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THE THREAT THAT NEVER SLEEPS: CAN SCIENCE STOP SUPERBUGS?

Traditional antibiotics are losing the battle with bacteria and Michigan engineers are stepping into the breach.| Long Read

They never released the woman’s name. News articles and government reports that came out in early 2017, months after her death, referred to her as “a Northern Nevada woman,” “a female Washoe County resident,” or something similarly vague.

Her killer, however, they didn’t miss that: Carbapenem-resistant Enterobacteriaceae.

Parse through those vowels and you’ll dig out the reason for the interest: a drug-resistant strain of bacteria. In this case, it proved to be something particularly tenacious.

Doctors in the United States had 26 approved antibiotics available to treat infections in their patients.

The bacteria that killed the unnamed Nevada woman were resistant to every one of them.

Health officials around the globe are tracking an alarming rise in cases of bacteria that no longer respond to treatments with antibiotics – the go-to remedy for infections since the mid-20th century. Common infections that today we brush aside with a vial of pills are increasingly overwhelming the treatments. And projections on where we’re headed are staggering.

In 2017, an estimated 700,000 people died from drug-resistant bugs. By the year 2050, that number could rise as high as 10 million in what Britain’s top medical officer described as a “post-antibiotic apocalypse.”

An obvious savior from such doom and gloom would be a new class of antibiotics – drugs bacteria have never encountered and have not mutated to resist.

But the pipeline has been nearly empty for years.

Major pharmaceutical manufacturers are far more interested in drugs that consumers take regularly – think antidepressants, erectile dysfunction pills or diabetes medications. The payoff on products patients take continually goes far beyond what a company would make developing and marketing antibiotics that are taken for a week or, at the most, a few months.

Big Pharma has basically given up on antibiotics.

A molecular biologist and former pharmaceutical company official wrote in Forbes last year: “Big Pharma has basically given up on antibiotics. It’s not that the risks are too high; it is that the rewards are too low.”

Engineers, including researchers at the University of Michigan, have stepped into the chasm between what we have and what we need. Work underway in labs across North Campus represents several new fronts in the fight, including killer nanomaterials and antibiotic combinations that mimic the immune system.

But we’re going to start this story elsewhere: with a U-M guy on the bottom of the Red Sea.

SEEKING SERENDIPITY

EnlargeDavid Sherman stands with his hands in his pockets
IMAGE:  David Sherman scours the waters of the world in search of organic material that could lead to new classes of antibiotics. It's a practice that has been largely abandoned by the pharmaceutical industry.

The two-sentence version of one of the most important scientific discoveries in history goes like this: penicillin, the first modern-day antibiotic, was discovered by accident. A Scottish scientist found mold growing in untended petri dishes that turned out to have bacteria-curbing properties.

A game-changing infection killer derived from nature – it’s exactly what David Sherman searches for on diving trips around the globe. The Hans W. Valtech Professor of Medicinal Chemistry in the College of Pharmacy is often found with a tank strapped to his back, eyes behind a facemask, scouring the seabed for organic material that could lead to the next class of antibiotics.

“There is so much opportunity to find new things because there has been very little exploration,” said Sherman, who grew up in Minneapolis, far from the warmer waters he likes to work in.

In lieu of seeking natural keys to new antibiotics, large pharmaceutical companies tinker with existing ones. By altering the chemical makeup of an antibiotic that is no longer effective against certain bacteria, chemists can jumpstart its killing power and create something new.

“Companies were investing in finding new materials up until 20 years ago,” Sherman said. “Then a new technology – combinatorial chemistry – came along. All of a sudden, robots started making millions of compounds very simply and very inexpensively – all based on known structural entities.”

But many of the old antibiotics, as well as their reconfigured upgrades, target similar weak points or processes in bacteria. And minor changes in an antibiotic’s makeup, according to a U-M biomedical engineer you’ll meet later, create minor new hurdles for bacteria to overcome on their way to drug-resistance.

In the lower levels of U-M’s Life Sciences Institute, Sherman houses the fruits of his underwater endeavors – a library of microorganisms he and his team have pulled from marine sediments around the globe.

