Tuberculosis researchers uncover a crucial clue that explains antibiotic resistance in bacteria

Reviewed by Emily Henderson, B.Sc.November 18, 2020

For a slow-growing microbe that multiplies infrequently, Mycobacterium tuberculosis, the pathogen that causes tuberculosis (TB) has long puzzled researchers as to how it develops resistance to antibiotics so quickly, within weeks or months.

Now, TB researchers at San Diego State University have uncovered a crucial clue to the mystery: the answer may lie in the epigenetic domain rather than the genetic domain in which most scientists have focused their efforts.

Their discovery could help advance new diagnostic, therapeutic and vaccine targets.

Epigenetics is the study of heritable changes in gene expression that do not involve a corresponding change to the underlying DNA sequence, i.e. changes in the phenotype but no changes in the genotype.

This only affects the physical structure of the DNA, through a process called DNA methylation in which a chemical “cap” is added to the DNA molecule, preventing or facilitating the expression of certain genes.

The SDSU researchers describe the rapid response phenomenon they discovered as “intercellular mosaic methylation,” a process by which Mycobacterium tuberculosis diversifies, creating multiple subpopulations each with their own phenotype.

While antibiotics could kill many of these mutant subpopulations, at least some survive and develop drug resistance.

We believe this also explains why diagnostic tests in some patients do not predict treatment failure and why some patients return months later with the disease re-emerging in a much more resistant state. This is also the reason why CT scans of the lungs of many “healed” patients show lesions with possible bacterial activity. “

Faramarz Valafar, tuberculosis expert, School of Public Health, San Diego State University

Valafar studies the genetics and epigenetics of lung diseases.

Worldwide, tuberculosis is among the top 10 causes of death. It killed 1.4 million people in 2019 and around 10 million people fall ill each year, according to the World Health Organization.

Valafar’s team has collected hundreds of samples of drug-resistant bacteria strains from patients in India, China, the Philippines and South Africa, as well as Europe, through collaborations with TB researchers around the world.

Their study was published in eLife in late October. Valafar and project scientist Samuel Modlin began exploring epigenetics for the tuberculosis bacterium in 2016 and PhD student Derek Conkle-Gutierrez joined them in 2018, in the Clinical Drug Resistance and Persistence Pathogenesis Laboratory .

Modlin, a former student of the SDSU, and Conkle-Gutierrez used the skills and knowledge acquired at the SDSU to carry out this research: data and statistical analysis, coding skills and bioinformatics knowledge.

“We have known for decades that bacterial epigenetics can affect the expression of certain genes, which can lead to a variety of phenotypes even when they have identical genotypes,” said Conkle-Gutierrez. “We found evidence of that phenomenon in the tuberculosis bacterium.”

Antibiotic resistance is typically caused by genomic mutations, but this bacterium is one of many that exploits alternative mechanisms in the epigenetic domain to allow for rapid adaptation.

“We found that some of them had mutations that led to variable DNA methylation and those strains had much more diversity in their epigenome, and therefore more potential to be drug resistant,” Modlin said.

The researchers found that there were no set patterns and the methylation was quite random. They used advanced comparative genomic and epigenetic techniques to identify variations between cells within a colony from a single isolate, from a single patient, including small variations that nevertheless impacted gene expression.

They were able to do this because, instead of assuming that the reference genome had a common structure, they rebuilt each genome from scratch and analyzed its epigenetic signatures.

They will now focus on testing and confirming the key genes they have identified with the methylation signatures. There is still a lot of work to be done before their discovery can finally be used for diagnostics.

“There is a lot of resistance in tuberculosis that escapes current molecular diagnostics and we don’t really know why. This is problematic,” Valafar said. “This study offers a new domain, new tools and a new approach to finding alternative mechanisms. We move away from the classical view of molecular diagnostics and use a new comprehensive approach to analyzing bacteria.”

Current standards of care use two types of antibiotics: bacteriostats that prevent bacteria from multiplying but don’t kill them, and bactericides that kill them.

“We have found a new mode of variation, and if we can inhibit that diversification mechanism, we can inhibit epigenetic resistance in the short term and kill bacteria before mutations in the genome develop and cause long-term genetic resistance,” Modlin said.

This may be how some bacterial populations survive treatment and make the patient ill again with much greater antibiotic resistance or hypervirulence.

The journal editors and reviewers consider their finding significant and asked the researchers to rename three of the genes for which they describe new functions: MamC, MamS, MamS1. The article appeared on the cover of the eLife magazine.

Valafar’s team has obtained two provisional patents for their discovery.