A Motion-Sensor Switch for Antibiotic Resistance: My New Paper in the Journal Structure

fx1

I’ve been working on my thesis for the last few months, squirreled away in libraries and coffee shops, but now I’ve submitted and waiting to defend, I’m happy to share what’s happened in the meantime! A research paper I’ve been working on for a long time has been accepted, and published in the journal Structure. You can find it online, here. This paper makes up a bulk of the work in my PhD thesis, containing 10 protein crystal structures, and I’m glad to have it finally available to the world!

In the paper I describe the structure of a protein that causes antibiotic resistance. This protein makes a bacterium resistant to a class of antibiotics called aminoglycosides. They are one of the oldest classes of antibiotics and include some well-known compounds such as streptomycin, kanamycin, tobramycin, and gentamicin. They are effective antibiotics used against a broad variety of bacteria, and resistance factors that make them ineffective are a serious problem in the treatment of infections.

The protein that I work with generates aminoglycoside resistance. It chemically alters the antibiotics, turning them into inactive byproducts, making any bacterium with the protein resistant to aminoglycoside antibiotics. The protein acts as a “resistance factor” – bacteria that carry the gene for this protein use the protein to deactivate the antibiotic. They can easily break it down and go about their bacteria business instead of being killed.

This protein is called APH(2”)-Ia (more on that name later). It inactivates several different aminoglycosides. To learn how it carries out this transformation, I looked at the structure of the molecule and how it changes when it interacts with the antibiotic. To understand why the structure is important, let’s talk about what this type of molecule really is.

Enzymes: biological molecules that make chemistry happen

Proteins that carry out chemical reactions are also called enzymes. They allow a chemical change to happen that normally happens at extremely low or nonexistent rates. The enzymes that act on aminoglycoside antibiotics are collectively referred to as aminoglycoside-modifying enzymes. They deactivate antibiotics by transferring part of a common, metabolic molecule to the antibiotic. This makes the antibiotic worthless, and lets the bacterium survive the toxic effects of the compound.

An enzyme drives a reaction by forcing these chemicals together. They do this by specifically binding to the molecules, in a mechanism sometimes referred to as a lock-and-key interaction. An enzyme is specific for the molecules that it acts on, like a lock only opens for a specific key. The keys of an aminoglycoside-modifying enzyme are the antibiotic and a cellular molecule that donates a chemical group to the antibiotic. In APH(2”)-Ia, that cellular molecule is guanosine triphosphate, or GTP. The enzyme binds the antibiotic and GTP tightly, and undergoes changes in structure to drive a chemical change between these molecules. This all happens in the most important part of the protein, the active site.

The active site is the most important part of an enzyme. This part of the protein is typically a deep pocket that the rest of the molecule wraps around, where the enzyme holds and manipulates the molecules, and where chemical bonds are broken and formed. The enzyme separates these molecules from water and other compounds, and in this isolated state, the enzyme drives the chemical change to occur.

The active site of any enzyme is typically extremely sensitive to the shape and properties of the molecules it acts upon. Evolution has driven enzymes to develop a high degree of specificity for these molecules, known as substrates. An enzyme typically has only a few different substrates that it acts upon. The combination of the specificity of chemicals that an enzyme acts on and the reaction it carries out gives it its name, in this case aminoglycoside phosphotransferase.

Aminoglycoside phosphotransferase

I try to avoid saying the name of the protein I work on unless I want to sound important.

APH(2”)-Ia stands for aminoglycoside (2”)-O-phosphototransferase type Ia. It’s usually preferable to just say “the enzyme” and that’s what I’ll mostly do here. As the “type Ia” might suggest, the enzyme is part of a larger group of enzymes that all carry out a similar reaction. They use magnesium atoms, held tightly in the active site, to move a phosphate group, PO₄^3-, from one molecule to another. In this enzyme, it moves a phosphate from GTP to the aminoglycoside antibiotic. GTP is a nucleoside triphosphate molecule, the cellular phosphate source in this reaction. You might be familiar with a similar molecule, ATP, the “energy currency of the cell”.

APH(2”)-Ia is somewhat unusual because most similar enzymes use ATP, but this enzyme uses GTP. Researchers in my lab and in other groups have studied the relationship between these proteins and GTP, and there’s still some interesting unsolved mysteries about the use of GTP in these proteins. However, for this work, I focused on the part of the molecule that is the same between ATP and GTP, the triphosphate.