They represent hope for a new antibacterial M.O.

“What we’re trying to do is actually identify new antibiotics that somehow target either a brand new part of a pathogen’s machinery, or bind to a new part of an old target,” Sherman said. “It’s a wide open area, and I think we’ve only really explored a small number of the potential effective targets.”

While Sherman investigates what can be found in nature, U-M engineers using nanotechnology are creating a new class of antibiotics – composed of materials hundreds of times smaller than bacterial cells – that are tailor-made to exploit new targets.

CRAFTING KILLERS

EnlargeNicholas Kotov, Angela Violi, and Scott VanEpps stand together in the lab
IMAGE:  Nicholas Kotov, Angela Violi, and Scott VanEpps are crafting and deploying nanoparticles - dubbed nanobiotics - to interrupt the inner processes that keep bacteria alive. Identifying weak spots in a bacterium's cell walls and shaping nanoparticles to take advantage of them is the equivalent of designing a key to fit a specific lock.

A high-stakes version of Tetris is underway on North Campus, played by a chemical engineer, a mechanical engineer and a biomedical engineer who happens to be an emergency room doctor.

And they’re cheating.

When an antibiotic does its killing work, it essentially shuts down a process the targeted bacterium needs to survive. That could be the ability to build cell walls or to generate proteins capable of turning food into energy.

Now, go back to Tetris and its falling puzzle pieces. If you could change the shapes of those pieces, it would be simple to fit them into a neat grid below and endless high scores would follow.

The U-M trio is using nanobiotics as their “cheat code.”

“Nanobiotics,” a riff on nanoparticles and antibiotics, uses a particle’s shape, size and chemistry to interrupt a bacterium’s survival processes. Endless configurations and sizes are possible with current technology, creating new pathways for the nanoparticles to insert themselves into those key processes. Once it’s there, the nanobiotic effectively disrupts and shuts down that process, causing the bacterium’s death – even where there is resistance to traditional antibiotics.

Nicholas Kotov, the Joseph B. and Florence V. Cejka Professor of Chemical Engineering, makes the particles in his lab.

“These are pieces of inorganic material, a few nanometers in size,” he said. “What we’ve learned along the way is how to use the biomimetic natural design of nanoparticles and attribute to them some biological functions.”

Angela Violi, a professor of mechanical engineering, as well as chemical engineering and biophysics, lays out the pathway for Kotov’s work via computer simulations. She probes the defenses of cell walls, known as the lipid bilayer, to find the best paths for a manufactured nanoparticle to find its way in.

“We try to change the size, shape and chemical composition of the particle to control how it interacts with a target’s biological systems and interferes with its processes,” she said. “Then the question becomes: can we fine-tune the design enough to achieve our goal?”

In one example, the team fashioned nanoparticles into pyramid shapes with long points. Those pointed ends interact with bacterial enzymes.

“Those enzymes also have shapes,” Kotov said. “Some have a hole in them, or grooves. Their geometry fits well with the sharp apexes of our nanoparticles.”

Bacteria, however, don’t like to leave themselves vulnerable. They dig in, setting up defenses. They’ll bunch and adhere to any surface they can find in the body. And it’s easier for them to hang on to a medical implant than living tissue.

In this strength-in-numbers approach, the bacteria grow in layers and produce a protective gel as a barrier between themselves and immune cells. That gel also keeps antibiotics at bay.

“Even if you assumed a perfect world going forward, one where you weren’t seeing this increase in resistance to antibiotics, biofilms would still be a major problem because regular antibiotics don’t work on them,” said Scott VanEpps, a biomedical engineer and ER doctor at Michigan Medicine. “But in reality, the mechanisms inside biofilms foster the development of antibiotic resistance because you have bacteria in close proximity transferring genes.”

VanEpps takes the materials provided by Kotov’s lab and tests them to see not only if they work, but how. He has seen first hand what drug-resistant bacteria can do. For patients, it can create a painful cycle of surgeries to implant devices, remove them once they cause an infection, and replace them with new ones. “Ultimately, taking the devices out of people, that winds up being the solution,” he said. “I can’t kill the bacteria infecting the device because it’s in a biofilm. So it has to come out, often repeatedly.”

