The response of bacteria to antibiotics can be quickly assessed by monitoring the fluctuations of cantilevers coated with the bacteria.
Infections that are resistant to antibiotics are one of the gravest threats to human health and have recently been classified alongside dangers such as global warming and terrorism (1). Such drug-resistant strains of bacteria can spread rapidly and unpredictably, and have the potential to cause enormous human and economic losses. The problem is acute because the pipeline of new antibiotics has dried to a trickle, and the widespread misuse of antibiotics has fuelled the rise in bacterial resistance through natural selection. The correct treatment of an infection relies on early detection with accurate diagnostic tests, but current gold-standard methods are slow requiring days to weeks to culture bacteria — a method used in laboratories to grow sufficient bacteria so that their sensitivity to antibiotics can be tested. The inherent delays between tests, results, follow-up appointments and treatment impacts on patient health outcomes and leads to ongoing transmission of serious infections. These delays also hinder public health efforts to tackle new threats. These major unmet clinical needs are driving the development of new rapid diagnostic tests to revolutionize the management of infections and improve the stewardship of antibiotics. Writing in Nature Nanotechnology, Giovanni Longo and colleagues have now shown that low-frequency fluctuations of atomic force microscopy cantilevers can be used to characterize bacteria, rapidly test their sensitivity to antibiotics and identify resistance within minutes (2).
Free-standing cantilevers, which function like miniature diving boards, are of interest in a range of diagnostic applications due to their sensitivity and microscopic dimensions, and their ability to be mass manufactured and operated in parallel. Researchers have, for example, previously used cantilevers to study bacteria with high sensitivity (3,4,5). However, tests of antibiotic sensitivity have typically relied on culturing cells on the cantilever (6). Longo and colleagues — who are based at EPFL, University Hospital of Lausanne and the University of Lausanne — have been able to move beyond these limitations by using a novel nanomechanical transduction mechanism to sense bacteria, which they correlate to cell metabolism.
To test their approach, the team optically track the fluctuations of cantilevers coated with Escherichia coli (E. coli) and Staphylococcus aureus in response to stimuli that alter the metabolism of the bacteria (Fig. 1). The bacteria were selected as models of motile and non-motile bacteria, respectively, and are both clinically important targets. S. aureus is a common hospital-acquired infection often associated with surgical procedures. Its infamous drug-resistant counterpart methicillin-resistant S. aureus (MRSA) commonly grabs newspaper headlines, but E. coli is the most frequent cause of infection in UK hospitals with increasing levels of multidrug resistance (1).
The measurements are carried out using cantilevers coated on both sides with several hundred physisorbed live bacteria. The cantilever analysis is not based on a simple static deflection signal or a change in resonant frequency but a time-dependent fluctuation chart, which is statistically analysed in terms of its variance. The signal appears highly variable and no well-defined oscillatory component is reported. However, on exposure to ampicillin (a broad spectrum antibiotic related to penicillin) there is a 20-fold reduction in cantilever variance in less than 5 min. The signal does not recover after flushing with bacterial growth medium, which Longo and colleagues associate with cell death. In comparison, cantilevers coated with a strain of E. coli that is resistant to ampicillin shows only a temporary reduction in variance, which is reversed after a few minutes.
The most convincing evidence that the fluctuations are due to the bacteria is provided through a systematic titration of antibiotic concentrations, which yields quantitative measurements of the minimum inhibitory concentration (the amount of antibiotic needed to prevent bacterial growth) and minimum bactericidal concentration (the amount needed to kill the bacteria) that are comparable with gold-standard methods. To rule out the influence of Brownian motion and mechanical resonances, the team test increasingly glucose-rich environments to enhance cell metabolism and find the variance increases accordingly. Optical microscopy suggests that the bacteria remain attached to the cantilever and there is no evidence of lateral movement. Moreover, the results have been independently verified by other researchers using a different experimental set-up.
The results are likely to be of particular value to the analysis of slow growing species of bacteria such as Mycobacterium tuberculosis (TB), which is an infection that is of significant concern in developing countries and can take several weeks to test by culture methods. The grand challenge now will be to integrate cantilevers into robust, cost-effective point-of-care devices that can examine clinically problematic bacteria in a variety of community settings, including doctors' surgeries and developing countries. However, over the past ten years, the successful commercialization of cantilever-based biosensors has proved challenging and alternate micro- and nanoelectromechanical systems may also be worth pursuing. Furthermore, the work is likely to spark new research into the role of nanomechanical forces in the systems biology of pathogens, which could in turn help to improve our understanding of how antibiotics actually work.
The exact origin of the fluctuations arising from cantilevers coated with bacteria remains unknown. The immediate response to ampicillin is surprising given that it inhibits cell wall crosslinking and is traditionally associated with a slow mechanical weakening of the cell followed by death due to cell lysis. Longo and colleagues cite a report of massive ampicillin-triggered changes in transcription profiles (7) and suggest that the fluctuations originate from the cell wall or bacterial envelope, highlighting the similar timescales of the movement of the molecular motors in the bacterial membranes and cell metabolism. A more fundamental understanding of the signal origins is limited as the fluctuations are generated by a population of bacteria and depend on the position and the attachment of the cells on the cantilever.
To address this, future research could utilize the atomic force microscope to track fluctuations of single bacterium. Similar studies on single yeast cells has detected periodic kHz mechanical oscillations linked to metabolic changes (8) and there is a rich body of work on the mechanics of single eukaryotic cells (9) suggesting this form of cantilever-diagnostics could potentially have wider applications. Future work should also investigate the role of the bacterial cytoskeleton, analogous to the complex force machinery in eukaryotes; the discovery of this cytoskeleton is relatively recent and is transforming our understanding of the nanomechanics of some of the simplest organisms on the planet and could lead to new targets for drug discovery (10).
The work of Longo and colleagues provides renewed hope for the development of rapid diagnostics to help improve the use of antibiotics and preserve their effectiveness for future generations. However, the battle is far from over and the risks are high. New antibiotics are urgently needed and our knowledge of pathogens must evolve faster than the emergence of new drug-resistant and untreatable infections.
Figure: Longo and colleagues (2) use microfabricated cantilevers (grey) that are coated on both sides with bacteria. The fluctuations in the cantilevers are tracked using a laser beam (red lines), which is reflected off the free-end of the cantilever onto a photodetector. In the absence of antibiotics, low-frequency mechanical fluctuations of the cantilever are detected and statistically analysed in terms of their variance. On exposure to antibiotics (light blue objects), the variance for drug-susceptible bacteria (dark blue objects; left image) is almost negligible. However, for cantilevers coated with antibiotic-resistant bacteria (red objects; right image), low-frequency mechanical fluctuations are observed in the presence of antibiotics. This nanomechanical approach can be used to identify antibiotic resistance within minutes.
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