Development of pBACpAK entrapment vector derivatives to detect intracellular transfer of mobile genetic elements within chloramphenicol resistant bacterial isolates

Abstract

Antimicrobial resistance disseminates throughout bacterial populations via horizontal gene transfer, driven mainly by mobile genetic elements (MGEs). Entrapment vectors are key tools in determining MGE movement within a bacterial cell between different replicons or between sites within the same replicon. The pBACpAK entrapment vector has been previously used to study intracellular transfer in Gram-negative bacteria however since pBACpAK contains a chloramphenicol resistance gene, it cannot be used in bacterial isolates which are already resistant to chloramphenicol. Therefore, we developed new derivatives of the pBACpAK entrapment vector to determine intracellular transfer of MGEs in an Escherichia coli DH5α transconjugant containing the chloramphenicol resistance plasmid pD25466. The catA1 of pBACpAK was replaced by both mcr-1 in pBACpAK-COL and aph(3')-Ia in pBACpAK-KAN, allowing it to be used in chloramphenicol resistant strains. The plasmid constructs were verified and then used to transform the E. coli DH5α/pD25466 transconjugants in order to detect intracellular movement of the MGEs associated with the pD25466 plasmid. Here we report on the validation of the expanded suite of pBACpAK vectors which can be used to study the intracellular transfer of MGEs between, and within, replicons in bacteria with different antimicrobial resistance profiles.

Keywords: Antimicrobial resistance; Entrapment vector; Insertion sequence; Intracellular; Mobile genetic elements; Plasmids.