Uli Klümper

Selection for antibiotic resistance at very low antibiotic concentrations has been demonstrated for individual antibiotics in single species experiments. Furthermore, selection in these focal strains is reduced when taking place in complex microbial community context. However, in the environment, bacteria are rarely exposed to single, but rather complex mixtures of selective agents. Here, we explored how the presence of a second selective agent affects selection dynamics between isogenic pairs of focal E. coli strains, differing exclusively in a single resistance determinant, in the absence and presence of a model wastewater community across a gradient of antibiotics. An additional antibiotic that exclusively affects the model wastewater community, but to which the focal strains are resistant to, was chosen as the second selective agent. This allowed exploring how inhibition alters the community’s ability to reduce selection. In the presence of the community, the selection coefficient at specific antibiotic concentrations was consistently decreased compared to the absence of the community. While pressure through the second antibiotic significantly decreased the activity and diversity of the community, its ability to reduce selection was consistently maintained at levels comparable to those recorded in absence of the second antibiotic. This indicates that the observed effects of community context on selection dynamics are rather based on competitive or protective effects between the focal strains and a small proportion of bacteria within the community, than on general competition for nutrients. These findings have implications for our understanding of the evolution and selection for multi-drug resistant strains.

Antimicrobial resistance (AMR) genes are widely disseminated on plasmids. Therefore, interventions aimed at blocking plasmid uptake and transfer may curb the spread of AMR. Previous studies have used CRISPR-Cas-based technology to remove plasmids encoding AMR genes from target bacteria, using either phage- or plasmid-based delivery vehicles that typically have narrow host ranges. To make this technology feasible for removal of AMR plasmids from multiple members of complex microbial communities, an efficient, broad host-range delivery vehicle is needed. We engineered the broad host-range IncP1-plasmid pKJK5 to encode cas9 programmed to target an AMR gene. We demonstrate that the resulting plasmid pKJK5::csg has the ability to block the uptake of AMR plasmids and to remove resident plasmids from Escherichia coli . Furthermore, due to its broad host range, pKJK5::csg successfully blocked AMR plasmid uptake in a range of environmental, pig- and human-associated coliform isolates, as well as in isolates of two species of Pseudomonas . This study firmly establishes pKJK5::csg as a promising broad host-range CRISPR-Cas9 delivery tool for AMR plasmid removal, which has the potential to be applied in complex microbial communities to remove AMR genes from a broad range of bacterial species.