Optimización de un sistema para cuantificar la eficiencia de la conjugación bacteriana in vitro basado en selección antibiótica

Abstract

Antibiotic resistance has become one of the major problems in health care nowadays. Bacteria can acquire antibiotic resistance by different natural mechanisms, but the most important is the acquisition through horizontal gene transfer (HGT). Despite HGT can occur in three main mechanisms, conjugation is the one that causes the greatest propagation of antibiotic resistance genes. Conjugation is based on the transmission of conjugative plasmids which carry these genes. One of the most promising strategies to fight antibiotic resistance is the investigation of conjugation inhibitors (COINs). The aim of this study was the optimization of an in vitro conjugation protocol for the reproducible quantification of conjugation frequencies, which permits the study of anticonjugation properties of candidate chemical compounds. Also, we tried to confirm the constitutively expression of the green fluorescent protein (GFP) in the transconjugant strain generated. The optimized protocol using nitrocellulose filters and antibiotic selection, employing the donor E. coli MDS42 strain carrying plasmid pJC01 and the recipient E. coli BL21 (DE3) strain, allowed to obtain reproducible conjugation frequencies in the order of 10-1 . Additionally, the system was useful for the preliminary study of the possible conjugation inhibitory activity of five candidate chemical compounds. Finally, the pJC01 plasmid in the transconjugant strain was stable under antibiotic selection and constitutively express the GFP. This strain will be used for the optimization of a high-throughput in vitro conjugation protocol for the rapid and cost-effective evaluation of COIN candidates