Keeping Bugs from Pumping Drugs

A new way to defeat antibiotic-resistant microbes?

Give a baby a spoonful of medicine and phttt! In an instant, he’s spit it all over the room. Getting the medicine down, however, isn’t the whole battle. Even when an antibiotic’s been swallowed, the microbe that it targets may eject it before the infection has been quelled.

In the chemical arms race between modern medicine and quickly evolving bacteria and fungi, some researchers are now opening a new front. They’re attacking the machinery, known as efflux pumps, that microbes use to rid themselves of toxic materials, including drugs. These scientists are beginning to develop compounds—efflux-pump inhibitors—that have no infection-fighting power of their own but can make current antimicrobial drugs more effective.

The introduction of modern antibiotics almost 60 years ago ushered in a time when previously untreatable infections—tuberculosis, gonorrhea, and syphilis, to name just a few—could be almost miraculously cured. However, many of the microbes that caused suffering in the pre-antibiotic era are again making people sick because these bugs have become adept at fighting off the modern medical armamentarium.

Various bacteria and fungi are now resistant to one or more widely used classes of antibiotics, such as penicillins, cephalosporins, tetracyclines, quinolones, aminoglycosides, and macrolides (SN: 6/7/97, p. 348: This may lead to deadly infections that no antibiotic drugs can overcome.

Bacteria and fungi can resist a drug in several ways. They can alter it so that it’s no longer toxic. They can modify their own components so that the antimicrobial compound can’t bind to them or have an effect. In recent years, microbiologists have realized that some bacteria and fungi can also expel drugs, lowering the internal concentration enough that the microbes escape the treatment’s intended effects.

“The growing number of publications regarding these [efflux] systems in bacteria, fungi, and parasites clearly indicates that they are widespread in nature and implicated in the resistance to many antimicrobial compounds,” says Patrick Plesiat of the Jean Minjoz Hospital in Besançon, France.

“If one considers the ever-growing resistance of bacteria worldwide, . . . efflux-pump inhibitors might well represent economically and therapeutically promising drugs,” he says.

Antibiotic pumps

Most efflux pumps probably evolved to handle toxins in the environment and only serendipitously pump out antibiotics, says Paul M. Tulkens of the Catholic University of Louvain in Brussels. Scientists are still working to determine the basic mechanisms of these pumps. Each pump is made up of one or several proteins that span the cell membrane of the microbe.

The two most likely explanations for their operation are that efflux pumps actively expel a drug through a local channel or that by flipping the drug they propel it across the cell membrane, says Tulkens.

Efflux-mediated antibiotic resistance was first documented in 1980, when Stuart B. Levy of Tufts University School of Medicine in Boston and his colleagues reported that some enterobacteria pump out tetracycline. The first multidrug efflux pump was reported in 1989 in Staphylococcus aureus.

“Although efflux systems have been known for many years, their importance, both in terms of number and variety of substrates, has been clearly recognized only very recently,” Tulkens says.

When efflux pumps were first discovered, many researchers assumed that they were important in just a few microbes, Tulkens says. But it’s now becoming an accepted concept that efflux pumps are responsible for at least a moderate level of resistance in many different species of bacteria and fungi and against several drugs, he says.

Not all microbes have efflux pumps, and those that do employ widely varying numbers and types. Some microbes always have abundant pumps, whereas others manufacture additional pumps after exposure to drugs.

Efflux pumps may help explain why some bacteria are inherently less susceptible to drugs than others are. For example, Pseudomonas aeruginosa has many efflux pumps of several different types and is intrinsically resistant to many common drugs, says C. Kendall Stover of PathoGenesis Corp. in Seattle. This bacterium causes pneumonia and infections of the skin, urinary tract, and bloodstream.

P. aeruginosa is a very big problem in hospital-acquired pneumonia and may be becoming more of a problem because it is becoming even more resistant over time,” he says. “Much of this [acquired] resistance appears to be due to revving up its efflux pumps.”

This and several other species of bacteria, says Tulkens, seem to use efflux pumps to resist tetracyclines, macrolides, and fluoroquinolones well enough to often make these antibiotics useless weapons. Since efflux pumps may act on more than just one kind of antimicrobial agent, microbes may develop resistance against several different drugs simultaneously, he warns.

Some recent studies indicate that between 40 and 90 percent of some bacterial pathogens, such as Streptococcus pneumoniae, Streptococcus pyogenes, and P. aeruginosa, carry efflux pumps for most of the major classes of available antibiotics. Tulkens calls this “alarming” and notes that clinical microbiology labs aren’t routinely testing for efflux pumps when they look for resistance.

“Most insidiously, antibiotic efflux may be found in association with other mechanisms, such as antibiotic inactivation, to confer high-level resistance to bacteria,” he says. Mutations that would not protect the bacteria from a full dose of antibiotics may be enough to save them from the lower effective drug concentrations that efflux pumps achieve.

Studies that genetically manipulate microbes by removing or adding genes for efflux pumps have confirmed that the pumps trigger resistance in common bacterial strains. For example, strains of P. aeruginosa lacking one of three types of intrinsic efflux pumps were more susceptible to a fluoroquinolone antibiotic, levofloxacin, than normal strains were. Olga Lomovskaya of Microcide Pharmaceuticals in Mountain View, Calif., and her colleagues reported this finding in the June 1999 Antimicrobial Agents and Chemotherapy.

