doi:10.1038/nindia.2014.9 Published online 7 February 2014
In a finding that promises to revolutionise the diagnosis and treatment of Tuberculosis (TB), scientists have come up with a technique that could end the need for long-duration conventional TB therapy. They have created the first ever sensor that can image the TB-causing bacteria in real time in pathological samples and make a clear distinction between drug-sensitive bacterial cells and their drug-resistant cousins1 .
The findings have large scale implication because, at present, TB patients have to undergo 6-12 months of drug therapy to effectively cure the disease. A large number of TB patients are unable to comply with such lengthy treatment regimen and end up developing multi-drug resistant (MDR), extensively-drug resistant (XDR), and totally drug resistant (TDR) TB.
Realising that a shorter drug regimen could be the magic trick, scientists from International Centre for Genetic Engineering and Biotechnology, New Delhi set out to understand the reason why a lengthy treatment is needed in the first place.
They found that a long-term multiple drug therapy is required to eliminate a small sub-population of the TB-causing bacteria Mycobacterium tuberculosis or Mtb can easily evade the current anti-TB drugs. This small population of bacteria called "persisters" (and the phenomenon called "persistence") are the real reasons why sustained treatment is needed.
"Persisters are genetically similar to their drug-susceptible counterparts but are able to survive the lethal effects of antibiotics," says lead scientist Amit Singh, who has now moved to the Indian Institute of Science, Bangalore. What makes these persisters different? "That was the question we were asking," Singh told Nature India. "Everyone has been thinking that they are genetically same, so how do they avoid drugs? Our surmise was they are obviously phenotypically different then."
Understanding the physiology of drug-tolerant persisters has been a critical challenge for developing new anti-TB drugs. Despite their clinical importance, there has been no technology that can capture the phenotypic or physiological diversity within Mtb populations during an infection. "This represents a major technological gap in our understanding of TB and drug-resistance," Singh says.
The team, therefore, created a biosensor that can do exactly this —differentiate these subpopulations of bacteria by measuring the level of antioxidants in them during an infection. They created mycobacteria that can express a green fluorescent protein (Mrx1-roGFP2) in varying degrees depending on the level of a specific antioxidant called mycothiol inside the mycobacteria. In essence, the sensor is a "meter of the antioxidant levels" of the mycobacteria.
"Using this biosensor we have tracked the mycothiol status of individual bacteria, including drug-resistant Mtb strains during infection of macrophages and upon exposure to current anti-TB drugs. We found a surprising and previously unrecognized variety in the antioxidant state of individual Mtb cells within a single macrophage," Singh adds. He says its the first time that a sensor has been able to differentiate drug-sensitive Mtb cells from drug-resistant persisters by providing a unique antioxidant "barcode" to each bacterial cell inside it.
"Since antioxidants are known to induce drug tolerance in other diseases such as cancer, we reasoned that the observed variations in the mycothiol levels may be the basis of emergence of drug-tolerant persisters in the mycobacterial population during infection," says Singh's co-researcher Ashima Bhaskar.
While most frontline anti-TB drugs deplete the antioxidant mycothiol to kill Mtb inside macrophages, in test tubes these drugs do not influence mycothiol levels and have entirely different mechanisms of killing. "Around the world, TB screening is done in a test tube or a plate and drugs can kill all TB cells in those conditions," Singh says adding that drug screening should, therefore, be on a real infection model to be absolutely precise.
Their results suggested that the macrophage environment cooperates with antibiotics to efficiently kill Mtb by depleting mycothiol levels during the natural course of chemotherapy. However, Singh says, it did not solve the problem of how and why some Mtb cells survive the combined onslaught of drugs and stressful host environment.
"To address this issue, we tried to find the response of antibiotics on changes in the membranes of individual bacteria with different antioxidant capacities. We found a small group of bacteria with higher antioxidant levels that preserve membrane integrity and survive antibiotics," he says.
The implications of the findings range from development of redox-based TB diagnostics and biomarkers to innovative screening approaches to identify redox-oriented anti-mycobacterial compounds, the team says.