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Nanoscale chip system measures light from a single bacterial cell to enable portable chemical detection

05/09/2017

Further development could open door to on-chip biological and chemical sensing applications, e.g. detecting chemicals in real-time continuous flow systems and even in an open-air environment

Researchers at the Hebrew University of Jerusalem have created a nanophotonic chip system using lasers and bacteria to observe fluorescence emitted from a single bacterial cell. To fix the bacteria in place and to route light toward individual bacterial cells, they used V-groove-shaped plasmonic waveguides, tiny aluminum-coated rods only tens of nanometers in diameter. The novel system, described in the journal Nano Letters, paves the way for an efficient and portable on-chip system for diverse cell-based sensing applications, such as detecting chemicals in real-time.

The field of on-chip photonic devices for biological and chemical sensing applications presents many powerful alternatives to conventional analytical techniques for applications ranging from “lab on a chip” to environmental monitoring.  However, these sensing schemes rely mainly on off-chip detection and require a cumbersome apparatus, even when measuring only single cells. 

The Hebrew University team looked for ways to integrate all system components, including light sources and detectors, on-chip at the nanoscale. This would result in a lab-on-chip system that is small, portable and can perform sensing in real-time.

To achieve this, they molecularly engineered live bacteria that emit a fluorescent signal in the presence of target compounds. They paired these on-chip with a nanoscale waveguide, which not only served the purpose of guiding light, but also allowed mechanical trapping of individual bacteria within the V-groove.

In three different illumination conditions, they experimentally demonstrated the interrogation of an individual Escherichia coli bacterial cell using a nanoscale plasmonic V-groove waveguide. First, they measured the light emitted from a bacterium flowing on top of the nanocoupler in a liquid environment by allowing the fluorescence from the bacterium to be coupled directly into the waveguide through the nanocoupler. Next, a bacterium was mechanically trapped within the V groove waveguide and was excited by laser directly either from the top or through the nanocoupler. In all cases, significant fluorescence was collected from the output nano coupler into the detector.

The system worked well both in wet environments, where the bacteria are flowing on top of the waveguide, and in dry conditions, where the bacteria are trapped within the waveguide.

The research was led by Prof. Uriel Levy, Director of The Harvey M. Krueger Family Center for Nanoscience and Nanotechnology at the Hebrew University in collaboration with Prof. Shimshon Belkin, at the Hebrew University’s Alexander Silberman Institute of Life Sciences, who genetically engineered the bacterial sensors, and Prof. Anders Kristensen from the Danish Technical University, who was in charge of fabricating the V-groove waveguides. Prof. Levy is the Eric Samson Chair in Applied Science and Technology, and Prof. Belkin is the Ministry of Labor and Social Welfare Chair in Industrial Hygiene, at the Hebrew University.

Unlike the more traditional plasmonic waveguides consisting of either silver or gold, the choice of aluminum was instrumental for being able to guide the fluorescent light emitted from the bacteria all the way to the output nanocoupler. Furthermore, the waveguide dimensions allow for efficient mechanical trapping of the bacteria and the multimode characteristics may become instrumental in gathering more information, e.g., on the specific position and orientation of the bacteria.

The results provide a clear indication of the feasibility of constructing a hybrid bioplasmonic system using live cells. Future work will include the construction of waveguide network, diversifying the system to incorporate different types of bacterial sensors for the detection of various biological or chemical analytes.

The research is a collaboration between scientists at the Department of Applied Physics, the Rachel and Selim Benin School of Engineering and Computer Science, the Harvey M. Krueger Family Center for Nanoscience and Nanotechnology, and the Alexander Silberman Institute of Life Sciences, at the Hebrew University of Jerusalem, Israel; and the Department of Micro- and Nanotechnology, Technical University of Denmark, Kongens Lyngby, Denmark. Additional researchers include Oren Lotan, Jonathan Bar-David, Cameron L.C. Smith, and Sharon Yagur-Kroll.

Support: The researchers acknowledge financial support from the Danish International Network Programme (grant no. 1370-00124B) with Israel. Work in the Belkin lab was partially supported by the Minerva Center for Bio-Hybrid Complex Systems and by the NATO Science for Peace and Security Programme project 985042.

Citation: Oren Lotan, Jonathan Bar-David, Cameron L.C. Smith, Sharon Yagur-Kroll, Shimshon Belkin, Anders Kristensen, and Uriel Levy*. Nanoscale Plasmonic V-Groove Waveguides for the Interrogation of Single Fluorescent Bacterial Cells. Nano Lett., Article ASAP. DOI: 10.1021/acs.nanolett.7b02132. Publication Date (Web): August 3, 2017. http://pubs.acs.org/doi/10.1021/acs.nanolett.7b02132

Videos for Download:

