How do scientists kill the bacteria they themselves made resistant?

How do scientists kill the bacteria they themselves made resistant?

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I was reading this article on researching bacteria resistance to silver by removing some of their genes.

Researchers then used "colony-scoring" software to measure the differences in growth and size of each plate's bacterial colony. E. coli strains with genes deleted involved in producing sensitivity, or toxicity, to silver grew larger colonies. Strains with genes deleted involved with resistance grew smaller colonies.

Once you end up with some resistant bacteria and you're done researching it, you can't just flush it down the toilet. How do you safely dispose those colony plates in a way that ensures those bacteria don't get out into the wild and reproduce?

You are absolutely right, flushing down the toilet (or the sink) or simply throwing them into the normal waste doesn't work for biosafety reasons. And it is also not allowed, depending on the country you would do this in, this can lead to hefty fines.

Biologically contaminated lab waste can be inactivated (=all potential dangerous organisms are destroyed) by two ways: Either by heat or chemically. Which ways is used, depends on the kind of waste.

The most commonly used way is autoclaving, meaning treating the waste with steam at high temperatures at higher pressure. The temperature used here is usually 121°C, the exposure time depends on the volume of the waste, since the temperature needs to be reached and kept for at least 20 minutes. See the references for more details.

Liquid wastes (like culture media) can also be inactivated chemically by adding chlorine bleach to decompose the cells. Bleach can also be used to decontaminate surfaces, although here more often alcoholic solutions (70% Ethanol or Isopropanol) are used. After chemical inactivation, the remaining solutions should not be autoclaved as the emerging fumes are either unhealthy (bleach) or explosive (alcoholic solutions) and this is unnecessary, too. Liquid wastes can also be autoclaved to inactivate them.

Autoclaving has the main advantage that it is rather simple (put the waste into the autoclave, close it and run a appropriate program), the waste can afterwards simply be discarded as normal waste, which may not be the case for chemically inactivated waste, which may need special precaution for disposal.


  1. Decontamination and Sterilization
  2. Decontamination of laboratory microbiological waste by steam sterilization.
  4. Decontamination of Laboratory Microbiological Wasteby Steam Sterilization

To address what seems to be the misconception underlying your question:

Killing pathogenic bacteria is not difficult; killing them without harming their (usually human) host is. This is why antibiotics are so precious: They are drugs that affect only bacteria¹ - by exploiting properties that are unique to them. It is usually resistance to antibiotics that we care about in bacteria, because it makes infections with such bacteria more difficult or even impossible to treat.

Once we investigate bacteria in vitro, e.g., in a Petri dish, there is no host that has to survive and thus we have far more options to kill them. The other answers have listed plenty of methods to do this - which are all lethal to humans as well.

¹ This is of course somewhat simplified: Every drug has a side effect, and so do antibiotics. But their side effects on humans are more lenient than their primary effect on bacteria.

Resistance Is… Reversible?

While people generally don't talk about bacteria losing antibiotic resistance, it does happen, and for a pretty obvious reason: the biochemical tools which confer resistance come at a metabolic/reliability/efficiency cost, or the bacteria would have had the resistance to begin with. In general, we can assume that bacteria are not adept at dealing with silver toxicity because silver does not occur often enough in their favorite environments. When you take the bacteria out of a silver-concentrated environment, there is no longer pressure to take extra measures to deal with it, and the resistance can be lost.

A Pound of Flesh

There are many ways to attack microbes, and even more ways for them to respond. The CDC provides a nice overview of some strategies.

Take fluoroquinolones, for instance. They kill bacteria by suppressing the enzymes which uncoil DNA. As you can imagine, this makes for a Very Bad DayTM for the bacteria. If you're a bacterium, and you end up with some ciproflaxin inside your membrane, the most obvious thing to do is get rid of it. Some bacteria, like P. aeruginosa, which are resistant to ciproflaxin do just that: they build pumps in their membranes to pump it out. But as any engineer will tell you, adding new pumps to a system costs both space and energy which could have been used for other things (like finding food, reproducing, etc.), so you wouldn't do this unless you absolutely had to.

How do we know that they actually lose resistance? Well, we've observed it happening! We literally know which mutations and gene transfers are necessary to confer various resistances for P. aeruginosa, because wild strains are both resistant and susceptible, depending on their exposure to antibiotics (or laboratory conditions).

Gray Goo

Be glad that biology tends to find the most efficient solutions. This energy minimization tendency might be characterized as a kind of laziness, but it's really an optimal strategy for allocating your energy where it gives you the most value. For a microbe, that would be finding food and reproducing. That means that once the lab-grown freaks escape into the wild, whatever super-powers they gained from our mad scientist experiments are likely to be lost over time, once we stop bathing them in mutagenic toxins.

Unlike nanotechnology-gone-wild, we actually don't have so much to fear from lab-induced antibiotic resistance. It is the pernicious and casual use of antibiotics in our daily lives that will kill us in the end. These flow down our drains and into our streams and rivers and evolve those critters night and day, virtually guaranteeing that we will encounter resistant types in the wild.

Resistant to WHAT?

Scientists generally deal with bacteria resistant to some concentration of some particular substance (or combination thereof).

Those strains are still pretty much sensitive to great deal of other substances (e.g. etanol 70%), high temperature (boiling water at normal pressure or higher), etc generic ways to kill bacteria.

Autoclave at 120 degrees Celsius for 30 minutes. [ref]

Don't ever flush biological material, living animals, or anything other than sanitary-code approved waste down the toilet.

The proper way to dispose of used culture material is to sterelize it first, then dispose in sealed bags.

