New Chip Makes Testing for Antibiotic-Resistant Bacteria Faster, Easier

It’s a device that could transform a doctor’s ability to treat infections: a test for antibiotic resistance that works in just one hour – instead of several days.

We live in fear of ‘superbugs’: infectious bacteria that don’t respond to treatment by antibiotics, and can turn a routine hospital stay into a nightmare. A 2015 Health Canada report estimates that superbugs have already cost Canadians $1 billion, and are a “serious and growing issue.” Each year two million people in the U.S. contract antibiotic-resistant infections, and at least 23,000 people die as a direct result.

But tests for antibiotic resistance can take up to three days to come back from the lab, hindering doctors’ ability to treat bacterial infections quickly. Now PhD researcher Justin Besant and his team at the University of Toronto have designed a small and simple chip to test for antibiotic resistance in just one hour, giving doctors a shot at picking the most effective antibiotic to treat potentially deadly infections. Their work was published this week in the international journal Lab on a Chip.

Resistant bacteria arise in part because of imprecise use of antibiotics – when a patient comes down with an infection, the doctor wants to treat it as quickly as possible. Samples of the infectious bacteria are sent to the lab for testing, but results can take two to three days. In the meantime, the doctor prescribes her patient a broad-spectrum antibiotic. Sometimes the one-size-fits-all antibiotic works and sometimes it doesn’t, and when the tests come back days later, the doctor can prescribe a specific antibiotic more likely to kill the bacteria.

“Guessing can lead to resistance to these broad-spectrum antibiotics, and in the case of serious infections, to much worse outcomes for the patient,” says Besant, pictured at right (photo courtesy U of T Engineering). “We wanted to determine whether bacteria are susceptible to a particular antibiotic, on a timescale of hours, not days.”

The problem with most current tests is the time it takes for bacteria to reproduce to detectable levels. Besant and his team, including his supervisor Professor Shana Kelley of the Institute for Biomaterials & Biomedical Engineering and the Faculties of Pharmacy and Medicine, and Professor Ted Sargent of The Edward S. Rogers Sr. Department of Electrical & Computer Engineering, drew on their collective expertise in electrical and biomedical engineering to design a chip that concentrates bacteria in a miniscule space – just two nanolitres in volume – in order to increase the effective concentration of the starting sample.

They achieve this high concentration by ‘flowing’ the sample, containing the bacteria to be tested, through microfluidic wells patterned onto a glass chip. At the bottom of each well a filter, composed of a lattice of tiny microbeads, catches bacteria as the sample flows through. The bacteria accumulate in the nano-sized well, where they’re trapped with the antibiotic and a signal molecule called resazurin.

Living bacteria metabolize resazurin into a form called resorufin, changing its electrochemical signature. If the bacteria are effectively killed by the antibiotic, they stop metabolizing resazurin and the electrochemical signature in the sample stays the same. If they are antibiotic-resistant, they continue to metabolize resazurin into resorufin, altering its electrochemical signature. Electrodes built directly into the chip detect the change in current as resazurin changes to resorufin.

“This gives us two advantages,” says Besant. “One, we have a lot of bacteria in a very small space, so our effective starting concentration is much higher. And two, as the bacteria multiply and convert the resazurin molecule, it’s effectively stuck in this nanolitre droplet – it can’t diffuse away into the solution, so it can accumulate more rapidly to detectable levels.”

“Our approach is the first to combine this method of increasing sample concentration with a straightforward electrochemical readout,” says Sargent. “We see this as an effective tool for faster diagnosis and treatment of commonplace bacterial infections.”

Rapid alternatives to existing antibiotic resistance tests rely on fluorescence detection, requiring expensive and bulky fluorescence microscopes to see the result.

“The electronics for our electrochemical readout can easily fit in a very small benchtop instrument, and this is something you could see in a doctor’s office, for example,” says Besant. “The next step would be to create a device that would allow you to test many different antibiotics at many different concentrations, but we’re not there yet.”

Source: University of Toronto

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AgriLife Research Engineer Develops Real-Time Listeria Biosensor Prototype

A Texas A&M AgriLife Research engineer and a Florida colleague have developed a biosensor that can detect listeria bacterial contamination within two or three minutes.

“We hope to soon be able to detect levels as low as one bacteria in a 25-gram sample of material – about one ounce,” said Dr. Carmen Gomes, AgriLife Research engineer with the Texas A&M University department of biological and agricultural engineering, College Station.

The same technology can be developed to detect other pathogens such as E. coli O157:H7, she said. But listeria was chosen as the first target pathogen because it can survive even at freezing temperatures. It is also one of the most common foodborne pathogens in the world and the third-leading cause of death from food poisoning in the U.S.

