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1S85623N|1S85625N#56402774

Abstract:

The rapid evolution and adaption of bacterial pathogens 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 explored, including microbiome-based therapeutics such as probiotics. These probiotics compete with and inhibit pathogens through various mechanisms, including both competition for shared resources (exploitative competition), as well as secretion of antimicrobials (interference competition). While evolutionary adaptation of pathogens to conventional antimicrobial therapies have been well studied, if and how pathogens also adapt to inhibition by probiotics remains less well understood. Understanding these evolutionary interactions is vital to assess the risk of resistance development against probiotics, and identify potential cross-resistance to other antimicrobial therapies. Moreover, the use of probiotics adds an additional layer of complexity. In contrast to chemical antimicrobials, probiotics are themselves living entities subject to natural selection. This opens the door co-evolutionary arms races between pathogens and probiotics, where both competitors continuously adapt to outcompete the other. While studying how pathogens evolve is key to understand the evolution of resistance, investigating how probiotics evolve and adapt to outcompete a pathogen can inspire the engineering of next-generation probiotics with enhanced anti-pathogenic abilities. Here, we investigated the co-evolutionary interactions between probiotic Escherichia coli Nissle 1917 (EcN) and the enteric pathogen Salmonella Typhimurium in a mixed-species biofilm model using both fluorescence microscopy and plate counting. We first demonstrate that EcN’s ability to produce microcins plays a key role in its anti-Salmonella activity, but only under iron-limiting conditions. Competition experiments with wild-type EcN and microcin-deletion mutants revealed that deletion of microcin M (ΔmcmA), but not microcin H47 (ΔmchB), significantly compromised EcN’s ability to inhibit Salmonella in iron-limiting conditions, whereas no such differences were observed in an iron-rich environment (Figure 1A-B). Next, we were interested in how both competitors co-evolve. On one hand, we aimed to explore whether Salmonella adapts to microcin-mediated inhibition by probiotic EcN, and if so, how. On the other hand, we wanted to unravel whether EcN can in turn adapt to competition with potentially more resistant Salmonella. To investigate these co-evolutionary dynamics, we grew mixed-species biofilms of EcN and Salmonella for 20 days and followed the ecological dynamics using fluorescence microscopy (Figure 1C). In agreement with the previous short-term experiments, we observed that Salmonella's growth was initially heavily restricted within the mixed-species biofilm, especially in iron-limiting conditions. However, after a few days, Salmonella consistently outgrew EcN at the biofilm edges. Interestingly, a few days later, EcN regained the ability to outcompete Salmonella in certain regions of the biofilm, leading to the formation of distinct sectors radiating from the center. In some sectors, EcN completely dominated, while at the edges of others, Salmonella appeared to regain control. In contrast, under iron-rich conditions, while Salmonella initially outgrew EcN at the biofilm edges after a few days, EcN failed to re-establish dominance, and no sectors emerged from the center. Taken together, these observations are highly reminiscent of an ecological arms race, indicating that Salmonella may be able to acquire resistance against microcin-producing EcN and that EcN may be able to adapt to more resistant Salmonella. To further unravel these co-evolutionary dynamics, we locally collected samples from the biofilm's center, interface, edge, and sectors. In future research, we aim to phenotypically and genotypically investigate both EcN and Salmonella recovered from these samples. This will help uncover the underlying mechanisms, providing deeper insights into potential resistance development of pathogens against probiotics and identifying ways to enhance probiotics' effectiveness in targeting specific pathogens.