Biofilms present one of the most persistent challenges in antimicrobial treatment. Encased in a dense, sticky extracellular matrix, bacteria living in these communities can tolerate much higher drug concentrations than their free-floating counterparts – yet precisely why this happens has remained frustratingly hard to pin down.
Now, by embedding gold nanostars inside living Staphylococcus aureus biofilms, Steven Bell and colleagues at Queen’s University Belfast (QUB) used surface-enhanced Raman spectroscopy (SERS) to observe the antibiotic levofloxacin arrive in real time. The result is a direct measurement of antibiotic diffusion inside a biofilm, revealing transport rates more than three orders of magnitude lower than in water, and offering quantitative evidence of just how formidable the biofilm matrix can be.
In this interview, Bell explains how gold nanostars overcame long-standing matrix challenges, and what measuring diffusion – rather than simply detecting presence – could mean for understanding antibiotic resistance and designing smarter antimicrobial strategies.
What was your main inspiration for this work?
The main incentive for starting this work was the recognition that although we have had some success using SERS to study planktonic bacteria, the really important target is biofilms, since they are the predominant form of bacterial life on Earth. For example, 80 percent of all human bacterial infections involve biofilms. Through our colleagues in the School of Pharmacy at QUB, we were already aware of the problem of biofilms on implanted medical devices of all types, but they are also important in everything from wound infections to UTIs. We hoped we could bring the power of SERS, which combines sensitivity with molecular specificity, to understanding some of the processes that occur within these structures.
What was the biggest challenge you had to overcome?
It is really difficult to use SERS inside biofilms because it involves making measurements in a matrix which is basically a thin layer of a sticky viscous slime: a complex mixture of polysaccharides, DNA, proteins and many other components. This is very far from the ideal SERS sample, which would have the target molecules dispersed in a nice clean aqueous medium. Interactions between the matrix component and the enhancing particles can significantly compromise the measurements by blocking access of the target molecules to the enhancing surface.
Why gold nanostars specifically?
Metal nanoparticles of the type we use for SERS interact quite strongly with biofilms. As a result, it is relatively straightforward to place the enhancing media at the surface, but difficult to get the particles to move inside. Similarly, growing biofilms on an enhancing substrate only allows us to probe the underside of the polymer layer. Neither approach allows access to the biofilm interior, which is where many of the most interesting processes occur.
Gold nanostars offer a way around this problem. Conventional spherical nanoparticles need to be aggregated to create plasmonically active hotspots at particle junctions, but the biopolymers in biofilms interfere with that process. In contrast, nanostars are intrinsically plasmonically active due to the spikes on their surface and do not require aggregation. This allowed us to grow biofilms to a desired thickness, introduce a layer of plasmonic gold nanostars, and then continue growth so the particles became embedded at a fixed depth within the living biofilm, where they could report on the local chemistry of the matrix.
Why is measuring antibiotic diffusion important?
We chose to study the rate of antibiotic diffusion through the biofilm partly because it’s an excellent way of demonstrating how much control we have over the system, but also because reduced penetration has been suggested as one of the factors that makes the bacteria in the biofilms less susceptible to treatment with antibiotics.
Could the method be extended to other systems?
We think this could be a very general platform technology – extending to other species and molecular targets (not necessarily antibiotics) should be quite straightforward. One exciting possibility is to move away from solid substrates to working with biofilms on tissue.
What do these findings mean for future antimicrobial strategies?
Our work is really at a very early stage; we have been concentrating on getting to the point where the approach gives repeatable and meaningful results. Now that we have a method that works well, we hope it will provide a new tool for investigating all kinds of problems associated with the increased tolerance to antimicrobials that bacteria in biofilms display. An obvious possibility would be to use it to study the effectiveness of the many novel antimicrobial delivery systems that are being developed.
What’s next for this work?
This first study focused on detecting added antibiotics, but it has already given us indications that we can also observe chemical changes occurring within the biofilm itself, reflected in changes in biomarker concentrations. This is particularly exciting, since it may provide a simple method for monitoring how biofilms respond to a range of challenges – including physical factors such as changes in pH, the action of antimicrobials, or even the host response to infection. SERS is a highly general technique, so we will have many options for what we choose to monitor.
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