Skin Of Frog

E. coli has evolved its resistance to antibiotics almost entirely out of view. It was not trapped in a laboratory flask, where a scientist could track every mutation from one generation to the next. Its flask was the world.

The pieces of evidence scientists have assembled are enough for them to reconstruct some of its history. The genes that now provide E. coli with resistance to antibiotics did not suddenly appear in 1950. They descend from older genes that originally had other functions. Some of the pumps that flush antibiotics out of E. coli probably evolved from pumps bacteria use to release signaling molecules. Others originally flushed out the bile salts E. coli encounters in our guts.

When E. coli first encountered antibiotics, its pumps probably did a poor job of getting rid of them. But on rare occasion the genes for the pumps mutated. A mutant microbe might pump out antibiotics a little faster than others. Before modern medicine, such mutants wouldn't have been any better at reproducing than other bacteria. Their mutations might even have been downright harmful. But once they began to face antibiotics on a regular basis, the mutants had an evolutionary edge.

That edge may have been razor thin at first. Only a few of the resistant mutants might have survived a dose of antibiotics, but that was better than getting exterminated. Over time, resistant mutants became more common in populations of E. coli. Their descendants acquired new mutations that made them even more resistant. In 1986, scientists discovered strains of E. coli that made an enzyme able to destroy a group of antibiotics called aminoglycosides. In 2003, another team discovered E. coli carrying a new version of the gene. It had two new mutations that made it resistant not just to aminoglycosides but also to a completely different antibiotic, called ciprofloxacin.

Even within a single person, E. coli can evolve to dangerous extremes. In August 1990, a nineteen-month-old girl was admitted to an Atlanta hospital with a fever. Doctors discovered that E. coli had infected her blood, probably through an ulcer in her intestines. Tests on the bacteria revealed that they were already resistant to two common antibiotics, ampicillin and cephalosporin. Her doctors gave her other antibiotics, each more potent than the last. Instead of wiping out her E. coli, however, they made it stronger. It acquired new resistance genes, and the ones it already had continued to evolve. After five months and ten different antibiotics, the child died.

Terrifying failures like this one leave scientists hoping that someday they will find new antibiotics that are immune to the evolution of resistance. Like Fleming before them, they find promising new candidates in unexpected places. One particularly promising group of molecules was discovered in 1987 in the skin of a frog.

Michael Zasloff, then a research scientist at the National Institutes of Health, noticed that the frogs he was studying were remarkably resistant to infection. At the time, Zasloff was using frogs' eggs to study how cells use genes to make proteins. He would cut open African clawed frogs, remove their eggs, stitch them back up, and put them in a tank. Sometimes the water in the tank became murky and putrid, yet his frogs—even with their fresh wounds—did not become infected.

Zasloff suspected the frogs were making some kind of antibiotic. He ground up frog skin for months until he isolated a strange bacteria-killing molecule. It was a short chain of amino acids known as a peptide. He and other researchers discovered that it is fundamentally different from all previously discovered antibiotics. It has a negative charge, which attracts it to the positively charged membranes of bacteria but not to the cells of eukaryotes such as humans. Once the peptide makes contact with the bacteria, it punches a hole in their membranes, allowing their innards to burst out.

Zasloff realized he had stumbled across a huge natural pharmacy. Antimicrobial peptides, it turned out, are made by animals ranging from insects to sharks to humans, and each species may make many kinds. We produce antimicrobial peptides on our skin and in the lining of our guts and lungs. If we lose the ability to make them, we become dangerously vulnerable. Cystic fibrosis may be due in part to mutations that disable genes for antimicrobial peptides produced in the lungs. The lungs become loaded with bacteria and swell with fluid.

Having discovered antimicrobial peptides, Zasloff now tried to turn them into drugs. They might be able to wipe out bacteria that had evolved resistance to conventional antibiotics. Antimicrobial peptides might even be resistance proof. In order to become resistant to antimicrobial peptides, bacteria would have to change the way they build their membranes. It was hard to imagine how bacteria could make so fundamental a change in their biology, and experiments seemed to back up this hunch. Some scientists randomly mutated E. coli to see whether it could produce mutants able to survive a dose of antimicrobial peptides. No luck.

But an evolutionary biologist named Graham Bell at McGill University in Montreal suspected that E. coli—and its evolutionary potential—might be more powerful than others had thought. Michael Zasloff, for one, didn't think so. But as a good scientist, he was willing to put his hypothesis to the test. He teamed up with Bell and Bell's student Gabriel Perron to run an experiment. Remarkably, his hypothesis failed.

The researchers began by exposing E. coli to very low levels of an antimicrobial peptide. A few microbes survived, which the scientists used to start a new colony. They then exposed the descendants of the survivors to a slightly higher concentration of the antimicrobial peptide. Again most of the bacteria died, and they repeated the cycle, raising the concentration of the drug even higher. E. coli turned out to have a remarkable capacity to evolve. After only six hundred generations, thirty out of thirty-two colonies had done the impossible: they had become resistant to a full dose of antimicrobial peptides. These results raise some serious concerns about how effective antimicrobial peptides will be when they hit the market. E. coli and other bacteria that are hit by low doses of antimicrobial peptides may evolve resistance. If they do, they will survive stronger and stronger doses until they can withstand the full strength of these drugs.

If E. coli can evolve resistance to antimicrobial peptides so quickly, then how did they protect Zasloffs dirty frogs? E. coli and other bacteria are locked in an evolutionary race with the animals they colonize. When an animal evolves a new antimicrobial peptide, natural selection favors microbes that can resist it. One common counterstrategy is for a microbe to make an enzyme that can cut the new peptide into pieces before it is able to do any damage.

Now the evolutionary pressure shifts back to the animal. Mutations that allow an animal to block the peptide-cutting enzyme may allow it to survive infections. It will pass down the mutation to its descendants. Animals defend against peptide-slicing enzymes by stiffening the peptides. The peptides are folded over on themselves and linked together with extra bonds. But microbes have evolved counterstrategies of their own. For example, some species secrete proteins that grab the antimicrobial peptides and prevent them from entering the bacteria.

One of the most potent ways for animals to overcome all of these strategies is by making lots of different kinds of antimicrobial peptides. New ones can be produced by gene duplication or by borrowing peptides with other functions. The more antimicrobial peptides an animal makes, the harder it is for bacteria to recognize them all. Thanks to this arms race, the genes for antimicrobial peptides have undergone more evolutionary change than any other group of genes found in all mammals.

Compared with this complex, ever-changing attack on antimicrobial peptides, Bell and Zasloffs experiment is child's play. They exposed E. coli to a single kind of antimicrobial peptide and created a strong advantage for mutants that could withstand it. Unfortunately, modern medicine works more like Bell and Zasloffs experiment than like our own evolution. Doctors have only a few antibiotics to choose from when fighting an infection, and they generally prescribe only a single drug to a patient. In a few years this practice gives us resistant bacteria. We might do a better job of fighting bacteria if new drugs came through the development pipeline faster and if doctors could safely prescribe several of them at once.

There are many lessons to be learned from E. coli's quick evolution of resistance. The most surprising of all is that our own bodies, and those of our ancestors, are actually drug-development laboratories.

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