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Danish scientist Hans Christian Gram in the late 1800s. In Gram-positive bacteria, the process colors the bacterium with dark purple crystals that attach to its single cell membrane. Gram-negative bacteria, however, have a second membrane overlaying that found in Gram-positives; as a result, the same crystals don’t stick to these microbes.

It’s this double-layered cell wall that makes Gram-negative bacteria particularly formidable against antibacterials, as it houses some of their specialized mechanisms of defense. Compounding this with the newly emerging mutations that confer antibiotic resistance, it can only get worse.

As it stands, Gram-negative bacteria account for more than 60% of microbial infections being treated worldwide, but the pipeline of new drugs against them is disproportionally small1. According to recent estimates, there are only 16 antibacterials currently in late-stage development. Eight of these act against both

too many have died already, and many more will too, unless new antibacterials are made available.

Antibiotic resistance is a chronic problem for the infectious disease field, and Spellberg and his fellow physicians know this all too well. Many pathogens will eventually garner mutations that enable them to fight off even the most powerful drugs in the medical arsenal, and unless contained, these strains can spread. One strain in particular, methicillin-resistant Staphylococcus aureus—better known as MRSA—is infamous, having grabbed headlines and the attention of the pharmaceutical industry for years.

But the problem of MRSA is only half the story, in a sense, when it comes to bacteria: whereas MRSA is a species of Gram-positive bacteria, there are also the Gram-negatives, which are presenting problems of their own.

Both types of bacteria are named in reference to a form of cell staining developed by the

Brad Spellberg leaned into the podium, hunching over to hide a strained face. It was clear to the captive audience at the congressional hearing last month that he had reached a difficult part of his presentation.

On the screen, Spellberg pulled up pictures of six patients affected by highly drug-resistant bacteria. After minutes of rattling off numbers and statistics to the group of congressional aides and reporters before him, Spellberg, an assistant professor of medicine at the University of California–Los Angeles, was now showing pictures of the people he was advocating for. Spellberg paused. He looked up at the screen. And he turned back, breaking the silence with a different tone of voice, one with a hint of sadness—and a hint of frustration.

Of the six pictured in family photos, four of them had passed away: a young model, a college football player, a recent high school graduate and a 12-year-old boy. After he briefly told each story, Spellberg returned to the matter at hand:

The notoriously drug-resistant bug MRSA has made headlines for years, but a whole other class of bacteria may prove even more troublesome. These microbes, Gram-negative bacteria, are increasingly a threat—and yet not a single late-stage drug in development specifically targets them. Christian Torres follows one man’s quest to get the antibacterial pipeline flowing once again.

Up against the wall Ilustrations by Marina Corral

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Gram-positives and Gram-negatives, but none are specific to Gram-negatives2.

The lack of new antibacterials is a complex, challenging issue that’s particularly a problem for Gram-negative bacteria. There’s a perfect storm, experts say, of poor economic incentives, regulatory challenges and, of course, tricky cellular defenses holding off development. Each problem builds on another, and even if the barriers come down, there probably won’t be a new antibacterial specific to Gram-negative bacteria for at least six years.

Pipeline problemsIt’s been six years, in fact, since Brad Spellberg first hit this wall of resistance.

Back when he was still in the midst of his fellowship in infectious diseases, Spellberg treated Mrs. B, a young mother of two who had a strain of Acinetobacter baumannii coursing through her blood, spreading toxins and “eating her alive,” as Spellberg recalls. Mrs. B’s infection was not just highly resistant—it was resistant to everything. Just one day after Spellberg found out Mrs. B’s infection had overcome the most powerful antibiotic then available, a drug called imipenem, the infection defeated her, too.

Spellberg remembers informing her incredulous husband that there were no further drug options to treat her with. And it remains one of Spellberg’s most traumatic experiences as a physician, evident from the slight tremble in his voice. Years after telling Mr. B “there’s nothing left,” he remains frustrated that the options for resistant infections are still few to none.

Gram-negatives have in essence “set us back 70 years, to a time when we didn’t have antibiotic options,” Spellberg says. Patients like Mrs. B are becoming all too common, with imipenem-resistant A. baumannii accounting for an estimated 34% of US healthcare-associated infections in 2008 (ref. 3). But Spellberg suspects that statistic has gone up to as much as 60%. The hospital where he practices, for example, now sees at least one highly resistant infection each week, and completely resistant infections are increasingly among them.

Even tigecycline, the last Gram-negative antibacterial approved, has encountered resistance within a mere five years of use. Fending off these bacteria largely depends on continued development of antibiotics, but that is now failing, too.

“Years ago, the pipeline was very robust,” says Bob Bonomo, an associate professor of medicine at Case Western Reserve University in Cleveland. “Now, however, it’s down to a trickle of drops.”

There are many factors working against development. Economics, for one, make

antibacterials a distant concern for industry. Compared to drugs for chronic illness, such as diabetes or heart disease, the antibacterial market is limited to small numbers of patients who only receive treatment for a couple of weeks at most. What’s more, when powerful antibacterials are developed and finally brought to market, they’re only used sparingly to protect against the development of further resistance.

