Researchers discover that TB bacteria can shield themselves from antibiotics and stay alive longer by changing their outer fat coating.

For more than a century, tuberculosis (TB), caused by the bacteria Mycobacterium tuberculosis, has remained a serious global health problem. Even with effective antibiotics and widespread vaccination campaigns, the disease continues to take lives. In 2023 alone, about 10.8 million people became sick with TB, and 1.25 million died from it. India carries the largest burden, with over 2.6 million cases in 2024. These numbers show that TB remains far from being eliminated, and scientists are still trying to understand why it continues to persist.

One reason TB is so difficult to control is that the bacteria can enter a resting state called latent or dormant TB after the initial infection. In this phase, the bacteria stay alive but inactive, sometimes for many years. People with latent TB have no symptoms and cannot spread the disease. However, the bacteria can become active again if the immune system weakens, such as with another infection or HIV or use of immunosuppressants. Most antibiotics work only on TB bacteria that are active and dividing; therefore, dormant TB cells, which grow very slowly or not at all, can survive treatment and persist in the infected person, exhibiting antibiotic tolerance.

In a new study under the guidance of Prof. Shobhna Kapoor from the Department of Chemistry, Indian Institute of Technology Bombay and Prof. Marie-Isabel Aguilar from Monash University, the research team set out to answer a key question: Why are dormant TB bacteria so unaffected by antibiotics? Their study, published in the Chemical Science journal, identified how the bacteria survive antibiotic treatment and what contributes to their drug tolerance. The study also suggests that interfering with this survival mechanism could make existing TB drugs more effective.

Building on existing knowledge, Prof. Kapoor’s team suspected that the key to this drug tolerance might lie in the bacteria’s membranes— complex barriers made mostly of fats, or lipids that protect the cell. To explore this, they examined the membrane properties under different conditions, including how they change as TB shifts from an active to a dormant state. They also investigated whether these changes affect how easily antibiotics can enter the cell.

Handling TB bacteria is dangerous. For their experiments, the researchers used its harmless relative called Mycobacterium smegmatis . It behaves similarly but can be studied safely in regular laboratories. The team grew the bacteria under two conditions: an active phase, when the bacteria were dividing rapidly as they do in an active infection, and a late stage mimicking dormancy, as seen in latent infections.

To test whether these conditions affected antibiotic effectiveness, the team exposed the Mycobacterium smegmatis bacteria to four common TB drugs: rifabutin, moxifloxacin, amikacin, and clarithromycin. They found that the concentration of drugs needed to stop 50% of bacterial growth was two to ten times higher in dormant bacteria than in active ones. In other words, “the same drug that worked well in the early stage of the disease would now be needed at a much higher concentration to kill the dormant/persistent TB cells. This change was not caused by genetic mutations, which usually explain antibiotic resistance,” says Prof. Kapoor. The genetic strain of Mycobacterium smegmatis used in the experiments lacked any mutations associated with antibiotic resistance, confirming that the reduced drug sensitivity could be linked to the bacteria’s dormant state and most likely their membrane coats rather than genetic changes .

To check if the two bacterial phases have distinct lipid profiles, the researchers used advanced mass spectrometry to identify more than 270 distinct lipid molecules in the bacterial membranes. “We found clear differences between the lipid profiles of active and dormant cells,” says lead author Ms. Anjana Menon from Prof Kapoor’s lab, who is a PhD student from IITB-Monash Research Academy, Indian Institute of Technology Bombay. In active bacteria, the membrane was rich in the type of lipids called glycerophospholipids and glycolipids; however, in dormant bacteria, fatty acyls (long, waxy molecules) dominated the membrane.

To explore the physical consequences of these lipid differences between active and dormant bacteria, the researchers then measured how tightly these lipids were packed, a property called membrane fluidity, using fluorescence-based methods. Active bacteria had loose, fluid membranes, while dormant ones had rigid, tightly ordered structures. For example, one key lipid, cardiolipin, dropped sharply in dormant cells. “Cardiolipin helps keep the membrane slightly loose. When its level falls, the membrane becomes tighter and less permeable,” Ms. Menon explains.

“People have studied TB from the protein point of view for decades,” says Prof. Kapoor. “But lipids were long seen as passive components. We now know they actively help the bacteria survive and resist drugs.”

Next, the team examined how the antibiotic, rifabutin, interacted with these membranes. They found that rifabutin could easily enter active cells but barely crossed the outer membrane of dormant ones. “The rigid outer layer becomes the main barrier. It is the bacterium’s first and strongest line of defence,” explains Prof. Kapoor about the dormant bacteria.

If the outer membrane blocks antibiotics, weakening it could make the drugs work better. That is what Prof. Kapoor’s team is now exploring. Current TB treatment lasts at least six months, and dormant bacteria often survive this long course. Instead of only developing new antibiotics, the researchers suggest improving existing ones. “Even old drugs can work better if combined with a molecule that loosens the outer membrane,” says Prof. Kapoor. This approach makes bacteria sensitive to the drugs again without giving them a chance to develop permanent resistance.

The team is further studying antimicrobial peptides (small proteins) that can make bacterial membranes slightly leaky. They believe that these peptides could be used as potential additions to treatment. “These peptides alone don’t kill the bacteria, but when combined with antibiotics, they help the drugs enter and act more effectively,” says Prof. Kapoor.

This study used a harmless version of the bacteria for experiments, and the next step will be to confirm the results with the real TB bacterium under higher safety conditions. “Our lipid analysis is very detailed. It can easily be applied in labs that work with the actual TB strain,” shares Ms. Menon, while explaining that their work can be expanded to actual TB bacteria.

Funding information: This study was supported by grants from DST-SERB, National Health and Medical Research Council Project, Australia.

In Hindi In Marathi