These enzymes that use nucleoside triphosphates as phosphate donor have a special name: kinases. We know quite a lot about kinases. Their importance for cell biology was discovered in the 1970s-1990s and researchers learned that they are critically important regulators of cellular metabolism, cell division, and many other processes. They add phosphate groups to proteins that generate molecular communication networks in the cell. In mammalian cells kinases are typically involved in important cellular decisions, and because many of these decisions impact how a cell grows and divides, many of these kinases are involved in cancer. When it was found that aminoglycoside phosphotransferases were kinase enzymes, there was already a large amount of research to compare to on similar enzymes. However, comparisons to other enzymes only gets you part of the way. To really learn how any molecule works, you have to look at it directly.

How do you look at something a few nanometers in size?

A mantra in the molecular sciences is that structure dictates function. I argue it needs a little updating, that structure and dynamics dictate function (more here and here), but you need to have a structure before you can study how it moves. Determining the structure is where we start.

Using the techniques of structural biology, we can get a direct look at the molecules that carry out biological functions. Techniques like nuclear magnetic resonance, and electron microscopy can provide excellent structural information about biological macromolecules, but the technique I used for these experiments was X-ray crystallography. Matt Kimber, the professor who taught my undergraduate structural biology course referred to crystallography as a “one-trick pony, but it’s a damn good trick”. Well, I’ve ridden that pony right to the end of my degree.

To determine a structure by crystallography, you purify the protein that you’re interested in, and run an array of experiments in parallel to try to find conditions under which it will form crystals. Not every protein can crystallize, and not every crystal gives you good data, which makes protein crystallography an intimidating technique. The perceived risk of protein crystallography experiments drives many in the molecular sciences to treat protein crystallographers with a sort of reverence. I don’t know that that reverence is particularly well-placed, but I’ll take it all the same.

302_B3_001 — Crystals of APH(2”)-Ia + GMPPNP. This is a ~3μL drop with protein and various chemicals, suspended upside-down on a glass cover slide. The bright colours are because I used a polarizing filter, crystals do cool things when you shine polarized light through them!

If you are able to make protein crystals of sufficient size and quality, then you can collect X-ray diffraction data with the crystals. Several companies sell instruments to collect this diffraction data, and there are dedicated facilities that conduct these experiments using high-intensity X-rays from accelerated electrons. The instrumentation for these experiments continues to dramatically improve, allowing us to get more and better data from our protein crystals all the time. The job of the X-ray crystallographer is much easier these days than it used to be.

Using a home-based or synchrotron source, a beam of X-rays are directed at the protein crystal. X-rays interact with the electrons of a molecule. Because of the physics of diffraction of rays from a crystal, the result is a pattern of diffraction from the x-rays that can be recorded. By measuring these diffraction spots, we can apply physical rules about how diffraction works to interpret the distribution of electrons within the crystal of proteins. From the intensity of diffracted X-ray spots, we can reconstruct the shape of the electron density in our molecule of interest.

281a31_testa_001 — X-ray diffraction from a crystal of APH(2”)-Ia. The spots are X-rays diffracted from the crystal, and the intensity of those spots is related to the shape of the molecule in the crystal. The further out from the centre, the lower the “resolution” of the data – the better quality structure you can build. This crystal diffracted to ~2.15 Å, or 0.215 nanometers.

At this point, the job still isn’t done. In some cases, you have to solve what we call the phase problem, although in this case it wasn’t too much trouble so I’ll jump past it. However, there is still a considerable amount of analysis required to interpret what the electron density means, and what the shape of molecules that fill this electron density truly are. It’s the crystallographer’s job to build a molecular model that recapitulates the observed electron density as closely as possible. The point at which you consider the model “done” is an ongoing struggle, similar to that experienced by artists and writers, where there is always another brushstroke, sentence, or water molecule that could improve the final product, but then at some point you stop and call your model “finished” and interpret what it says about the chemistry of the molecule you’re studying.

Screenshot from 2016-06-23 13:20:04 — Part of the model-building process for APH(2”)-Ia. The blue/purple mesh represents the electron density of the molecule, through which I build the model of the protein molecule, with yellow (carbon), red (oxygen) and blue (nitrogen) atoms linked together to form the structure of the protein. The mesh are a transformation of the experimental data, while the sticks built into it are the model of connected atoms that we interpret from this data. The cross in the bottom left represents a water molecule.

