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HU Researchers: Simple Method Measures How Long Bacteria Can Wait Out Antibiotics

21/06/2017

The efficient classification of bacterial strains as tolerant, resistant, or persistent could help to guide treatment decisions, and could ultimately reduce the ever-growing risk of resistance

A growing number of pathogens are developing resistance to one or more antibiotics, threatening our ability to treat infectious diseases. Now, according to a study published in Biophysical Journal, a simple new method for measuring the time it takes to kill a bacterial population could improve the ability of clinicians to effectively treat antimicrobial-tolerant strains that are on the path to becoming resistant.

“These findings allow measurement of tolerance, which has previously been largely overlooked in the clinical setting,” says senior study author Prof. Nathalie Balaban, the Joseph and Sadie Danciger Professor of Physics at the Hebrew University of Jerusalem. “Routinely measuring tolerance could supply valuable information about the duration of antibiotic treatments, reducing the chance of both under- and over-treatment. Furthermore, data compiled from such measurements could give an estimate of how widespread the phenomenon of tolerance really is, which is currently a complete unknown.”

According to the World Health Organization, antibiotic resistance is one of the biggest threats to global health and is putting the achievements of modern medicine at risk. Due to selective pressure, pathogens acquire resistance through mutations that make the antibiotic less effective, for example, by interfering with the ability of a drug to bind to its target. Currently, clinicians determine which antibiotic and dose to prescribe by assessing resistance levels using a routine metric called minimum inhibitory concentration (MIC)—the minimal drug concentration required to prevent bacterial growth.

Although resistant strains continue to grow despite exposure to high drug concentrations, tolerant strains can survive lethal concentrations of an antibiotic for a long period of time before succumbing to its effects. Tolerance is often associated with treatment failure and relapse, and it is considered a stepping stone toward the evolution of antibiotic resistance. But unlike resistance, tolerance is poorly understood and is currently not evaluated in healthcare settings.

“The lack of a quantitative measure means that this aspect of the treatment relies largely on the experience of the individual physician or the community,” says first author Asher Brauner, a PhD student in Balaban’s lab at the Hebrew University’s Racah Institute of Physics. “This can lead to treatment being either too short, increasing the risk of relapse and evolution of resistance, or much too long, unnecessarily causing side effects, release of antibiotic waste into the environment, and additional costs.”

To address this problem, Balaban and her team developed a tolerance metric called the minimum duration for killing 99% of the population (MDK99). The protocol, which can be performed manually or using an automated robotic system, involves exposing populations of approximately 100 bacteria in separate microwell plates to different concentrations of antibiotics for varied time periods, while determining the presence or lack of survivors.

The researchers applied MDK99 to six Escherichia coli strains, which showed tolerance levels ranging from 2 to 23 hr under ampicillin treatment. MDK99 also facilitates measurements of a special case of tolerance known as time-dependent persistence—the presence of transiently dormant subpopulations of bacteria that are killed more slowly than the majority of the fast-growing population. Like other forms of tolerance, time-dependent persistence can lead to recurrent infections because the few surviving bacteria can quickly grow to replenish the entire population once antibiotic treatment stops.

“A take-home message from this is that it is important to complete a course of antibiotic treatment as prescribed, even after the disappearance of the symptoms,” Balaban says. “Partial treatment gives tolerance and persistence mutations a selective advantage, and these, in turn, hasten the development of resistance.”

In future studies, Balaban and her team will use MDK99 to study the evolution of tolerance in patients. Moreover, the ability to systematically determine the tolerance level of strains in the lab could facilitate research in the field. “If implemented in hospital clinical microbiology labs, MDK99 could enable the efficient classification of bacterial strains as tolerant, resistant, or persistent, helping to guide treatment decisions,” Balaban says. “In the end, understanding tolerance and finding a way to combat it could significantly reduce the ever-growing risk of resistance.”

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Scientists involved with this research are affiliated with The Racah Institute of Physics and The Center for NanoScience and NanoTechnology at The Hebrew University of Jerusalem, and The Broad Institute of Harvard University and Massachusetts Institute of Technology (MIT).