These concerns have made pharmaceutical companies largely unwilling to spend hundreds of millions of dollars only to see their products sit on a shelf. And in the early 2000s, many companies began dropping their antibacterial development programs entirely, just as the numbers of such medicines being approved by the US Food and Drug Administration (FDA) went into a tailspin.

Spellberg, who also consults for several drug companies, was among those who first delivered concrete data on the pipeline. Shortly after seeing Mrs. B slip away, he was asked by members of the Infectious Diseases Society of America (IDSA), a group representing around 9,000 infectious disease physicians, to contribute to their advocacy efforts. Spellberg teamed with FDA officials, and together they authored a paper analyzing the research and development programs of 15 major pharmaceutical companies and seven major biotechs4.

Out of the hundreds of drugs that Spellberg and his coauthors found in early-stage development, a mere six were new antibacterials, and none of these represented a truly novel mechanism. Those results were a bad sign for already declining FDA approvals: only seven new antibacterials were approved between 1998 and 2002, down more than 50% from 1983–1987. In a follow-up study, analyzing 2003–2007, that number was just five5. And since 2008, Spellberg says, there has been only one approval. The last new antibacterial approved for Gram-negatives, tigecycline (marketed as Tygacil), was approved in 2005.

Pacing progressThe evidence was, and remains, clear: antibacterials are far from keeping pace with drug resistance. And physicians like Spellberg have to deal with the complications.

“At this point, [resistance is] just a part of daily life,” he says. For years there’s been “a constant angst and worry,” and, at the end of any given day, Spellberg might solemnly return to his office, frustrated over the loss of yet another patient, or he might be left commiserating with other physicians, tired of their impotence against the onslaught of infections.

“Things were going from bad to worse” back when Spellberg wrote the 2004 paper,

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and though that opportunity fell into his lap, he says he could no longer passively watch the antibacterial arsenal stagnate. Both on a personal and a statistical level, Spellberg knew something had to be done.

Later in 2004, he joined the IDSA’s Antimicrobial Availability Task Force (AATF), which that same year cited Spellberg’s analysis in a major report, “Bad Bugs, No Drugs: As Antibiotic R&D Stagnates, a Public Health Crisis Brews.” The AATF added that companies can’t be forced to develop antibacterials—they have to want to develop them. This is where the US government can step in, the group said. For example, companies that produce successful new drugs might be offered tax exemptions and enhanced patent protections. The AATF has also advocated for setting up a federal office that would set priorities for research among various pathogens, and it has pushed for greater funding for the National Institute of Allergy and Infectious Diseases.

However, like the pipeline itself, these ideas have moved slowly. For years, the IDSA has held meetings with Congress and the pharmaceutical industry, given hundreds of talks to the public and medical community, spread the word through the media (Spellberg, for example, has written a book titled Rising Plague) and published several more letters and reports. One report even rephrased the AATF’s message to “Bad Bugs, No Drugs:

No ESKAPE!”—a clever acronym for six kinds of problem bacteria, four of which are Gram-negative: Enterococcus faecium, S. aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa and Enterobacter species2.

This year, hopes are high that at least one piece of legislation will finally make its way

through. The Strategies to Address Antimicrobial Resistance (STAAR) Act, though not focused on the development incentives IDSA has sought, would establish an Antimicrobial Resistance Office under the Department of Health and Human Services, as

well as create a resistance-monitoring network that would keep track of both infections and the distribution and use of antibiotics across the country. Antibacterial stewardship, a term for conservative use of what’s available, is the STAAR Act’s goal.

“This is an important step in strengthening the national response” to antibacterial resistance, said Democrat Representative Jim Matheson at the same congressional briefing Spellberg spoke at last month. Matheson introduced the House bill in May 2009 and said that its passage could lead to further reforms, especially as the government comes to terms with this “major blooming health crisis.”

That message is exactly what the AATF has been trying to get across, and, with further hearings expected this summer, the group is optimistic.

“My sense is both the White House and Congress are starting to appreciate how important this is, and they’re giving a signal,” Spellberg says. “They’re ready to act.”

Double troubleEconomics and legislation aren’t the only barriers that drug developers face with Gram-negative bacteria. These pathogens posses a fortified defense system, and much of this toughness is summed up in their name.

The secondary membrane that prevents these bacteria from becoming purple in Gram staining also serves as tough armor. For example, the membrane drastically decreases permeability to hydrophilic compounds, including available drugs, when compared with the membrane of Gram-positives. Certain Gram-negative species, such as P. aeruginosa and A. baumannii, for example, have as little as 1% the permeability of other bacteria.

The secondary membrane also creates what is called a periplasmic space—between the first and second membranes—that can prevent invading drugs from immediately entering the cytoplasm and reaching their respective targets. And, adding insult to injury, membrane proteins called efflux pumps can actively remove drugs from the cell; among Gram-negatives, these pumps can remove drugs from the periplasmic space before they even reach the cytoplasm and accrue an effective concentration.

“The bugs vexing us are very difficult,” says Lou Rice, a professor of medicine at Case Western and AATF member. “If it were easy to find new antibacterials, then the pharmaceutical industry would still be involved.”

