Electrically Induced Arrangement of Bacteriophages Improves Bacteria Biosensors

Viruses that attack bacteria - bacteriophages - can be fussy: they only inject their genetic material into the bacteria that suit them. The fussiness of bacteriophages can be exploited in order to detect specific species of bacteria. Scientists from Warsaw have just demonstrated that bacteriophage-based biosensors will be much more efficient if prior to the deposition on the surface of the bacteriophage sensor their orientation is ordered in electric field.

In the future, an effective method of detecting a particular species of bacteria will be a bacteriophage-based biosensor. The sensitivity of current sensors coated with bacteriophages, that is, viruses attacking bacteria, is far from ideal. In the journal Sensors and Actuators B: Chemical, researchers from the Institute of Physical Chemistry of the Polish Academy of Sciences (IPC PAS) in Warsaw, Poland, have presented a method for creating layers of bacteriophages which significantly increases the efficiency of detection. This achievement, funded by the Polish National Centre for Science within SONATA and MAESTRO grants, paves the way for the production of low-cost biosensors, capable of rapidly and reliably detecting specific species of bacteria.

The late detection and identification of bacteria have been - and, unfortunately, still are - the causes of many a tragedy. The lack of reliable and rapid medical tests that, even these days, doctors only find out after several hours which bacterial species is wreaking havoc in the body of the patient. As a result, instead of administering the optimal antibiotic at an early stage of the disease, they have to guess - and often get it wrong, with disastrous consequences for the patient.

"Hospital-acquired infections, to which 100 thousand patients in the United States alone succumb each year, are just some of the problems arising from the lack of good methods for the detection of undesirable bacteria. Industrial contamination is no less important. Nobody wants to sell - much less buy - for example, carrot juice with the addition of dangerous bacteria causing typhoid fever or sepsis. However, such cases continue to occur," says Dr. Jan Paczesny (IPC PAS).

Attempts have been under way for some time to construct sensors to detect bacteria in which the key role is played by bacteriophages. A single phage, with a length of about 200 nanometers, consists of a head (capsid) containing DNA or RNA and a tail through which genetic material is injected into the interior of the bacteria. The mouth of the tail is surrounded by fibrils. They perform a very important function: they are receptors detecting the presence of bacteria and recognizing their species. The bacteriophage cannot take any risks: its genetic material must reach the interior of only those bacteria that have suitably matching genetic machinery. If the phage were to make a mistake and inject its genetic code into the wrong bacteria, then rather than duplicating itself, it would self-destruct.

The specific structure of bacteriophages means that when they are deposited on the surface they are arranged at random, and most of them cannot effectively penetrate the space around them with their receptors in search of bacteria. As a result, only a few bacteriophages in the detection layer of current biosensors can fulfill their role and the equipment's sensitivity is greatly reduced.

"Phage heads are electrically negatively charged, whereas the filaments penetrating the surroundings are positive. The bacteriophage is therefore an electrically polarized entity. This gave us the idea of 'ordering' the bacteriophages using an electric field," says PhD student Kinga Matula (IPC PAS).

The idea was simple, but its implementation proved to be far from trivial.

"There is a high pressure of up to 50 atm in phage heads. This is what enables the bacteriophage to inject its genetic material. That's fine, only that this means that bacteriophages like highly saline solutions, because then the pressure difference between the head and the environment is reduced. Such solutions are highly conductive, and therefore the electric field inside them is present only in a thin layer at the surface, further on it drops to zero. And there is a problem. Fortunately, we have managed to solve it," explains PhD student Lukasz Richter (IPC PAS).

During their experiments, the Warsaw-based scientists, led by Prof. Robert Holyst, used an appropriately selected constant electric field. Bacteriophages were deposited on a carefully constructed glass substrate, coated first with titanium and then with gold. The titanium served as the glue binding the gold with the glass, while the gold was the main 'bait' to which the bacteriophages bound. Unfortunately, not only bacteriophages like gold, so do bacteria. To prevent the binding of random bacteria with the gold layer, the empty spaces between the deposited bacteriophages were covered with a neutral protein (casein).

