OVERCOMING RESISTANCE

In the face of bacterial threats that can evade modern medicines,
researchers are trying every trick in the book to develop new, effective antibiotics.

BY THE SCIENTIST STAFF
Although researchers and drug developers have been
sounding warnings for years about bacteria out-evolving
medicine’s arsenal of antibiotics, the crisis is coming
to a head. In the United States alone, some 23,000 people
are killed each year by infections caused by drug-resistant bacteria,
according to the Centers for Disease Control and Prevention’s
2013 Threat Report. Many more patients die of other
conditions complicated by infection with resistant pathogens.
Such maladies cost the health-care system more than $20 billion
annually, in part because patients suffering from drugresistant
infections require more than 8 million additional
hospital days.
The statistics are sobering, and they’re made even more so by
the fact that the US Food and Drug Administration (FDA) has
only approved two new classes of antibiotics since 1998. In fact,
only five new classes have hit the market in the last 45 years; the
vast majority of today’s antibiotics were developed before 1968.
Overuse—and not just in people, but in animals, too—is a primary
driver of the antibiotic-resistance epidemic. One of the most
controversial antibiotics practices has been the “nontherapeutic”
treatment of farm animals with low doses of the drugs to promote
growth and prevent disease in crowded factory-farm conditions.
(See “Antibiotics in Animals We Eat,” The Scientist, April 2012.)
Up to 80 percent of the antibiotics used in the U.S. is fed to animals,
and the Natural Resources Defense Council recently criticized
the FDA for allowing livestock producers to include 30 different
antibiotics in the animals’ feed and water, 18 of which the
agency itself had rated as “high risk” for introducing antibioticresistant
bacteria into the human food supply.
While debates rage over what is driving the recent onslaught
of antibiotic-resistant pathogens and how to best stem the bacterial
tide, many researchers are now focused on developing new
treatment regimens to combat these deadly superbugs. One thing
most of these scientists on the front lines can agree on is that
antibiotic resistance is not a single-solution problem. Here, The
Scientist surveys four strategies being explored to overcome even
the most resistant bacteria: tweaking old compounds into entirely
new classes of antibiotics; combining modern antibiotics in a onetwo
punch against infection; supplementing existing antibiotics
with adjuvants that can render resistant pathogens susceptible
once more; and reviving the field’s roots by combing the globe for
novel antimicrobial compounds.

BLASTS FROM THE PAST
The golden era of antibiotic discovery is well behind us. In the
mid-20th century, numerous new classes of antibiotics came on
the market, and scientists tinkered with these molecules to create
ever more powerful versions of the drugs. Since then, however, the
well has dried up. Even genomics has failed to rescue the stalled
antibiotics field, says Anthony Coates, an antibiotic researcher at
St. George’s, University of London, and the founder of Helperby
Therapeutics.
One approach researchers are undertaking to break the dry
spell is to alter old drugs, including those that have been abandoned
by Big Pharma, using new techniques. Richard Lee at St.
Jude Children’s Research Hospital in Memphis, Tennessee, for
example, is studying an old antibiotic called spectinomycin that
was introduced in the 1960s to treat gonorrhea. While the drug
worked against the sexually transmitted bacterium, large doses
were required, and, eventually, drug makers developed more
potent antibiotics. Although spectinomycin only has weak effects
against most microbes, Lee saw potential in remodeling it to treat
certain bacterial infections, thanks to its ability to bind the bacterial
ribosome and clog protein synthesis.
“What we could take advantage of, which wasn’t available 20
years ago, is the crystal structure of spectinomycin bound to the
ribosome,” Lee says. With a tweak to adjust how the molecule
binds to the ribosome, the modified drug was able to fight off
tuberculosis in vitro and in mice (Nat Med, 20:152-58, 2014).
The changes not only maintained the affinity of the drug for the
ribosome, but allowed the antibiotic to avoid the efflux pump
that normally ejects this drug from the tuberculosis bacterium.
“It works better than we would have dreamed,” Lee says, although
its potency against gonorrhea was not improved.
Jason Sello, a biochemist at Brown University, has also found
that slight chemical tweaks can make a profound difference to
antibacterial activity. His group has been tinkering with the structure
of ring-shape compounds called acyldepsipeptides (ADEPs).
Discovered in the 1980s, ADEPs were initially of interest to pharmaceutical
companies because of their antibacterial activity, but
were ultimately set aside in pursuit of other endeavors and never
brought to market. One unfavorable aspect of ADEPs is that bacteria
tended to become resistant to them quite quickly, rendering
the drugs incapable of clearing an infection. But because ADEPs
work in a way that no other antibiotic does—they activate widespread
protein degradation by the bacterial enzyme ClpP, which
normally clears misfolded proteins—they’re extremely appealing
for development as a drug to fight bacterial infections, says Sello.
He and his colleagues have focused on the rigidity of the cyclic
structure of ADEPs and found that strengthening the hydrogen
bonds of the ring can increase the antibacterial power of the compound,
seemingly by allowing the drug easier entry into target
cells (J Am Chem Soc, 136:1922-29, 2014). “Why it’s better at killing
bacteria is not because it has a better mechanism of action, but
we think it’s more cell-permeable,” says Sello. Preliminary studies
in mouse models show that the modified ADEP is good at treating
staph and Enterococcus infections, and so far, there’s no evidence
that the drug is toxic.
At Oregon State University, microbiologist Bruce Geller has
picked up on yet another discovery that was made decades ago.
Phosphorodiamidate morpholino oligomers (PMOs) are short,
synthetic versions of genetic material that were invented in the
1980s. Their molecular backbone makes them resistant to nucleases,
so they can sneak past bacteria’s defenses against foreign
DNA, and the sequence of each PMO is custom-designed to
interfere with mRNA expression by a particular gene. Geller’s
group bonded the PMOs to membrane-penetrating peptides to
enhance entry into bacteria. The resultant peptide-conjugated
PMOs (PPMOs) are “the ultimate narrow-spectrum therapeutic,
because they’re species- and gene-specific,” Geller says.
Geller has designed PPMOs to target a variety of bacterial
genes, including acpP, a gene required for lipid biosynthesis. “If
you knock it out, it’s a lethal event,” he says. Sure enough, when
treated with acpP-specific PPMOs, mice infected with multidrug-
resistant Acinetobacter baumannii survived for at least
one week, while control mice died, most within a day (J Infectious
Diseases, 208:1553-60, 2013).
Geller and the other researchers hope their work will one
day bear fruit in the clinic, but for now, such new drugs emerging
from old discoveries remain merely a preclinical glimmer
of hope, with many years of work ahead before medicine gets a
desperately needed novel class of antimicrobials. “The scientific
difficulty [of developing a new antibiotic] is not to be underestimated,”
says Coates. —Kerry Grens

