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Biomedicine

Battling Bacterial Resistance

Challenged by the tendency of disease-causing microbes ot mutate into breeds that defy conventional antibiotics, researchers are pursuing a variety of high-tech schemes to give humans the upper hand in the war on infectious diseases.

Back in the 1980s, a false sense of security that infectious diseases were under control helped spur the drug industry to shift its resources away from creating new antibiotics. Since the discovery of penicillin, hailed in the ’40s as the miracle drug, scientists had developed new generations of antibiotics that cured a wide range of diseases.

“There was a sense that the market and the clinical needs were already pretty well satisfied with existing agents,” remembers Keith Bostian, who was a researcher at Merck Research Laboratory in the ’80s. “The hurdle to qualify for a new drug candidate that could be competitive and take market share was getting higher and higher.” As a result, many drug companies turned their basic research efforts to antiviral and antifungal medicines, few of which were on the market.

What a difference a decade makes. Today Keith Bostian is the founding scientist and chief operating officer of Microcide Pharmaceuticals, Inc., a California company created in 1992 to develop novel antibiotics for serious infectious diseases. Bostian typifies the rejuvenated attitude of the pharmaceutical industry, which sees opportunities that didn’t exist in the ’80s to make and market new antibiotics as well as new types of bacterial killers. Across the country, researchers are competing furiously to uncover the private lives of bacteria, probing their genes to learn which are necessary for survival and which are involved in infecting people, and what mechanisms the microbes use to survive antibiotics. And pharmaceutical and biotech companies are nearly stampeding to find significant bacterial targets, create novel methods of attacking them, and be first to bring the new drugs to market.

The Seeds of Change

There isn’t just one reason this rush is on. There are at least four motivators. Foremost is the growing sense of urgency to find ways to stem the advancing strains of bacteria no longer killed by major antibiotics. “Drug resistance is just an increasing problem in essentially every kind of bacterium that causes infection,” says Michael Lancaster, section chief for antimicrobial resistance in the division of hospital infections at the U.S. Centers for Disease Control and Prevention (CDC). Among the diseases caused by drug-resistant bacteria are pneumonia, tuberculosis, ear infections, sexually transmitted diseases, diarrhea, and bloodstream and wound infections. “We are clearly in a public health crisis,” says Stuart B. Levy, director of the Center for Adaptation Genetics and Drug Resistance at Tufts University School of Medicine, “and on the road to an impending public health disaster.”

“Vancomycin-resistant enterococci (VRE) probably are the biggest resistance problem,” says Lancaster. The VRE microbe, which can kill patients with weakened immune systems if it enters their bloodstreams, has been causing growing problems in hospitals. Among patients who got enterococci infections while in hospitals, the incidence of VRE rose from 0.3 percent in 1989 to 14.2 percent in 1996, according to the CDC. What makes VRE so significant is that vancomycin is not the first drug of choice to treat the microbe, but the last. Not only is VRE resistant to vancomycin, it is usually also resistant to all other drugs commonly used to treat it, says Lancaster. The fallback treatment is investigational drugs or high doses of combinations of drugs already on the market.

Microbiologists believe much antibiotic resistance is the result of natural selection. Resistant microbes tend to appear where an antibiotic is used frequently. As the bacteria reproduce, some mutations occur; eventually one bacterium is changed in a way that allows it to survive the drug. For instance, the mutant might have grown a thicker cell wall, making it impossible for the drug to permeate it. Or tiny cellular pumps that were used to get rid of waste products may acquire a new instruction that will now dump the antibiotic right out. This mutant, which can shield itself from the lethal drug, is the one that lives on.

Unfortunately for humans, mutating is not the only way bacteria develop antibiotic resistance. What scares public health officials even more is that bacteria can also transfer their genetic instructions for avoiding an antibiotic to other bacterial species. They have been most concerned that VRE will pass its high resistance to vancomycin over to a common and even more virulent and more aggressive bacterium, Staphylococcus aureus. That could be a huge problem because there are cases of staph bacteria that have proved resistant to every drug except vancomycin, Lancaster explains. Staph, which causes a range of problems from boils to pneumonia, toxic shock syndrome, and bloodstream infections, is the leading cause of infections patients get while they are in hospitals.