After counting, petri dishes should be secured in a plastic autoclave bag and autoclaved prior to disposal. During multi-day field trips, place used petri dishes in plastic autoclave bags, and close the bags for transport to the office. Then, following autoclaving, place the closed bags of petri dishes in dumpsters or other containers that will be emptied mechanically, to prevent accidental contact with people or animals. Never place petri dishes in motel or office wastebaskets.

On a more budget-restricted level, when I worked in a high school, the science lab tech would take all the old Petri dishes out of a dedicated disposals freezer, and take them to the campus coal fired boiler.

Since the boiler only ran in Winter, there was a lot of stuff to dispose in the first few cold days in Autumn. As such, the science department scheduled much of this work over winter. Work involving dissection also produced a lot of biowaste, but that was less-bad and could be disposed of in the general waste (it was the 80s, not a lot of recycling at the time.)

Once the furnace part of the boiler was at temperature, the thing was shuttered (somehow) and the access hatch opened. The frozen plastic dishes were thrown in, inside either paper or plastic shopping bags for ease of handling, then the hatch was closed and the dampers opened.

As for PPE, it wasn't much of a thing. Disposable gloves were worn for handling the dishes, and they went into the furnace once the dishes were all gone.

I remember once looking in the furnace when it was cold, before the ashes were raked out. There was zero trace of any material left behind, even metal items like staples or clips were vapourised.

In short - Burnination

Antibiotic resistance: How do antibiotics kill bacteria?

This is a multi-part series on antibiotic resistance in bacteria.

Eventually, we'll reach the ways in which bacteria develop antibiotic resistance, but before we get there, we'll spend a little more time on antibiotics themselves.

What have we learned so far?

1. Antibiotics are natural products, made by bacteria and some fungi. We have also learned about the difference between antibiotics and synthetic drugs. There isn't always a clear distinction since chemical groups can be added to antibiotics, making them partly synthetic and partly natural.

2. Antibiotics are a chemically diverse group of compounds. Antibiotics are not DNA. Neither are they proteins, although some antibiotics contain amino acids, which are the building blocks of proteins. In a way, antibiotics are kind of luxury molecules, since they aren't essential for life.

Bacteria don't even make them until the population reaches a certain density and phase of growth. Even though we can describe all antibiotics with a single word, there is no single description that does justice to these fascinating compounds.

We can group antibiotics into classes, either by chemical similarity - peptide antibiotics all contain amino acids held together by peptide bonds, ß-lactams all have a ß lactam ring - by the range of organisms they can kill - broad spectrum antibiotics kill a wide variety of bacteria, where narrow spectrum antibiotics have more specific targets or by the metabolic pathway that they target.

Perhaps the easiest way to categorize antibiotics is by the organisms that produce them. Even there, antibiotics defy easy groupings. The majority are made by denizens of the dirt, but both fungi and bacteria get into the act.

But what do antibiotics do?
How do they fulfill their role as agents of warfare? They do kill bacteria - but how? In a bacterial population, do all members get killed? Unfortunately, no, many antibiotics work by preventing bacterial growth. This means that most antibiotics only kill growing bacteria.

They keep bacteria from getting bigger?
No. When we talk about animals, plants, or people growing, we're really describing individual organisms getting larger. But, when we talk about bacterial growth, we're referring to the size of a bacterial population and not just the size of a cell.

In order for bacteria to grow, then, they need to make all the parts necessary for building new bacterial cells. DNA must be copied. New RNA, ribosomes, and proteins must be made. Cell walls must be built. Membranes have to be synthesized. And, then, of course the cells must divide. Many, if not most, antibiotics act by inhibiting the events necessary for bacterial growth. Some inhibit DNA replication, some, transcription, some antibiotics prevent bacteria from making proteins, some prevent the synthesis of cell walls, and so on. In general, antibiotics keep bacteria from building the parts that are needed for growth.

There are some antibiotics that act by attacking plasma membranes. Most antibiotics, though, work by holding bacterial populations in check until the immune system can take over. This also brings us, to our first mechanism of antibiotic resistance.

Persistance is resistance.

If bacteria need to grow in order to be killed by antibiotics, then bacteria, can escape from antibiotics, by NOT growing or by growing very slowly. This phenomenon has been observed with biofilms (colonies of bacteria living on a surface) (1), E. coli in urinary tract infections (2), and most notably in the slow growing bacteria, that cause tuberculosis, Mycobacterium tuberculosis, and leprosy, Mycobacterium leprae (3).

It seems funny to think that not growing can be a mechanism for survival. But if you're a bacteria, and you can hang around long enough in an inactive, non-growing state, enventually your human host will stop taking antibiotics, they will disappear from your environment and you can go back to growing.

1. P.S. Stewart 2002. "Mechanisms of antibiotic resistance in bacterial biofilms." Int J Med Microbiol. 292(2):107-13.

2. Trülzsch K, Hoffmann H, Keller C, Schubert S, Bader L, Heesemann J, Roggenkamp A. 2003. "Highly Resistant Metabolically Deficient Dwarf Mutant of Escherichia coli Is the Cause of a Chronic Urinary Tract Infection." J Clin Microbiol. 41(12): 5689-5694.

3. Gomez JE, McKinney JD. 2004. Tuberculosis (Edinb). 84(1-2):29-44.

Other articles in this series:
1. A primer on antibiotic resistance: an introduction to the question of antibiotic resistance.
2. Natural vs. synthetic drugs: what is the difference between an antibiotics and synthetic drugs?
3. How do antibiotics kill bacteria? a general discussion of the pathways where antibiotics can act and one characteristic that helps some bacteria survive.
4. Are antibiotics really only made by bacteria and fungi? It depends on what you'd like to call them.
5. The Five paths to antibiotic resistance: a quick summary

Pathogenic bacteria rendered almost harmless

Pseudomonas aeruginosa is an opportunistic pathogenic bacterium present in many ecological niches, such as plant roots, stagnant water or even the pipes of our homes. Naturally very versatile, it can cause acute and chronic infections that are potentially fatal for people with weakened immune systems. The presence of P. aeruginosa in clinical settings, where it can colonise respirators and catheters, is a serious threat. In addition, its adaptability and resistance to many antibiotics make infections by P. aeruginosa increasingly difficult to treat. There is therefore an urgent need to develop new antibacterials.