In the case of APH(2”)-Ia, a lot of the challenge for me was making the models as good as possible, and after a long struggle, they were of sufficient quality that I could use them to gain some interesting insights about how the protein works and suggested a new feature of an antibiotic resistance enzyme.

Determining the structure of APH(2”)-Ia

When I started working with APH(2”)-Ia, others had already determined the structures of three related molecules: APH(2”)-IIa, -IIIa, and -IVa. These structures give us the shape of the enzymes and some of their interactions with their substrates, but a key factor always missing was a well-defined active site set of the triphosphate ligand.

Without the triphosphate in the enzyme, we can’t get any sense of how the enzyme interacts with that molecule. And if we can’t see it, we can’t predict how it works, or understand how to affect it.

Building the shape of the first versions of APH(2”)-Ia weren’t as hard as I’d expected. I had a few nights up late, excited to carry out my next rounds of model-building and improving the data, and was proud to build models with some pretty excellent statistics for the quality of X-ray diffraction data I was working with. The challenge came when I had to start interpreting what was in the active site of the enzyme.

overview — Overview of the structure of APH(2”)-Ia. There are four copies (A-D) within the crystal structure, and I blow up one here for illustration. The three regions of the protein, the N-terminal lobe and core and helical subdomains of the C-terminal lobe are indicated. The nucleoside triphosphate binds between the N-lobe and core subdomain, while aminoglycosides bind between the core and helical subdomains.

Probing the active site of an antibiotic resistance enzyme

Remember how I talked about how the enzyme takes a phosphate group from GTP and moves it to the antibiotic? Well, in the first structures I looked at, that really didn’t seem possible. I used a GTP-like analogue molecule called GMPPNP to make the protein crystals, and in the structures, the phosphate groups of the GTP analogue were stuck in a position where they couldn’t react with the antibiotic. I named this the stabilized conformation, because it seems to be sitting in a position where it can’t react with anything.

There were several similar enzymes I could look to for comparison, and none of them show this stabilized triphosphate form. They have a different, activating conformation which directs it toward the other substrate. I was able to make the molecule adopt the activated conformation, but I had to break the protein by removing an important amino acid to make it let go of the stabilized phosphate.

conformations_equilirbrium — Stabilized and activated forms of the GMPPNP molecule in the APH(2”)-Ia active site. In both cases the magnesium atoms in the active site stay the same, but the phosphate groups (yellow) of the molecule switch to a different location, far from or close to the aminoglycoside, which contacts D374. By removing the S214 contact from the enzyme, the normal enzyme gave us the activated conformation, which suggests that the enzyme normally holds the compound in a stabilized form for some reason. Why?

So why didn’t the protein in my structures normally activate the triphosphate?

The answer came when I added antibiotic molecules to the crystals. Introducing the antibiotic after the crystals were grown could let us track the changes driven by the introduction of the antibiotic. I had a good idea from other aminoglycoside kinases that the antibiotic would bind in the same position, and initially was just trying to confirm that the antibiotic bound in the same position. As a fortuitous observation, the addition of aminoglycoside substrate drove changes in the shape of the protein, as well as the GMPPNP molecule. The antibiotic activated the triphosphate by binding to the enzyme.

The flip between these states gives us a clear way to understand how the enzyme could turn itself “on” when it encounters an antibiotic.

A motion sensor switch for antibiotics

gentamicin_induction — Gentamicin binding to the APH(2”)-Ia active site pulls the equilibrium of conformations in the active site from the stabilized to the activated form of the triphosphate group. A hydroxyl group (dark red) of gentamicin lies closest to the activated triphosphate, where it could then be phosphorylated.

So, we see a new shape of the triphosphate group that can’t modify the antibiotic. It is held in an awkward, non-reactive form in the back of the enzyme active site. When an antibiotic comes in, the enzyme shifts its shape to respond. In the process of making this change, the GTP triphosphate changes and becomes active. This catalytic switch helps the enzyme keep the GTP inactive until the antibiotic is bound and then activates its triphosphate to a shape that lets it carry out the reaction.

But why is this necessary?

