IISc’s potent molecules show promise for TB therapy

SEM photo of Mycobacterium tuberculosis bacteria. - Photo NIAD-Optimized

The two molecules were able to prevent biofilm formation and even disrupt biofilms that had formed.

Scientists at the Indian Institute of Science (IISc) Bengaluru have developed two new, potent molecules that can severely impact the survival of mycobacteria, including Mycobacterium tuberculosis that causes TB. The results were published in the journal Antimicrobial Agents and Chemotherapy.

Unlike most antibiotics that target the bacterial metabolism by aiming at the cellular components, the novel molecules inhibit the stress response pathway of mycobacteria. The stress response pathway is crucial for bacteria to survive during hostile conditions such as lack of nutrients and the presence of antibiotics, to name a few. So any inhibition of this pathway will lead to its death.

The master regulator of stress pathway in the case of mycobacteria is (p)ppGpp (Guanosine pentaphospahte or Guanosine tetraphosphate). Though a molecule that inhibits the (p)ppGpp formation has already been synthesised, the efficacy is not much. “Very high concentration of Relacin molecule is needed to inhibit the pathway and, therefore, the efficacy is low. So we synthesised two new molecules — acetylated compound (AC compound) and acetylated benzoylated compound (AB compound) — by bringing about a modification in the base of the Relacin molecule,” says Prof. Dipankar Chatterji from the Division of Biological Sciences at IISc and the corresponding author of the paper.

“We found both the molecules to be very good inhibitors of stress response. The two compounds affected the rate of synthesis of (p)ppGpp and also reduced the cell survival,” he says. Laboratory studies showed that the two molecules were not toxic to human cells and were able to penetrate the human lung epithelial cells.

IMG_0508

Inhibiting (p)ppGpp synthesis would target the survival of the bacteria, says Dr. Kirtimaan Syal.

“We found our compounds were targeting the Rel gene. The Rel gene makes Rel protein, which in turn synthesises (p)ppGpp. When the Rel gene is knocked out, the long-term survival of Mycobacterium smegmatis decreases,” says Prof. Chatterji.

“The Alarmone molecule “(p)ppgpp”, a modified nucleotide, is ubiquitous in bacteria and absent in humans. Inhibiting (p)ppgpp synthesis would specifically target the survival of bacteria without having any effects on humans,” says Dr. Kirtimaan Syal from the Division of Biological Sciences, IISc and the first author of the paper.

Earlier studies have shown that when the rel gene is deleted, the long-term survival ability under stress was lost; the M. tuberculosis bacteria was unable to persist in mice and unable to form tubercle lesions in guinea pigs.

“The major reason for prolonged treatment of TB is the bacterium’s ability to persist in dormant form, which is tolerant to most antibiotics used in the treatment regimen. So inhibition of (p)ppGpp-mediated persistence could help in shortening the treatment regime, dealing with the emergence of multiple drug resistance and treatment of chronic infections, Dr. Syal says.

Inhibiting biofilm

Under hostile conditions, bacteria tend to form biofilms, which protect the bacteria from stress and induce tolerance to antibiotics. Recent studies have shown that tuberculosis bacteria that cannot form a biofilm cannot survive inside the host. Evidences have shown that at the time of infection, the M. tuberculosis display a biofilm-like phenotype and this helps the bacteria to survive inside the host.

Studies carried out by the researchers showed that both the molecules were able to inhibit biofilm formation by M. tuberculosis and M. smegmatis and also disrupt the already formed biofilm. “The biofilm formed by TB bacteria is very dangerous. The ability of the molecules to destroy the biofilm and even prevent its formation is a very important achievement,” says Prof. Chatterji.

Since there are very few antibiotics that target the stress response pathway of the bacteria, the two molecules offer great promise. “The next step is to test the molecules on animals. We have not thought about it. It will also be interesting to see if the bacteria develop resistance against these molecules,” Prof. Chatterji says.

Published in The Hindu on April 15, 2017

Pharmacies in India may not be causing TB drug-resistance

Pharmacy - Photo R. Prasad

Thirty-seven per cent of 622 pharmacies in Mumbai, Delhi and Patna handed out antibiotics to TB ‘patients’ with symptoms. – Photo: R. Prasad

If an earlier study revealed the tendency of private practitioners to liberally use antibiotics to treat tuberculosis leading to a delay in TB diagnosis and treatment and increase the chances of TB spreading within a community, pharmacies in Delhi, Mumbai and Patna are no better. A study published on August 25, 2016 in the journal The Lancet found that a majority of 622 pharmacies in the three cities dispensed antibiotics to TB patients even when they did not carry a prescription.

