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New Strategy Makes Bacteria More Vulnerable to Antibiotics

New Strategy Makes Bacteria More Vulnerable to Antibiotics

Scientists at MIT have discovered a way to make bacteria more vulnerable to a class of antibiotics known as quinolones, which include ciprofloxacin and are often used to treat infections such as Escherichia coli and Staphylococcus aureus.

The new strategy overcomes a key limitation of these drugs, which is that they often fail against infections that feature a very high density of bacteria. These include many chronic, difficult-to-treat infections, such as Pseudomonas aeruginosa, often found in the lungs of cystic fibrosis patients, and methicillin-resistant Staphylococcus aureus (MRSA).

“Given that the number of new antibiotics being developed is diminishing, we face challenges in treating these infections. So efforts such as this could enable us to expand the efficacy of existing antibiotics,” says James Collins, the Termeer Professor of Medical Engineering and Science in MIT’s Institute for Medical Engineering and Science (IMES) and Department of Biological Engineering and the senior author of the study.

Arnaud Gutierrez, a former MIT postdoc, and Saloni Jain, a recent Boston University PhD recipient, are the lead authors of the study, which appears in the December 7 online edition of Molecular Cell.

Overcoming bacterial defenses

Bacteria that have become tolerant to a drug enter a physiological state that allows them to evade the drug’s action. (This is different from bacterial resistance, which occurs when microbes acquire genetic mutations that protect them from antibiotics.) “Tolerance is not well-understood, and we don’t have the means to circumvent it or overcome it,” Collins says.

In a study published in 2011, Collins and his colleagues found that they could increase the ability of antibiotics known as aminoglycosides to kill drug-tolerant bacteria by delivering a type of sugar along with the drug. The sugar helps to boost the metabolism of the bacteria, making it more likely that the microbes will undergo cell death in response to the DNA damage caused by the antibiotic.

However, aminoglycosides can have serious side effects, so they are not widely used. In their new study, Collins and his colleagues decided to explore whether they could use a similar approach to boost the effectiveness of quinolones, a class of antibiotics used more often than aminoglycosides. Quinolones work by interfering with bacterial enzymes called topoisomerases, which help with DNA replication and repair.

With quinolones, the researchers found that it wasn’t enough to add just sugar; they also had to add a type of molecule known as a terminal electron acceptor. Electron acceptors play an essential role in cellular respiration, the process that bacteria use to extract energy from sugar. In cells, the electron acceptor is usually oxygen, but other molecules, including fumarate, an acidic organic compound that is used as a food additive, can also be used.

In tests in high-density bacterial colonies grown in a lab dish, the researchers found that delivering quinolones along with glucose and fumarate could eliminate several types of bacteria, including Pseudomonas aeruginosa, Staphylococcus aureus, and Mycobacterium smegmatis, a close relative of the bacterium that causes tuberculosis.

“If you simply add a carbon source like glucose, that’s not enough to enable the quinolone to kill. If you simply add oxygen, or another terminal electron acceptor, that by itself is not enough to cause killing either. But if you combine the two, you can eradicate the tolerant infection,” Collins says.

Metabolic state

The findings suggest that high-density bacterial infections rapidly consume nutrients and oxygen from their environment, which then provokes them to enter a starvation state that helps them to survive. In this state, they greatly reduce their metabolic activity, which allows them to avoid the cell death pathway that is normally triggered when DNA is damaged by antibiotics.

“This finding highlights that the metabolic state of the bug significantly influences how the antibiotic will impact the bug. And, for the antibiotic to be effective as a killing agent, it requires downstream cellular respiration as part of the process,” Collins says.

The researchers now hope to test this approach in bacterial infections in animals, and they are also exploring how to best deliver the drug combination for different types of infections. A topical treatment could work well for Staphylococcus aureus infections, while an inhaled version could be used to treat Pseudomonas aeruginosa infections of the lungs, Collins says.

Collins also hopes to test this approach with other types of antibiotics, including the class that includes penicillin and ampicillin.

“This study encourages work to find new ways to stimulate bacterial respiration and thereby enhance the production of reactive oxygen (or even non-oxygen) species during antibiotic treatment, for better eradication of bacterial pathogens, particularly those having low metabolic activity that may render them tolerant to antimicrobials,” says Karl Drlica, a professor at the Public Health Research Institute at Rutgers New Jersey Medical School, who was not involved in the research.

The research was funded by the Defense Threat Reduction Agency, the Broad Institute of MIT and Harvard, and a gift from Anita and Josh Bekenstein.