Last year a strain of staph that was moderately resistant to vancomycin showed up in Japan. This past August the CDC reported that for the first time in the United States a slightly different, moderately resistant strain had surfaced in Michigan. The CDC warned that this could be the harbinger of a fully resistant strain. Later, another moderately resistant strain showed up in New Jersey. The strains are all thought to be rare mutants. VRE has not yet passed its high resistance on to staph, but the watch is on.

Drug-resistant bacteria are not confined to hospitals and nursing homes. In the last decade, says Alex Rakowsky, a medical officer for the Food and Drug Administration (FDA), health officials have learned that certain resistant bacteria occur more heavily in the suburbs, probably because affluent residents there take large numbers of antibiotics. Other pockets of highly resistant bacteria have been found in poor rural areas, for reasons not clear to health officials.

Learning Bacteria’s Secrets

While doctors and public health officials were becoming aware of the extent of the drug-resistance problem, scientists and drug companies were taking note of the growing stockpile of knowledge pouring in from gene-sequencing projects. J. Craig Venter, chief executive officer of The Institute for Genomic Research in Rockville, Md., estimates that the biochemical codes for up to 40,000 new microbial genes already exist in public databases and that researchers will discover as many as 500,000 genes in the next decade, mostly in microbes. Now, says Venter, industry must winnow its interest to the few that will make the best drug targets.

Of particular interest to the pharmaceutical industry are the fully sequenced genomes already available for several bacteria. Understanding which genes, and therefore which proteins, make a bacterium function is essential to learning how to fight it. Having complete genomes “permits entirely new forms of analysis,” says David Searls, a vice president at SmithKline Beecham Pharmaceuticals. “With a whole genome at your disposal, you have in a sense a closed world. You can analyze an organism in terms of biochemical pathways, for example, and be assured that you’re going to have represented in your database every possible biochemical reaction.” And that is why gene sequencing became the second motivator for drug makers to develop new antibiotics and rescue those in distress on the market.

Last summer at a forum on resistant bacteria held by the National Academy of Science’s Institute of Medicine, gene sequencing pioneer Venter described data released from four of the first completely sequenced genomes of bacteria. Then he told his colleagues: “The chief breakthrough that allowed this to take place was not molecular biology. It was actually bioinformatics that allowed us to deal with thousands and thousands of sequences.”

In August 1987 no one had heard of bioinformatics. Ten years later an Internet search engine came up with more than 22,000 references to it. This field of study was born when scientists began to realize that the volumes of data to come from gene sequencing would be of little use unless a systematic process could be devised to organize and analyze them. Using computer programs, researchers can compare sequences of newly discovered genes with known ones from other species. To experts, a close match may offer clues to the functions of the “new” genes. Genes that code for proteins are the target of much gene sequencing. Scientists can also use bioinformatics to determine “where and when the specific messages coding for these proteins are made,” explains George S. Michaels, one of the early bioinformatics specialists.

And so this tool quickly became the third factor prompting the drug industry’s renewed research interest in antibiotics. Almost overnight, bioinformatics is becoming a profession. Michaels, who now heads George Mason University’s graduate program in bioinformatics, says his four-year-old program awarded its first Ph.D. last year. And in recognition of the importance the technology plays at SmithKline, last year that company named David Searls vice president for bioinformatics. “I think that genomic sequencing and bioinformatics opened up really unparalleled opportunities in antimicrobial research,” says George H. Miller, presidential fellow-vice president for microbiology at the Schering-Plough Research Institute. “You can select new targets now.”