This is where we have to speculate a bit. In the stabilized position, the enzyme can’t carry out its normal reaction, but it also can’t carry out a second reaction, called hydrolysis. Hydrolysis is the breakdown of a molecule caused by water. As all biological molecules are found in water, it is always available to react with an activated molecule. Normally, the kinase enzyme should transfer the phosphate the antibiotic substrate. However, when there is no antibiotic around, it’s possible that a water molecule can sneak into the active site and react with the GTP instead. The result of this interaction is that the GTP is broken down, and no antibiotic is inactivated. This wastes precious GTP for the bacterium, so any enzyme that cuts back on rates of hydrolysis will be preferable for the survival of the cell. This mechanism may have been developed to reduce this energy wastage by the APH(2”)-Ia enzyme. In environments where there is a lot of competition and resources are scarce, enzymes that conserve energy are an enormous benefit for a bacterium.

This switch between stable and activated forms of the GTP molecule turns parts of the enzyme into a molecular motion sensor for the presence of antibiotics. When they aren’t around, the enzyme hangs out and holds on to the GTP, inactive. It’s only when the antibiotic shows up and sticks to a different part of the protein, that the enzyme undergoes changes that activate the GTP. Like a motion sensor-based system to turn your lights off when there’s no one around, flipping between these states might be an interesting way for the enzyme to turn its activity off and conserve energy when it doesn’t have an antibiotic to modify.

So what’s the big picture?

There’s a lot of places this work leads. I’ve skipped over another interesting finding that two different classes of aminoglycoside interact with the protein despite the fact that only one of those classes can actually be modified. I’ll save that for another blog post. There’s also a lot more detail on the specifics of how APH(2”)-Ia works that I’ve glossed over, which we could also explore some time later.

This mechanism isn’t too different from other mechanisms in proteins that carry out various functions in biology. This switch can be considered a type of induced fit or conformational selection of the enzyme, a well-established model for protein behaviour. The thing that makes it different is that this enzyme is an antibiotic resistance enzyme. Usually antibiotic resistance enzymes aren’t thought to be very complicated. They are thought to be inefficient but highly active machines to turn off antibiotics as fast as possible. This work shows us that an antibiotic resistance factor can be modulated and subject to regulation in a way that reduces its energetic waste.

How about the bigger picture?

Taking the inference a step further, this induced activation of the enzyme indicates that there is a greater complexity in the action of this enzyme than we previously might have expected. However, it isn’t as surprising as we might think when we remember that many antibiotic resistance factors have been around for millions of years, with a very long time to optimize their catalytic activity. We treat antibiotic resistance as something that pops up fresh when we use antibiotics, but the truth is, like we’ve discussed before, there are some forms of ancient antibiotic resistance are highly fine-tuned and regulated to respond to challenges in their natural environment.

APH(2”)-Ia is one resistance factor among many. This paper shows us that resistance factors need not just be catalytically optimized machines, that they can contain a degree of fine-tuning that regulates their activity. This nuanced activity is supported by long periods of evolutionary selection to produce highly effective resistance enzymes, ones that lead to terrifyingly effective antibiotic resistant microbes. We now understand one factor a little bit better, and hopefully that helps us just a little bit more in our efforts to beat back the surge of antibiotic resistance.

Citation:

Caldwell SJ, Huang Y, & Berghuis AM (2016). Antibiotic Binding Drives Catalytic Activation of Aminoglycoside Kinase APH(2″)-Ia. Structure, 24 (6), 935-45 PMID: 27161980

Antibiotic Resistance as a Force of Nature

My research focuses on antibiotics – specifically antibiotic resistance. Last week I gave a seminar on my work, which was followed by some excellent questions about the origins and evolution of resistance. While I don’t personally get my hands dirty studying molecular evolution or microbial ecology, I think about these topics often, for a couple reasons. First, the origins and evolution of resistance factors have interesting implications that contextualize the structure and function of resistance factors – it helps me make sense of the molecules I study. Second and more importantly, the evolution of antibiotic resistance gets at a fundamental understanding of the environment and the world around us. We tend to focus on the problems that resistance create in medicine, but in nature, the relationship between microbes is much more complicated than we might assume. Antibiotics and resistance give us a window into the fascinating world of microorganisms and their strange and complicated existence.

In the discussion after my talk, I brought up a recent paper in Nature. In this report, a group at Harvard Medical School modelled the community dynamics of antibiotic-producing and -resistant microbes. The headline finding was that antibiotic production and resistance can stabilize microbial environments. The production and degradation of antibiotics are an intrinsic feature of mature microbial communities.

