Squashing Superbugs

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It’s like calling the fire department, police department and a nearby town’s fire department.John Foster

Medical professionals and researchers warn that hospitals are becoming hotbeds for antibiotic-resistant bacteria, and without action, mortality rates could rise to early-20th century levels or higher. Michigan engineers are taking action – exploring approaches that can prevent bacteria from gaining a foothold or help antibiotics work more effectively.

While antibiotics stop bacteria in a variety of ways, no method is foolproof. Bacteria can pick up drug resistance from one another, from genes that happen to be loose in their environment, and from viruses that infect them. There might be relatively few drug-resistant bacteria in an initial infection, but after antibiotics have decimated the population, only the resistant bacteria are around to reproduce. If the body’s immune system can’t pick up the slack on those resistant types, the disease becomes difficult – if not impossible – to treat.

But antibiotics aren’t the only tool in the box. Antiseptics kill bacteria on skin and in wounds, and sterilization destroys them on medical equipment. If bad bacteria get into the body, then antibiotics usually give the immune system the edge it needs to clear the infection. The work of Michigan engineers could impact each of these three tactics.

A collaboration of nuclear engineers and chemists is investigating the effects of using an exotic state of matter to kill bacteria and help heal wounds, and a team of chemical engineers is developing a method for producing custom molecules that could make medical equipment inhospitable to bacteria. Finally, a joint project between chemical engineers and medical researchers explores not how to kill a single bacterium, but how to conquer a colony.

The ultimate antiseptic?

Immaculate standards for surgical cleanliness allow antibiotics and the patients’ immune systems to handle bacteria that get into most surgical sites, but with multi-antibiotic-resistant strains on the rise, doctors are interested in methods that could completely sterilize wounds.

While chemical antiseptics help kill bacteria, concentrations that totally eradicate them could seriously aggravate the already-damaged tissue. But in the last few years, a new antiseptic has emerged that is much gentler toward mammalian tissue, annihilating the bacteria while accelerating the healing of the wound. It’s not a liquid, gel or paste – it’s a plasma.

Plasma is a glowing gas containing free electrons, charged atoms and atoms with excited electrons. In nature, plasmas typically exist in extremely hot environments, such as a bolt of lightning or the sun. The group of John Foster, an associate professor of Nuclear Engineering and Radiological Sciences, specializes in making plasma by giving smaller amounts of energy directly to the electrons, avoiding temperatures that could vaporize human flesh.

“Those free radicals always attack something, break something up,” said Raoul Kopelman, the Richard Smalley Distinguished University Professor of Chemistry, Physics, Biophysics, Biomedical Engineering and Applied Physics, and Foster’s collaborator. “There is a good chance it will get rid of bacteria.”

NERS Professor John Foster demonstrates a plasma beam. A pair of high-voltage electrodes turn air into free radicals that can help heal cells by killing bacteria or other potentially harmful cells such as cancer. The metal in the pen attracts the charged gas.

Photo: Joseph Xu, Michigan Engineering Communications & Marketing

Plasma-produced radicals are thought to be beneficial for healing because, in addition to breaking up the cell membranes of bacteria, the nitrogen oxide echoes the body’s own call for repairs.

“It’s like calling the fire department, police department and a nearby town’s fire department,” said Foster. “This precursor radical essentially initiates the healing response of the body.”

Studies have shown that plasma encourages mammalian cells to grow and spread, and nitrogen oxide plays an important role in guiding the formation of new blood vessels to feed the regenerating tissue. Still, the details of what plasmas do to cells are largely unexplored. To discover these effects, Foster’s team is bringing their plasma beam to Kopelman’s lab.

The collaboration is starting by applying plasma to cancerous human cells since they tend to be hardier than healthy cells. Foster’s team makes the plasma by flowing helium through a quartz tube with one electrode running through the center. The other electrode coils around the end of the tube, creating a powerful electric field when the electrodes are charged up to about 2,000 volts.

The brief, high-voltage pulses from the electrodes accelerate the few loose electrons in the gas so that they ram helium atoms with enough energy to free more electrons. Those electrons then strike other atoms, and so on. This process also boosts electrons in the atoms into energetic states, causing the plasma to glow as these atoms release the energy as light.

When the avalanche of electrons reaches the air, it creates those reactive molecules in a blue-white shaft of captive lightning. Unlike the plasma of natural lightning, it is at room temperature – only the free electrons are hot, and they’re too small in mass and in number to make much difference. “You could put your hand in it,” said Foster.

