Protective effects of antibiotic resistant bacteria on susceptibles in biofilm: Influential factors, mechanism, and modeling.

In environmental biofilms, antibiotic-resistant bacteria facilitate the persistence of susceptible counterparts under antibiotic stresses, contributing to increased community-level resistance. However, there is a lack of quantitative understanding of this protective effect and its influential factors, hindering accurate risk assessment of biofilm resistance in diverse environment. This study isolated an opportunistic Escherichia coli pathogen from soil, and engineered it with plasmids conferring antibiotic resistance. Protective effects of the ampicillin resistant strain (AmpR) on their susceptible counterparts (AmpS) were observed in ampicillin-stress colony biofilms. The concentration of ampicillin delineated protective effects into 3 zones: continuous protection (<1 MIC of AmpS), initial AmpS/R dependent (1-8 MIC of AmpS), and ineffective (>8 MIC of AmpS). Intriguingly, Zone 2 exhibited a surprising "less is more" phenomenon tuned by the initial AmpS/R ratio, where biofilm with an initially lower AmpR (1:50 vs 50:1) harbored 30-90 % more AmpR after 24 h growth under antibiotic stress. Compared to AmpS, AmpR displayed superiority in adhesion, antibiotic degradation, motility, and quorum sensing, allowing them to preferentially colonize biofilm edge and areas with higher ampicillin. An agent-based model incorporating protective effects successfully simulated tempo-spatial dynamics of AmpR and AmpS influenced by antibiotic stress and initial AmpS/R. This study provides a holistic view on the pervasive but poorly understood protective effects in biofilm, enabling development of better risk assessment and precisely targeted control strategies of biofilm resistance in diverse environment.