ABSTRACT
AIMS: Antimicrobial resistance genes (ARGs) are often associated with mobile genetic elements (MGEs), which facilitate their movement within and between bacterial populations. Detection of mobility is therefore important to understand the dynamics of MGE dissemination and their associated genes, especially in resistant clinical isolates that often have multiple ARGs associated with MGEs. Therefore, this study aimed to develop an entrapment vector to capture active MGEs and ARGs in clinical isolates of Escherichia coli. METHODS AND RESULTS: We engineered an entrapment vector, called pBACpAK, to capture MGEs in clinical E. coli isolates. It contains a cI-tetA positive selection cartridge in which the cI gene encodes a repressor that inhibits the expression of tetA. Therefore, any disruption of cI, for example, by insertion of a MGE, will allow tetA to be expressed and result in a selectable tetracycline-resistant phenotype. The pBACpAK was introduced into clinical E. coli isolates and grown on tetracycline-containing agar to select for clones with the insertion of MGEs into the entrapment vector. Several insertion sequences were detected within pBACpAK, including IS26, IS903B and ISSbo1. A novel translocatable unit (TU), containing IS26 and dfrA8 was also captured, and dfrA8 was shown to confer trimethoprim resistance when it was cloned into E. coli DH5α. CONCLUSIONS: The entrapment vector, pBACpAK was developed and shown to be able to capture MGEs and their associated ARGs from clinical E. coli isolates. We have captured, for the first time, a TU encoding antibiotic resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that a TU and associated resistance gene has been captured from clinical E. coli isolates using an entrapment vector. The pBACpAK has the potential to be used not only as a tool to capture MGEs in clinical E. coli isolates, but also to study dynamics, frequency and potentiators of mobility for MGEs.