IT TAKES TWO
Given the difficulties in bringing an entirely new class of antimicrobials
to market, some researchers are setting their sights on
what they see as a more readily attainable goal: to combine existing
drugs into more effective therapies. “Finding a brand-new
chemical scaffold that has all the wonderful chemical properties
of the [antibiotics] we have now is going to be extremely hard to
do,” says McMaster University chemical biologist Gerard Wright.
“All future antibiotics should be developed as combinations.”
To explore the potential of new combination therapies, microbiologist
Kim Lewis, director of the Antimicrobial Discovery Center
at Northeastern University in Boston, looked to one of the
seemingly failed ADEP antibiotic compounds, ADEP4. Bayer
Healthcare scientists discovered the drug in 2005 but later
dropped it after in vitro experiments showed that bacteria rapidly
developed resistance to it. But, like Sello, Lewis and his colleagues
thought that it just needed a little help. So they combined
ADEP4 with a conventional antibiotic, rifampicin, in the hopes
that the treatment would be effective—and stay effective—against
Staphylococcus aureus, which readily forms antibiotic-resistant
biofilms harboring dormant cells known as persisters. (See “Bacterial
Persisters,” The Scientist, January 2014.)
The therapy worked better than anyone had dared to hope:
while ADEP4 and rifampicin each reduced microbial populations
in vitro and in mice, administered together they obliterated the
bacteria (Nature, 503:365-70, 2013). “What we discovered unexpectedly
was that with the combination of this ADEP compound
with another antibiotic we got complete sterilization,” Lewis says.
Lewis and his colleagues don’t yet know the precise mechanism
of vulnerability that the ADEP4/rifampicin combination
exploited, but it likely involved ADEP4’s activation of the ClpP
protease. Triggering ClpP to degrade proteins nonspecifically in
persister cells within biofilms may have caused the breakdown of
hundreds of proteins, forcing the cells to self-digest, Lewis says.
While some bacteria could have evolved to lack functional ClpP
and therefore resist ADEP4’s strike, rifampicin, which inhibits
RNA polymerase, likely stepped in and killed those cells. “We have
to do some additional toxicity testing, but the goal is to move this
into clinical studies,” Lewis says.
Yanmin Hu, a medical microbiologist at St. George’s, University
of London, and director of research at Helperby Therapeutics,
also had recent success with a combination antibiotic therapy.
Hu and Helperby founder Coates used high-throughput screening
to identify HT61, a small antibiotic compound that exhibited
selective bactericidal activity against methicillin-susceptible S.
aureus (MSSA) and methicillin-resistant S. aureus (MRSA) by
depolarizing the bacterial cell membranes. “We thought, ‘OK, if
we combine our compound with existing antibiotics, let’s see what
we can get,’” Hu recalls. The result in vitro and in mouse models:
HT61 enhances the antimicrobial activities of traditional antibiotics,
especially aminoglycosides such as neomycin, gentamicin,
and chlorhexidine, against MSSA and MRSA (J Antimicrobial
Chemother, 68:374-84, 2012).
Hu says that the combination therapy likely worked so well—
far better than either antibiotic administered alone—because
HT61 was essentially punching holes in the membranes of nondividing
bacterial cells, allowing the aminoglycosides to flood in.
Used as a topical agent in combination with the antibiotic mupirocin,
HT61 has cleared Phase 1 and 2 trials for the treatment of
latent MRSA infections, Hu says. She and Coates have also identified
a plethora of other potential compounds that might serve
to enhance the effects of existing antibiotics. “We have about 300
similar compounds that show very good activity against persistent
organisms,” Hu says.
Antibiotics can also be combined with existing, nonantibiotic
drugs, as Wright is doing. In 2011, he and his colleagues
screened more than 1,000 approved drugs for compounds that
augmented the ability of the antibiotic minocycline to fight
infection. They identified a suite of promising nonantibiotic
drugs—for indications as diverse as Parkinson’s disease, irritable
bowel syndrome, cancer, and diarrhea—which, in combination
with minocycline, were able to fight infections of Pseudomonas
aeruginosa, E. coli, and S. aureus in vitro and in mice (Nat
Chem Biol, 7:348-50, 2011). “We’ve really missed a whole section
of antimicrobial target space,” says Wright, who adds that
he feels strongly that combination therapies are the best way to
tackle the antibiotic resistance threat. “The idea of a magic bullet
is gone. We need a magic shotgun.” —Bob Grant
The idea of a magic bullet is gone. We need
a magic shotgun.
—Gerard Wright, McMaster University

