A bug’s journey

So you think you have food poisoning.

You go to the doctor. The source? Salmonella. Do you go home with an antibiotic? Hopefully not, said Jorge Galán, Ph.D., D.V.M., chair and the Lucille P. Markey Professor of Microbial Pathogenesis and professor of cell biology. The frequency of antibiotic resistance tends to be high, Galán said, so there is a chance that such a drug may not work. More importantly, an antibiotic will wipe out your intestinal flora—the gut bacteria that otherwise would compete with the pathogenic intruders, leaving the resistant bugs to thrive unimpeded. A good doctor will not prescribe antimicrobials in this case, Galán said, because that could turn a few days’ discomfort from a self-limiting infection into a lethal outcome.

It’s for that same reason that if you do end up needing antibiotics, doctors urge you to finish the course even after you feel better in order to kill every last bug. Not doing so could leave you—and the world—with resistant infections.

From the human perspective, the emergence of antimicrobial resistance is a public health nightmare. For microbes, however, the ability to flourish in the face of a drug challenge is a success story and one of resilience: the ability to bounce back from challenges.

We tend to think of human resilience as the way in which individuals recover from such setbacks as the loss of a job or a loved one. Microbial resilience, in contrast, operates at the population level. When faced with an environmental change or selective pressure, individual microbes that are genetically unprepared simply die—but those that remain, such as the resistant Salmonella enterica Galán speaks of, are suited to their circumstances and can flourish, benefiting the microbial population. Through natural selection, bugs can evolve to withstand almost anything.

“Masters of evolution”

Galán likes to engage his students in a thought experiment. If you wanted to perpetuate your genes forever, he asks, and could choose between having a brain the size of Connecticut or being able to reproduce every 20 minutes, which would you pick? “To me, it’s a pretty easy choice,” Galán said. “The replication every 20 minutes combined with genetic plasticity to change the genetic content will ‘outsmart’ the brain the size of Connecticut. We are no match for these creatures.”

Bacteria exhibit impressive genetic diversity, but such RNA viruses as hepatitis C, poliovirus, influenza, and HIV are diverse to a gargantuan degree. Brett D. Lindenbach, Ph.D., associate professor in the department of microbial pathogenesis, calls RNA viruses “masters of evolution.” Their populations are so varied that no matter what situations they face, some will have the genetic makeup to survive.

At work behind this microbial resilience are the concepts of genetic diversity followed by natural selection. We often think of mutations as mistakes and sources of human disease, but they are simply genetic changes, and sometimes they are beneficial. Mutations are in fact critical to microbial survival, assuring that in any population at least one bug will survive any situation. Bacteria and viruses share high genetic diversity, but they achieve it in different ways.

First, some background. Bacteria have DNA genomes, the genetic blueprints that they pass on to their offspring; viruses may have either RNA or DNA genomes. Lindenbach studies RNA viruses. The enzymes that make copies of the genetic material, either RNA or DNA, during microbial replication are called polymerases.

How mistakes make bugs stronger

To generate diversity, RNA viruses like those Lindenbach studies depend largely on replication mistakes. The RNA polymerase of the hepatitis C virus, for example, makes so many mistakes that every new virus particle produced contains on average one mutation. “That’s an incredibly high error rate,” Lindenbach said. In comparison, the error rates for bacterial DNA polymerases are 1,000 times smaller.

Because of this high error rate, the evolution of RNA viruses diverges from the traditional “survival of the fittest” model, which predicts that for any gene, the version that confers the greatest selective advantage will become dominant. But that’s not how it works for RNA viruses. Because their polymerases are so prone to error, rather than having a single “correct” sequence, RNA viruses exist in what microbiologists call “swarms” or “clouds,” hovering around theoretical master sequences but constantly deviating from them, the way individual bees in a swarm shift in space relative to the general path of their group.

“RNA viruses really are masters of evolution,” Lindenbach said, “because [in] fairly short generation times they give rise to large populations of divergent daughter genomes, and then evolution can just take over.”

Not only do RNA viruses tolerate their faulty polymerases; they actually depend on their “mistakes.” Lindenbach noted that the Kirkegaard laboratory at Stanford compared the survival of different poliovirus clones whose polymerases had different error rates. The researchers found that mistakes were valuable to the viruses. “The idea is that the virus needs a certain amount of variability in order to adapt to the environments that it’s encountering in an animal,” Lindenbach explained.

