Bacteria go dormant to survive antibiotics and restart infections

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Salmonella living in macrophages can survive antibiotic treatment and potentially give rise to resistance by two different mechanisms that slow or arrest their growth.

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Antibiotic-resistant bacteria thrive in the face of drug treatment, which can lead to life-threatening and often incurable infections. Other bacteria may exist in a pre-resistant state. To survive, these bacteria slow their growth in response to antibiotics, allowing mutations to appear that help insects tolerate the drug, or alter their phenotypes to help them persist in its presence. After the antibiotic treatment is finished, the few remaining bacteria can grow again, reestablishing the infection.1

Infections that cannot be treated are a significant problem. To research ways to combat this growing health threat, Peter Hill and his colleagues at Harvard Medical School explained how antibiotic tolerance and persistence occur.2 They recently reported in Cell host and microbe this tolerance comes from mutations in genes related to nutrient production while persistent bacteria activate a specific DNA repair pathway to survive.

“Antibiotic resistance is, in some ways, the final stage of a problem,” said Peter Hill, now a researcher at Imperial College London and co-first author of the recent work. “If you can stop these…initial processes from happening, you can stop the final process.”

Play dead to survive

To find out which mutations were responsible for tolerance, Hill’s team infected macrophages with a strain of Salmonella which causes recurrent diarrheal diseases,3 induced tolerance by exposing them to antibiotics and sequenced the genomes of surviving bacteria. They found mutations that prevented bacteria from making certain molecules essential to life. Bacteria that cannot grow these compounds slowly, which makes them survive antibiotics that target dividing cells. Since tolerant bacteria only grow in nutrient-rich environments, Hill found that tolerant bacteria Salmonella were sensitive to antibiotics once they moved to favorable conditions.

Next, the team studied antibiotic persistence, which results from a phenotypic change that temporarily slows or stops the growth of a small portion of antibiotic-susceptible bacteria. Like tolerant insects, persistent bacteria grew slowly, if at all, in macrophages treated with antibiotics. But the researchers found that persistent Salmonella became alive in macrophages once they cleared antibiotic stress.

Because persistence arises from a phenotypic change rather than a mutation, Hill and his colleagues performed RNA sequencing on persisters to see how antibiotic treatment affected gene expression in this dormant population. They observed an increase in a bacterially-induced stress-response pathway in response to DNA double-strand breaks (DSBs), a common side effect of genome replication in hostile macrophages.

“It’s generally assumed that when you have DNA replication, bacteria will then go through cell division,” Hill said. “We found that DNA replication, or at least a form of DNA replication, occurs in these non-proliferating bacteria.”

Additionally, Hill’s team found that to survive against drugs and macrophages, Salmonella the persisters required a DNA repair mechanism to activate the stress response and repair the DSBs. After damage repair, persisters reset infection in new host cells, which mimics recurrent clinical infections.

From laboratory to clinic

Testing macrophages in a lab offers a lot of information, but Hill and his team wanted to know what happens during infections in the human body. They obtained patient samples from a Salmonella strain and sequence bacterial genomes. To their surprise, they did not identify any of the tolerance mutations that appeared in their cell culture experiments. Instead, the growth of the isolates in the antibiotic-treated macrophages mimicked that of the persistent bacteria; there was a subpopulation that was not as sensitive to antibiotics and had robust activation of the DNA repair response.

“The observation that clinical isolates seem to behave closer to persistent ones in terms of [DNA repair] answer is very interesting,” wrote Nathalie Balaban, a professor at the Hebrew University of Jerusalem who was not involved in this study, in an email. “It would also be good to see if [the clinical isolates] better reinfect macrophages.

Understanding the role of DNA repair in persistence and tolerance will help researchers develop alternative treatment strategies for bacterial infections. Block DNA repair needed to Salmonella survival in combination with antibiotic treatment can arrest relapse of infection and slow the development of antibiotic resistance.

The references

  1. PWS Hill, S. Helaine, “Antibiotics Persist and Relapse Salmonella enterica infection”, in Persistent Cells and Infectious Diseases, K. Lewis, ed., Springer International Publishing, 2019, p. 19-38.
  2. PWS Hill et al., “The vulnerable versatility of Salmonella the antibiotic persists during the infection”, Cell Host Microbe, 29:1757-73.e10, 2021.
  3. CK Okoro et al., “High-resolution single nucleotide polymorphism analysis distinguishes recrudescence and reinfection in non-typhoid invasive Salmonella typhimurium relapsing disease”, Clin Infect Dis, 54:955-63, 2012.

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