Gen-directed re-sensitization of carbapenem-resistant microorganisms in biofilms

International audience ; Background: Antibiotic resistance is a major concern in public health. The control of environmental reservoirs is necessary to limit the spread of resistant bacteria and resistance genes. Hospital effluents are considered as hotspots of antibiotic-resistant bacteria, mainly within biofilm communities. CRISPR/Cas9 system can be used to specifically kill resistant bacteria within such complex communities or to re-sensitise them when the resistance is plasmid mediated.The goal of this work is to decrease the burden of carbapenem resistance in complex communities such as hospital effluents, by constructing a conjugative plasmid harboring CRISPR-Cas9 system targeting carbapenemase-encoding genes.Materials: A conjugative plasmid carrying the CRISPR/Cas9 system targeting carbapenemase-encoding genes was engineered. To construct this tool, the pB10 plasmid (64 508 bp), belonging to the IncP-1β family and originally isolated from waste-water treatment plant, was used because of its environmental origin and its high conjugation frequency. The single guide RNA (sgRNA) specifically targeting oxa48 (a carbapenemase gene) was designed using conserved region of about fifty oxa48 genes. The re-sensitisation capacities of the CRISPR-Cas9 tool was then assessed using planktonic carbapenem resistant bacteria.Results: Two regions of pB10 encoding antibiotic resistance genes were removed and replaced by the cas9 gene and the sgRNA targeting oxa48 gene, a gfp gene and a spectinomycin-resistance gene. The conjugative capacities of the resulting plasmid, pB10-CRISPR[oxa48] (47 522 bp) remained elevated (transfer rate of about 10-2). Re-sensitisation assays performed in planktonic conditions showed efficiencies of about four-log compared to the control.Conclusions: The CRISPR-Cas9 (Oxa48) conjugative plasmid engineered in this study showed a high efficiency of re-sensitisation when tested with carbapenem resistant planktonic bacteria. The next step will be to assess the efficiency of this tool in mono-species ...