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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’

Lighting up antibiotic resistance

Lighting up antibiotic resistance

Carbapenems are among the “antibiotics of last resort” and can fight infections for which other drugs have long lost their effectiveness. However, even carbapenem-resistant pathogenic strains have emerged over the last decades. To find out whether a pathogen contains carbapenem-cleaving enzymes, the carbapenemases, Chinese scientists have developed a simple and fast assay based on a fluorescent probe and optical detection. They introduce their approach in the journal Angewandte Chemie .

Carbapenems are a class of β-lactam antibiotics similar to cephalosporins and penicillins. Although some bacterial strains have found powerful strategies to resist β-lactam antibiotics by producing a class of cleaving enzymes, the β-lactamases, most β-lactamases cannot affect the carbapenems. Therefore, these substances, which are called “antibiotics of last resort,” are the drug of choice for several diseases such as urinary-tract and abdominal infections as well as hospital-acquired pneumonia, if they are caused by multidrug-resistant bacteria. But there is growing evidence of even carbapenem resistance, and some pathogens were found to produce carbapenem-cleaving enzymes, the carbapenemases. Now, Hexin Xie at East China University of Science and Technology and his team have set up a strategy to identify those pathogens that carry the carbapenemases.

The researchers developed a molecule that has the same structure as the carbapenems but has a fluorogenic dye attached. If this carbapenem-mimicking compound, CVB-1, is recognized by a carbapenemase, for example, in an bacterial extract, CVB-1 is cleaved and undergoes spontaneous degradation. As this destroys the electronic interaction of the attached dye with the carbapenem compound, the dye turns into a green fluorescent molecule, which means, if it is irradiated with light of a certain wavelength, it emits intense green light. Thus, the assay in principle works as follows: If there is an active carbapenemase present, for example, in a bacteria extract, a couple of minutes later the sample glows green upon excitation. Xie and his colleagues said: “CVB-1 […] is essentially non-fluorescent […], and the addition of [the carbapenemase] triggers the turn-on of the fluorescent signal upon excitation […] with over 200-fold enhancement ratio.”

This technique allows the detection of antibiotic resistance activity by fluorescence. Thus, using this fluorescence-based assay system, it would be possible to find out in very short time whether carbapenem-resistant bacteria (such as certain Enterobacteriaceae and Klebsiella pneumoniae strains) are indeed present during an infection. More specific treatment strategies could be designed and an overuse of noneffective drugs could be avoided. The scientists have performed several tests to prove that their CVB-1 assay is specific, that the detection limit is low, and that it can indeed be used in live systems. This fast and simple fluorescence-based assay is certainly a remarkable approach in the ongoing and urgent fight against the fast spread of antibiotic resistance.

This article was published in Science Daily dated ‘Mar. 24, 2017’