Among P. aeruginosa with mutations known to cause resistance, those strains missing genes for the efflux pumps were significantly less resistant to levofloxacin than strains with the normal amount of efflux pumps were, Lomovskaya says.

In the January Antimicrobial Agents And Chemotherapy, Levy and his colleagues show that when they genetically engineer fluoroquinolone-resistant Escherichia coli to lack a normal efflux pump known as AcrAB, the bacteria become susceptible to the antibiotic again. This indicates that the pumps strengthen resistance that results from other mechanisms, Levy says.

A widely used tool in the fight against disease may actually rev up microbes’ production of efflux pumps. Herbert P. Schweizer at Colorado State University in Fort Collins and his colleagues have shown an increase in pump production in P. aeruginosa exposed to triclosan, a general bactericide used in antibacterial soaps, kids’ toys, and clothing. Could this mean, asks Schweizer, that antiseptic additives intended to make products safer are actually engendering microbes that can resist a wide range of drugs?

Disrupting activity

The new understanding of efflux pumps opens up opportunities for pharmaceutical companies to find compounds that will disrupt this microbial activity. As drugs, efflux-pump inhibitors aren’t expected to have a significant antimicrobial effect on their own. Companies now developing these compounds expect them to reverse acquired drug resistance in microbes that used to be susceptible to antibacterial and antifungal drugs, says George H. Miller of Microcide.

Efflux-pump inhibitors might also make some microbes that are intrinsically drug resistant vulnerable to antibiotics, he says. Miller predicts, too, that efflux-pump inhibitors will reduce the chance that bacteria will successfully reproduce enough times to select for a drug-resistance mutation.

The work on efflux-pump inhibitors remains preliminary. At the Interscience Conference on Antimicrobial Agents and Chemotherapy, held in San Francisco last September, Lomovskaya and her colleagues reported on some of the first animal tests of bacterial and fungal efflux inhibitors. They’ve been developing efflux-pump-inhibiting drugs against four organisms—staphylococci, enterococci, psuedomonas, and streptococci. Together, these account for 44 percent of the 2 million infections that occur each year in U.S. hospitals.

In test-tube studies, Microcide researchers had found several efflux-pump inhibitors that reduced the resistance of a strain of P. aeruginosa to fluoroquinolones. The inhibitors reduced the intrinsic resistance to about one-eighth and the acquired resistance to as little as one-sixty-fourth of that in untreated bacteria.

In mice infected with this strain of bacteria, a combination of the antibiotic levofloxacin and an inhibitor known as compound 4 significantly reduced the concentration of bacteria in the animals’ muscles compared with mice given no treatment, Lomovskaya reported at the San Francisco meeting. Mice given either levofloxacin or compound 4 alone showed no reduction in bacterial growth.

The Microcide researchers have found that their efflux inhibitors increase the intracellular concentration of antibiotic 8- to 16-fold. This allows the antibiotic to overcome resistance triggered by other types of mutations, Miller says.

At the meeting, Lomovskaya and her colleagues also reported that several efflux-pump inhibitors useful against a variety of fungal efflux-pumps increased 64- to 128-fold the efficacy of antifungal drugs against drug-resistant Candida albicans. The fungal efflux-pump inhibitors reduced intrinsic resistance in Candida glabrata to one-eighth to one-sixteenth of normal, they said.

Multiple targets

One major issue is whether to design inhibitors that target just one type of efflux pump or that block a number of different pumps. The pharmaceutical researchers are concerned that inhibiting a single type of efflux pump might not overcome resistance in bacteria and fungi with multiple efflux pumps. However, developing drugs that block a variety of efflux pumps raises another concern.

Mammalian cells have their own efflux pumps, which are primarily responsible for clearing environmental toxins and metabolites from cells. Drugs that block a wide range of efflux pumps may have unforeseen side effects, Stover cautions.

Miller reports that researchers have seen antibiotics accumulate in animal tissues during tests with efflux-pump inhibitors. So far, he says, “none of the bacterial efflux-pump inhibitors we’ve found inhibit mammalian efflux-pumps. However, some of the fungal efflux-pump inhibitors do inhibit a mammalian pump.”

To avoid inhibition of mammalian efflux-pumps, scientists might be able to interfere with the regulation of the bacterial or fungal genes that control the production of the microbes’ efflux pumps, Lomovskaya suggests.

Efflux-pump inhibitors have “real promise,” Tulkens says. If they turn out to be effective adjuncts to new antibiotics, “the spread of resistance will be slower,” he adds.

Miller suggests that efflux-pump inhibitors may make it easier for researchers to develop new antibiotics and antifungal drugs. “When you made an antibiotic in the past and it didn’t work, you assumed it didn’t get into the bacteria,” he says. “Now, we think these antibiotics are probably effluxed.” He suggests that drugs that have failed as antibiotics in the past may be effective in combination with an efflux-pump inhibitor.

Although enthusiastic, researchers studying efflux pumps still have some reservations about the prospects for pump-inhibiting drugs. Plesiat cautions that the preliminary research can’t yet predict which compounds might have future clinical applications or even what those applications might be.

William M. Shafer of Emory University in Atlanta raises a broader caution. He says, “My concern with pump inhibitors is that history has shown us that with any agent that interferes with a biologic process, it is only a matter of time before resistance develops.”