Among the drugs currently available are aminoglycosides, which prevent bacterial protein synthesis and halt the development of defensive structures in the cell membrane. Another class of drugs, the quinolones, prevent DNA transcription and replication. But, for a sense of how bacteria have evolved, one need only look at the beta-lactams. These drugs, which block synthesis of the peptidoglycan layer in bacterial cell walls, are increasingly rendered powerless by destructive enzymes in bacteria that have acquired resistance mutations. (Many drugs in development are actually meant to block these beta-lactamases and restore antibacterial function.)

Gram-negative bacteria, in particular, are showing these defensive mechanisms, along with mutations that prevent drug target binding and some that even further reduce membrane permeability. There’s a desperate need for new compounds, then, even if they are just new versions that can evade the slightest mutation. Several companies have these new-in-class drugs in development, including

“Years ago, the pipeline was very robust. Now, however, it’s down to a trickle of drops.”

—Bob Bonomo

Accentuate the positive: only the Gram-negative bacterium (bottom) has a secondary membrane.

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stems from a lack of clarity with regards to FDA expectations for clinical trials, ranging from sample sizes to endpoints to statistical significance. Large trial populations, for example, are hard to come by, especially among the still rare, highly resistant infections. Currently, there is no clinical trials network available for garnering patients, and study sponsors instead have to rely on word of mouth.

As Helen Boucher, another AATF member and previous pharma consultant, describes, there’s far from a “cookbook approach”

available for antibacterial trials, and companies become afraid of large investments that falter in the final stages. Many companies are unsure of how to handle the FDA-prescribed noninferiority trials. In these trials, new drugs are tested to show efficacy on par with already available medicines, but matters

such as statistical significance and endpoints remain up for debate.

“You also can’t enroll patients if their infections are resistant to the standard,” adds Talbot. “It makes it difficult, if not impossible, to conduct trials given the current situation [with resistance].”

The FDA, however, is working to at least clear up the process. Edward Cox, director of the Office of Antimicrobial Products, says the agency is working to spell out the expectations for clinical trials.

“We recognize the importance of developing clear pathways and providing guidance,” Cox

says, noting how the past four years have seen the release of five draft documents concerning various types of antibacterial infection. The agency hopes to release two more draft guidances by the end of the year, and once these are released, Cox says, the office will return to the released drafts and renew the conversation over various concerns.

Spellberg, although agreeing with other AATF members that the process needs to speed up, understands the FDA’s difficult position in handling the concerns of statisticians, physicians and the agency itself. And he fully recognizes that regulators are only one part of the problem: “There isn’t a single step that’s broken—the entire process is broken.”

Still, whether the FDA, legislators, pharmaceutical companies or Gram-negative bacteria themselves are the final gatekeepers to new antibacterials, something has to be done. When Spellberg was finishing his May presentation on Capitol Hill, there came another moment in which he paused with emotion. This time it was to give his sternest words yet: “the time for debate has passed. The time for action is now.” And from the stark silence that followed Spellberg’s remarks, it’s apparent Congress is, at least, listening.

Christian Torres is a former intern at Nature Medicine who lives in San Diego.

1. Vincent, J.-L. et al. J. Am. Med. Assoc. 302, 2323–2329 (2009).

2. Boucher, H.W. et al. Clin. Infect. Dis. 48, 1–12 (2009).3. Kallen, A.J., Hidron, A.I., Patel, J. & Srinivasan, A.

Infect. Control Hosp. Epidemiol. 31, 528–531 (2010).

4. Spellberg, B., Powers, J.H., Brass, E.P., Miller, L.G. & Edwards, J.E. Jr. Clin. Infect. Dis. 38, 1279 –1286 (2004).

5. Spellberg, B. et al. Clin. Infect. Dis. 46, 155–164 (2008).

Massachusetts-based Cubist, which has two compounds entering phase 2 trials, including a new cephalosporin (a type of beta-lactam) and another drug of a known, but undisclosed, class.

As for truly novel compounds, those are few and far between—or, at least, not yet disclosed. Among the few that have been publicized is a compound by Switzerland-based Basilea that falls into the beta-lactam class but has unique activity against multi-drug resistant bacteria. Another early-stage drug is an antibody by South San Francisco–based KaloBios; it helps by blocking the toxin-secreting structures on the outer membrane of P. aeruginosa. Meanwhile, Trius, based in San Diego, is conducting preclinical studies of a compound that would block two different targets involved in DNA replication at once, allowing for the drug to hit one kill-switch, even if not the other.

Still, these drugs, like many others, might fail to make it through the early stages of development. As George Talbot, an AATF member and pharmaceutical consultant, describes, “there’s no magic bullet, so the focus has to be on augmenting the pipeline.”

Reaching regulatorsOne of the largest factors keeping companies from antibiotic development is also the last stage: regulatory approval.

In discussions with IDSA, many companies have cited uncertainty over the approval process as a major impediment. Much of this

“If it were easy to find new antibacterials, then the pharmaceutical industry would still be involved.”

—Lou Rice

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