Given the resistance problem and the new tools to try to do something about it, “there’s a lot more enthusiasm” to create new antibiotics, says Allan Weinstein, vice president for international medical and regulatory affairs at Eli Lilly and Company. “You can look at current antibiotic therapy as sort of a sledgehammer,” he explains, while the new approach “directed at specific genes necessary for bacterial life would be more of a stiletto.”

The fourth factor behind the enthusiasm to search for new antibacterial drugs involves two other new tools that have been developed in the last 5 to 10 years that serve to speed up the hunt. In the past, chemists randomly prepared compounds one at a time to see if they had any usefulness as a drug. Now, explains Ted McDonald, director of chemistry at Pharmacopia, Inc., in Princeton, N.J., most drug companies have replaced that method with a much faster one. Through a process called combinatorial chemistry, pharmaceutical chemists create collections of small organic compounds in which they systematically vary the different units or building blocks in the compound’s molecular structure, preparing many possible combinations.

Another technology called high-throughput screening allows these synthetic molecules to be screened in large groups. In one method, plates of 96 miniature wells containing a molecule and a protein target from a bacterium are passed over a detector that shows a color change when a molecule binds to a protein. Some companies prefer attaching molecules to polystyrene beads because of the ease of purifying the product after several chemical reactions by simply washing reagents and byproducts off the beads. Whatever method is employed, the bottom line for drug makers is speed. In the time it used to take to screen a few hundred compounds for potential drug activity, pharmaceutical companies now can screen tens of thousands.

Everybody’s Responsibility

As one antibiotic after another loses ground to a new resistant strain of bacteria, government officials and scientists from industry, academia, and professional scientific and medical organizations have met to examine the problem and discuss what can be done. New alliances and partnerships are forming among public health departments and scientists based at universities, and between giant pharmaceutical companies and small biotechnology firms. Now the question is: Can we beat antibiotic resistance?

The answer involves almost everybody. Drug manufacturers are one significant part of the solution.

Pharmaceutical and biotech firms are placing in the pipeline many potential new drugs intended to help meet the challenge of resistant bacteria. One is a class of antibiotics known as everninomicin that the Schering-Plough Research Institute is developing. George Miller is glad his company hung on to a soil organism called Micromonspora carbonacea, which was shown to have antibiotic activity nearly 20
years ago. From 1979 to 1984 company researchers worked with the organism and found it could make an antibiotic that was not toxic and was active against a range of disease-causing microbes known as gram-positive bacteria. But the scientists quit working on it, Miller says, because in the ’80s there was no need for a new antibiotic of this type.

In 1990, when they noticed that some gram-positive bacteria were beginning to develop resistance to existing drugs, Schering-Plough scientists plucked the organism from its home in the freezer and started back to work on it. Everninomicin contains seven different sugars that are not involved in any class of medicine used in people, Miller says. This means that no current form of drug resistance can transfer to
the new antibiotic, which in lab and animal studies has killed resistant strains of staphylococci, streptococci, and enterococci, three of the most problematic bacteria. Miller points out, however, that “resistance will probably happen to all new drugs eventually.”

Clinical trials of the first everninomicin drug have begun in South Africa and Latin America. And they may start soon in the United States, Miller says. If those trials succeed, the FDA may take another two to three years to approve the drug, he adds.

Even sooner than that, another new class of antibiotics-the oxazolidinones-may be ready for use, courtesy of Pharmacia & Upjohn, Inc. Scientists at E. I. Du Pont de Nemours actually discovered oxazolidinones more than 10 years ago but couldn’t overcome a toxicity problem with the compound, so they stopped trying to develop it into a usable antibiotic. Gary Zurenko, a senior research scientist at Pharmacia & Upjohn, was sitting in a scientific meeting in New York in 1987 when Du Pont researchers described the product. Zurenko was intrigued by its unique mechanism of action, which unlike other inhibitors stops the initiation of protein synthesis in bacteria. This was significant because its way of preventing protein synthesis in bacteria implied that “organisms resistant to the known protein-synthesis inhibitors would likely not be resistant to the oxazolidinones,” Zurenko explains. The compound also looked attractive as a drug candidate because it could be given by mouth as well as intravenously.