This article was published in Scitechdaily dated ‘Dec. 07, 2017’

Understanding antibiotic resistance

Understanding antibiotic resistance

Central to understanding why bacteria become antibiotic resistant is knowing how bacteria respond to the drugs trying to kill them. In a new study, Boston College researchers report that antibiotics disrupt the genetic defensive responses in lethal bacteria.

When facing a common — or historic — threat such as deprivation of nutrients, the deadly bacterium Streptococcus pneumonia exerts a highly organized response — one influenced by the bacteria’s genetic evolution and powered by genes that respond cooperatively to stress, the researchers found. But when confronted with antibiotics — a relatively new form of stress — the bacterium mounts a confused defense, according to the study “Antibiotics Disrupt Coordination between Transcriptional and Phenotypic Stress Responses in Pathogenic Bacteria,” published today in the journal Cell Reports.

“We show that nutrient stress results in a highly organized response from the bacterium,” said Associate Professor of Biology Tim van Opijnen, the study’s principal investigator. “It basically seems to recognize the stress and knows how to deal with it.

“But the response to antibiotics was highly disorganized, showing that the organism has difficulties with this stress, trying out all kinds of unrelated things to come to a solution and overcome the stress,” he said. “This shows that the bacterium is far less familiar with antibiotics, not ‘knowing’ how to respond appropriately.”

Van Opijnen, whose data-driven research has helped to establish an understanding of how bacteria rely on underlying genetic networks to function, said the findings may advance the development of new drugs and also help predict how bacteria evolve, adapt and become resistant to antibiotics.

Van Opijnen and co-authors Karen Zhu, a BC doctoral student, and former post-doctoral researcher Paul Jensen, now at the University of Illinois, combined large quantities of experimental data with a new, large-scale computational model they developed to produce new insights and challenge some long-held assumptions about the interplay between bacteria and the drugs designed to treat them.

S. pneumoniae kills approximately 1.5 million people annually. In prior research, van Opijnen and colleagues have revealed that different strains of the bacterium respond uniquely to antibiotics. This time, the research team looked at how strains respond to different stressors. The team employed two analytical approaches — one in use for many years and one developed in the van Opijnen lab.

The team used a process known as RNA sequencing, or RNA-Seq, to assess bacterial genes that are provoked to change, a process known as transcription. This activity has long been viewed as central to understanding how bacteria combat antibiotics and other stressors.

The team paired that analysis with its own technique: transposon insertion sequencing, or Tn-Seq. Developed by van Opijnen, Tn-Seq combs through millions of genetic sequences and singles-out gene functions in bacteria. The advantage of Tn-Seq is that it is able to begin to pinpoint which genes play the most important defensive roles.

During the course of more than two years, the team’s RNA-Seq experiments analyzed 800 million genetic sequences and produced 150,000 data points. Tn-Seq analyzed 1.2 billion sequences and produced 300 million data points, Zhu said.

“RNA-Seq looks at the activity of every gene in the genome of the organism,” said van Opijnen. “The activity of every gene has always been associated with importance. The assumption for nearly three decades has been if you take an organism and stress it out, and a gene’s activity changes, it must be important.”

That assumption has been difficult to test, said van Opijnen. But with Tn-Seq, van Opijnen has the ability to assess the importance of a gene in a specific condition.

“We found that you cannot assume that change in the activity of a gene means the gene is important,” said van Opijnen. It was a surprising finding, he said. After all, why would a change in gene activity take place if it was not important to the organism’s survival?

Part of the answer may be in the collaborative nature of the way genes work across the entire genome of a bacterial strain, van Opijnen said. “They collaborate and corroborate to perform functions, to resolve into a specific phenotype,” he said. “So there is a relationship as genes all work with each other in pathways, or networks; they cooperate with each other.”

The researchers constructed a metabolic model of the coordinated response to deprivation, which placed the responding genes in close proximity to each other. When challenged with antibiotics, the model shows that the response of the physical network breaks down in disorganization and those genes are no longer in close proximity.

“Bacteria use these types of regulation to fight against stress,” Zhu said. “In terms of nutritional depletion, because they have responded to this type of stress over the course of their evolution, the bacteria ‘know’ how to coordinate this activity. But with a relatively new invasive stressor, like an antibiotic, bacteria may not be able to figure out a way to produce a coordinated response.”

In a search for reasons why certain types of bacteria become resistant to antibiotics, the researchers have established a new approach to understanding why antibiotics succeed in combatting bacterial infections.

“By combining computational and large-scale experimental work, we’ve built a model to get the first, basic understanding of how known and unknown stress is processed by bacteria,” said van Opijnen. “That’s critical because it makes it a stepping stone to a new intervention at some point in the future. If we can understand how stress is processed, we can come up with a better way to develop a new stressor to break an organism, or eradicate it.”

This article was published in Science Daily dated ‘Aug. 15, 2017’