I have to admit to liking a good mystery. Even more so when it's set in a past where I can learn a little about a different time and place while enjoying a good story. And while I've never attempted writing my own mystery novel (unlike my talented colleague Joanna), I do enjoy puzzling out what might have happened and making my own guesses about what will happen next.

So, when I got the opportunity to do a little bit of scientific sleuthing of my own, I jumped at the chance.

First, I should explain that the Trends journals are in the midst of celebrating our 40th anniversary. As part of this, the editors selected a list of Best of Trends articles that we felt best represented our respective journals. For my journal, Trends in Microbiology, one of the first reviews that popped into my mind was a review by Thien-Fah Mah and George O'Toole on mechanisms of biofilm resistance to antimicrobial agents, published in 2001. This was a review that I've been aware of for many years because it's our highest cited review, and even now as I write, 15 years later, it's still one of our most downloaded reviews.

I was curious to try and understand why this review had such an enduring impact. And so it was time to have my own Nancy Drew moment, digging into where biofilm research was at during the early 2000s and reflecting on its progress. At the point that Mah and O'Toole were writing, we knew that communities of bacteria readily formed on surfaces. Where this becomes a problem is when biofilms form in places where we wouldn't want them—such as on in-dwelling medical devices including catheters, artificial heart valves, and joints.

The problem with biofilms comes when trying to treat these infections, because biofilms are more resistant to antimicrobial agents than planktonic bacteria. In their review, Mah and O'Toole delved into causes for this increased biofilm resistance to antimicrobials and focused on several different contributing mechanisms, including lack of diffusion of antimicrobials into the biofilm, slower growth and heterogeneous growth within a biofilm, general stress response, quorum sensing, and induction of a biofilm phenotype involving multidrug efflux pumps. In the end, they conclude that all these mechanisms most likely play some role in the overall resistance that is seen by biofilms, and all these proposed mechanisms have stood the test of time.

But what really struck me when I got to the end of the review was the box where Mah and O'Toole discussed outstanding questions that they felt should be addressed by future research. With the benefit of hindsight, I could see that these proposed questions have all blossomed into mature research areas.

Although there's been much research in areas related to these questions including work on multispecies biofilms, searching for a biofilm-specific phenotype in the genes and proteins expressed by biofilms and finding new signals that are involved in regulation of the general stress response of biofilms, what intrigued me was tracing the body of research after this review on the role of quorum sensing broadly in biofilms and the role that efflux pumps play in antimicrobial resistance.

At the time of the Mah and O'Toole review, the field was studying the involvement of quorum sensing during biofilm formation and virulence. Initial work had already begun in examining how quorum sensing could be therapeutically targeted during infections, and this work greatly expanded in regards to biofilm infections in subsequent years. The idea of inhibiting quorum sensing has grown to include blocking quorum sensing at different stages and a range of types of compounds used to inhibit quorum sensing.

Work on the expression of particular efflux pumps during biofilm formation also moved toward how these could be therapeutically targeted. For example, efflux pump inhibitors can block biofilm formation in bacteria and act synergistically with antibiotics to reduce biofilm tolerance to antibiotics.

I could also see how these outstanding questions from Mah and O'Toole's review could be linked conceptually to later work that was aimed at understanding biofilm dispersal and identifying broad biofilm inhibitors, trying to circumvent the many resistance mechanisms that biofilms have to antimicrobial agents. Though these have not yet made it to the market, continued efforts are still aimed at how we might be able to treat biofilm infections. To this aim, we continue to see patent applications that draw on Mah and O'Toole, such as one for inhibitors of bacterial diguanylate cyclase to inhibit biofilm formation.

In the end, my sleuthing came back to the idea that Mah and O'Toole's review exemplified what we want a Trends review to be—insightful, visionary, and shining a light on what we know now while highlighting areas where research needs to continue to grow into the future.