Stopping Superbugs in Their Tracks

“The problem [of antibiotic resistance] is so serious that it threatens the achievements of modern medicine. A post-antibiotic era – in which common infections and minor injuries can kill – is a very real possibility for the 21st century.” The opening words of the World Health Organization’s global report on antimicrobial resistance (1) emphasize the magnitude of this issue, then call for action in developing methods to detect and monitor multiple-drug-resistant bacterial pathogens (2). But detecting these “superbugs” is no easy task, and overcoming them is even more difficult.

One of the first pathogens to be given “superbug” status, methicillin-resistant Staphylococcus aureus (MRSA), is also one of the best-known multi-drug-resistant bacteria. MRSA can strike anywhere, but is especially deadly in immunocompromised patients or when it enters the internal organs. Though a problem worldwide, the danger of “superbugs” like MRSA is emphasized in environments where many people live in close quarters. This includes hospitals, prisons and the military – one reason why the United States Air Force has chosen to collaborate with Auburn University on a new method to test for drug-resistant pathogens.

Current methods of detecting drug resistance take hours; biochemical and microbiological assays are long and labor-intensive, whereas DNA- or antibody-based methods require considerable sample preparation and purification, along with time-intensive sequencing protocols. Labs that use plate testing for MRSA need two plates for each test, which are read at 24 and 48 hours so that the final results aren’t ready for two full days. Those that use molecular analyzers can speed the process up; PCR-based instruments take only a few hours, but even they require a substantial amount of setup at the bench. To prevent these kinds of testing delays and the waste of lab resources, a team of researchers at Auburn University have devised a new technique that takes only minutes to identify antibiotic-resistant strains of Staphylococcus (3). Designed for the specific recognition and detection of MRSA, the technique includes both the identification of the bacteria and the verification of its drug resistance in real-time. While the technologies involved are not new to biosensor science, they have never before been married in a tandem technique like this one for bacterial testing.

The 10-minute test

The new method takes about 10–12 minutes to identify MRSA strains, a task it accomplishes by taking advantage of bacteriophages. These simple viruses target and kill bacteria, but are benign in humans; the MRSA test uses a strain of lytic phage that specifically targets Staphylococcus bacteria while excluding all others. The novelty of the test is in the first step, which uses this bacteriophage as a sensor probe – the lytic phage is transformed into spheroids (which maintains high bacterial capture efficiency, but makes them more suitable for sensors), then transferred onto a quartz crystal microbalance (QCM) sensor as a spheroid monolayer using the Langmuir-Blodgett technique. Once the monolayers were prepared, the researchers tested their biosensors with bacterial water suspensions while measuring changes in resonance frequency and energy dissipation; using those numbers, they were able to determine whether or not the mass density of the monolayer was increasing as bacteria bound to the phage spheroids. They found that all strains of S. aureus bacteria interacted with the spheroids to bind to the sensor, whereas other kinds of bacteria did not.

A second step exposes the biosensors to a flow of latex beads, which are conjugated to a penicillin-binding protein (PBP2a)-specific antibody. In this step, the beads will bind to sensors previously exposed to methicillin-resistant strains of S. aureus, but not to sensors that were exposed to methicillin-sensitive strains (MSSA). As the sensors are exposed to the bead flow, the changes in resonance frequency and energy dissipation (Figure 1, left) are measured again to capture the change in mass as beads bind to resistant bacteria; it is also possible to obtain a scanning electron micrograph of the bound bacteria and, where applicable, anti-PBP2a-conjugated beads (Figure 1, right), though it is not a necessary component of the test. This second step provides unambiguous discrimination between resistant and sensitive strains, so that if both steps of the test yield a positive result, it signals specific detection of MRSA.

Can it help avert a crisis?

The need for rapid, effective and sensitive detection of antibiotic-resistant bacteria is growing rapidly as more and more pathogens develop resistance to our most effective drugs. “A crisis has been building up over decades,” the World Health Organization warns, “so that today many common and life-threatening infections are becoming difficult or even impossible to treat,” (2). The tandem approach can be used not only with MRSA, but with other drug-resistant bacteria as well, and could provide medical laboratories with a quick, cost-effective way of diagnosing multi-drug-resistant infections in patients. Because of its speed and reliability, the test is particularly useful in settings with high population density, where MRSA and other drug-resistant infections are most likely to spread – and where early diagnosis can make a major difference, allowing doctors to treat their patients with the appropriate antibiotics from the start, rather than “flying blind.”

Could pathologists use it?

Melissa Andreas, a medical laboratory scientist at a core clinical lab in Oregon, USA, feels that current testing methods are somewhat outdated. “I think molecular techniques are where we’re headed,” she says, but warns that in order to implement a biosensor test like this one in the lab on a commercial basis, “it needs to be rock solid and easy to use.” Even working in a small laboratory, Andreas sees as many as 10 samples a day for MRSA testing and adds that a rapid protocol would ease the burden of testing not only patients showing signs of infection, but also potential carriers of the superbug. “Most of the MRSA assays we do are to check if people are carriers while they’re preoperative, and those are a two-day test,” she says. “But we also see MRSA show up in the normal course of microbiology testing, especially in wound cultures and urinary tract infections.” In standard infectious disease evaluation, the workflow is slightly different, but the time taken to culture bacteria and run panels of tests still results in a turnaround of up to two days. For all applications, a quick test for drug-resistant bacteria would save time and work in the lab and speed up the overall pipeline.

Michael Prystowsky, chair of the Department of Pathology at Albert Einstein College of Medicine, New York, says that his priority is to get patients the best treatment possible, as early as possible. With that in mind, he’s interested in seeing testing times reduced by any means as long as sensitivity and specificity are preserved – whether that’s through point-of-care testing, faster sample processing, or other forms of new technology. With this new test, however, he cautions that the nature of the samples used for testing will determine its value; though the test itself may only take 10 minutes, potential requirements for bacterial culturing or other preparative steps may extend the period between taking the initial sample and returning a result. The key to good patient care when dealing with multi-drug-resistant pathogens, he says, is “the right test at the right time to optimize treatment decisions” – and whether or not this new, biosensor-based diagnostic test will become “the right test” still remains to be seen.

Certainly, the test still needs evaluation with real patient samples before it’s ready for widespread use. When it can be implemented in clinical labs, the test offers the chance to greatly reduce the time from laboratory to point-of-care without losing the effectiveness or sensitivity of current methods; it’s even fast enough for use during surgical procedures. And it paves the way for the development of other applications using biosensors and bacteriophages – for instance, phages might be used as a treatment for drug-resistant infections, or transformed into spheroids to create antimicrobial surfaces for clinical use. Most importantly, the current test was designed to detect MRSA in particular, but its success outlines a new approach to screening.