According to government guidelines, “pharmacies are required to counsel patients with TB, identify and refer persons with tuberculosis symptoms to the nearest public health facilities for testing” and dispense TB drugs. Much like the private practitioners, pharmacies tend to be the first point of contact for primary care in India.

Srinath Satyanarayana, the first author of the paper from McGill University, Montreal, Canada used standardised TB patients — healthy individuals trained to pose as TB patients and interact with pharmacists — to understand how pharmacies in the three cities treated patients presenting with TB symptoms or microbiological confirmation of pulmonary TB. The other main objective was to determine whether the pharmacies were contributing to the inappropriate use of antibiotics.

The standardised patient 1 presented with 2-3 weeks of cough and fever and was directly seeking drugs from a pharmacy. The standardised patient 2 presented with one month of cough and microbiological confirmation of TB from a sputum test.

Only 13 per cent of simulated patients with TB symptoms and 62 per cent of patients with microbiological confirmation were correctly managed.As expected, liberal dispensation of antibiotics was seen in the case of standardised patient 1. Only 96 of 599 pharmacies (16 per cent) refereed such patients to health-care providers. But ideal case management was in only 13 per cent of the cases as a few pharmacies handed out antibiotics to the patients even while referring them to a physician. Antibiotics (37 per cent), steroids (8 per cent) and fluoroquinolones (10 per cent) were given to standardised patients with symptoms.

“That nearly 37 per cent of the pharmacies are handing our antibiotic to persons presenting with TB symptoms is really worrisome,” says Dr. Satyanarayana in an email to me. But more worrying is the dispensation of fluoroquinolones. “Fluoroquinolones are an essential part of MDR-TB treatment regimen and emerging regimens, so quinolone abuse is a concern,” they write.

In stark contrast, in the case of standardised patient 2 who had a microbiological confirmation of TB disease 67 per cent (401 of 601) of pharmacies referred the patient to a health-care provider.  Like in the earlier case, ideal case management was seen in only 62 per cent as the standardised patient did receive antibiotics (16 per cent) or steroids (3 per cent) even while being referred to a health-care provider.

“In case of TB patients with microbiological confirmation of TB disease, antibiotics (without anti-TB properties) will be ineffective and un-necessary, and can delay the initiation of proper therapy for patients. These patients will continue to spread the disease in the community and TB disease will continue to progress in the concerned individual. Steroids reduce body immunity, suppress symptoms temporarily and can worsen the TB disease,” Dr. Satyanarayana says.

Silver lining

The only silver lining is that none of the pharmacies in all the three cities handed out first-line anti-TB drugs to these “patients.” So pharmacies are unlikely sources of irrational drug use that contributes to multidrug-resistant tuberculosis. “Also, pharmacies are not trying out high end antibiotics such as fluoroquinolones when they realise that the patient has some underlying illness such as TB,” he says.

“TB Drug resistance occurs primarily due to incorrect regimens, intake of drugs irregularly or intake of drugs for very short duration of time. From our study, it appears that pharmacies are not playing a role in deciding the anti-TB regimens and are also not dispensing anti-TB drugs over-the-counter, at least in the three cities that we studied. So the drug resistance in India could be due to either patient related factors or provider related factors or due to health system related factors (which has not created a system for all TB patients in country to access quality assured diagnosis and treatment free of cost and seamlessly),” Dr. Satyanarayana says.

One reason why pharmacies did not dispense anti-TB drugs could be because they belong to a more stringent Schedule H1 category of drugs where details of the prescription and name of the doctors and patients have to be documented and the registry has to be retained for two years.

A novel, powerful antibiotic found inside human nose

MRSA - Photo National Institute of Allergy and Infectious Diseases (NIAID)

The novel antibiotic ludunin, found inside human nose, has antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). – Photo: NIAD

A novel antibiotic — lugdunin — produced by a bacterium found inside the human nose has been found to kill the bacterium Staphylococcus aureus, including drug-resistant forms such as the methicillin-resistant S. aureus (MRSA). The study found S. aureus does not develop resistance against the novel antibiotic. The findings could aid the development of new therapies for hard-to-treat bacterial infections. The results were published today (July 28, 2016) in the journal Nature.

The human body is home to an immense variety of microorganisms known collectively as the microbiota. Several bacteria species including Staphylococcus are found inside the human nose and these bacterial species are fewer in number when the pathogenic S. aureus bacterium is present. However, in about 70 per cent of human population, colonisation by S. aureus inside human nose is absent. The reasons for this were not clear.