  • http://media.huji.ac.il/new/multimedia/hu170816_nano1.mp4 -A pulsed laser illuminates bacteria which are trapped in a plasmonic waveguide (the waveguide is the dark rectangle seen in the film, bacteria are invisible). The presence of the bacteria causes light coupling into the waveguide. The light then propagates in the waveguide until it is coupled back out by the nano-mirrors and appears as bright pulses on the waveguides' ends.  (Photo credit: Hebrew University)
  • http://media.huji.ac.il/new/multimedia/hu170816_nano2.mp4 - A laser beam excites fluorescent bacteria swimming in a micro-fluidic device. The fluorescent light emitted by these bacteria radiates in all directions, and some of it couples into a plasmonic waveguide and is directed toward the waveguide's end. The direct fluorescence from the bacteria is seen most clearly on the right-hand side, while light which was coupled and propagated in the waveguide appears as a bright spot blinking on the left-hand side. The blinking is a result of the bacteria's changing position, and is correlated to the location of the bacteria relative to the nano-mirror in the waveguide's right side. (Photo credit: Hebrew University)

 

Nanoscale chip system measures light from a single bacterial cell to enable portable chemical detection
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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.”

###

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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Glowing bacteria detect buried landmines

23/04/2017

Researchers remotely detect buried landmines using fluorescent bacteria encased in polymeric beads illuminated by a laser-based scanning system

The need for safe and efficient technologies for detecting buried landmines and unexploded ordnance is a humanitarian issue of immense global proportions. About half a million people around the world are suffering from mine-inflicted injuries, and each year an additional 15 to 20 thousand more people are injured or killed by these devices. More than 100 million such devices are still buried in over 70 countries.

The major technical challenge in clearing minefields is detecting the mines. The technologies used today are not much different from those used in World War II, requiring detection teams to risk life and limb by physically entering the minefields. Clearly, there is a critical need for an efficient solution for the remote detection of buried landmines and unexploded ordnance.

Researchers from the Hebrew University of Jerusalem now report a potential answer to this need. Writing in the journal Nature Biotechnology, they present a novel, functional system combining lasers and bacteria to remotely map the location of buried landmines and unexploded ordnance.

The system is based on the observation that all landmines leak minute quantities of explosive vapors, which accumulate in the soil above them and serve as markers for their presence. The researchers molecularly engineered live bacteria that emit a fluorescent signal when they come into contact with these vapors. This signal can be recorded and quantified from a remote location.

The bacteria were encapsulated in small polymeric beads, which were scattered across the surface of a test field in which real antipersonnel landmines were buried. Using a laser-based scanning system, the test field was remotely scanned and the location of the buried landmines was determined. This appear to be the first demonstration of a functional standoff landmine detection system.

"Our field data show that engineered biosensors may be useful in a landmine detection system. For this to be possible, several challenges need to be overcome, such as enhancing the sensitivity and stability of the sensor bacteria, improving scanning speeds to cover large areas, and making the scanning apparatus more compact so it can be used on board a light unmanned aircraft or drone,” said Prof. Shimshon Belkin, from the Hebrew University’s Alexander Silberman Institute of Life Sciences, who was responsible for genetically engineering the bacterial sensors.

Research groups participating in this study at the Hebrew University were led by Prof. Shimshon Belkin at the Alexander Silberman Institute of Life Sciences (genetic engineering of the bacterial sensors); Prof. Aharon J. Agranat at the Department of Applied Physics and the Brojde Center for Innovative Engineering and Computer Science (design and construction of the remote scanning system); and Prof. Amos Nussinovitch at the Robert H. Smith Faculty of Agriculture, Food and Environment (bacteria encapsulation in polymeric beads).

CITATION: Remote detection of buried landmines using a bacterial sensor. Shimshon Belkin, Sharon Yagur-Kroll, Yossef Kabessa, Victor Korouma, Tali Septon, Yonatan Anati, Cheinat Zohar-Perez, Zahi Rabinovitz, Amos Nussinovitch, Aharon J Agranat. Nature Biotechnology, Volume 35, Number 4, April 2017. doi:10.1038/nbt.3791 (link: http://dx.doi.org/10.1038/nbt.3791)

Glowing bacteria detect buried landmines
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‘Smart’ bacteria remodel their genes to infect our intestines

22/02/2017
Hebrew University researchers describe how infectious bacteria sense they’re attached to intestinal cells and remodel their gene expression to exploit our cells and colonize our gut

Infectious diarrhea, a common disease of children, is responsible for over 2 million infant deaths annually in developing counties alone. A primary cause of this and other devastating conditions is enteropathogenic bacteria, which attack the intestinal tract when contaminated food is consumed.

The infection process involves hundreds of genes and proteins, both in the infectious bacteria and the human host. However, the processes by which the pathogens establish themselves in our gut are poorly understood.

Now, a new study published in the prestigious journal Science, by researchers at the Hebrew University of Jerusalem’s Faculty of Medicine, describes how pathogens sense their host, and tailor their gene expression to exploit their host to cause disease. The research was led by led by Prof. Ilan Rosenshine, the Etta Rosensohn Professor of Bacteriology at the Hebrew University.

Working with a pathogenic strain of E. coli, the researchers found that the bacteria can sense attachment to the human intestinal cells and activate gene expression in response.  This was demonstrated by engineering one of these genes to express a protein that stains the expressing bacteria to appear green under the microscope. Under microscopic examination, the researchers observed that only the attached bacteria fluoresce in bright green, whereas non-attached bacteria remain dark.