“It can grow under refrigeration, but it will grow rapidly when it is warmed up as its optimum growth temperature ranges from 30 to 37 degrees Celsius — 86 to 98 degrees Fahrenheit,” Gomes said. “This makes it a particular problem for foods that are often not cooked, like leafy vegetables, fruits and soft cheeses that are stored under refrigeration.”

Currently, the only means of detecting listeria bacteria contamination of food requires highly trained technicians and processes that take several days to complete, she said. For food processing companies that produce and ship large quantities of foodstuff daily, listeria contamination sources can be a moving target that is often missed by current technology.

The biosensor she is working on is still in the prototype stage of development, but in a few years she envisions a hand-held device that will require hardly any training to use.

Gomes is collaborating with Dr. Eric McLamore at the University of Florida at Gainesville.

“I do the biological and polymer engineering; he does the electrochemistry and nanostructures,” she said.

As for the biological component, Gomes said she is using “nanobrushes” specially designed to grab particular bacteria.

The nanobrushes utilize “aptamers,” which are single-stranded DNA or RNA molecules that bind to the receptors on the target organism’s cell outer membrane, Gomes said. This “binding” is often compared to the way a key fits into only one lock.

In this manner, the nanobrushes select for only a specific type of cell, which in the case of her work is the listeria bacterium.

Gomes noted that the inspiration for the nanobrushes comes from the Hawaiian bobtail squid, a football-sized creature that forms a symbiotic relationship with bioluminescent bacteria. Microscopic, hair-like structures, called cilia, on the squid’s light organ select and capture the bacteria from a very complex microbial soup of the ocean.

“The squid feeds the bacteria sugar and amino acids and in return, the bioluminescent bacteria allow the squid to produce light, which then allows the squid to escape from things that might want to eat it,” she said. “To predators, the bioluminescence is very similar to the light coming from the moon and stars at night, which acts as a ‘camouflage’ when observed from below.

“The selection process the polymers use to select for specific bacteria in the listeria biosensor is very similar to the squid’s cilia. We are trying to mimic the same mechanism of bacteria’s capture used by the squid’s cilia.”

Currently, the listeria biosensor is about the size of a postage stamp, with two wires leading to two etched conductive areas. After a few minutes, when the polymer nanobrushes have had time to grab the selected bacteria, the rest of the sample is washed away and the impedance, or resistance, between the two surfaces is measured electronically.

Gomes and McLamore are moving on to refining the electronics to something that can be handheld and easily used. Also in the works is a disposable paper-based biosensor that can be disposed of after one use.

In early April, they were awarded a three-year $340,000 National Science Foundation grant to continue their work on nanobrushes for pathogen detection.

Source: AgriLife TODAY

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Colorado Researchers Fighting to Get Ahead of the Next Ebola Outbreak

ReEBOV may be one of the most important technologies developed by Corgenix Medical Corp., but it's not a financial boon for the 25-year-old diagnostic test firm.

Company officials would just as soon keep it that way.

ReEBOV this year became the first rapid diagnostic test to gain U.S. and international approval for use to detect the deadly Ebola virus.

In the past couple of weeks, more than 10,000 of the kits — which bear similarity to pregnancy tests and can detect the presence of Ebola in blood in less than 15 minutes, instead of hours or days — were shipped to clinics and hospitals across the globe.

"It would be highly unlikely that this product would ever be an enormous commercial success," said Douglass Simpson, the former Corgenix chief executive who now oversees the Broomfield firm's infectious-diseases group. "Certainly, we would hope not."

Since last summer, Ebola, which has no known cure or vaccine, has killed more than 11,000 people, mostly in West Africa, according to the Centers for Disease Control and Prevention.

But the worst-ever outbreak is waning, and that has Corgenix and other Colorado researchers racing to test their products before the disease goes dormant again.

Their aim: to develop an artillery of therapies and technologies along with a robust, data-driven network to track and combat Ebola the next time it rears its head. (Some countries, including Liberia, have been declared Ebola-free.)

"You can imagine that even before (the Ebola outbreak) that the health care infrastructure there needed some upgrading," said Asher Greenberg, Ebola project coordinator and communications officer for Fio Corp., a mobile diagnostics firm based in Canada. "The crisis has brought out a number of companies with solutions, but all of this needs to be integrated."

Corgenix's work, funded mostly by grants, includes adapting its ReEBOV test to be compatible with Fio's Deki Reader. The reader is an 8-inch-by-4-inch mobile device that analyzes and transmits data from immunoassay tests, like ReEBOV, that have been fit into a plastic housing about the size of a microcassette tape.

Researchers from Fio and Corgenix are in West Africa now, testing the new products in the lab and the field.

"This is a great time, with all of the attention still on West Africa, to update the infrastructure so that the next outbreak can be handled with less loss of life," Greenberg said.

Although the Ebola epidemic was thousands of miles away from the U.S., its severity spurred agencies such as the National Institutes of Health and organizations including the Bill & Melinda Gates Foundation and the Paul G. Allen Family Foundation to pour billions of dollars into the fight.