This seems counterintuitive – how would antibiotics, substances that kill bacteria, bring stability to an ecosystem?

It isn’t as ridiculous as it may sound. While a majority of antibiotics research focuses on the medical applications and repercussions, a less celebrated contingent of microbiologists look at the environmental role of antibiotics. These researchers find that antimicrobials play a more nuanced role than we have been led to believe. Rather than the chaotic battle royale we picture when we think of the microscopic struggle for survival, they find that antibiotic resistance often plays subtler roles in the microbial world. Let’s look into that world.

Microbes live in complex communities, pass toxins and signalling molecules back in forth in a complicated web of interactions. Image adapted from the Lewis Lab at Northeastern University. Image created by Anthony D'Onofrio, William H. Fowle, Eric J. Stewart and Kim Lewis. — **Microbes live in complex communities, pass toxins and signalling molecules back in forth in a complicated web of interaction.**
*Image adapted from the Lewis Lab at Northeastern University. Image created by Anthony D’Onofrio, William H. Fowle, Eric J. Stewart and Kim Lewis.*

What is Antibiotic Resistance?

We should get some definitions out of the way. An antibacterial is a chemical compound that kills or stops the growth of a bacterium. Antimicrobials are less well defined, and include antibacterials as well as antifungal and antiparasitic compounds, sometimes antivirals as well. Antibiotic was coined to specifically refer to a chemical that kills or stops bacteria but doesn’t affect animal cells – a nontoxic antibacterial. It since expanded to include some compounds that also target fungi and protists, but not antiviral compounds. In common speech, “antimicrobial” and “antibiotic” frequently mean “antibacterial”, so I’ll use them interchangeably here.

Antibiotic resistance is a catch-all term we give to many different mechanisms a bacterium can use to survive and/or grow, in the presence of an antibiotic. Every antibiotic has a specific target it interacts with, and anything that keeps the antibiotic and target from binding will result in resistance. Common mechanisms of antibiotic resistance include:

Chemical breakdown of the antibiotic
Molecular pumps that kick it out of the cell
Changes to the microbial target that block the antibiotic
Bypass the target molecule to allow the microbe to grow even when the target is productively blocked

Resistance factors are the molecules that give the bacterium resistance, by any of the above mechanisms. These resistance factors have diverse origins. In some cases, they’re still controversial. But broadly speaking, there’s two types of antibiotic resistance: new resistance and ancient resistance.

Two Origins of Antibiotic Resistance

New resistance makes sense. It is what we think of when we talk about antibiotic resistance as an example of Darwinian evolution. A spontaneous mutation emerges that confers resistance, and selective pressures drive it to succeed and take over the population. This is a common phenomenon that we have seen in the clinic and can induce in the lab, but is far from the only means by which antibiotic resistance happens.

Today I’ll focus on a second type of antibiotic resistance – the transfer of ancient antibiotic resistance factors from environmental bacteria into the strains that cause human disease. It was found in the 1970s that some antibiotic resistance factors appear to come from the microbes that produce the antibiotic. The thinking was that they act as a means of self-protection for the bacteria from their own toxin. This is one origin for resistance factors, although the original origin of many of these environmental resistance factors remain unknown.

Transfer of an environmental resistance factor to disease-causing bacteria results in resistance within the pathogen. In cases where this has happened multiple times, we get superbugs with resistance to multiple antibiotics. The collective environmenal pool of antibiotic resistance factors has been dubbed the “antibiotic resistome“. These resistance factors form a latent environmental reservoir, ready to jump into the strains that make us sick.

Antibiotic Resistance as a Healthcare Menace

Most of our concerns about antibiotic resistance come from the impact it has on medicine. Antibiotics are critical to our treatment of infectious disease, and are also necessary in prophylactic use for surgeries, cancer treatment, neonatal care, and many other intensive medical procedures. The spread of antibiotic resistance in pathogens could remove our ability to treat these infections, or care for many of our society’s most vulnerable members. This could lead to a transition to a “post-antibiotic era” where once again these miracle drugs are not available to us. A minor infection from a scraped knee, sore throat, or scratch off a rosebush could be fatal.

While spontaneous emergence of antibiotic resistance occurs, resistance frequently comes from environmental cross-over. A benign soil microbe meets a pathogen, shares some genetic material, and that pathogen becomes resistant. A notorious recent example of this is the emergence and worldwide spread of the New Delhi beta-Metallolactamase, a resistance factor that knocks out some of our last-resort antibiotics.