On screening a collection of nasal Staphylococcus species for antimicrobial activity against S. aureus, a team of scientists led by Andreas Peschel from the University of Tubingen, Germany, found that Staphylococcus lugdunensis bacterial strain had a particularly strong capacity to prevent the growth of S. aureus.

On screening S. lugdunensis mutants, Dr. Peschel and colleagues identified the compound that resisted the growth of S. aureus and called it lugdunin. Lugdunin causes the breakdown of S. aureus.

Peptide antibiotic

The novel compound represents the first known example of a new class of peptide antibiotics.

Nose

The bacteria producing the novel antibiotic reduced the S. aureus colonisation by six times. – Photo: R. Prasad

The lugdunin’s role in inhibiting S. aureus growth was first proved in the lab. To test the efficacy in animals, the researchers infected mice skin with S. aureus and then treated it with the novel antibiotic 24, 30 and 42 hours after infection had set in. Lugdunin was found to completely clear all viable S. aureus from the surface and in the deeper layers of the skin.

Next, lugdunin’s ability to inhibit S. aureus’ growth in the nose was tested using cotton rats. When both S. aureus and S. lugdunensis bacteria were introduced into the cotton rat nose, relatively fewer S. aureus cells were retrieved proving that lugdunin production effectively prevented S. aureus colonisation inside the nose.

The nasal swabs of 187 hospitalised patients were tested for the presence of S. aureus, S. lugdunensis or both. When both the bacterial species were found in a patient’s nose, S. aureus colonisation was nearly six times lower than in those people who had only S. aureus.

The researchers found lugdunin had bacterial activity against many major pathogens, and S. aureus isolates had “pronounced susceptibility” to lugdunin in all nasal and clinical samples. So it may be quite difficult for S. aureus to develop resistance against lugdunin, they say.

Published in The Hindu on July 28, 2016

Will mcr-1 gene cause a new superbug?

E. coli - Photo Janice Haney Carr, CDC

A highly magnified view of E. coli bacteria. – Photo: Janice Haney Carr/CDC

Alarm bells have been sounded after a woman in the U.S. was detected with bacteria resistant to a last-resort antibiotic. The 49-year-old was carrying E. coli bearing a new gene, mcr-1, which is resistant to colistin, the last available antibiotic that works against strains that have acquired protection against all other medication. This is the first reported case of the mcr-1 gene in an E. coli strain found in a person living in America, but it raises worries about how far it may have spread. The results of mcr-1 gene identification were published recently in the journal Antimicrobial Agents and Chemotherapy. Though resistance to colistin have been detected for about 10 years in several countries, the danger of this has been somewhat played down since such resistance was brought about by gene mutations that cannot spread easily between bacteria. But mcr-1 poses a threat of an entirely different order; in this case a small piece of DNA (plasmid) found outside the chromosome carries a gene responsible for antibiotic resistance. Since the gene is found outside the chromosome, it can spread easily between different types of bacteria, as well as between patients. In the case of E. coli, the colistin resistance is not insurmountable, as it is still treatable by other known drugs. But were the gene to spread to bugs treatable by only last-resort antibiotics, we could be facing the dreaded — and indeed, long anticipated — superbug. Thus the discovery of mcr-1 gene in more countries and settings increases the chances of the emergence and spread of resistance against all available antibiotics. It could well lead to an era without effective drugs to treat bacterial infections — the post-antibiotic age, as it were. Though Colistin has been around for more than 50 years, it has not been used widely due to its serious adverse effects. But the increase in carbapenem resistant bacteria resulted in increased use of colistin.

The mcr-1 gene was first identified in China in November last year following which there were similar reports from Europe and Canada. In each case, including the latest one, it is important to investigate if the mcr-1 gene emerged locally or if the patient had acquired it from outside the country as all efforts to contain its spread will depend on this information. The unchecked use of antibiotics in livestock is a major reason for the development of drug resistance. Indeed, given the widespread use of colistin in animals, the connection to the drug-resistant mcr-1 gene appears quite clear. A November 2015 paper in The Lancet noted that a significantly higher proportion of mcr-1 positive samples was found in animals compared with humans, suggesting the mcr-1 gene had emerged in animals before spreading to humans. Besides being administered for veterinary purposes, colistin is used in agriculture as well. The global community needs to urgently address the indiscriminate use of antibiotics in an actionable manner and fast-track research on the next generation of drugs.

UPDATE: July 12, 2016:

A second case of E. coli with mcr-1 gene has been discovered in the U.S. The results of this discovery was published on July 11, 2016 in the journal Antimicrobial Agents and Chemotherapy

Published in The Hindu on May 30, 2016