Several million dollars went to Corgenix, a 55-employee company that has developed a cache of more than 50 diagnostic products spanning a variety of diseases.

Financial backing

Corgenix, in partnership with the Viral Hemorrhagic Fever Consortium, had success in bringing to market a rapid diagnostic test for Lassa, another deadly hemorrhagic fever, and dipped its toes in developing a similar test for Ebola.

When the latest Ebola outbreak worsened, Corgenix's Ebola research was brought to the frontline.

The Gates and Allen foundations both provided financial backing to integrate the Corgenix and Fio technologies. The aim not only was to identify and then isolate Ebola-infected individuals but also to gather data that would allow health officials to monitor potential hot spots and get a leg up on containment.

Matt Boisen, Corgenix's program director for infectious disease and emerging technologies, last week began his ninth research trip to West Africa. Before the Ebola outbreak, Boisen conducted research in Kenema, Sierra Leone, related to Corgenix's Lassa rapid test.

Boisen and other Corgenix employees and consortium research partners volunteered for the work, said Simpson, who moved from the role of CEO to consultant after Corgenix's acquisition last year by Orgentec.

"Thank goodness for people who are willing to go," said Simpson, who also has traveled to the region on behalf of Corgenix. "I don't think anyone is not fully aware of the dangers. If something were to happen, I'd carry that. I'd blame myself forever."

There are elements of risk in traveling to countries roiled by political unrest and to regions battling deadly diseases with limited health infrastructure. Despite a strict regimen of taking medications and applying skin protection from mosquitoes, Boisen returned from one of his trips and landed in a hospital with a case of malaria, said his wife, Shelly.

"He did go to Nigeria one time, and Matt and (other researchers) were escorted by several men with AK-47s," she said. "The whole time, all I'm thinking of is 'Are you safe? Are you safe? Are you safe?' "

On his trip earlier this year, Boisen saw how Ebola's spread overwhelmed the Kenema Government Hospital, effectively shutting it down temporarily.

Ebola killed several of his friends and associates at the Kenema hospital and lab.

"But that's why we take the precautions that we do," he said.

Boisen's work in Kenema will be centered entirely in the Lassa Fever Laboratory at the Kenema hospital, and any samples tested will take place under a protective hood.

Boisen will be wearing protective gear as he works, which helps lift some of the emotional weight that builds when he travels to hot zones, his wife of 14 years said.

"I know he's safe," said Shelly Boisen, who works as a certified nursing assistant at Longmont United Hospital. "He actually did a video for us the last time he went down. I'm going to give it to our hospital (as an example of) precaution training. It took him about 20 minutes to put on the first layer; he duct-tapes the shirt, puts on gloves, duct-tapes the gloves onto the sleeves, and puts on the helmet."

She's also buoyed by the understanding that the dangerous work being done by her husband and others is for the greater good.

"There are good people over there that are also helping and putting their lives on the line, and he's just one of them," she said. "He's done a lot of this in the company, too, in the lab — test after test after test after test to perfect it."

Complementing the on-the-ground work in Africa by Corgenix, Fio and others is Ebola-related domestic research such as that occurring at Colorado State University.

Grants for research

Mathematics professor Michael Kirby and the university's Biopharmaceutical Manufacturing and Academic Resource Center, or BioMARC, each received grants to conduct Ebola-related research.

BioMARC was awarded a $2 million subcontract from the U.S. Department of Defense in October to develop and manufacture a vaccine to protect against filoviruses, including Ebola and Marburg.

"The project is moving forward with successes that are in-line with product expectations," Dennis Pierro, BioMARC director, said in a statement. "We are advancing the product toward the objective of the U.S. Department of Defense."

BioMARC officials said they expect to provide more information in the coming months.

The separate mathematics-focused study has generated some positive initial results, said Kirby, a professor in CSU's departments of Computer Science and Mathematics.

Kirby previously applied mathematical algorithms to data collected from patients infected with the H1N1 influenza virus to try to understand how the flu virus moved through and took root in the immune system.

Analyzing the behavior of the more than 1,400 genes involved, Kirby helped to show the genetic pathway of the virus and identify the point at which an affected person became symptomatic.

"What we discover with influenza we hope will carry over to the Ebola virus," Kirby said.

Kirby's team does not collect the data but rather uses data sets that already are in the public domain, including that of infected mice and nonhuman primates.

The early results from the mice data showed some pathways that were comparable to human influenza infection.

"For Ebola, it's very interesting," he said. "There are no tests for Ebola until you've become symptomatic."

And it can be weeks after infection before symptoms such as a fever start to show.

Finding those pathways could bolster diagnostic and early warning tests and, ideally, limit the spread of the disease, he said.

"It's kind of like a canary in a coal mine," he said.

SOURCE: The Denver Post

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