Scientists from the University of Geneva (UNIGE), Switzerland, have identified a previously unknown regulator of gene expression in this bacterium, the absence of which significantly reduces the infectious power of P. aeruginosa and its dangerous nature. These results, to be published in the journal Nucleic Acid Research, could constitute an innovative target in the fight against this pathogen.

RNA helicases perform essential regulatory functions by binding and unwinding various RNA molecules to perform their functions. RNA helicases are present in the genomes of almost all known living organisms, including bacteria, yeast, plants, and humans however, they have acquired specific properties depending on the organism in which they are found. "Pseudomonas aeruginosa has an RNA helicase whose function was unknown, but which was found in other pathogens," explains Martina Valentini, a researcher leading this research in the Department of Microbiology and Molecular Medicine at UNIGE Faculty of Medicine, and holder of an SNSF "Ambizione" grant. "We wanted to understand what its role was, in particular in relation to the pathogenesis of the bacteria and their environmental adaptation."

A severely reduced virulence

To do this, the Geneva team combined biochemical and molecular genetic approaches to determine the function of this protein. "In the absence of this RNA helicase, P. aeruginosa multiplies normally in vitro, both in a liquid medium and on a semi-solid medium at 37°C," reports Stéphane Hausmann, a researcher associate in the Department of Microbiology and Molecular Medicine at UNIGE Faculty of Medicine and first author of this study. "To determine whether the infection capacity of the bacteria was affected, we had to observe it in vivo in a living organism."

The scientists then continued their research using Galleria mellonella larvae, a model insect for studying host-pathogen interactions. Indeed, the innate immune system of insects has important similarities with that of mammals. Moreover, these larvae can live at temperatures between 5°C and 45°C, which makes it possible to study bacterial growth at different temperatures, including that of the human body. Three groups of larvae were observed the first, after injection of a saline solution, saw 100% of its population survive. In the presence of a normal strain of P. aeruginosa, less than 20% survived at 20 hours after infection. In contrast, when P. aeruginosa no longer possessed the RNA helicase gene, over 90% of the larvae remained alive. "The modified bacteria became almost harmless, while remaining very much alive," says Stéphane Hausmann.

Inhibiting without killing

The results of this work show that this regulator affects the production of several virulence factors in the bacteria. "In fact, this protein controls the degradation of numerous messenger RNAs coding for virulence factors," summarises Martina Valentini. "From an antimicrobial drug strategy point of view, switching off the pathogen's virulence factors rather than trying to eliminate the pathogen completely, means allowing the host immune system to naturally neutralise the bacterium and potentially reduces the risk for the development of resistance. Indeed, if we try to kill the bacteria at all costs, the bacteria will adapt to survive, which favours the appearance of resistant strains."

The Geneva team is currently continuing its work by screening a series of known drug molecules in order to determine whether any of them have the capacity to selectively block this protein, and to study in detail the inhibition mechanisms on which the development of an effective therapeutic strategy could be based.

A 'plug-and-play' system

Now, in their most recent study, the team made one final tweak to their E. coli by deleting genes that code for two specific tRNA molecules — the molecules that read the codons and collect all the appropriate amino acids. These tRNAs would usually recognize TCG and TCA codons. The team also deleted genes for a so-called release factor that normally recognizes the TAG stop codon. These changes made the new bacterial strain invulnerable to viruses, the team found.

Virus genomes contain TCG, TCA and TAG codons, but without the right tRNA and release factors, the designer E. coli can't read these viral genes and therefore can't fall prey to the pathogens. "When the virus infects, it doesn't have the same genetic code as our [modified E. coli] cells, and then it can't make its own proteins and it can't propagate," Robertson said.

But again, the main goal of the study was to reprogram the freed codons in order to generate new proteins. To do so, the team generated tRNA molecules that paired with unnatural amino acids of their own design these tRNAs were programmed to recognize the TCG, TCA and TAG codons now missing from the modified E. coli strain. The team reintroduced the missing codons by placing them within small loops of DNA, called plasmids, which can be inserted into the bacterium without altering its genome.

The plasmids, tRNA and unnatural amino acids provided all the blueprints, tools and materials the cells needed to build designer proteins for the researchers. "So you can make proteins in a cell in a programmable fashion, based upon the DNA we provide to the cell, with 23 building blocks," rather than 20, Robertson said. "It's quite a plug-and-play system."

Other research groups have attempted to introduce unnatural amino acids into proteins in the past, but these strategies were not very efficient, Chatterjee and Delilah Jewel, a graduate student in Chatterjee's lab, wrote in a commentary published in the same issue of Science. For example, Chatterjee's lab successfully paired unnatural amino acids with the stop codons in E. coli, but this method only allowed them to insert these unnatural amino acids at a single site in the final protein, they reported in a 2019 study in the Journal of the American Chemical Society.

Now, with the new method, scientists can begin pushing the boundaries of what proteins and polymers they can build, Chatterjee told Live Science. "It's kind of up for imagination. What could those amino acids look like?" he said. "What kind of chemistry could they have, functionalities could they have, that nature never had access to?"

Looking into the future, scientists could potentially remove even more codons from the E. coli genome, freeing up even more channels for designer protein construction, Robertson said. But for now, three open channels are likely plenty to work with, he said. "Do we need seven open channels? Or is three open channels enough to really expand what we can do, in terms of providing new applications?" he said. "It's beneficial to just focus on the applications now."


