Many patients die due to bacterial infections. Almost 10 thousand deaths occur due to antibiotic-resistant bacteria. Understanding how antibiotics work is difficult for creating effective treatment procedures both to target new superbugs and to make existing medications more viable against their objectives.

Scientists at the Wyss Institute at Harvard University have recently discovered that these bacteria respond very distinctly to antibiotics. They act exactly opposite to them. Indeed, scientists suggest that some of our current assumptions about antibiotics may be incorrect.

According to most of the clinicians, antibiotics work by killing actively dividing bacteria. And those nondividing bacteria resist treatment and cause infections to persist.

Assumptions of How Antibiotics Work may be Incorrect
Credit: Wyss Institute at Harvard University
“The image most clinicians have is that antibiotics work by killing actively dividing bacteria, and nondividing bacteria are the ones that resist treatment and cause infections to persist,” said Laura Certain of the Wyss Institute. “I wanted to know whether that’s actually true.”

Laura Certain, a clinical fellow said, “I wanted to know whether that’s actually true, does the proportion of dividing bacteria change over the course of an infection, and how do antibiotics impact that?”

“Synthetic biology is widely used to engineer bacteria so that they produce useful products or diagnose diseases, and we used that same approach to create a microbiology tool that can tell us how bacteria are behaving in the body.”

Scientists mainly used a genetically engineered strain of E. coli bacteria. The bacteria actually composed of a genetic toggle switch encoded into their DNA that changes from the ‘off’ to the ‘on’ position when the bacteria are exposed to a chemical called anhydrotetracycline (ATC).

When the switch is ON, a genetic change occurs that makes the bacteria to digest the sugar lactose. The key to this system is that the toggle switch can only be flipped if the bacteria are actively dividing when ATC is added. In the case of nondividing bacteria, the toggle switch remains OFF. Thus, the toggle switch offers a snapshot in time that can indicate whether bacteria were active or dormant at the moment of ATC exposure.

Scientists evaluated their bacteria in vivo, where they embedded a small plastic rod into the legs of mice. They then inoculated their built bacterial strain into the legs to mimic the incessant bacterial contaminations. This usually emerges in people when medicinal gadgets and simulated joints are embedded.

Assumptions of How Antibiotics Work may be Incorrect
Colonies of engineered E. coli bacteria that were actively dividing at the time ATC was added turn blue when grown on a medium containing lactose, while those that were not dividing when ATC was added remain white. Credit: Wyss Institute at Harvard University

Later on, they injected the mice with ATC at different times throughout the course of the infection to flip the toggle switch in any dividing bacterial cells to the “on” position. After extracting bacteria from mice, they found that all the bacteria were actively dividing for the first 24 hours. But, after 4 days, they found that the fraction dropped to about half. It next remained the same for rest of the infection.

Means, the number of bacteria being killed by the body was balanced by new bacteria created by cell division. This result differed from the in vitro response, in which all the bacteria stopped dividing once they reached the carrying capacity of their environment.

Scientists then tested the bacteria in contrast to antibiotics. They then injected the mice with the antibiotic levofloxacin. After analyzing, they found that while the total amount of bacteria in the mice decreased. Additionally, the proportion of the living bacteria were actively dividing actually increased. It kills dividing cells more than nondividing cells.

While screening the bacterial colonies, scientists did not find any evidence that the bacteria had evolved to better withstand the killing effects of the levofloxacin.

Certain said, “There are several possible reasons why we saw a higher proportion of dividing bacteria in the presence of an antibiotic. We find it most likely that dormant cells are switching into an active state in order to ‘fill the gaps’ that arise when antibiotics reduce the overall bacterial population. If bacteria continue to actively divide throughout an infection, as our study suggests, they should be susceptible to antibiotics.”

Jim Collins said, “If an antibiotic isn’t working, we should focus on finding ways to deliver more of it to the infection site or identifying other tolerance mechanisms that might be at play, rather than assuming that a bastion of nondividing bacteria is the culprit.”

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