T4 bacteriophages that attack Escherichia coli bacteria were used to construct the new detection layer at the IPC PAS. The phages for the studies were prepared by the team of Prof. Marcin Los from the Department of Biology, University of Gdansk.

"Virtually all of the bacteriophages in our detection layers stand on the substrate's surface, so they can easily spread out their receptors. The situation is somewhat similar to what is seen at a rock concert, where fans often raise their hands high above their heads in unison and wave them cheerfully in all directions. We have the impression that our phages are even happier, because we try not to place them too close to each other. After all, the neighbours' receptors should not interfere with each other," says Prof. Holyst with a smile.

Meticulous laboratory tests have established that the bacteriophage layers produced using the method developed at the IPC PAS trap up to four times more bacteria than existing layers. As a result, their sensitivity is close to that of the best biosensors that use other, more time consuming and expensive, methods for the detection of bacteria.

The method of preparing layers of ordered bacteriophages developed in Warsaw has numerous advantages. The creation of an external electric field, which is necessary to put the bacteriophages in order, is not very costly. The field acts through space and therefore direct contact of the electrodes with the solution is not required. The presence of an external electric field also means there is significantly less physicochemical interference than in the situation where current is passed through the solution. At the same time, the method is fast and universal: it can be used not only for bacteriophages but also for other electrically polarized molecules.

Source: Institute of Physical Chemistry of the Polish Academy of Sciences  

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Department of Homeland Security Bioterror Detection Program Unreliable, GAO Says

Although the US has made significant strides towards improving preparedness for a bioterrorist attack, the nation’s biosurveillance capabilities are a far cry from adequate, according to a recent report by
the Government Accountability Office (GAO), which found he nation’s billion dollar biosurveillance detection system can’t be counted on to actually work.

In April, the House Committee on Homeland Security’s Subcommittee on Emergency Preparedness, Response and Communications said a biological terrorist attack on the US is an "urgent and serious threat."

The US began to recognize a bioterrorist attack as a serious and urgent threat just days after the terrorist attacks of Sept. 11, 2001 when anonymous letters laced with deadly anthrax spores were sent through the mail, sickening 17 people and killing 5 others. The anthrax attacks awakened the nation to the catastrophic impact of a bioterrorist attack in the US.

Shortly thereafter, the Department of Homeland Security (DHS) quickly rolled out a biosurveillance program known as BioWatch to provide early warning of a biological weapon attack in the US. Deployed in more than 30 metropolitan areas throughout the country, the system uses aerosol collectors to detect the intentional release of select aerosolized biological agents.

However, GAO determined the rapid deployment of the program in 2003 did not allow for sufficient testing and evaluation of the system’s capabilities. The report said that without sufficient testing, DHS could not support the claim that the program could meets its operational objective to detect catastrophic attacks, which they define as attacks large enough to cause 10,000 casualties.

“DHS officials told us that in the 12 years since BioWatch’s initial deployment, they have not developed technical performance requirements against which to measure the system’s ability to meet its objective,” GAO stated.

Over the years, numerous false alarms have plagued the program, exasperating local and state officials where the detection system is deployed. From 2003 through 2014, BioWatch generated 149 mistaken detections — all of which have been termed false positives by scientists at the US Centers for Disease Control and Prevention and other experts GAO consulted.

False positives are more than just a mere annoyance. They can lead to the shutdown of major transportation and economic facilities, such as airports and shopping centers, as well as the unnecessary medication of an uninfected public.

“I am supportive of efforts in early detection and mitigation of a biological attack against our homeland,” said Senate Committee on Homeland Security and Governmental Affairs Chairman Ron Johnson (R-Wis.). “However, GAO raises serious questions about the uncertainty in the capabilities of the current BioWatch system. We may be missing opportunities to properly support our biodefense infrastructure.”