But you wouldn’t want to leave your hand in there for long. At higher doses, the plasma coaxes cells into undergoing programmed cell death, the body’s way of getting rid of unwanted cells without inflaming the surrounding tissue. Other researchers have used plasma to remove tumors by killing off the cells. Foster's and Kopelman’s work may clarify where the line between tissue healing and cell death lies for human cells exposed to plasma.

Their first experiments, in April, measured changes in the cells’ internal acidities. Preliminary results suggest that plasma pushes the cells toward higher acidities, which may affect their growth. Kopelman and Foster intend to measure the pH levels within cell compartments and study the concentrations of reactive molecules containing oxygen. With this information, they can begin to understand the mechanisms that allow low doses of plasma to kill bacteria while healing mammalian tissue.

Although plasma seems to be good for sterilizing wounds, it’s not suitable for maintaining bacteria-free surfaces. That might be a job for frog-inspired coatings.

Killer coatings

If you synthesize peptides chemically, just one of them may cost a couple thousand dollars… So we use nature to do the legwork.Erdogan Gulari

Tubes, catheters and other artificial points of access into patients’ bodies allow the direct delivery of drugs and nutrients, help with breathing and more, but these conduits also pose a risk. If bacteria can get into them, they have a shortcut past the patient’s defenses. Erdogan Gulari, the Donald L. Katz Collegiate Professor of Chemical Engineering, believes that these devices could be made to fight off the bacteria with a coating of antimicrobial molecules.

When humans get sick, we usually absorb viruses or bacteria through mucus membranes, such as the moist tissues of our eyes, noses and mouths. These sites are reasonably protected, but a frog’s skin is like one big mucus membrane. So why aren’t they sick all the time?

It turns out that frogs produce a huge range of microbe-killing peptides, which are small protein-like molecules, on their skins. These peptides open holes in the cell membrane of a bacterium, killing them. Humans use antimicrobial peptides as well – some immune cells that swallow bacterial invaders slay them with peptides.

Gulari’s team is developing a way to tweak natural peptide designs to fight target microbes more effectively. Because designing peptides is still a trial-and-error affair, this means producing and testing thousands to millions of them.

Saadet Albayrak examines a test plate to see which peptides have effectively eliminated bacteria. Albayrak and Professor Erdogan Gulari have been studying with peptides for fighting bacteria, either for coatings that keep surfaces sterile or as alternatives to antibiotics.

Photo: Joseph Xu, Michigan Engineering Communications & Marketing

“If you synthesize peptides chemically, just one of them may cost a couple thousand dollars, whereas if you use a yeast or E. coli cell to synthesize them, you can get tens of thousands of them at almost no cost,” said Gulari. “So we use nature to do the legwork.”

They started with the peptide Plantaricin-423, which is harmless to humans but capable of killing the food-borne pathogen Listeria. Peptides and proteins are strings of molecules called amino acids, and Plantaricin-423 contains 37 of them. The first 18 bind the peptide to the target cell membrane. Since Plantaricin-423 already attaches to Listeria but not human cells, Gulari’s team left those alone.

To make the peptide deadlier, they changed the 19-amino-acid hole-poking section. A computer program randomly assigned one of six amino acids to each spot, with the option to shorten the chain by one or two amino acids. That resulted in about 12,000 different peptides. They then made DNA blueprints for each of these designs and slipped them into E. coli cells, where the bacteria’s machinery could begin churning out peptides.

“We can do large petri dishes of 4,000 or 5,000 colonies, so that’s 4,000 or 5,000 different peptides being produced,” said Saadet Albayrak Guralp, a research associate in Gulari’s lab. With those production levels, she isn’t fazed by the idea of testing hundreds of thousands of peptides, up to around a million.

To find out how well the peptides worked, the team grew the Listeria on the same petri dish. The laboratory E. coli bacteria were designed with leaky cell membranes, so the peptides escaped into the petri dish, creating Listeria-free zones around the E. coli colonies.

The sizes of those zones gave Gulari’s team a general idea of how well the peptide discouraged the growth of the Listeria, but that measure is dependent on how well the E. coli hosts produce the peptides. The group isolated the most promising peptides to find out how potent they were at killing the Listeria. It turned out that the best could keep the bacteria at bay with half the concentration of the original Plantaricin-423.

This first study was stage one of finding the best Plantaricin-inspired anti-Listeria peptide, Albayrak explained. “As soon as we start seeing a pattern of increased activity based on mutations, then we can really focus on that part [of the peptide],” she said. “It’s like screening and designing.”