Other nanoparticles can be designed to attack biofilms specifically. Graphene, a single layer of carbon atoms Kotov describes as “chicken wire,” can be designed at the microscopic level, two to five nanometers in size.

“We can coat the edges of the particles with some chemistry and it turns out these graphene particles have a different type of activity in connection with bacteria,” Kotov said. “It turns out they can prevent and destroy the formation of biofilms.”

That discovery has led the research team to wield nanobiotics in a different way – preventing the formation of biofilms in the first place. By coating medical implants in graphene nanoparticles prior to implantation, researchers are arming them with bacteria repellant that could block infections from taking hold.

But nanobiotics have years of experimentation and clinical trials ahead of them. New options for challenging drug resistant bacteria are needed now. And for that, our own immune systems may show the way.

MULTI-PRONGED ATTACKS

EnlargeSriram Chandrasekaran faces the camera with an open computer screen behind him
IMAGE:  Sriram Chandrasekaran uses the blueprint provided by bacteria-killing T-cells and the study of proteomics to design drugs and treatments to combat drug resistance. Knowing which proteins the body naturally targets informs how new drugs or new combinations should work to be most effective.

Cricket didn’t bring Sriram Chandrasekaran fame as a bowler or batsman the way he imagined growing up in Chennai, along India’s southeastern coast. Yet somewhere, in the sport’s ranking systems and statistical analyses, it still nudged him toward his future.

Early on, cricket’s numbers – particularly odds, probabilities and averages – spoke to Chandrasekaran in a way other youthful interests did not. Now an assistant professor of biomedical engineering, the numbers tell a different story today – hinting at strategies in a struggle more important than anything played on the pitch.

In his North Campus lab, Chandrasekaran and his team are harnessing numbers via the large-scale measurement of proteins – a field given the name proteomics two decades ago. Their work takes place not under a microscope, but on the backs of microprocessors.

Computer simulations predict the impact of changing protein levels in cells before and after stimuli are introduced. Applied to bacteria, these simulations can identify which proteins to disable if you want to kill the cell.

Chandrasekaran’s early work in the arena provided our closest look to date at how the body’s defenders – killer T-cells – target and destroy bacteria. When the immune system recognizes the presence of harmful bacteria, killer T-cells deploy the protein perforin, which opens up holes in the bacterium’s protective membrane. With that door opened, Chandrasekaran’s team found that T-cells simultaneously attack multiple processes in the bacteria with protein-degrading enzymes to kill it.

That contrasts with how antibiotics work. For example, amoxicillin, among the most widely-used antibiotics on the market, halts a single process – the bacteria’s formation of cell walls.

Mirroring the body’s approach, Chandrasekaran said, could aid the battle against drug-resistant bacteria. His proteomics research creates a roadmap for combining drugs already on the market in a regimen that recreates the multi-pronged approach of T-cells.

But roadmaps, while a help, do little to shorten the journey by themselves. And in many ways, researching treatments for drug-resistant bacteria is a race against time.

Technological advances within the last decade have accelerated what researchers like Chandrasekaran can accomplish. And that’s key because they face a mountain of variables in their search for better drug formulations.

“Twenty years back, researchers normally measured just one protein at a time,” Chandrasekaran said. “You would see papers come out saying, ‘We measured the level of Protein X in something like E.coli, and we observed its levels change over time.’ That would be a whole study in itself.”

“Because E. coli has something like 4,000 proteins, just measuring one protein doesn’t tell us much – doesn’t give us the big picture.”

In recent years, proteomics technology and improved computational methods have allowed for this kind of deep dive. The experimental tech identifies what proteins T-cells target in the bacteria and the computer modeling helps show why specific proteins are attacked and what the outcome is.

And the data generated creates all kinds of possibilities.