Living systems must sometimes, temporarily, stay in one place, climb or otherwise move around, or hold things together. This entails attaching temporarily with the ability to release, which minimizes energy and material use. Some living systems repeatedly attach, detach, and reattach for an extended time, such as over their lifetimes. Despite being temporary, these attachments must withstand physical and other forces until they have achieved their purpose. Therefore, living systems have adapted attachment mechanisms optimized for the amount of time or number of times they must be used. An example is the gecko, which climbs walls by attaching its toes for less than a second. Other examples include insects that attach their eggs to a leaf until they hatch, and insects whose wings temporarily attach during flight but separate after landing.

Protect From Microbes

In living systems, microbes play important roles, such as breaking down organic matter and maintaining personal and system health. But they also pose threats. Bacteria can be pathogens that cause diseases. Some bacteria create colonies called biofilms that can coat surfaces, reducing their effectiveness–for example, inhibiting a leaf’s ability to photosynthesize. Living systems must have strategies for protecting from microbes that cause disease or become so numerous that they create an imbalance in the system. At the same time, living systems must continue living in harmony with other microbes. Some living systems kill microbes. Others repel without killing to reduce the chances that microbes will adapt to the lethal strategy and become resistant to it. For example, some pea seedlings exude a chemical that inhibits biofilm buildup.

Break Down Living Materials

Living materials are those that are part of living systems (whether currently or formerly alive). For example, a fallen log, although dead, is considered a living material. Breaking down or breaking up living materials is important for living systems that feed on them, as well as in facilitating the decomposition of organic matter. Breakdown increases the material’s surface area exposed to moisture, fungi, bacteria, and other living systems, many of which use enzymes and other chemicals to further break it down. But living materials can be difficult to break down because, for their own survival, their composition must provide support and protection. Therefore, living systems require mechanical means (such as grinding, tearing, or chewing) to manipulate these materials, as well as strong materials that can overcome resistance. For example, small beetles that chew through wood have large, strong jaws that enable them to cut through this tough material.

Modify Size/Shape/Mass/Volume

Many living systems alter their physical properties, such as size, shape, mass, or volume. These modifications occur in response to the living system’s needs and/or changing environmental conditions. For example, they may do this to move more efficiently, escape predators, recover from damage, or for many other reasons. These modifications require appropriate response rates and levels. Modifying any of these properties requires materials to enable such changes, cues to make the changes, and mechanisms to control them. An example is the porcupine fish, which protects itself from predators by taking sips of water or air to inflate its body and to erect spines embedded in its skin.


Class Insecta (“an insect”): Flies, ants, beetles, cockroaches, fleas, dragonflies

Insects are the most abundant arthropods—they make up 90% of the animals in the phylum. They’re found everywhere on earth except the deep ocean, and scientists estimate there are millions of insects not yet described. Most live on land, but many live in freshwater or saltwater marshes for part of their life cycles. Insects have three distinct body sections: a head, which has specialized mouthparts, a thorax, which has jointed legs, and an abdomen. They have well-developed nervous and sensory systems, and are the only invertebrate that can fly, thanks to their lightweight exoskeletons and small size.


Every summer, annual cicadas emerge from underground all over the world, thickening the air with their distinctive clacking and rattling. Seventeen-year cicadas, which exist only in North America, slurp plant root sap beneath the ground for nearly two decades before surfacing and molting their shells to become breeding adults.

According to the Onondaga nation’s oral tradition, a brood of 17-year cicadas sprouted from the ground after George Washington’s 1779 scorched-earth campaign left the people with no crops, giving them an emergency food source that enabled them to survive.

Now, cicadas may provide another means for human survival. Recent science shows that tiny structures on cicada wings kill bacteria, which could give us another way to fight germs that kill millions of people each year.

The Strategy

In 2012, scientists observed that cicada wings kill several types of harmful bacteria, but it wasn’t immediately clear how it worked. Were the wings coated in an antibiotic? Was there a rapid immune response? Using powerful microscopes to get an extremely close view of the wings, the scientists observed tiny cone-shaped bumps called nanopillars covering both sides in a hexagonal arrangement.

They hypothesized it was actually the cones themselves that were killing the bacteria, and they used “the Midas touch” to prove it. They coated cicada wings with a super thin layer of gold to inhibit any biochemical reactions. When exposed to the gold-plated cicada wings, bacteria still died, proving there was no chemical killer—the unique nanopillar structures were directly responsible.

To understand how the cones kill bacteria, think of a bacterial cell like a water balloon. With a diameter several times larger than the distance between cones, one cell rests on many nanopillars. It’s tempting to think of these nanopillars as a bed of nails that simply pop the water balloon. However, in 2013, the same group of scientists developed a model that told a different story.

The nanopillars bond with the bacterial cell membrane, stretching and eventually rupturing it.

Consider what’s happening between just two of the cones. The water balloon would sag around both cones, while the membrane between would extend across the gap like a bridge. However, at the nanoscale, the cell membrane isn’t just sagging—it is physically attracted to the nanopillars’ surfaces, essentially sticking onto them. As the membrane adheres farther down on both cones, the membrane spanning between them stretches, eventually snapping like a rubber band.

Now consider ruptures ripping between every pair of cones the cell touches—cytoplasmic guts spilling out of a shredded membrane spells bacterial death.

This mechanism is only effective on gram-negative bacteria—the type of bacteria that tends to cause infections. It doesn’t work on gram-positive bacteria, which tend to be beneficial “probiotic” bacteria. Instead of pliable water balloons, beneficial bacteria are more like hard-shelled eggs with rigid membranes. They are protected from the nanopillars’ rupturing effect because the physical forces that attract their membranes to the cone surfaces aren’t strong enough to overcome their stiffness.

