A novel biofilm model to investigate co-evolutionary arms races between pathogens and probiotics

The ability of bacterial pathogens to rapidly evolve and adapt to antimicrobial therapies has become an area of increasing interest, especially given the clinical consequences of antimicrobial resistance. Against the backdrop of such a global resistance crisis, novel antimicrobial approaches are being actively explored. One promising strategy is the use of microbiome-based therapeutics, including the use of beneficial microbes or probiotics. These probiotics can compete with and inhibit pathogens through various mechanisms, including both competition for shared resources (exploitative competition), as well as secretion of antimicrobial compounds (interference competition). While the evolutionary adaptations of pathogens to conventional antimicrobial therapies such as antibiotics have been well studied, if and how pathogens can also adapt to inhibition by probiotics remains less well understood. Getting more insight in these evolutionary adaptations of pathogens is essential to assess the risk of resistance development against probiotics, and if resistance can develop, acquire mechanistic insights to avoid cross-resistance with other therapies. Moreover, the use of probiotics as an antimicrobial approach adds an additional layer of complexity. In contrast to chemical antimicrobials, probiotics are themselves living entities subject to natural selection. This opens the door for co-evolutionary arms races between pathogens and probiotics, where both competitors can continuously adapt to outcompete the other. While studying how pathogens evolve is essential to understand the evolution of resistance, investigating how probiotics evolve and adapt to outcompete a target pathogen can provide inspiration to design and engineer next-generation probiotics with enhanced anti-pathogenic abilities. Here, we develop a new fluorescence-based biofilm model to investigate potential co-evolutionary arms races between the model probiotic Escherichia coli Nissle 1917 (EcN) and the enteric pathogen Salmonella Typhimurium. Using a combination of fluorescence microscopy and plate counting, we first demonstrate that microcin M, but not microcin H47, is essential for the anti-Salmonella activity of EcN. While Salmonella is strongly inhibited in mixed-species biofilms with the bacteriocin producing wild-type EcN, knocking out the precursor peptide for microcin M (ΔmcmA) allows Salmonella to recover to similar levels as in mono-species biofilms. In contrast, knocking out the precursor peptide (ΔmchB) for the other major bacteriocin of EcN, microcin H47, does not substantially affect the inhibition of Salmonella, and similar results as with the wild-type strain are obtained (Figure 1A). Currently, we are performing long-term experiments with these mixed-species biofilms to investigate how both competitors co-evolve. At one hand, we are interested to see if and how Salmonella adapts to microcin-mediated inhibition by the probiotic EcN. Preliminary data demonstrates that while Salmonella growth is initially strongly confined in the mixed-species biofilms, Salmonella starts outgrowing EcN at the edges of the biofilm after a few days (Figure 1B). Even more intriguingly, a few days later EcN is able to overtake the Salmonella outgrowth again in some regions of the biofilm, resulting in clear sectors emerging from the biofilm center. While for some of these sectors EcN completely overtakes Salmonella, the latter seems to take over again in some of these regions. Taken together, these observations are highly reminiscent of an ecological arms race, and future efforts will attempt to unravel the underlying mechanisms to gain more insight in the potential resistance development of pathogens to probiotics, as well as how probiotics can be improved to inhibit a target pathogen.