cases, S. aureus, is usually a harmless hitchhiker on healthy human skin. But sometimes it invades its host, often through a cut or abrasion. The boils, carbuncles, pimples, yellow crusts and milky pus associated with staph infections, while unsavory, are usually treatable. However, adding MRSA strains to this mix leads to “more and more stubborn infections at which doctors throw more and more drugs,” Rockey says. Sometimes, patients fail to respond. Sometimes, they die.

This trend gives a sense of urgency to researchers like Jessina McGregor, an OSU microbiology graduate who came back to join the faculty after getting her Ph.D. from the University of Maryland School of Medicine. Like David Bearden, her colleague down the hall, McGregor is captivated by bacteria’s warp-speed knack for adaptation.

“You can actually observe evolution happening!” She leans forward in her chair, the energy in her voice rising as she contemplates the awesome power of microbial communities. “You can directly observe the bacteria’s response to evolutionary pressure. With larger organisms, you would have to watch for decades or millennia to witness evolutionary change.”

As an epidemiologist, McGregor scans, not populations of microbes in Petri dishes, but populations of humans in all sorts of settings — cities and states, hospitals and doctors’ offices, residential-care facilities and outpatient clinics. She studies long-term data and looks for trends. She plumbs the numbers to stem the threat of resistant disease.

One solution is outreach. Hand-in-hand with the Oregon Department of Health, she and OSU pharmacy students have spearheaded a local project under the national AWARE (Alliance Working for Antibiotic Resistance Education) umbrella, which fans out across the state with brochures, games, videos and face-to-face conversations for local communities. Other solutions may emerge from her current studies of urinary-tract infection patterns at Kaiser Permanente Northwest and OHSU, as well as infection rates among inpatient-versus-outpatient settings.

So far, McGregor says, Oregon and Portland have dodged the full force of resistance hammering other states and cities. “What’s happening in Oregon is very different than in other places in the U.S.,” she explains. “We have very different prescribing patterns here. Oregon is one of the lowest antibiotic utilizers per capita, behind only Alaska. That definitely helps us out.

“Having locally specific information to guide our policies is important. We don’t need to be reactionary ahead of the curve.”

Now More Than Ever

Ironically, the escalating demand for new drugs coincides with declining dollars for antibiotic research. That’s because pharmaceutical companies are concentrating on “blockbuster” medicines for patients with long-term conditions, like elevated cholesterol and high blood pressure. Antibiotics don’t pencil out on the balance sheet, say six sponsors of a bipartisan bill currently before Congress.

“The development of any new pharmaceutical costs hundreds of millions of dollars for basic and clinical research,” write the sponsors, three Democrats and three Republicans from six states. “For antibiotics, revenue is limited because they tend to be prescribed for short-course therapies that are completed in days or weeks.” If it passes, the GAIN Act (Generating Antibiotic Incentives Now) will create incentives to encourage R&D and speed up drug discovery.

The bill’s sponsors mince no scary words, spare no sobering statistics. “The antibiotic pipeline is dwindling, and a global crisis looms,” they write. “Each year, antibiotic-resistant infections are responsible for tens of thousands of deaths, hundreds of thousands of hospitalizations and up to $26 billion in extra costs to the U.S. health-care system. Just when we need innovation the most, the pipeline of drugs to replace ineffective antibiotics has dwindled to a trickle.”

To turn that trickle into a life-giving river, infectious-disease research at OSU and other universities is more urgent than ever. Taifo Mahmud sums it up simply: “We are running out of drugs. We have no other choice than to keep moving.”

After all, a hero perseveres, no matter the cost, no matter the odds.

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