The Potential

For centuries, the concept of bacterial infection went undiscovered, postponing the war we’d eventually wage against it. In the 1860s, French microbiologist Louis Pasteur kicked the battle off by finally proving that germs cause infection. Shortly after, he invented pasteurization to make some beverages safer to drink.

Joseph Lister, a surgeon, quickly applied Pasteur’s work to hospitals, developing the first sterilizing technique to cleanse instruments, hands, and wounds with carbolic acid. Then in 1928, Scottish researcher Alexander Fleming accidentally discovered penicillin, sparking decades of antibiotic research.

Now that some bacteria are developing resistance to antibiotics, we need to look to nature to help us discover new ways of fighting infection.

Cicada wing nanopillars may be the next weapon in our germ arsenal. Surgical instruments, biomedical implants, door handles, and food preparation surfaces might one day be coated with microscopic cones to kill bacteria before it can invade.

Where Are We Now?

Now that more and more bacteria have developed resistance to antibiotics, scientists around the world have a renewed interests in phages. The European Union invested 5 millions euros in Phagoburn, a project that studies the use of phages to prevent skin infections in burn victims (Figure 2). In the USA, the FDA approved ListshieldTM, a food additive containing phages, that kills Listeria monocytogenes, one of the most virulent foodborne pathogens and one cause of meningitis. Currently, many clinical trials using phage to treat or prevent bacterial infections such tuberculosis and MRSA are undergoing.

Despite the fact that phage therapy is not yet approved by FDA, phages have already been used to save lives in experimental treatments. A miraculous recovery of a patient who suffered from antibiotic-resistant bacteria was reported in San Diego. While on a vacation in Egypt, Tom Patterson was infected by a multidrug-resistant strain of Acinetobacter baumannii. He was flown back to California and treated with antibiotics for over 100 days, but Patterson did not get better and fell into coma. He was finally saved by a cocktail of phages purified from sewage in Texas.

In the near future, as antibiotics lose their effectiveness, we may begin to hear more stories like this. And one day, phage might move from our last resort against antibiotic-resistant bacteria to our first line of defense.

Bugs Vs. Superbugs: Insects Offer Promise In Fight Against Antibiotic Resistance

Scientists have isolated a molecule with disease-fighting potential in a microbe living on a type of fungus-farming ant (genus Cyphomyrmex). The microbe kills off other hostile microbes attacking the ants' fungus, a food source. Courtesy of Alexander Wild/University of Wisconsin hide caption

Scientists have isolated a molecule with disease-fighting potential in a microbe living on a type of fungus-farming ant (genus Cyphomyrmex). The microbe kills off other hostile microbes attacking the ants' fungus, a food source.

Courtesy of Alexander Wild/University of Wisconsin

Nobody likes a cockroach in their house. But before you smash the unwelcome intruder, consider this: that six-legged critter might one day save your life.

That's right. Insects—long known to spread diseases—could potentially help cure them. Or rather, the microbes living inside them could. Scientists have discovered dozens of microorganisms living in or on insects that produce antimicrobial compounds, some of which may hold the key to developing new antibiotic drugs.

They can't come too soon. More infections are becoming resistant to common antibiotics, and the pipeline of new antibiotic drugs has slowed to a trickle.

"There is a growing demand [for antibiotics], and a diminishing supply," explains Gerry Wright, who directs the Michael G. DeGroote Institute for Infectious Disease Research at McMaster University.

Most antibiotic drugs have been discovered from bacteria living in the soil. But Cameron Currie, professor of bacteriology at the University of Wisconsin-Madison, says that searching the soil for new antibiotics has become increasingly futile.

"They keep finding already known antibiotics," Currie says. "There's a common sentiment that the well of antibiotics from soil. is dry."

Fortunately, there may be another well. Currie and a team of 28 researchers recently published a paper in Nature Communications showing that some of the bacteria living in insects are really good at killing the germs that make people sick.

"There's an estimated 10 million species [of insect] on the planet," Currie says. "That implies a huge potential for a lot of new [antibiotic] compounds."

Microbial fighters

Each insect contains an entire ecosystem of microorganisms, just like the microbiome found in humans. And there's one quality that many of those insect-associated microbes have in common, says Jonathan Klassen, assistant professor of molecular and cell biology at the University of Connecticut and an author on the study.

They don't get along with each other very well.

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'Predatory Bacteria' Might Be Enlisted In Defense Against Antibiotic Resistance

And by don't get along, he means they're constantly trying to kill each other through biochemical warfare. Many of the microorganisms in insects make compounds that are toxic to other microbes—essentially, natural antibiotics.

Some of those natural antibiotics attracted Currie's attention while he was a student, researching leaf cutter ants.

Leaf cutter ants are among nature's most prolific gardeners. They actually don't eat the leaves they cut — instead they use them to cultivate a special type of fungus for food. Still, it's not easy being a fungus farmer.

"Like human agriculture, the ants have problems with disease," Currie says. "I found a specialized pathogen that attacks their fungus garden."

Fortunately, the ants have a tool to deal with the problem. A species of bacteria living on the ants' exoskeletons produces a toxin that kills the pathogen. Like the pesticides a gardener uses, the toxin keeps the ants' garden disease-free.

The discovery inspired Currie's curiosity. If ants could use these bacterial compounds to treat disease in their fungus gardens, could doctors use them to treat disease in people? If so, what other insects might also be carrying disease-fighting microbes?

To answer those questions, Currie and his team spent years collecting thousands of insects, including cockroaches, from Alaska to Brazil.

"Every few months somebody would be going out somewhere to collect something," remembers Klassen, who was working at the time as a postdoctoral researcher on the project.