RESENSITIZING
BACTERIA
Rather than combining antibiotics with new compounds found
to have antibiotic activity, some researchers are looking to simply
add adjuvant compounds. Although adjuvants themselves are
unable to kill bacteria, when added to antibiotic regimens they
render resistant microbes susceptible once again.
“We are developing agents to sensitize bacteria to the agents
we already have,” says microbiologist Anders Hakansson of the
State University of New York at Buffalo, who in 2012 found that
treatment with a protein-lipid complex from human milk could
potentiate the effect of common antibiotics against drug-resistant
Streptococcus pneumoniae (PLOS ONE, 7:e43514, 2012).
From a financial standpoint, antibiotic adjuvants make sense.
Developing and validating a small-molecule sensitizer to be used
in conjunction with an existing antibiotic should cost far less than
developing and validating a completely new drug. The aim is “to
extend the utility and lifetime of existing antibiotics,” says biomedical
engineer James Collins of Boston University. “There is
still some activity, in some cases, of the antibiotic, it just doesn’t
get to the lethality threshold. The adjuvant allows one to shift
that threshold.”
One way microbes are evolving resistance to first-line antibiotics
is by blocking entry of the drug into the cell. Many gramnegative
bacteria pose the additional challenge of producing
􀁠-lactamase enzymes that block antibiotics containing a 􀁠-lactam
ring, such as penicillins, cephamycins, and some carbapenems,
from inhibiting bacterial cell-wall biosynthesis. And even if an
antibiotic is able to penetrate the bacterial cell wall and avoid
degradation by 􀁠-lactamases in the cytoplasm, the drug must
also fight against efflux pumps to stay inside the bacterium long
enough to kill the cell.
It’s a tall order, says Laura Piddock, a professor of microbiology
at the University of Birmingham, who leads the Antimicrobials
Research Group there. “These molecules have to not only get
through the outer [bacterial cell] membrane, they then have to
get past all these enzymes, and then they’re almost certainly going
to be pumped out,” she says. “These three things together make a
very, very tough challenge.”
But new adjuvant sensitizers can target any one of these bacterial
defenses—by damaging cell walls, inhibiting 􀁠-lactamase,
or stopping efflux pumps—and a handful of biotech companies
now have antibiotic adjuvants in their discovery and development
pipelines. For example, the Boston-based firm Collins
cofounded, EnBiotix, is working on potentiators such as silver
compounds that sensitize persistent bacteria to existing antibiotics
by increasing bacterial membrane permeability. Oklahoma
City-based Synereca is working to validate inhibitors of
the bacterial protein RecA, which plays a role in recombinational
DNA repair. And Venus Remedies in Chandigarh, India,
secured approval in a handful of countries last year to sell
Elores, a 􀁠-lactamase inhibitor combined with the antibiotic
adjuvant disodium edetate.
By and large, however, progress has been slow, limited in part
by the toxicity of these small molecules. “People are starting to
look [for antibiotic adjuvants],” says Hakansson, “but right now,
there’s not really a critical mass of molecules that really work”
without causing unacceptable side effects.
Nevertheless, he and others continue to search for new adjuvants
that could render increasingly useless antibiotics effective
once again. “Different drugs synergize with each other,” says
Hakansson, who envisions a future in which antibiotic-resistant
bacterial infections are treated much like HIV, with a cocktail of
drugs. Once identified and validated, adjuvant sensitizers could
be as common to pharmacy shelves as the antibiotics themselves.
—Tracy Vence