Beyond mutation, bacteria have additional ways to achieve genetic diversity that viruses lack. Different strains of bacteria can exchange genetic material with each other in what’s called horizontal gene transfer—horizontal because it occurs within a generation rather than during reproduction. There are three ways in which horizontal gene transfer can happen: transformation, in which a bacterium takes up a closed loop of DNA called a plasmid from the environment; transduction, in which a bacteria-infecting virus, a bacteriophage, moves DNA from one bacterium to another; and conjugation, in which two bacteria connect physically and DNA passes from one to the other. Bacteria use all three methods to achieve genetic diversity, Galán said, making mutation relatively less important to bacteria in comparison to RNA viruses.

Then there is the issue of replicating every 20 minutes—or rapidly, anyway. The more frequently microbes replicate, the more opportunities they have to experiment with genetic possibilities and the faster they can adapt. It’s hard to compare the replication rates of viruses and bacteria, Lindenbach said; they replicate very quickly within a cell, but outside their hosts they can spend periods of time not replicating at all. In the scheme of things, viruses also have short life cycles.

Though genetic diversity is useful overall, individual mutations are usually harmful. For example, a study of one RNA virus, vesicular stomatitis virus, found that 60 percent of mutations were deleterious. There is, therefore, a limit to the frequency of mistakes that RNA viruses can tolerate. If viruses accumulate so many mistakes that they destroy their essential genes, Lindenbach said, they will go extinct in what’s termed an error catastrophe.

Finding unknown strengths

It’s a common theme in movies. Our heroes live quiet and unremarkable lives until disaster strikes. Suddenly, the characters find strength that they didn’t know they had. In an analogous way, the importance of genetic diversity becomes clear when environmental conditions change and the pressure of natural selection is applied. The most familiar kind of selection, and the one that gets public health attention, is that of an antimicrobial drug.

Resistant strains of tuberculosis, a disease that is topped only by HIV in the number of people it kills annually worldwide, emerged as soon as doctors began treating TB with antibiotics in the 1940s. Since then, drug resistance has spread in frequency and geographic scope—450,000 cases of multidrug-resistant TB were reported around the world in 2012. Drug-resistant strains of malaria, gonorrhea, and Staphylococcus aureus (better known as MRSA) also threaten global public health. Take it from the World Health Organization (WHO): Antibiotic resistance is “a problem so serious that it threatens the achievements of modern medicine,” states a 2014 report on antimicrobial resistance. “A post-antibiotic era—in which common infections and minor injuries can kill—far from being an apocalyptic fantasy, is instead a very real possibility for the 21st century.”

Of course, not only bacteria but also RNA viruses like hepatitis C become resistant to drugs. For example, nucleoside analogs are one class of drugs that have been developed against hepatitis C; they work by interfering with the viral RNA polymerase. Mutations in the polymerase that prevent the drugs from binding can confer resistance to the drugs, Lindenbach said.

There is a silver lining, however, because in this case, the mutations that confer drug resistance come at a cost to viral fitness. “Those mutations also attenuate the normal function of the polymerase, even in the absence of the compound,” Lindenbach said, “so that those viruses that have that mutation are resistant to the polymerase, but they’re very weak viruses.”

Hope for future antimicrobials

Galán believes that, in the future, antimicrobial drugs will be designed not to test microbial resilience. “In terms of dealing with pathogens,” he said, “a strategy that is beginning to gain some traction, and that some of us have been preaching for a long time, is instead of developing drugs to kill the bug, the notion is to develop drugs that will hamper its ability to cause disease and then let our defense mechanisms deal with it.” By chance, some bugs may well resist the drugs, but others will be susceptible. The key is that with this new kind of drug, the resistant microbes will not be the only ones left alive, and they will therefore not come to dominate the way they would under the selective pressure of a microbe-lethal agent.

Designing drugs that tweak microbial machinery to prevent disease will require a specific understanding of the way in which that machinery works. This is exactly what Lindenbach and Galán spend their days doing. “As we know more and more how pathogens engage the host, cause disease, enter cells, attach to cells,” Galán said, “then we can develop highly specific drugs that will target those abilities.”