A suite of modular, all-synthetic suicide vectors for allelic exchange mutagenesis in multidrug resistant Acinetobacter strains

Abstract

Background: Acinetobacter baumannii is an opportunistic human pathogen that causes a variety of infections in immunosuppressed individuals and patients in intensive care units. The success of this pathogen in nosocomial settings can be directly attributed to its persistent nature and its ability to rapidly acquire multidrug resistance. It is now considered to be one of the top priority pathogens for development of novel therapeutic approaches. Several high-throughput techniques have been utilised to identify the genetic determinants contributing to the success of A. baumannii as a global pathogen. However, targeted gene-function studies remain challenging due to the lack of appropriate genetic tools. Results: Here, we have constructed a series of all-synthetic allelic exchange vectors – pALFI1, pALFI2 and pALFI3 – with suitable selection markers for targeted genetic studies in highly drug resistant A. baumannii isolates. The vectors follow the Standard European Vector Architecture (SEVA) framework for easy replacement of components. This method allows for rapid plasmid construction with the mutant allele, efficient conjugational transfer using a diaminopimelic acid-dependent Escherichia coli donor strain, efficient positive selection using the suitable selection markers and finally, sucrose-dependent counter-selection to obtain double-crossovers. Conclusions: We have used this method to create scar-less deletion mutants in three different strains of A. baumannii, which resulted in up to 75% deletion frequency of the targeted gene. We believe this method can be effectively used to perform genetic manipulation studies in multidrug resistant Gram-negative bacterial strains.

Characterisation of the carbapenem-resistantAcinetobacter baumanniiclinical reference isolate BAL062 (CC2:KL58:OCL1): resistance properties and capsular polysaccharide structure

“…Additional studies on spermidine/spermine efflux (11), a comparison to other clinical and environmental isolates (12,13), and demonstration of the utility of novel suicide vectors (14) have also used BAL062.…”

“…The BAL062 library has been used to identify genes that contribute to resistance to the last-resort antibiotic colistin (8, 9), and several clinically relevant biocides (7, 10). Additional studies on spermidine/spermine efflux (11), a comparison to other clinical and environmental isolates (12, 13), and demonstration of the utility of novel suicide vectors (14) have also used BAL062.…”