Over time, Pharmacia & Upjohn researchers developed a set of oxazolidinones far less toxic than the Du Pont compound. Zurenko re-ports that the first drug his group created, Linezolid, works against methicillin-resistant staphylococci, vancomycin-resistant enterococci, and penicillin-resistant streptococci and retains other key features of the early oxazolidinones. His company is now testing Linezolid on patients at several sites around the world, including in the United States.

Resisting Resistance

Another new way to attack antimicrobial resistance is to design drugs that disable the antibiotic-resistance mechanisms in bacteria. Patients would take these drugs along with antibiotics already on the market, making them effective again. For instance, Microcide Pharmaceuticals, the antibacterial company begun in 1992, is focusing on this approach against four organisms-staphylococci, enterococci, pseudomonas, and streptococci-which together account for 44 percent of the 2 million infections that occur each year in U.S. hospitals.

One resistance mechanism Microcide is concerned with is the elaborate method Pseudomonas aeruginosa has developed to resist many antibiotics. This bacterium causes a variety of diseases, including pneumonia and infections of the skin, urinary tract, and bloodstream. The resistant microbe has evolved in such a way that it has hundreds of pumps that bind to antibiotics as they enter the bacterial cell. The pumps literally eject the medicine from the cell. Each bacterium can have as many as four variations of the basic pump.

To target and disable the pumps, the researchers first had to thoroughly understand the pumps’ molecular biology. The investigators learned that often the pumps “recognize more than one type of antibiotic,” says Keith Bostian, Microcide’s founding scientist. “Some pumps recognize all the antibiotics.” A crucial factor therefore became making sure that the inhibitor Microcide was developing would inactivate all four pumps.
This past fall Microcide screened and tested a variety of chemicals that inactivate the pumps. Bostian says the company is close to picking its best candidate for drug development and that clinical trials might occur next year, with the product possibly on the market three to four years later.

These are just a few examples of the potential new drugs many pharmaceutical companies are hoping will get to market and prove successful in holding the line against antibiotic-resistant bacteria. But just how much help the public can expect and how soon is hard to predict. “I couldn’t honestly say whether I’m optimistic or not that they are going to fill the gap in time,” says Joshua Lederberg, who chairs the Institute of Medicine’s Forum on Emerging Infections and is a Nobel laureate. Because details of companies’ work are private, he says “there is no way to gauge how far along they are.”

Meanwhile, one approach to controlling infectious diseases that has been underutilized, Lederberg says, is preventive vaccines. He points to the success of the vaccine against Haemophilus influenzae type b (Hib). While the microbe sounds as if it causes the flu, it actually was the leading cause of meningitis, a potentially deadly disease, in U.S. infants during the 1980s. It also causes blood poisoning and pneumonia. Claire Broome, deputy director of the CDC, notes that the number of U.S. Hib cases in the ’80s was on the same order of magnitude as polio cases before the polio vaccine. Now, she says, “The [Hib] vaccine has almost entirely wiped out Hib meningitis.”

At least two drug companies are developing another bacterial vaccine, this one against pneumococcus, the leading cause of pneumonia and meningitis for all age groups in the United States and the most common bacterial cause of otitis media, middle-ear infections in children. Ear infections are a major reason for office visits to pediatricians, the prime cause of emergency-room visits for kids, and a common reason doctors pass out antibiotics. “If this vaccine is successful in preventing otitis media, that would be a tremendous step forward,” says Broome.

Back to the Future

While most of the attention and competition is focused on creating new antibiotics and rescuing those now on the market, a few researchers advocate returning to the anti-infection remedies used before the introduction of antibiotics in the 1940s. One such treatment is bacteriophage. Commonly known as phage, this is a virus that grows in bacteria until it finally pops out and kills its host.