In the face of this ongoing menace, what do we do? For decades, the answer has always been “find more antibiotics”. This is important to do, but not enough. We search the world for more obscure microbes that might produce a new antibiotic, and we’re beginning to find a few, but we are still falling behind. Searching for new antibiotics is a game of whack-a-mole, finding new drugs as nature sends more sources of resistance to knock down. We’ve kept up for a while, but now we’re falling behind.

Some of the search for new ways of killing microbes involves looking for new targets to bind antibiotics to – reading the antibiotics literature, everyone and their grandmother wants to sell you a new potential antibiotic target. Other strategies include directly inhibiting antibiotic resistance factors, blocking bacterial toxins to “disarm” the bacterium, or more obscure methods like bacteriophages. But none of these strategies have yet led to sustainable long-term solutions for treating resistance. We may be doomed to fail – we’re going up against a fundamental feature of nature.

Antibiotic Resistance as a Force of Nature

Almost as long as we’ve had antibiotics, we’ve had antibiotic resistance. In his 1945 Nobel Prize Lecture for the 1928 discovery of penicillin, Alexander Fleming said:

It is not difficult to make microbes resistant to penicillin in the laboratory by exposing them to concentrations not sufficient to kill them

Since Fleming’s time, any newly-developed antibiotic has had only a few years of use before a case of antibiotic resistance was identified. This observation alone suggested that antibiotic resistance existed in the environment before we began to use these compounds to cure ourselves. Recent studies of permafrost cores and isolated cave systems have also produced compelling evidence that antibiotic resistance factors have existed in the environment long before humans came around.

The idea of transfer of bacterial resistance factors took some time to catch on. Discovered in the 1950s in Japan, the greater scientific establishment received this finding with disbelief and scorn. It took many additional reports for researchers to believe that resistance could move from one bacterial species to another. By the time the greater scientific community clued in, antibiotic resistance was already running rampant in some clinical strains. Since that point we have been in a constant search for new antibiotics, often falling behind the spread of resistance.

A Microbial Cold War

Starting with the first discoveries of antibiotics, we’ve considered them to be weapons in an ongoing environmental war between microbes. Selman Waksman, Nobel laureate for the discovery of streptomycin*, developed his entire research program on this assumption – that there are microbes in the environment that produce antibiotics in order to kill those around them and gain selective advantage. This program was replicated in many labs, resulting in the 1940’s-1960’s golden age of antibiotic discovery.

Waksman envisioned the environment as an ongoing microbial war. An antibiotic is the bacterium’s sword, resistance is it’s opponent’s shield. But what if that’s the wrong way to think about it?

What if this microscopic battle between bacteria was more of a Cold War? An ongoing stand-off that only occasionally breaks out into active conflict? That’s what microbial researchers seem to keep finding. Microbes play games of rock, paper, scissors using antibiotics. They punish freeloaders, engage in brinksmanship and even cooperate in difficult times. These bacteria are in competition, but this competition generates a kind of rolling stability as they hold each other in check.

Things get even more interesting when we decrease the concentration of an antibiotic below the range at which it kills – to “sub-therapeutic”, “sub-lethal”, or “sub-inhibitory” concentrations. In toxicology, we talk about how “the dose makes the poison” – the same thing applies for bacteria. At low concentrations of antibiotic, bacteria can trigger adaptive stress responses, go into dormant states, adjust their metabolism, trigger complex growth modes (biofilms) or change their behaviour in even more subtle ways. At the extreme of this concentration range, it’s even been suggested that antibiotics might instead be thought of as signalling molecules rather than toxins.

With an environment full of these compounds at various concentrations, microbes are in constant cross-talk with each other. Studies like the most recent paper on community stability find that even in mixtures of a few strains of bacteria, antibiotics and resistance can keep competing stains in check, stabilizing a community and keeping any particular one from growing out of control. With 10-50 000 different species in a single gram of soil, the interrelationships are almost limitless.

As metagenomics studies and other work teach us more about the diversity of resistance in the environment, we find that microbial communities are complex, with different producers and resistant strains in constant rolling flux. Add to this an understanding of things like quorum sensing, and a picture of a complex, dynamic ecosystem emerges. A network of chemical cross-talk forms, and we are only starting to scratch the surface of this environmental complexity.