The team tested bacteria from each insect to determine if they could kill common human pathogens, such as E. coli and methicillin-resistant Staphylococcus aureus (MRSA). They then compared the results from strains of insect bacteria to strains drawn from plants and soil.

"We were really surprised that [insect strains] were not just as good, but apparently better at inhibiting [pathogens]," Currie says.

Testing a new antibiotic

Once a scientist has discovered that a strain of bacteria can kill germs, the next step in drug development is to determine what bacterial compound is responsible for the antimicrobial activity—like a cook searching for the secret ingredient in a particularly delicious soup.

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Currie's team had found dozens of promising bacterial strains in insects. And each could yield a secret ingredient that might be a new antibiotic compound.

That in itself was a big accomplishment. But the researchers went a step further. They isolated one compound from one particularly promising bacterial strain and showed that it could inhibit fungal infections in mice, an important step in drug development.

The compound, cyphomycin, is found on Brazilian fungus-farming ants, close relatives of the ants Currie studied as a PhD student. Though it's far from becoming an approved drug, the research shows that antibiotic compounds new to science can be isolated from insects.

Wright, an antibiotic researcher who did not participate in the study, says that previous research has shown that single insect species contained antimicrobial compounds. But this is the first study to comprehensively demonstrate that insects as a group are a promising source of new antimicrobials.

"No one's ever done something on this scale before," Wright explains.

Currie is hopeful that cyphomycin may one day be approved to treat yeast infections in people. But before that happens, it must undergo years of further testing.

"It [cyphomycin] is a million miles away [from approval]," Wright says. "That's the reality of drug discovery."

Still, Wright says the researchers have already overcome one of the toughest hurdles in drug development by demonstrating that the compound works in mice.

For Klassen, the stakes are too high not to try.

"Efforts such as this study are crucial to keeping the antibiotic pipeline flowing so that disease doesn't gain the upper hand," he says.

In the end, the consequences of a world without antibiotics are enough to make scientists look for new drugs in unconventional places—even if that means looking in a cockroach.

Self-Sterilizing Plastics Kill Drug-Resistant Bacteria

Despite the proliferation of antibiotics and assorted antibacterial hand lotions and wipes, bacteria remain a moving target for hospitals and clinics seeking to protect their patients from infections. One approach gaining traction in the effort to banish bacteria is to mimic the way the human body attacks these microorganisms by punching holes in bacterial cell membranes and hobbling their ability to morph into antibiotic-resistant pathogens.

Bacteria are becoming increasingly resistant to antibiotics due to a combination of their overuse (which allows bacterial cells to become familiar with these drugs and purge them before they can do their work) and the pathogens' ability to quickly adapt to new conditions. Bacteria divides every 20 minutes, speeding evolution and creating the equivalent of a changing keyhole for makers of antibiotics, says Nicholas Landekic, CEO and founder of PolyMedix, Inc., a Radnor, Pa., biotech firm. "Seventy percent of bacterial infections are resistant," he says, "which is a big problem."

Indeed, bacterial infections are now the fourth leading cause of death in the U.S., killing about 100,000 people each year, according to the U.S. Centers for Disease Control. Ironically, one of the best places to get an infection is the hospital&mdashmore than two million cases of hospital-acquired infections are reported in the U.S. annually, according to a paper published recently in The Society of Chemical Industry's journal, Polymer International.

PolyMedix is, with the help of scientists at the University of Pennsylvania, developing drugs and polymers that behave much like the body's own defenses. Among those in the works: medications that can kill bacteria without the need to actually enter the cells themselves as well as new polymers that the company hopes will be used in paints, plastics and textiles to create self-sterilizing products and surfaces. The polymer is not a coating like silver, ammonium salts or phenols, which Landekic says dissolve over time and lose their effectiveness. "Our compounds become part of the surface," he says, and can kill bacteria in a matter of seconds. "If you make the antibiotic part of the material, the effect is long lasting."

The company has no time frame for delivering the antibacterial polymer to store shelves because it is focusing its resources primarily on getting its antibiotic drug to market. The company would need approval from the U.S. Environmental Protection Agency&mdasha process that takes up to 16 months once an application is filed&mdashto include its antibacterial polymer in bedding, carpeting, countertops and towels.

"We've developed a lot of different prototype materials and proven [the polymer] works when added to different materials"&mdashas long as the surface is clean, Landekic says. "The polymers are self-sterilizing, not self-cleaning," he adds. "You have to allow them to interact with the bacteria."

He says the antimicrobial polymers may also be successful in wiping out Stachybotrys chartarum, or "black mold," in residential and commercial buildings, a fungus that can cause lung disease and exacerbate allergies. The polymers also may prove significant in the war on terrorism, which explains why the U.S. Department of Defense handed the company more than $1.6 million to develop drug compounds and polymers able to combat biowarfare pathogens that cause infectious diseases such as anthrax, plague and tularemia. The company received another $3 million for research and development from the U.S. Department of Health and Human Services.

PolyMedix needs the approval of the Food and Drug Administration before it can test meds in development that may one day supplant antibiotics. This new crop of microorganism fighter would, much like the polymers, kill bacteria by punching their membranes full of holes. PolyMedix recently announced that the Health Canada&mdashthe country's national health care watchdog&mdashhas given the company the green light to begin a human clinical trial to assess the safety and effectiveness of a synthetic compound dubbed PMX-30063, designed to treat systemic infections while avoiding the pitfalls of normal antibiotics no longer effective against bacteria.

Overcoming Resistance

The Scientist Staff
Apr 1, 2014

© JUSTIN GABBARD A lthough researchers and drug developers have been sounding warnings for years about bacteria out-evolving medicine&rsquos 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&rsquos 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 drug-resistant infections require more than 8 million additional hospital days.

The statistics are sobering, and they&rsquore 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&rsquos.

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 antibiotic-resistant bacteria into the human food supply.