The researchers also deciphered how upon sensing that it has attached to intestinal cells, the pathogen reorganizes its gene expression, including genes involved in virulence and metabolism, to exploit the host cell. These findings may lead to the development of new strategies to combat bacterial infection.

"The next steps include mapping in detail the genes that change their expression upon attachment, and describing the precise effects of this expression remodeling,” said Prof. Ilan Rosenshine. “Another important issue is testing whether similar regulation is involved in the infection processes of other pathogens."

CITATION: Host cell attachment elicits posttranscriptional regulation in infecting enteropathogenic bacteria. Naama Katsowich, Netanel Elbaz, Ritesh Ranjan Pal, Erez Mills, Simi Kobi, Tamar Kahan, Ilan Rosenshine. Science, 17 Feb 2017: Vol. 355, Issue 6326, pp. 735-739. DOI: 10.1126/science.aah4886. Link: http://science.sciencemag.org/content/355/6326/735

FUNDING: The work was funded by a grant from the Israel Academy of Sciences and Humanities. N.K. is a recipient of a fellowship from the Carol and Leonard Berall Endowment.

The Hebrew University of Jerusalem is Israel’s leading academic and research institution, producing one-third of all civilian research in Israel. For more information, visit http://new.huji.ac.il/en.

- Dov Smith

‘Smart’ bacteria remodel their genes to infect our intestines
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Bacteria Sleep, Then Rapidly Evolve, to Survive Antibiotic Treatments

13/02/2017

Hebrew University biophysicists used quantitative approaches from Physics to understand issues in Biology

Antibiotic resistance is a major and growing problem worldwide. According to the World Health Organization, antibiotic resistance is rising to dangerously high levels in all parts of the world, and new resistance mechanisms are emerging and spreading globally, threatening our ability to treat common infectious diseases. But how these bacterial resistance mechanisms occur, and whether we can predict their evolution, is far from understood.

Researchers have shown (http://new.huji.ac.il/en/article/22060) that one way bacteria can survive antibiotics is to evolve a “timer” that keeps them dormant for the duration of antibiotic treatment. But the antibiotic kills them when they wake up, so the easy solution is to continue the antibiotic treatment for a longer duration.

Now, in new research published in the prestigious journal Science, researchers at the Hebrew University of Jerusalem report a startling alternative path to the evolution of resistance in bacteria. After evolving a dormancy mechanism, the bacterial population can then evolve resistance 20 times faster than normal. At this point, continuing to administer antibiotics won't kill the bacteria.

To investigate this evolutionary process, a group of biophysicists, led by Prof. Nathalie Balaban and PhD student Irit Levin-Reisman at the Hebrew University’s Racah Institute of Physics, exposed bacterial populations to a daily dose of antibiotics in controlled laboratory conditions, until resistance was established. By tracking the bacteria along the evolutionary process, they found that the lethal antibiotic dosage gave rise to bacteria that were transiently dormant, and were therefore protected from several types of antibiotics that target actively growing bacteria. Once bacteria acquired the ability to go dormant, which is termed “tolerance,” they rapidly acquired mutations to resistance and were able to overcome the antibiotic treatment.

Thus, first the bacteria evolved to "sleep" for most of the antibiotic treatment, and then this "sleeping mode" not only transiently protected them from the lethal action of the drug, but also actually worked as a stepping stone for the later acquisition of resistance factors.

The results indicate that tolerance may play a crucial role in the evolution of resistance in bacterial populations under cyclic exposures to high antibiotic concentrations. The key factors are that tolerance arises rapidly, as a result of the large number of possible mutations that lead to it, and that the combined effect of resistance and tolerance promotes the establishment of a partial resistance mutation on a tolerant background.

These findings may have important implications for the development of new antibiotics, as they suggest that the way to delay the evolution of resistance is by using drugs that can also target the tolerant bacteria.

Unveiling the evolutionary dynamics of antibiotic resistance was made possible by the biophysical approach of the research team. The experiments were performed by a team of physicists, who developed a theoretical model and computer simulations that enabled a deep understanding of the reason behind the fast evolution of resistance that were observed.

Researchers involved in the research are affiliated with the Racah Institute of Physics and the Harvey M. Kruger Family Center for Nanoscience and Nanotechnology at the Hebrew University of Jerusalem, and the Broad institute of Harvard and MIT.

FUNDING: The work was supported by the European Research Council (Consolidator Grant no. 681819) and Israel Science Foundation (492/15). ILR acknowledges support from the Dalia and Dan Maydan Fellowship.

CITATION: Antibiotic tolerance facilitates the evolution of resistance. Irit Levin Reisman, Irine Ronin, Orit Gefen, Ilan Braniss, Noam Shoresh and Nathalie Q. Balaban. Science, February 9, 2017. doi: 10.1126/science.aaj2191. (Paper will be published online at http://science.sciencemag.org/lookup/doi/10.1126/science.aaj2191.)

Bacteria Sleep, Then Rapidly Evolve, to Survive Antibiotic Treatments
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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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