FUNDING: This work was supported by the European Research Council (ERC) (grant 681819) and the Israel Science Foundation (ISF) (grant 492/15).

CITATION: Biophysical Journal. Asher Brauner, Noam Shoresh, Ofer Fridman, Nathalie Q. Balaban.: “An Experimental Framework for Quantifying Bacterial Tolerance” http://www.cell.com/biophysj/fulltext/S0006-3495(17)30551-9 / doi: 10.1016/j.bpj.2017.05.014

HU Researchers: Simple Method Measures How Long Bacteria Can Wait Out Antibiotics
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Bacterial Survival Strategy: Splitting Into Virulent and Non-Virulent Subtypes

12/02/2017

Scientists find long-term epigenetic memory switch that controls different modes of bacterial virulence

Researchers at the Hebrew University of Jerusalem have discovered a survival strategy that harmful bacteria can use to outsmart the human immune response, resulting in more severe and persistent infections and more effective spreading from person to person.

Bacteria have an array of strategies for coping with the harsh and changing environments in the organisms they invade. These include developing adaptive mutations as they evolve, and activating specific genes to respond to changes. However, sometimes these defenses aren’t enough, and alternative strategies are needed.

One such alternative strategy recently discovered is the generation of non-genetic variability, in which bacteria develop sub-populations that are each pre-adapted to a different environment or task. This pre-adaptation could give invading bacteria an extra advantage during invasion and in overcoming the immune system. Called phenotypic variability, this variation involves the development of sub-populations of bacteria with altered traits such as size or behavior.

To better understand this survival strategy, researchers Irine Ronin, Naama Katsowitz, Ilan Rosenshine and Nathalie Q. Balaban, led by Dr. Irine Ronin from the Balaban lab at the Hebrew University of Jerusalem’s Racah Institute of Physics, examined whether non-genetic variability plays a role in the virulence of a human-specific pathogen, enteropathogenic E. coli (EPEC), responsible for many infant deaths worldwide. Their goal was to uncover whether exposing EPEC to challenging conditions similar to the ones they would encounter in a human can trigger EPEC to spontaneously differentiate into different bacterial sub-populations. To do this, they used mathematical modeling and genetic analysis.

Their analysis revealed that EPEC spontaneously differentiates into two sub-populations, one of them particularly virulent, when exposed to conditions that mimic the host environment. Surprisingly, they found that once triggered, this hyper-virulent state maintains a very long memory and remains hyper-virulent for many generations. In addition, they identified the specific regulatory genes that control the switch between the non-virulent and hyper-virulent states in EPEC bacteria. 

“These results shed new light on bacterial virulence strategies, revealing the existence of pre-adapted EPEC subpopulations which can remain primed for infection over weeks,” said Prof. Nathalie Q. Balaban. “They also show that long-term memory drives the expression of the pathogen’s major virulence factors, even upon shifting to conditions that do not favor their expression.”

“The unique memory switch we identified may be common in pathogenic bacteria, resulting in increased disease severity, higher infection persistency and improved host-to-host spreading,” said Prof. Ilan Rosenshine. “Further research to characterize the switching mechanism may point to strategies for tuning down EPEC virulence and fighting infections, and our approach can provide a framework to search for similar switches in other pathogens.”

PHOTO: http://media.huji.ac.il/new/photos/hu20170207_bimodalbacteria.jpg - The image shows the spontaneous differentiation of the bacteria into two states (shown in the histogram on the left as two peaks). One of these peaks represents the hypervirulent state that is seen on the right infecting human cells (bacteria are marked in green, the human cells in red and violet). (Photo credit: Irine Ronin)

FUNDING: European Research Council (ERC) 681819; Israel Science Foundation (ISF) 492/15 and 617/15; Minerva Foundation (Minerva Stiftung).

CITATION: A Long-term Epigenetic Memory Switch Controls Bacterial Virulence Bimodality. Irine Ronin, Naama Katsowich, Ilan Rosenshine and Nathalie Balaban. eLife, online publication February 7, 2017. doi: 10.7554/eLife.19599. Link: http://dx.doi.org/10.7554/eLife.19599.

Bacterial Survival Strategy: Splitting Into Virulent and Non-Virulent Subtypes
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