Phage treatments used in the ’20s and ’30s often failed.

At the time scientists didn’t understand that each kind of phage is highly specific; only one type will attack a particular species-and even strain-of bacteria. Re-ports of the treatment in journals were lacking in controls and filled with anecdotes, and many scientists looking at the studies today say they are not professionally acceptable.

But Carl R. Merril, chief of the Laboratory of Biochemical Genetics at the National Institute of Mental Health, is enthusiastic about the potential for using phage therapy to treat serious infectious diseases in humans. Merril, who reported in the April 1996 Proceedings of the National Academy of Sciences (PNAS) that he had successfully used phage therapy in mice, says several procedures must be followed for phage treatments in people to be successful. First, the bacterium causing the infection must be cultured to determine the exact type and strain. Next, the strain has to be tested against different phage to find the one phage specific to that strain. Then a mutant phage must be grown in a manner that ensures that the patient’s body will not see the virus as foreign and remove it. Finally the phage needs to be purified to eliminate any toxic bacterial fragments left after bursting free of its bacterial host. Merril says he has found that spinning phage in a centrifuge separates out bacterial particles.

Merril is collaborating with Exponential Biotherapies, a start-up biotech firm in New York, to develop phage therapies for use in people with major infectious diseases. Richard Carlton, a psychiatrist, founded the firm after meeting Merril and becoming impressed with his work. Carlton says that the first therapy the company hopes to market aims to kill a strain of vancomycin-resistant enterococcus called VREf, which causes skin, bloodstream, wound, and heart-valve infections.

At least one other American company reports that it is pursuing phage treatments. Formed in the fall of 1996, Phage Therapeutics is trying first to develop a treatment against resistant Staphylococcus aureus, according to Richard Honour, a microbiologist and the company’s president and chief executive officer.

Because of the lack of rigorous scientific proof that phage treatment works against infectious diseases, the companies hoping to market it in the United States have taken on a big selling job.

Last November, Elizabeth Kutter, a biophysics professor, visited the Eliava Institute of Bacteriophage, Microbiology, and Virology in Tbilisi in the Republic of Georgia, where phage therapy is heavily used. She says the institute’s clinicians most often administer phage treatment topically to treat wounds and burns, or orally for intestinal disorders and some other infections. “What I’ve seen is enough good data to make me think it’s definitely worth further exploration,” says Kutter, of Evergreen State College in Olympia, Wash.

Honour, whose company has a relationship with the Eliava Institute, says the work of researchers and doctors in Tbilisi and other parts of eastern Europe is not readily transferable to the United States. “They’ve saved thousands of lives, but you could never take those products or techniques and submit them to Western regulators,” he says. When a treatment “works, it works well, but when it doesn’t work, they don’t know why.”

Other Western experts are even more skeptical. Bruce Levin, a population and evolutionary biologist at Emory University who has conducted some successful animal experiments with phage therapy, says its main drawback for human use is its specificity. Identifying the particular species and strain of an infecting bacterium before treating patients could be a problem, he suggests. Also, he says, researchers have not yet proven that phage treatment works throughout the body. But despite his skepticism, Levin says that given the potential crisis antibiotic-resistant bacteria could cause, researchers should examine phage therapy in light of modern scientific knowledge.

Attacking on Other Fronts

Regardless of what treatments may be used once bacteria have infected people, scientists and public health officials agree that one crucial step in developing an effective offense against the microbes must be the establishment of a strong surveillance system. The system should pinpoint resistance problems early, give clues as to why they are happening, and quickly provide critical information to public health officials worldwide. But no worldwide system is in place, and surveillance in the United States is uneven and has major problems, according to public health officials themselves.

Rosamund Williams, a bacteriologist who is coordinating the new Antimicrobial Resistance Monitoring Program for the World Health Organization (WHO), spoke openly at last summer’s Institute of Medicine forum about the outcome of the current lack of such a setup. “We know that we have a big problem with resistance to antimicrobial agents, but we don’t know how big it is,” she said. WHO is therefore starting a system that will attempt to strengthen the ability of laboratories within its 191 member countries to monitor resistance problems, build national reporting operations, and provide international coordination.

