With a pivotal study in the battle against antibiotic resistance, scientists at The Rockefeller University in New York have identified a pair of genes governing penicillin resistance in Streptococcus pneumoniae. The findings, published in a recent issue of the Proceedings of the National Academy of Sciences, suggest novel targets against the bacteria that may give new life to penicillin as a tool against infection.

Since the 1990s a rapid proliferation of drug-resistant strains of S. pneumoniae has emerged as a significant public health threat. According to recent data from the Centers for Disease Control, as many as 30 percent of clinical isolates of S. pneumoniae from a nationwide survey were resistant to penicillin, and 17.4 percent were resistant to penicillin and at least two other classes of antibiotics. S. pneumoniae is a virulent microbe responsible for an estimated one million annual deaths worldwide. In the United States, it is the leading cause of death from infection—believed to cause at least 6,000 cases of meningitis, 50,000 blood infections, half a million cases of pneumonia, and several million childhood ear infections every year.

Magnification of penicillin

Penicillin kills S. pneumoniae by interfering with the synthesis of the bacterial cell wall; specifically by inhibiting the enzyme that catalyzes cross linkage of cell wall macromolecules called peptidoglycan. The peptidoglycan are composed of a linear arrangement of building blocks called muropeptides. Scientists at The Rockefeller University have already shown in the late 1980s that resistant strains of S. pneumoniae have abnormal cell walls characterized by a high percentage of branched rather than linear muropeptides—although how this difference might account for resistance was unclear.

In the present study, Sergio R. Filipe and Alexander Tomasz of The Rockefeller University identify the genes that code for enzymes involved in the synthesis of branched muropeptides and provide experimental evidence showing that inactivating the genes (called murM and murN) corrects cell wall abnormalities and re-sensitizes the bacteria to penicillin toxicity.

"These are novel genes, a new target for drug development," says Stuart B. Levy, Director of the Center for Adaptation Genetics and Drug Resistance at Tufts University. "It's a very exciting finding, especially with such a worthwhile antibiotic as penicillin."

According to Tomasz, the findings are intriguing because they show that resistance in S. pneumoniae can be eliminated without any apparent effect on the low affinity penicillin binding proteins (PBPs), which are critical parts of the resistance mechanism in these bacteria. "It's really a puzzle," he says. "We are eliminating penicillin resistance without touching the affinity of PBPs at all."

Tomasz suggests these findings could eventually lead to a combination drug therapy he likens to a two-strike military strategy: "One missile inactivates the bacteria's defense mechanism (the murMN function), while the other (penicillin) hits the target (the PBPs)," he says. This suggested strategy is similar to at least one combination therapy currently in use. Augmentin, for example, is a popular drug that combines amoxicillin with clavulanic acid—a chemical that inactivates an antibiotic-neutralizing enzyme secreted by resistant bacteria.

The approach suggested by Tomasz could add to the clinician's arsenal a drug that targets cell wall branching enzymes. Says Barry Kreiswirth of the Public Health Research Center in New York, "What we have now are bacteria that are resistant to every drug that we throw at them. So using old drugs in a new context, or in combination with other drugs, is a clever way to deal with this problem."

Tomasz suggests that the principle could be extended to what he calls a variety of "partner drugs": agents that could pair-up with and facilitate the performance of already existing anti-microbial agents. Targets for such future "partner drugs" may be among the host of "auxiliary genes," he says, that seem to be essential for high-level beta-lactam antibiotic resistance in Staphylococci. "We should compare resistance to a complex stress response analogous to what bacteria mobilize when they face an adaptive challenge such as an encounter with viruses or the defenses of an invaded host," he explains. "They turn on a whole battery of genes, not just one. All of these auxiliary genes could serve as potential targets against resistant bacteria."

With the impact of antibiotic resistance on medical care becoming more daunting by the day, scientists are becoming especially interested in how bacteria acquire resistance in the first place. Abigail A. Salyers, of the University of Illinois, says that two important methods of acquiring resistance are spontaneous mutations in DNA—a lengthy process that can be lethal to the organism—or direct gene transfer using plasmids, which are extra-chromosomal genetic elements that can be passed from one bacteria to another. Of the two, Salyers says, the latter (known as horizontal gene transfer) is vastly more rapid and stable. "With horizontal transfer, resistance genes can be passed within an hour. They are easy to get and hard to lose," she says. According to Tomasz, there is no doubt that horizontal gene transfer is the source of penicillin resistance in S. pneumoniae, which, he says, has an almost unparalleled capacity to absorb DNA molecules released by other bacteria. The real question, and one that is currently being addressed in his research, is the identity of the donor organism.

Salyers says that research has shown some bacteria to possess a worrisome capacity for transferring resistance against multiple antibiotics simultaneously. This has been demonstrated in her own work on tetracycline resistance genes in Bacteroides, which are common bacteria in the gut that can be lethal if inadvertently released to the bloodstream. Bacteroides exposed to low levels of tetracycline quickly transfer multiple resistance genes that are effective not only against tetracycline, but several other antibiotics as well. Also of interest are genes that govern synthesis of bacterial efflux pumps, which eject antibiotics from resistant bacteria. Research conducted in Levy's laboratory has recently shown that efflux pumps, once thought effective only against tetracycline, are actually effective against several antibiotics. "This shows that the transfer of a single gene allows a bacteria to acquire simultaneous resistance to more than one drug. This is bad news, and a source of great concern," says Salyers.

Already, antibiotic resistance is having a major impact on medicine that will only increase in the coming years. According to Salyers, some large pharmaceutical companies became so discouraged by resistance microbes wiping out their R&D investment during the 1990s that they simply stopped investigating new antibiotics altogether. Only recently has the current resistance crisis pushed these companies back into antibiotic research. In the meantime, clinicians are looking for new ways to use existing antibiotics, a trend that may be advanced by the recent findings at The Rockefeller University.