She and Gulari hope that the method may also uncover peptides that can fight off other hospital bugs such as staph and those that cause pneumonia in patients on ventilators.

But no preventative method is completely successful, so doctors will continue to deploy antibiotics to fight infections. Designer peptides may become new drugs as well if they can be coated so that the body doesn’t break them down before they reach the infection. But new antibiotics aren’t the only options – another team is exploring ways to make the current antibiotics work better.

Divide and Conquer

With even the most skilled surgeon and fastidious care, one to two percent of devices that are implanted will become infected...it’s just going to happen.John Younger

“Bacteria are survivors,” said Michael Solomon, a professor of Chemical Engineering. Fossil evidence suggests that they showed up on Earth about 3.5 billion years ago in the acidic oceans of a planet still seething with volcanic eruptions. They soon defended themselves from the harsh environment by banding together and surrounding themselves with a protective gel. Carpets of bacteria are thought to have spread over the ocean floor and quietly ruled the world for a couple billion years until burrowing animals evolved and began disrupting the seabed.

The empire of microbial mats has long since fallen, but bacteria in smaller collectives, including those inside the human body, did not forget how to leverage the strength of the colony. And the strategy is particularly successful on implanted devices, such as artificial joints or heart valves.

“Bacteria contaminating the surfaces of medical devices like being hunkered down in communities, and living in those communities gives them advantages over bacteria living in isolation,” said John Younger, a professor and associate chair for research in Emergency Medicine. Those benefits include the option to distribute metabolic activity so that more bacteria survive on less food, the ability to communicate and even exchange genes with their neighbors, a barrier that keeps antibiotics and patient immune defenses from reaching the inner layers of the colony, and importantly, a way to tether themselves to surfaces.

These advantages may seem obvious now, but the colony aspects of bacterial infections are only recently coming to the fore in medicine. “Ironically, the father of microbiology planted a false assumption about single-celled life that persisted for almost 400 years, namely that single-celled organisms prefer to live as single cells,” said Younger. “They don’t.”

Bacterial communities in the body typically take the form of thin films, about a tenth to a twentieth of a millimeter thick, known as biofilms. Researchers have thoroughly explored the biology and chemistry of these films, but Solomon and Younger are looking into how they behave as materials. Their ultimate aim is to use the mechanical properties of biofilms to fight bacteria that have colonized implanted medical devices.

“With even the most skilled surgeon and fastidious care, one to two percent of devices that are implanted will become infected,” said Younger. “That’s everything from artificial knees to pacemakers to shunts in the central nervous system – it’s just going to happen.”

An infection around an implanted device often doesn’t respond well to antibiotics, so clearing bacteria typically requires another surgery to take out the old device.

“With some medical devices, there’s only a certain number of times you can insert them,” said Solomon, so one less surgery can make a huge difference to patients.

Students in the Solomon and Younger groups rigged up a device for growing bacteria in a rheometer, a machine that measures properties such as how elastic a material is or how much you can push on a gel before its structure breaks up. They grow bacterial strains similar to what Younger sees in the emergency room, carefully controlling the environment so that it is a close mirror of infection sites – a catheter in the bloodstream, for instance.

Because the colony is grown right on the measurement device, they avoid disturbing the bacteria by relocating them. And it’s a good thing they took that precaution. They’ve found that the bacteria react to all kinds of stress – physical prodding or chemical attacks – by producing more of that protective gel. The higher gel concentration makes the film harder to break up.

The team has also characterized bacterial gels on the microscopic and molecular levels. The gel is made of strings of sugar molecules, called polysaccharides. Bacteria do much of their biofilm building with one type of sugar molecule, but they can create chains of various lengths and adorn them with different chemicals that serve different purposes such as promoting strong adhesion to artificial surfaces and blocking attacks by human white blood cells. They may even be using proteins in a patient’s own blood to help link up the polymer chains, firming up the gel.

Younger and Solomon are just beginning to venture from taking basic measurements to applying that knowledge, finding ways to break up biofilms in the body. They are keeping their ideas under wraps as they continue the research, but the general plan is to find ways to weaken the biofilms so that they are easier for the body’s internal forces to break up. Perhaps with the right strategies to loosen the biofilm, the squeeze of heart muscles around infected pacemaker electrodes could disperse the bacterial community without the need for another surgery.

Superbugs are forcing us to up our game in the fight against bacterial infections, but Michigan Engineers and their collaborators are rising to the challenge. Their work helps to ensure that the dire predictions for hospital mortality rates won’t come to pass – rather, doctors may have a better arsenal for clearing up infections and protecting their patients against harmful bacteria.