The effect of synthetic microbial spatial self-organization on the fate of antibiotic resistance genes

Biofilms are considered as hotspots for the transfer of antibiotic resistance genes (ARGs), but very few studies have investigated the fate of ARGs (e.g. proliferation or elimination) in situ given different microbial spatial self-organization (SSO). SSO refers to a pervasive process during biofilm formation when microbes arrange themselves non-randomly across surfaces. So far the causes of SSO have been uncovered in a sense, however, the consequences of SSO were largely overlooked. Here, I hypothesize that the magnitude of inter-species intermixing, as one fundamental character of SSO, will determine the fate of ARG-carrying conjugative plasmid in both absence and presence of antibiotic selection. I evaluated this by performing range expansion experiments on agar plates to develop an artificial biofilm using a synthetic microbial community consisting of two isogenic Pseudomonas Stutzeri A1501 who are facultative denitrifiers in anaerobic condition. By knocking out different functional genes responsible for different steps of denitrification I am able to modify the metabolic interactions between these two strains from competing (without trophic interaction) to cross-feeding (with trophic interaction), which will further result in different magnitude of inter-species intermixing. Competing group has lower magnitude due to demixing of two, while cross-feeding group has higher magnitude due to mixing. I observed that in the absence of antibiotic selection plasmid experienced faster pace of elimination in competing group than cross-feeding group, whereas in the presence of antibiotic selection plasmid proliferated more efficiently in cross-feeding group than competing group. These results suggest that SSO is a determining factor of the fate of ARGs in biofilms, which provides a novel perspective of better understanding ARGs-related pressing problems facing our society.