Scientists capture live images of bacteria battling antibiotics

Physicians have relied on a class of antibiotics called polymyxins to fight potentially life-threatening Gram-negative bacterial infections for more than 80 years. These drugs are typically reserved as the last line of defense. Even after all those years on the shelves, however, scientists never fully understood how polymyxins work: killing off target bacteria. A new study is shaking what we thought and revealing why the antibiotics fail to act.

The research, conducted by scientists at University College London and Imperial College London, shows that polymyxin B is not acting alone. Instead, the antibiotic hijacks the energy and metabolism of the bacteria to breach their outer defenses. That surprising twist might be able to explain why such drugs are so potent in the laboratory, but do not live up to expectations against long-term infections in the clinic.

Gram-negative bacteria are especially hard to treat because they have two layers of protection. The outer membrane, composed primarily of molecules called lipopolysaccharides (LPS), is a suit of armor that shields the cell from antibiotics. Polymyxin B has long been known to target this armor, but how it gets in and kills the cell remained a mystery.

To have a grasp of it, the researchers used advanced imaging methods to watch polymyxin B in action. By means of atomic force microscopy, a needle only a few nanometers in thickness "felt" the outside of intact E. coli cells, revealing what happened when the antibiotic struck. Within minutes, the smooth bacterial surface broke out into little bumps and bulges. Soon afterward, the cells began shedding bits of their armor.

At first glance, it appeared as though polymyxin B was simply battering down the wall using brute strength. But the researchers noticed something peculiar: the effect only happened when active bacteria were present. The resting cells, which had shut down most of their internal functions, didn't respond. This suggested polymyxin B wasn't simply pushing its way in. It was, in a sense, tricking the bacteria into helping it get the job done.

To determine whether the bacterial activity was involved, the scientists compared a growing E. coli with a resting one and treated both with polymyxin B at levels comparable to those administered to patients. The growing cells were killed instantly regardless of where they were. But the resting cells were killed only when glucose, a monosugar, was added to the solution. Without added sugar, the antibiotic barely affected them.

Throwing in a sugar that bacteria wouldn't be able to metabolize made no difference. That confirmed what the scientists had suspected: polymyxin B needs energy from the bacteria themselves in order to reach through the outer defenses. Specifically, the drug is based on a mechanism by which the bacteria shed LPS molecules from the outer membrane. Without energy to power that shedding, the antibiotic gets stuck outside.

"It was incredible to see the antibiotic's effect on bacteria's surface in real time," said Carolina Borrelli, a PhD student at UCL's London Centre for Nanotechnology. "It's as if the cell is forced to produce bricks for its outer wall at such an incredible rate that the wall collapses, with gaps for the antibiotic to get through."

What the research found is that killing by polymyxin B is a two-stage process. Step one is that the bacteria burn energy to dislodge LPS from their outer membrane. That compromises the protective covering. Step two, and only then, can the antibiotic sneak in and disrupt the inner membrane and kill the cell. Step two is an energy-free process-but without step one, it can't occur.

That is why polymyxin B generally does not succeed in the event of body infections. In true infections, the majority of bacteria are dormant. Some become dormant and remain low in expectation that things will improve. These resting bacteria are not sufficient to generate enough energy for LPS shedding, and so the antibiotic fails to finish the job. That allows bacteria to survive treatment and then "awaken" and create new infections.

“For decades we’ve assumed that antibiotics that target bacterial armor were able to kill microbes in any state,” said Dr. Andrew Edwards of Imperial College London. “But this isn’t the case. Our images show that these antibiotics only work with help from the bacterium. If the cells go into a hibernation-like state, the drugs no longer work—which is very surprising.”

Aside from this natural tolerance, bacteria have evolved genes to render polymyxins even weaker. The most feared among them is mcr-1, which modifies the LPS molecules by adding a chemical group to them that blocks polymyxin binding. When the researchers exposed bacteria with mcr-1 to the drug, the medication was still able to bind on to the cells, but failed to induce the cells to release LPS. Without the initial step, polymyxin B would be incapable of reaching the inner membrane, and the bacteria would not be damaged.

UCL Professor Bart Hoogenboom said, "Polymyxins are an essential defense against Gram-negative bacteria, which cause most lethal drug-resistant infections. It is vital we know how they work. The next challenge for us is to take these discoveries and make the antibiotics work better."

The discovery that polymyxin B requires bacterial energy in order to work suggests new ways to make the drug more effective. One suggestion is to combine it with drugs that "wake up" dormant bacteria and drive them into a state of activity where the antibiotic can take effect. Another strategy is to design helper drugs that destabilize the outer membrane by direct means, independent of bacterial energy.

Noted Nottingham University professor Boyan Bonev, and one of the authors of the study, said, "Collaboration has provided us with long-hushed secrets for decades about bacterial physiology and morphology when under stress. Now we know more about bacteria's weak points."

The results reveal in this study can be meaningfully added to changes in how polymyxins are utilized by doctors to treat infections. The fact that they depend on bacteria activity is the reason they sometimes fail with patients, especially with resting cells.

Clinicians in the future may combine polymyxins with drugs that augment bacterial metabolism or destabilize the outer membrane more directly. These findings also offer new ways to circumvent resistance genes like mcr-1, which block the crucial step of LPS loss.

As a whole, this work brings us closer to preserving the usefulness of polymyxins—one of the fallback defenses against multidrug-resistant bacteria that already kill more than a million people on the planet every year.

Research findings are available online in the journal Nature Microbiology.

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