The current BioWatch system in use is referred to as Gen-2, which expanded deployment of the system to additional jurisdictions and included the addition of indoor monitoring capabilities in three high-threat jurisdictions. While DHS has taken steps to mitigate the limitations associated with not testing the Gen-2 system in an operational environment with live biothreat agents, GAO determined DHS did not systematically test the Gen-2 system under the most realistic possible conditions.

“Because it is not possible to test the BioWatch system directly by releasing live biothreat agents into the air in operational environments, DHS relied on chamber testing and the use of simulated biothreat agents, which limit the applicability of the results,” GAO’s audit report report stated. “These limitations underscore the need for a full accounting of statistical and other uncertainties, without which decision makers lack a full understanding of the Gen-2 system’s capability to detect attacks of defined types and sizes and cannot make informed decisions about the value of proposed upgrades.”

As soon a DHS deployed the BioWatch program in 2003, they began working on an autonomous detection capability known as Gen-3 in order to reduce operational costs, as well as the time required to detect biothreat agents. DHS envisioned that the system would automatically collect air samples, conduct analysis to detect the presence of biothreat agents every 4 to 6 hours, and communicate results to public health officials via an electronic network without manual intervention.

However, after a GAO audit recommended DHS examine the acquisition process, DHS subsequently commissioned an analysis of alternatives, which was interpreted by DHS as showing that any advantages of an autonomous system over the current manual system were insufficient to justify the cost of a full technology switch.

DHS cancelled the Gen-3 acquisition in April 2014, and made Gen-2 the official program of record for aerosol biological threat detection. In the next year, some Gen-2 equipment will reach the end of its lifecycle; consequently, DHS will need to make decisions about reinvesting in the program.

DHS is considering autonomous detection as an upgrade to Gen-2, with possible benefits including reduction in casualties and clean-up costs. But, GAO said, “The extent of these benefits is uncertain because of several assumptions, such as the speed of response after a detection, that are largely outside of DHS’s control.”

GAO added, “As a result, the effectiveness of the response—and the number of lives that could be saved—is uncertain.”

Consequently, GAO recommended DHS not pursue upgrades or enhancements for Gen-2 until it reliably establishes the system’s current capabilities. Additionally, DHS should incorporate best practices for testing in conducting any system upgrades.

DHS generally concurred with GAO’s recommendations.

“The findings by the GAO bring into focus shortcomings in the BioWatch program at a time when concerns about the threat of a bioterrorism event are elevated,” said House Homeland Security Committee Chairman Michael McCaul (R-Texas), ranking member Bennie G. Thompson (D-Miss.), Emergency Preparedness, Response and Communications Subcommittee Chairman Martha McSally (R-AZ), and Emergency Preparedness, Response and Communications Subcommittee ranking member Donald Payne, Jr. (D-NJ) in a joint statement.

“Earlier this month, the co-chairs of the Blue Ribbon Study Panel on Biodefense testified before our Committee on the threat posed by bioterrorism,” McCaul, Thompson, and McSally added. “They made it clear that that we must act aggressively and deliberately to bolster our ability to detect and rapidly respond to a bioterror event. We also know terrorist groups, like ISIS, aspire to conduct attacks using biological agents. These facts make the GAO’s findings about BioWatch all the more concerning.”

Just weeks ago, Homeland Security Today reported that the bipartisan Blue Ribbon Panel on Biodefense strongly encouraged renewed focus on the need for rapid diagnostics and prioritization of the development of a fully functional environmental detection system to replace BioWatch.

“The entire BioWatch system is dying for lack of innovation,” the report stated. “DHS attempted and failed to acquire next-generation BioWatch technology (Generation 3) that could have reduced time to-detection to as few as six hours. Even if the acquisition had been successful, the system would still have been flawed: like the current system, it would have addressed only a small number of biological agents, inactivated them, and relied on non-random air currents.”