“We can now expose different bacteria to immune enzymes in simulations and track what proteins the enzymes go after,” Chandrasekaran said. “This gives us a huge amount of data to work with and it’s allowing us to develop computer models of the bacteria before and after T-cells attack.

“When data shows that the enzyme from T-cells blocks a specific protein, I should be able to predict what happens to the cell.”

That includes being able to simulate how the enzymes impact key cell processes bacteria need to survive. Chandrasekaran likens the work to using route-finding software. Ask those apps to get you home and they’ll sort through all of the possible routes to settle on the one most likely to get you there the quickest.

With drug-resistance treatments as the destination, sorting through the options via conventional means of trial and error under the microscope is daunting. Computer simulations point Chandrasekaran’s team in the right direction.

“The system of equations we’ve built mirrors Google Maps; we create a map of how all the proteins within a bacterium interact with each other,” he said. “This can tell us if the bacteria has a backup option when a protein is blocked, which they usually do for important proteins.

“So when a drug or enzyme blocks the backup protein, I can now say confidently that blocking that protein is a way to slow the bacteria down or, possibly, kill it.”

While Chandrasekaran brings this approach to fighting bacteria in general, one ancient bug is already getting the multi-pronged treatment. And that research may be giving us a look at the future of fighting bacterial infections.

VETERAN OF THE DRUG WARS

They’ll tell you tuberculosis can be harmless. On initial infection, it may not produce symptoms at all, or it can remain in the body for years in its latent form.

But its innate tenacity has put it ahead of the game in drug resistance, requiring combinations of antibiotics for treatment as a matter of course.

And when it kicks into its highest destructive gear, it still demonstrates the ferocity of earlier centuries when it was referred to as “captain of the men of death.” In 2006, the disease’s power was on full display in the small South African town of Tugela Ferry.

An “extensively drug-resistant tuberculosis” (XDR-TB) took hold among the population of roughly 3,000. Early on, the local hospital reported 53 cases. All but one died.

A year later, 314 cases had been reported, eventually resulting in 215 deaths.

Tugela Ferry’s region of South Africa is essentially the backyard of Elsje Pienaar’s youth – a long way from her current life in America’s Midwest. Her academic path wound from a university in Pretoria to a research team headed by Jennifer Linderman, a professor of chemical engineering.

Linderman has spent years examining cell behavior and internal processes such as diffusion, the movement of particles in the body, and chemical kinetics, with a particular interest in immunology. With collaborator Denise Kirschner, a Michigan Medicine researcher in microbiology and immunity, the team has painstakingly crafted a computer simulation of the disease as a means of studying it and, it is hoped, finding new ways to treat it.

With the introduction of tuberculosis, Linderman said, a “battle” begins in the body. One of the hallmarks of that confrontation is the creation of granulomas – dense groupings of immune cells surrounding the bacteria to protect the host. But they also protect the bacteria.

EnlargeClose up of Jennifer Linderman
IMAGE:  Jennifer Linderman's team utilizes highly-detailed computer simulations to wade through the "zillions" of drug combinations and treatment variables that can impact the progression of tuberculosis. In recent years, the team has focused on TB's drug-resistant forms.

“In some cases, the body’s immune system can eliminate the bacteria there completely, sterilizing the granuloma,” Linderman said. “That’s if you’re lucky.

“If you’re not so lucky, the bacteria are growing and dividing and the immune system is fighting them, keeping the levels of bacteria low and in check within the granulomas. Or worse, the immune system is not able to keep the bacteria in control and you develop active tuberculosis.”

That constant battle in the body has been a focus of Linderman’s research group in recent years. Quickly, basic questions came to the fore, like: Why doesn’t the immune system simply wipe out every last bacterium? Which cells and molecules of the immune system are most important to keeping the bacteria in check?

To get to the heart of such questions, the team developed computer simulations of the immune response. A powerful resource for such work lies in data collection, and Linderman uses everything she can get her hands on.

Studies of how bacteria behave in a petri dish? Got it.

Animal studies, from mouse to macaque, of tuberculosis progression? Check.