The use of chemical compounds to influence each other are not an exception, but the rule. The diversity of microbial species also drive a diversity of chemical compounds used to fight, defend, and communicate with each other. These countless microbial compounds have a name: the parvome. We harvest chemicals from this source for use in medicine, but must remain aware that any environmental molecule will also have corresponding mechanisms of resistance.

Rather than the all-out war that Waksman envisioned, the microbial world works like international diplomacy or a financial market. Every interaction trickles through a network and affects everything else. Booms and busts happen, but on average, the system selects for a kind of greater stability, so the entire community gains as a whole. Outright conflict is a zero-sum game. Most microbes prefer a tense collaboration, quietly manipulating their neighbours but avoiding actual battle. A microscopic Cold War. That war only rarely comes to active conflict, when we strip away diversity and release the bacteria from their self-imposed order.

Fear in a Handful of Dust

When we realize that the microbial world works this way, it means that antibiotic resistance is everywhere. Screen any environmental sample for resistance and you’ll find it. If an antibiotic exists, so do its resistance factors. Even antibiotics we haven’t yet discovered have resistance in the environment. This is a fascinating and terrifying thought at the same time.

It’s terrifying because antibiotic resistance is an enormously urgent public health concern. We’re running out of time. And as we understand that resistance is all around us, we realize that we’ll never eliminate it. We can only beat it back, and we can only play the game of antibiotic-resistance whack-a-mole for so long. Resistance seems to be an intrinsic property of the microbial world that we’ll never escape. As a great mathematician once said: Life finds a way.

I’m cynical whenever I see headlines about new breakthrough antibiotics or antimicrobial game-changers. All these do is kick the eventual resistance down the road a little bit farther. Even though I study mechanisms of resistance in hopes of blocking them, I think the most important solutions to antibiotic resistance will come from systems approaches: policy, sanitation, rapid diagnostics/response, and surveillance programs. We can’t control what resistance is out there, but we can take steps to limit the transfer of that environmental resistance to pathogens. Agricultural antibiotic use requires urgent action. Improved sanitation and means of reducing the spread of pathogenic microbes is critical.

As we struggle to deal with our impending antibiotic crisis, we are starting to realize how inevitable it probably was. It emerges from a complicated network of microbial cross-talk. Countless microbes silently jostle against their neighbours, subtly nudging with chemical signals, and being poked back with molecular weapons. This microscopic opera happens around us at all times, silently shaping our world and occasionally making our worst diseases even harder to fight.

The complexity is beautiful, and it is terrible.

_{* It should be noted that Waksman’s student, Albert Schatz, was heavily involved in the discovery of the compound, and by many accounts was snubbed by the Nobel committee when they presented the award to Waksman alone.}

Antibiotic usage regulations – too little, too late?

This post is cross-posted from a blog post I wrote for the Science and Policy Exchange. For more insightful writing on science and how it relates to government, the media, and society at large, visit their site http://www.sp-exchange.ca

In Dr. Seuss’ The Lorax, the narrator reflects sadly upon his past. He recounts arriving at a pristine forest of Truffula trees, which he cuts down to make thneeds, a product that “everybody needs”. Ignoring warnings and later pleadings from the titular Lorax, he cut down trees one-by-one, later 4-by-4, growing his business enormously in the process. He continues to cut down trees until all of the sudden, the last one is gone. Without any more Truffula trees, his business collapses and he falls into poverty, and tells his story so future generations won’t make the same mistake. He realizes how he was blinded by the promise that his business would only grow bigger, and overlooked the fact that he was rapidly removing the key resource he and others depended upon. By the time he realized his wrongs, it was already too late – the forest was gone, along with its animals – the Brown Bar-ba-loots, Swomee-Swans, Humming-Fish, and the Lorax himself.

Sure, The Lorax is a children’s story, but it illustrates some profoundly adult concerns. Take too much from a public good, and it will collapse, leaving everyone worse off in the long term. This story obviously parallels real-world issues like deforestation, pollution, and overfishing, but also applies to abstract public resources like transportation networks, taxation, and stability of financial markets. To protect common resources for the good of the public as a whole, regulations are needed to prevent individuals from exploiting these resources for short-term personal gain.