“To date, no fully automated, tested, and evaluated autonomous detection system has been deployed that adequately addresses the airborne biological threat or sufficiently provides operational response information,” the report added.


Bioterrorist attack: hype or reality?

GAO’s report emerged amid a time of heightened concern over the nation's vulnerability to biological terrorism. Moldovan police working alongside the Federal Bureau of Investigation recently uncovered multiple attempts by gangs with suspected Russian connections to sell radioactive material to Islamic State militants.

Homeland Security Today recently reported that the possibility of a bioterrorist event is not simply hype—it is a reality. Of particular concern is the threat of terrorist use of a “dirty bomb,” a type of radiological dispersal device (RDD) that combines conventional explosives such as dynamite with radioactive material like Cobalt 60.

Although terrorists have yet to launch a successful RDD attack on US soil, the threat is real and terrorists have shown an interest in RDDs. The material needed to make a dirty bomb is almost everywhere and the technical sophistication required to create such a device is minimal. Moreover, the intent to create and use such a device is certainly there.

“There can be absolutely no doubt as to the aspirations of terrorist groups, particularly Islamic organizations like ISIS, to acquire and use any and all weapons of mass destruction they can,” said former CIA WMD counterterrorism official Charles Faddis told Homeland Security Today. “ISIS holds an apocalyptic worldview. It is not simply in a battle with the West -- it is in the final battle. They believe the world is literally coming to an end, and any and all means necessary must be employed to ensure they emerge victorious. There is no such thing as too far or too horrific.”

Just last month, the House Committee on Transportation and Infrastructure Subcommittee on Coast Guard and Maritime Transportation recently held a hearing to discuss the vulnerability of US ports to terrorist attacks using a dirty bomb.

During the hearing, Dr. Stephen Flynn, director of the Center for Resilience Studies at Northeastern University, testified that if a dirty bomb ends up in the wrong hands, our country is at grave risk. Currently, there is a real and present danger that containers will be used as modern-day Trojan horses, since the reality is no one really knows what is inside a container except those who are there when the container is packed.

“Should a dirty bomb that originated overseas be set off in a US port, it would represent a major security breech in the global supply system that will result in US port closures,” Flynn said. “This, in turn, will place the intermodal transportation system at risk of widespread economic disruption generating tens of billions of dollars in losses, and potentially endangering lives as the shipments of critical time-sensitive goods such as medical supplies and defense-related materials are interrupted.”

Furthermore, earlier this year, Homeland Security Today conducted an exclusive interview with Brig. Gen. JB Burton, the commanding general of the United States Army 20th Chemical, Biological, Radiological, Nuclear and Explosives Command (CBRNE), in which he stated both ISIS and Al Qaeda’s leaders and their determined affiliates around the world have made it exceedingly clear they seek weapons of mass destruction – especially chemical, biological, radiological and nuclear – to use in attacks on the West – in particular, the United States.

CBRNE and security experts are all saying the same thing: terrorists are intent on using a biological weapon to attack the United States. The failure of US bio-preparedness efforts like BioWatch is not only wasting taxpayer dollars, it is putting the safety of the American people at risk.

“Now more than ever we need reassurances that our efforts to combat and prevent bioterrorism are successful and trusted. It is clear that BioWatch has not lived up to the job it set out to do, and we must put our efforts toward finding a program that will be successful in detecting and preventing these catastrophic attacks,” said full committee chairman Fred Upton (R-Mich.), Oversight and Investigations Subcommittee Chairman Tim Murphy (R-Penn.), and Oversight and Investigations ranking member Diana DeGette (D-Co.) in a statement.

Source: Homeland Security Today.US

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Researchers Propose Rapid Ebola Test Using Nanotechnology

Just as Ebola was finally fading from the headlines, it came back in the news with shocking reports: a Scottish nurse rehospitalized nine months after beating Ebola is now suffering from meningitis caused by the virus. A recent study also confirms the virus can live in semen for up to nine months, maybe more.