In the United States alone, some 23,000 people are killed each year by infections caused by drug-resistant bacteria. Many researchers are now focused on developing new treatment regimens to combat these deadly superbugs.

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 one-two 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

© JUSTIN GABBARD 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

© JUSTIN GABBARD 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.

The idea of a magic bullet is gone. We need a magic shotgun. —­Gerard Wright,
McMaster University

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

Resensitizing Bacteria

© JUSTIN GABBARD 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 gram-negative bacteria pose the additional challenge of producing β-lactamase enzymes that block antibiotics containing a β-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 β-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 β-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 β-lactamase inhibitor combined with the antibiotic adjuvant disodium edetate.

We are developing agents to sensitize bacteria to the agents we already have. —­Anders Hakansson,
State University of New York at Buffalo

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

© JUSTIN GABBARD 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 hypericums—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

Only a superweapon can kill superbacteria, and humanity finally found it

In 1945, in New Mexico, the researchers of the Manhattan Project performed the first detonation of a nuclear weapon it bathed the desert with light, and cast a pall over the world for decades after. In 2016, the Southwest saw another harbinger of destruction.

As a 2017 Center for Disease Control (CDC) report explained, a woman died in a Nevada hospital after contracting an infection from carbapenem-resistant Enterobacteriaceae (CRE). This “super bacteria” was resistant to all 26 antibiotics available in the United States.

Although the Nevada case may have been a wake-up call for some in the United States, for years now, researchers have been watching the crisis grow worldwide. In 2014, Dr. Keiji Fukuda, the Assistant Director for Health Security at the World Health Organization (WHO), warned of the already present danger, saying “Without urgent, coordinated action by many stakeholders, the world is headed for a post-antibiotic era, in which common infections and minor injuries which have been treatable for decades can once again kill.”

The CDC estimates that there are more than 23,000 deaths in the United States each year due to antibiotic resistant bacteria. India — where the Nevada woman was traveling when she sustained her fatal injury — has the highest rate of resistance to E. Coli in the world, according to the Center for Disease Dynamics, Economics, and Policy. In China, resistance to the drug colistin is spreading. This is particularly troubling, as colistin is already considered a last resort it is an old drug which can cause kidney damage, and physicians only pulled it out of retirement because modern drugs have become steadily less effective.

Antibiotic resistance will continue to spread, and it represents one of the great health crises of our time. Thankfully, there are researchers working to stop it.

A genetic solution

“Bacteria will develop resistance to any one antibiotic or antimicrobial given enough time,” Dr. Bruce Geller, a professor of microbiology at Oregon State University, told Digital Trends. “Because they’ve had a 4 billion year head start in the evolution of mechanisms to adapt to changing environments, they’re very, very good at getting around any antimicrobial they might encounter.”

For years, biologists like Geller have been playing evolutionary whack-a-mole with bacteria. Although researchers are armed with the collective knowledge of the scientific community, bacteria have the cunning flexibility of nature. For every tool humans use against them, bacteria develop a countermeasure. While antibiotics were a revolution in medicine, the moment we first employed them, bacteria began to reshape themselves.

Geller is exploring a unique approach: rather than developing yet another way to kill bacteria — to which they will eventually become resistant — why not make them vulnerable to already existing antibiotics again?

To this end, Geller’s weapons of choice are synthetic molecules called peptide-conjugated phosphorodiamidate morpholino oligomers — PPMOs, for short. As you may have guessed from the outrageously long name, PPMOs are fairly complex to understand how they work, you first need to wrap your head around how antibiotics work, and how bacteria have learned to fight them off.

How bacteria and antibiotics function

Bacteria are microscopic, single-celled organisms that come in a variety of shapes. Like other single-celled organisms, bacteria cells have a cell wall surrounding them in bacteria specifically, these walls contain a substance called peptidoglycan, and this can be essential to the use of antibiotics.

An antibiotic is designed to destroy microorganisms like bacteria. For an antibiotic to work effectively, it must kill bacteria cells without destroying human cells, so biologists engineer antibiotics to target aspects unique to bacteria cells. For example, penicillin prevents the peptidoglycan in bacteria cells from linking, leaving the cell walls weak and prone to collapse. Another class of antibiotics – sulfonamides — inhibits the ability of cells to produce folic acid. This is fine for human cells, which can absorb folic acid from outside sources, but it means death for bacteria cells, which must produce folic acid on their own. A third type of antibiotic, tetracycline, inhibits protein synthesis in cells but doesn’t accumulate in human cells enough to harm them.

However inventive antibiotics might be, bacteria always adapt. Some use protein structures called “efflux pumps” to push antibiotics out of their cells. Others can rearrange themselves, effectively hiding parts of the cell that are vulnerable to antibiotics. Still others produce enzymes — such as Geller’s target, New Delhi metallo-beta-lactamase (NDM-1) — that can neutralize antibiotics.

The human gut alone holds more bacteria than there are cells in the human body.

As if the mercurial nature of bacteria were not frightening enough, researchers must contend with the fact that bacteria also have a useful, if unwitting, accomplice: us. Resistance develops and spreads through natural, evolutionary processes, but human behavior gives it a helpful nudge.

How does resistance develop? Some bacteria cells develop random mutations that result in these resistance mechanisms. When a round of antibiotics kills a population of bacteria, resistant cells are left alive, able to reproduce. Making matters worse, non-resistant bacteria can acquire resistance from cells that have it, receiving a copy of the gene that provides the resistance mechanism.

This process is entirely natural — bacteria will inevitably develop resistance to an antibiotic used against them — but it moves faster due to human behavior. The first trend that has accelerated the spread of resistance is that society simply uses too many antibiotics. A report by the CDC estimates that at least 30 percent of antibiotic prescriptions in the U.S. are unnecessary many of these prescriptions go to patients suffering from viral infections, against which antibiotics are completely useless!

