Study reveals unexpected mechanism driving extreme antibiotic resistance

A patient with a severe infection is prescribed a potent antibiotic. Hopes for improvement are high, but just after treatment is given, resistance to the drug increases thousands of times. How could this happen?

Scientists at the University of Washington School of Medicine report an unexpected mechanism in a paper published July 23 in Nature Microbiology.

Resistance developing inside patients was thought to be caused by the accumulation of mutations in the bacterial genome. Because this process is gradual, physicians have time to react."

Dr. Sardar Karash, research assistant professor of microbiology and study's lead author

However, the researchers noticed people with cystic fibrosis, or CF, who did not fit this pattern. People with CF develop devastating lung infections and get intensive antibiotic treatment. In some patients antibiotic resistance increased by more than 10,000-fold, right after antibiotics were started.

This didn't make sense. The researchers wanted to find out how this could happen.

Karash sequenced hundreds of bacteria isolated from patient samples to look for mutations that might explain the extreme resistance. He came up empty-handed.

"The approach we used was state of the art," said Karash, "but it sequences short DNA strands and then strings them together. This is great for finding mutations, but it can miss new pieces of DNA that are acquired."

On a hunch, Karash switched strategies. He used a method that sequenced long, continuous DNA strands.

"That gave us the answer," said Dr. Colin Manoil, a research professor in genome sciences who collaborated on the project. "The long-strand sequencing showed that the lung pathogens had acquired circular pieces of DNA called plasmids that included new resistance genes."

Karash inserted the new genes into drug-sensitive bacteria and resistance jumped.

But how did these plasmids enter patients' lungs and get inside the bacteria?

"Many bacteria can't take up plasmids on their own, so we suspected they had help," said Dr. Pradeep Singh, professor of microbiology and medicine and the senior author of the study.

The team then studied other bacteria collected from the patients. The researchers observed that environmental bacteria transiently appeared in lung samples from some patients' right before resistance developed.

Karash sequenced these bacteria and found the smoking gun: The environmental bacteria contained the identical resistance-producing plasmids. He then found they could transfer the plasmids to antibiotic-sensitive pathogens that had already established infections in the patients' lungs. This instantly increased antibiotic resistance thousands of times.

"We see environmental bacteria in patient samples from time to time," said Singh, "but we thought they were pretty harmless as they aren't very virulent and only appear transiently."

Now the researchers think differently.

"If environmental bacteria can ferry resistance genes inside human organs, we have to worry about what other genes might be transferred," Karash said.

"Environmental bacteria are highly diverse," added Singh. "They can carry genes that can do almost anything."

In addition to antibiotic resistance, genes from environmental bacteria could help pathogens acquire more nutrients, block immune responses, and break down barriers that limit the spread of infection.

The researchers hope approaches like environmental monitoring and containment, as well as new strategies to block gene transfer, could mitigate this danger.