These stories, added to the continuing trickle of new cases out of Guinea, remind us of the persistence of the deadly virus and the need for quick diagnosis and treatment.

“Recent Ebola virus infection has taken more than 11,000 lives globally and there is still no way to target the virus and kill the disease,” says Ajeet Kaushik, assistant professor in the Center for Personalized Nanomedicine in the Institute of Neuroimmune Pharmacology at the Herbert Wertheim College of Medicine (HWCOM).

Kaushik decided to undertake an extensive review of the Ebola literature to see where and how there may be an improvement in the diagnosis and or treatment of the disease. Ebola Virus Disease (EVD) is lethal. When a patient gets infected with Ebola, the right diagnosis is not, unfortunately, a guarantee that the person will be saved. But recognizing the virus early greatly increases the chance the person will live, and catching it at later stages skyrockets the likelihood they will die.

Kaushik was aided in the review by colleagues Sneham Tiwari, Rahul Dev Jayant, and Center director Madhavan Nair, as well as by Dr. Aileen Marty. The paper,  titled “Toward detection and diagnosis of Ebola virus disease at point-of-care,” was recently published in the journal Biosensors and Bioelectronics: 75, 2016, 254–272.

Marty, a professor of Infectious Diseases in the Department of Medicine, Family Medicine and Community Health at the HWCOM, specializes in tropical medicine, infectious disease pathology and disaster medicine. She took two trips to Africa in 2014 and 2015 to offer aid and expertise in containing and treating the virus and offered a front-line perspective to the review.

The team’s conclusions after summarizing these articles: Early detection of the disease would be a critical tool in helping control the virus and save lives.

“Yes, recent vaccine trials have demonstrated effectiveness in preventing clinical disease, but only with fast detection and equally fast distribution of the vaccine to all those exposed, and the vaccines being tested only exist in limited supplies and have not yet been licensed,” Marty says.

It currently takes six to eight hours to perform the diagnostic testing that will prove or disprove that a patient has Ebola. This must be performed in a laboratory. In the meantime, the caregivers are facing shortages of resources, such as IV fluids and hospital beds, to help the sick while they await their fate.

“Conventional methods of detection are good but slow and should be performed at Biosafety Standard Level 3 laboratories which are expensive to maintain and require skilled personnel,” Kaushik says.

Marty, who was recently appointed to the Presidential Advisory Council on Combating Antibiotic-Resistant Bacteria, also noted that the lack of appropriate safety, work stations and effective selective rapid diagnostics methodologies will continue to be significant challenges in the fight against EBOV. But a cost-effective, rapid, sensitive and selective sensor that can detect Ebola at point-of-care could literally be lifesaving.

“It is important to devise more rapid, more sensitive, but also more specific and appropriate diagnostic assays for Ebola because there are grave social, economic and medical – even lethal consequences –to a misdiagnosis,” Marty says. “This requires that only diagnostics tests that have undergone independent, comprehensive assessment of quality, safety and performance be used in confirming the diagnosis of infection with Ebola virus. While this new nano-technology is extremely promising, we must be very careful to assure that a definitive diagnosis is given only if the specificity is extremely high – a criteria that becomes even more important as the incidence and prevalence of Ebola disease continues to decrease. This nanotechnology is currently likely to be a good screening test but it should still be backed up with nucleic acid testing using technologies such as polymerase chain reaction (PCR).”

Kaushik’s lab has been working on miniaturized sensing technology and integrating that into devices that are capable of detecting virus levels for quite some time. Combining the collaborative strengths of a biologist, nanotechnologist and engineers to develop smart compartments to integrate point-of-care (POC) Ebola sensing devices in a BSL-4 environment should be the aim of future research approach in this area.

“We believe that if we explore this option, the length of time it will take to detect the Ebola virus will be reduced to 40 minutes instead of multiple hours,” Kaushik says.

That’s time well-spent.

Source: Florida International University (Ileana Varela and Robyn Nissim)

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