Despite our obsession with hygiene, humans are walking bacteria farms. The human gut alone holds more bacteria than there are cells in the human body. When a patient takes antibiotics, the bacteria in his or her intestines can develop resistances, which can then spread to other people.

People aren’t the only creatures taking an excess amount of antibiotics even farm animals have contributed to the problem. For years, farmers have given antibiotics to food animals such as cows, chickens, and pigs. Not only does this keep the livestock healthy (sick animals are bad for business) but antibiotic use has also been shown to increase the growth of these animals. Good news for farmers, but terrible for anyone worried about the rise of superbacteria. The Food and Drug Administration has been trying to curtail the use of antibiotics in livestock, cracking down on growth promotion.

The wonderful world of PPMOs

Changing societal behavior is often a slow and difficult process. The CDC hopes to cut down on antibiotic prescriptions by 15 percent over the next few years, an ambitious goal given how often patients demand prescriptions for their ailments. Thanks to the work of researchers like Geller, the war on bacteria may flip without sweeping reforms.

Geller’s megaweapon is a PPMO designed to neutralize resistance mechanisms in bacteria, leaving them vulnerable to antibiotics. “This molecule can restore sensitivity to standard, already-approved antibiotics in bacteria that are now resistant to those antibiotics,” Geller says, which eliminates the need to invest time and money in developing new antibiotics. So how does this PPMO work?

A PPMO is a type of synthetic molecule that mimics DNA and can bind to the ribonucleic acid (RNA) of a cell. RNA takes the information stored in the DNA of a cell, translating it into proteins that carry out the various functions of that cell.

Imagine a gene as instructions, written in a letter. Normally, the RNA receives this letter and carries out the instructions, creating the appropriate proteins. The PPMO instead intercepts the letter along the way, replacing it with one that commands the RNA to do nothing. So Geller’s team can create a PPMO that binds to the gene that produces NDM-1 — an enzyme that neutralizes antibiotics — and silences it. Suddenly, the bacterium has no defense mechanism.

“Most standard antibiotics don’t target genes or gene expression, they bind to cellular structures like ribosomes or membranes,” Geller explains. “Our approach is to target the genes themselves, or more specifically, target the messenger RNA that’s made from the genes. Our molecules bind to a specific messenger RNA, and that prevents its translation into protein.”

Although the PPMOs are synthetic, they are not conjured from “earth, wind, and fire,” as Geller puts it. The process begins, as many a great night does, with brewer’s yeast. Chemists take the yeast from fermentation vats and extract the DNA.

Geller’s team can create a PPMO that binds to the gene that produces NDM-1 — an enzyme that neutralizes antibiotics — and silences it.

Chemists then break the DNA down, extracting some of the more valuable parts, and use their pieces as the building blocks of the molecule. Although bacteria are the target for the molecule, they are not the only obstacle it faces. The human body, with all its natural defenses, poses a threat, so the chemists make modifications to the resulting compound, protecting it from the enzymes in the human body that could disintegrate it.

The process may sound time-consuming, but it’s actually remarkably quick. “The real beauty of this technology,” Geller says, “is that it really shortens the discovery time for a new drug. One of the most time-consuming and laborious steps in drug development is discovery. When scientists go out and try to discover a new drug, it can take many years before they find a hit, something that they think might be a good medicine.” Since the PPMO “can really target any gene, all we have to do is change the sequence of our oligomer we can make a new drug in a matter of days, if not hours.”

Geller has been working on his research since 2001, and the results did not come easily. He works with Gram-positive bacteria, which have a thick peptidoglycan layer in their cell walls. Early in his research, his molecules — which were then just PMOs — could not penetrate the cell walls. How did he eventually break through?

If you’re a medieval warlord trying to crack a fortress, you use a trebuchet. Geller settled for peptides. His team attached membrane-penetrating peptides to the PMOs — creating PPMOs — allowing them to pierce the cell wall. Once inside, the molecule gets to work, binding to RNA and stopping it from translating genes.

Perhaps the most useful aspect of the PPMO is that, because it silences a gene rather than directly killing the bacteria, it could be less likely to trigger resistance mechanisms. To be safe, Geller thinks physicians should play the odds, using two antimicrobials or compounds in unison, to lessen the chances that any bacterium will survive treatment.

Nothing is perfect

Despite their virtues, PPMOs are not without flaws. For starters, Geller’s team has observed bacteria displaying resistance to the peptide portion of the molecule. The strength and frequency of resistance differs greatly based on the peptide used.

Beyond the cellular level, there are other drawbacks. Geller emphasizes that they are not broad-spectrum solutions because a PPMO is designed to target a specific gene, a physician will need to know the exact affliction. In cases where a patient has a long-term illness, like tuberculosis, a doctor would know exactly what to target. If the physician is unsure what the cause of illness is, the PPMO would be virtually useless.

Finally, Geller’s project faces the same constraints that any medical research does: time and money. Although his team can produce a PPMO quickly, Geller points out that the molecule will be subject to the same regulatory process that any drug must go through before it can be used on humans. “It takes many years to actually test these compounds and develop them to make them effective and safe, so that they can be ultimately tested in humans,” he says. “We’re still in the development stage.”

The testing process will last as long as it needs to, but the sword above our heads is dangling ever more precariously. The fight against super bacteria is not new humanity’s front has been inching back for years now, and the enemy is crawling over the gates. It will take all the ingenuity of the medical world to stem the tide, and without wise decision-making from politicians and society at large, even that may not be enough.

Watch the video: Τι Βρέθηκε Μέσα Στη Μεγαλύτερη Θαλάσσια Καταβόθρα Στον Κόσμο; (August 2022).