In fact, the team collaborates with JoAnne Flynn, a professor of microbiology and molecular genetics at the University of Pittsburgh, and Veronique Dartois of the Public Health Research Institute at Rutgers University, who examine the disease in animals.

“We were developing simulations where we’d take a piece of lung tissue and start with an infected cell, or infected macrophage and see the bacteria dividing,” Linderman said. “We could watch a granuloma grow in simulation.

“Then you start working with variables. What if the immune system came in later? What if the bug didn’t divide as well? You can play around with all of the different parameters.”

With a detailed simulation of tuberculosis in hand, Linderman’s group put together simulations of two antibiotics – isoniazid and rifampin – which are among the most widely used to treat the disease.

And most recently, the team has factored in drug resistance to their tuberculosis simulations.

What if this one bacterium mutates so it’s no longer susceptible to a drug or less susceptible to a drug?

“Now that we’ve built a model that has the bacteria and the antibiotics in it, we can start asking ‘What if this one bacterium mutates so it’s no longer susceptible to a drug or less susceptible to a drug?’” said Pienaar, who continues to collaborate with the team after taking a post at Purdue University last year. “We can change the bacteria’s susceptibility to the drug and its growth rate.”

The work has given researchers a glimpse of tantalizing treatment possibilities, as well as an idea of the Herculean task ahead.

“The problem is that when you treat tuberculosis, you use combinations of drugs,” Linderman said. “Let’s say there were 15 drugs that could be of use. Each can be given in multiple ways – once a week, twice a week, over a month or several months, or in multiple concentrations.

“We’re talking zillions of combinations. And you cannot test them all. It’s impossible to test all of the possible drug combinations, dosage combinations and regimens – from animal models through to human testing.”

Even high-powered computer simulations can’t slog through every potential combination. But they can point researchers in promising directions – toward the drugs and drug combinations that are most likely to produce results.

It’s the general area of the haystack to look in to find that needle.

“Using these simulations, we can predict which direction we should be moving in,” Linderman said. “We’re not only looking at existing therapies, including immunotherapy options, but we’re thinking what drugs – new, repurposed or existing – could be used in different combinations that might be effective.”

There’s a sad familiarity to many of the conferences Pienaar attends on drug resistance. Inevitably, a presenter will pull up a heat map of areas around where resistance crops up most often.

South Africa and its surrounding countries always stand out.

“It’s a huge problem back home,” she said. “That’s why I kind of can’t let it go.”

THE ROAD AHEAD

The threat of the problem at hand – and its truly global implications – has some of the best minds pivoting from other areas and bringing their talents to bear on antibiotic resistance. Angela Violi’s previous work centered on the chemistry of combustion. Scott VanEpps was, at one point, heading down the path of vascular biomechanics. And Sriram Chandrasekaran’s work on proteins could have led him anywhere.

I am always attracted by intelligence in nature, and bacteria have clearly shown they can outsmart humans.

“I am always attracted by intelligence in nature, and bacteria have clearly shown they can outsmart humans,” Violi said. “They are tiny and yet they understand that if they work in unison, they can launch attacks we can’t stop.

“If this work is successful, we can make a real difference and impact lives.”

From his doctor’s vantage point, VanEpps sees that the research being done at U-M is a vital cog in what will need to be a much larger machine.

“I can’t expect Congress to go up to a drug-maker like Merck or GlaxoSmithKline and order them to start developing and making new antibiotics,” he said. “There needs to be a diverse portfolio approach.”

A real solution, he said, lies in a convergence between philanthropic groups, university support and private investment.

It’s a sentiment echoed by undersea explorer and medicinal chemist David Sherman.

“There is a lot of discovery underway that is seeing promising success,” he said. “The next phase is the really hard part – getting these discoveries to a point where we can decide if a big investment in the next step is worthwhile.”

David Sherman stands with his hands in his pockets
Nicholas Kotov, Angela Violi, and Scott VanEpps stand together in the lab
Sriram Chandrasekaran faces the camera with an open computer screen behind him
Close up of Jennifer Linderman
Portrait of Jim Lynch.

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