A somewhat obscure public good, but one that medicine depends upon is the efficacy of antibiotics. Antibiotics are only effective when the microbes they are used to treat are susceptible to the drug. If resistance to antibiotics spreads, antibiotics lose their utility, and this public resource is lost. We need these drugs not just to treat infections, but also to facilitate surgery, cancer therapy, and to protect immunocompromised patients, so losing our effective antimicrobials would be an unmitigated disaster.

Unfortunately, to retain antimicrobial effectiveness, we fight against formidable evolutionary forces. Add a strong selective pressure (antibiotic), and evolution drives the selection of the most fit (resistant) microbes. These resistant microbes will thrive where their non-resistant counterparts die, and take over the competition-free environment left behind. For this reason, we instruct patients to take the full course of antibiotics to completely eradicate an infection. Otherwise it could come back, stronger and tougher, and spread to other patients as well.

This example is usually framed around medical patients, but when we look into the emergence of antibiotic resistance, the more important subjects of antimicrobial use aren’t humans, but chickens, pigs, and cattle. Almost 80% of antibiotics are used in agriculture. These antibiotics are used in three ways: to treat acute infections, in feed as an additive for “prevention and control”, and lastly for use as growth promoters. The last usage doesn’t make much sense – why would chemicals that kill microbes alter the growth of livestock? The short answer is that don’t really know, this question is at the forefront of the burgeoning field of microbiomics. What matters for farmers and feedlot managers, however, is that it works, and their animals grow faster, bigger, or both, which means better profitability. It makes perfect economic sense to routinely add antibiotics to feed as growth promoters.

The problem is that this constant background of growth-promoting antibiotics in agriculture generates a selective pressure that drives the emergence and spread of antibiotic resistance. This was long suspected based upon our understanding of natural selection. Careful research has now shown that indeed, these growth-promoting antibiotics lead to increased emergence and spread of antimicrobial-resistant bacteria, and further, that these resistant bacteria cross over to human patients. Knowing this to be true, the widespread use of antimicrobials for growth promotion in agriculture is now viewed as a considerable public health hazard.

Recognizing this hazard, in 1977 the US Food and Drug Administration (FDA) committed to putting restrictions on antibiotic use in agriculture, a crucial step to limit the spread of resistance. The agency took the first step last December, 36 years later. Their Final Guidance for Industry 213 recommends that antimicrobial manufacturers discontinue indication of antibiotics as growth promoting agents. Some are celebrating this as an important first step. Many are rolling their eyes at what appears like a mostly empty gesture.

The FDA recommendation took far too long to happen. It also lacks teeth. First, it is voluntary, and it doesn’t seem likely that many manufacturers will embrace the opportunity to cut their revenues in half. Farmers operate on such narrow margins that discontinuing antibiotic use could force them out of business, and so change won’t happen there either. The recommendation also has a gigantic loophole: by simply rebranding antibiotic use from “growth promotion” to “prevention and control”, farmers and feedlot managers can continue business, fully compliant with the recommendation, without changing their de facto usage at all.

The FDA recommendation is a first step, but it’s a feeble one, and one that took far too long to happen. A knot of competing interests seems to be preventing much from happening. The medical community and food purity movements want antibiotics removed from agriculture. While the FDA seems to concur, its hands are often tied – the agency is caught between protecting public health and strong economic incentives to keep the status quo in agriculture. The US congress has meddled considerably in the matter, and no doubt lobbyists are pulling strings along the way. Comparatively low food costs have also contributed to the problem, as they push producers to squeeze every drop of efficiency they can from the system, growth promoters forming an important part of their plan. Given these opposing forces, it becomes less surprising that it took the FDA 36 years to publish their recommendation, but at this pace, by the time any substantial change happens, it may be far too late.

The Lorax in this story is the scientific and medical community, who have been pleading with industry for decades to adopt more responsible agricultural antibiotic practices. Individuals within agriculture, like Dr. Seuss’s tragic narrator, are mostly concerned with day-to-day operation of their business, and not esoteric concerns about the future of medicine. But as a collective, the industry needs to realize the loss of antibiotics will have long-term consequences for their business as well and take steps towards more sustainable use. In the absence of this kind of industry action, firm regulations on responsible antibiotic use are essential to protect antibiotics as a public resource. Otherwise, our antibiotics will drop one-by-one from pharmacists’ shelves like Truffula trees, until none remain, and like the animals in the Lorax’s forest, other life-saving innovations of medicine could be lost as well.