Some strains of nasty bacterial infections, such as MRSA (methicillin-resistant Staphylococcus aureus), come loaded with resistance to antibiotics built right into their genes. But certain infections seem to acquire an ability to persist in the face of drugs that should knock them out—without developing the genetic hallmarks of antibiotic resistance. For decades, researchers have thought this holdout occurred because many antibiotics target cell growth, so even though most of the bacteria were killed by the drug, a select group simply shut down, going into a sort of hibernation, thereby allowing the infection to persist. In other words: if the bacteria aren't growing, they're also not dying.
But a new study suggests that quite the opposite is occurring: some surviving bacteria are actually flourishing and multiplying while under antibiotic attack. The findings were published online January 3 in Science.
"We thought that surviving bacteria made up a fixed population that stopped dividing," Neeraj Dhar, of the Swiss Federal Institute of Technology in Lausanne and study co-author, said in a prepared statement. Instead, a stable overall population was hiding a "very dynamic" colony, he said.
The researchers studied Mycobacterium smegmatis, a species closely related to the bacterium that causes tuberculosis (Mycobacterium tuberculosis), which often resists antibiotic treatment and remains a major health threat in many countries. The traditional analysis of a persistent culture of these bacteria would reveal that the population was not growing, which is what had led scientists to think that the colony had been reduced to non-proliferating "persister cells" that could better ride out the antibiotic attack by laying low. But using time-lapse images taken through a microscope of cells in a microfluidic culture (allowing study of small-scale changes), the researchers saw quite a different story.
"Using microfluidics, we can now observe every bacterium individually, instead of having to count a population," John McKinney, also of the Swiss Federal Institute of Technology, said in a prepared statement. Not only were the surviving cells not playing dead, they were just as likely to be growing as cells that died off.
McKinney and his team observed that even after the introduction of an antibiotic and the death of most bacterial cells, a large percentage of the so-called persister cells continuing to divide—129 of 153 progenitor cells they followed divided at least once in the face of antibiotic treatment. And this cycle of growth, division and death kept up for at least 10 days of exposure to the antibiotic.
The antibiotic was isoniazid (known by the drug names Laniazid and Nydrazid), which has been a common first-line treatment for tuberculosis. This antibiotic becomes an activated bacterium killer when it comes into contact with an enzyme called KatG that the bacteria produces. The enzyme, however, was not produced consistently, the researchers found. Instead, individual cells generated it in seemingly random spurts. So the cells that happened to have had pauses in their KatG production at just the right time were often able to avoid activating the antibiotic—and thus were saved from certain death.
Tuberculosis cells that should have been essentially identical, genetically, showed different propensities for survival, pointing to a possible role of epigenetic differences (such as those in gene expression) in determining cell survival. "This diversity is critical for microbial persistence in fluctuating environments because it ensures that some individuals may survive a lethal stress that would otherwise extinguish the population," the researchers explained in their paper.
Because cell survival does not seem to be tied to permanent genetic change in this case, it means the bacterial colony should remain susceptible to future antibiotic treatment, which could be good news for treating infections. However, given the low level of continued growth and change during exposure to antibiotics, "the bacteria can mutate and thus develop resistance in the presence of the antibiotic," Dhar said.
This discovery now paints a clearer picture of how antibiotic resistance can develop in persistent bacterial infections. With so many individual bacteria reproducing, "some of them can adapt to stressors that they have not previously encountered, thanks to the selection of persistent individuals," McKinney said. Such findings could help inform the creation of more effective antibiotics and perhaps expand to other illnesses, such as the persistence of cancer cells, although the researchers acknowledge that the persistence behavior of other bacterial infections might be quite different.
Nevertheless, the insights offer “a new approach for trying to figure out why some infections are so difficult to eliminate," McKinney said.