DISCOVERY ZONE
Since Alexander Fleming’s serendipitous 1928 observation that a
Penicillium fungus prevented growth of staphylococci bacteria,
the search for new antibiotics has largely been focused on fungi
and microbes living in the soil, in the hopes of discovering another
natural product with the broad effectiveness and low toxicity of
penicillin. But as more recent searches result in disappointment,
some investigators are turning to new sources—plants, insects,
and marine organisms—to find antibiotics that can kill our most
common and persistent pathogens.
When chemist Simon Gibbons of the University College London
School of Pharmacy went in search of plants harboring compounds
with antimicrobial properties in 2008, he paid particular
attention to those that have been used in traditional medicine—
especially for wound healing. “If a plant is used as a wound-healing
agent, it’s quite likely that it contains chemicals that kill the bacteria
in the wound,” he says. Although best known for its psychoactive
properties, cannabis, historically ingested in parts of Afghanistan
and India to treat infection, fit the bill. Gibbons and his colleagues
isolated five cannabinoids from Cannabis sativa and found that
each one was effective against MRSA (J Nat Prod, 71:1427-30,
2008). “It hasn’t been confirmed in vivo, but certainly in the lab,
we know that these things kill drug-resistant bacteria,” he says.
Gibbons has also found chemicals with antibiotic properties
in other familiar plant groups. For instance, plants in the Allium
genus, which includes garlic and onions, produce sulfur-containing
compounds that have activity against MRSA and Mycobacterium
(J Nat Prod, 72:360-65, 2009). And many of the hyperihypericums—
the family that includes St. John’s wort—make chemicals
called acylphloroglucinols that also effectively kill MRSA in vitro
(J Nat Prod, 75:336-43, 2012). “We’ve had leads . . . and a series of
compounds, which have been patented,” says Gibbons, and those
compounds are now being synthesized and modified to improve
their activity.
Andreas Vilcinskas of Justus Liebig University Giessen in Germany
is using another vast resource to identify novel antibacterial
compounds: insects. “Insects are considered the most successful
group of organisms in the world,” says Vilcinskas, who suspects
that one of the keys to their success is the ability to manage
microbes. And it’s likely, he adds, that different insects have different
strategies for protecting themselves against pathogens. “I’m
convinced that the biodiversity that you see at the species level is
also reflected at the molecular level.”
In 2012, he decided to home in on invasive insect species,
which he hypothesizes have a particularly strong immune system
to allow them to succeed in new environments. Harvesting
hemolymph from harlequin ladybird beetles (Harmonia axyridis),
which have successfully outcompeted native beetles the
world over, Vilcinskas discovered more than 50 novel antimicrobial
compounds. One compound, called harmonine, demonstrated
activity against both Mycobacterium tuberculosis and
MRSA (Biology Letters, 8:308-11, 2012), and Vilcinskas’s group
is now making chemical modifications to harmonine and other
compounds to produce even more potent antibiotics.
Other researchers, such as William Fenical of the Scripps
Institution of Oceanography in San Diego, California, have
moved the quest for antibiotics away from terrestrial environments
entirely. From offshore shallows to depths of nearly 6,000
meters (more than 19,000 feet), Fenical and his colleagues collect
ocean-floor samples, then culture the microorganisms contained
within and test the compounds they produce against antibiotic-
resistant microbes such as MRSA. “Seventy percent of the
Earth is the ocean,” Fenical says. “We feel the ocean has enormous
potential.”
Last year, the group detailed its discovery of a unique antibiotic
made by a species of Streptomyces bacteria isolated from
marine sediments off the coast of Santa Barbara. They named
the compound anthracimycin because of its high activity against
the potential bioterrorism agent Bacillus anthracis, but the compound
also demonstrated inhibition of MRSA in nutrient broth
assays (Angew Chem Int Ed, 52:7822-24, 2013).
Moving from the lab to the clinic is not trivial, however, Fenical
notes. Improving the compound’s solubility and activity, lowering
its toxicity, and scaling up its production can all present challenges.
And in such early stages, the impact of these discoveries
on the problem of antibiotic resistance remains to be seen. “To be
completely honest, the jury’s out,” says McMaster’s Wright, whose
group has explored compounds made by microbes found in an isolated
Mexican cave and in a Cuban mangrove forest. Nevertheless,
given the diversity of natural products now being discovered, he
adds, “it’s certainly worthwhile exploring.” —Abby Olena


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