Even U.S. surveillance is spotty, David Bell, assistant to the director of the CDC’s National Center for Infectious Diseases, pointed out during the meeting. The CDC has several systems that collect different information from various sources. The systems monitor hospital-acquired infections, pathogens in food, tuberculosis, sexually transmitted diseases, malaria, and others. Managed-care organizations also run private surveillance systems, while networks of universities-with funding largely from the drug industry-oversee others. “One of the problems we have,” Bell said, “is that virtually none of these systems provides anywhere close to nationwide coverage.”

There are other problems, too. “We really need to know more than which drugs are becoming resistant to which bugs,” Bell said. “We need to know who are these patients with the resistant infections, and are they randomly distributed across a population group or do they fall into certain risk groups-for example, hospital patients, or people who travel abroad, or people who use a lot of antibiotics.”

Fred Tenover, chief of the CDC’s hospital-infections laboratory branch, said the resistance problem could be much worse than is already known because not all organisms are tested for resistance and some labs rely on improper testing methods. In one case last year, he said, 30 percent of the 2,100 labs looking for resistance in Streptococcus pneumoniae used the wrong test.

Effective treatments and strong surveillance systems that spread early warnings about resistant bacteria are still only part of a solution to fight drug-resistant bacteria. Public-health officials, doctors, and members of the drug industry also agree that preventing overuse of antibiotics is critical. “The more and more we use these antibiotics, the more selection we have, and these mutants will emerge,” says Stuart Levy, president-elect of the American Society for Microbiology. “There is inadequate physician, veterinarian, farmer, and patient education,” says Mitchell L. Cohen, director of the CDC’s division of bacterial and mycotic diseases. He says doctors admit to overusing antibiotics by 15 to 20 percent.

Doctors in managed-care settings, where pressures can mount to see high numbers of patients quickly, are among those who often over-prescribe antibiotics, says S. Michael Marcy, a Kaiser Foundation staff pediatrician and pediatrics professor at the University of Southern California and University of California at Los Angeles Schools of Medicine. Patients who don’t receive an antibiotic on the first visit and are convinced they need one often return-a problem for doctors at managed-care facilities that try to reduce return visits, according to Marcy. Patient satisfaction surveys also contribute to generous prescriptions for antibiotics: “Now, up to 30 percent of our salary will be determined by our satisfaction rating,” he says. The message becomes: give patients “what they want.”

In answer to the criticism of antibiotic overuse, some professional medical and scientific organizations have begun distributing brochures for patients and doctors on the proper use of antibiotics. “It would be much more powerful and effective,” urges Frederick Sparling, chair of the department of medicine at the University of North Carolina and president of the Infectious Diseases Society of America, if major professional organizations would all get behind one set of guidelines.

In the end, so many players exist in the saga of resistant microbes and their invasion of people that answering the question “Can we beat antibiotic resistance?” is impossible.

If scientists reveal the inner workings of all virulent microbes and design novel ways to overcome their drug resistance; doctors quit over-prescribing antibiotics and patients learn proper respect for the drugs; hospitals, nursing homes, and doctors’ offices adhere to strict infection-control policies; and a strong global surveillance system is established; then humans would seem to have the advantage.

Except that bacteria have a much longer track record at adapting and surviving than humans. And scientists, left to wonder what unknown paths of mutations lie ahead, say they aren’t willing to bet against the microbes. “I don’t think any of us will ever develop an agent [for which it is] totally impossible to have resistance,” says Zurenko of Pharmacia & Upjohn. “There are still a lot of imponderables,” points out Joshua Lederberg. Yet based on the recent level of activity in antibiotic-resistance research, he says, “At least some of the right people are paying close attention. That was not true a couple of years ago.” That, he says, is “progress.”

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