Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) Acinetobacter baumannii HUMC1

Abstract

Multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) strains of Acinetobacter baumannii have frequently been characterized. The ability of A. baumannii to develop resistance to antibiotics is a key reason this organism has been difficult to study using genetic and molecular biology approaches. Here we report selectable markers that are not only useful but necessary for the selection of drug-resistant transformants in the setting of drug-resistant backgrounds. Use of these selectable markers can be applied to a variety of genetic and molecular techniques such as mutagenesis and transformation. These selectable markers will help promote genetic and molecular biology studies of otherwise onerous drug-resistant strains, while avoiding the generation of pathogenic organisms that are resistant to clinically relevant antibiotics.

New Shuttle Vectors for Gene Cloning and Expression in Multidrug-Resistant Acinetobacter Species

“…Here, we demonstrated that Gm concentrations that cannot be reached in vivo can be used in vitro (up to 200 g/ml) for selection of A. baumannii MDR clinical strains, like ACICU, whose MIC for Gm is 64 g/ml in LB and LA media. Recent studies have shown that other nonclinically relevant antimicrobials, such as tellurite and Zeo, can be used for in vitro selection (39,40). Zeo is not intended for clinical use, though it has already been employed as a selectable marker for XDR A. baumannii strains (40).…”

“…Recent studies have shown that other nonclinically relevant antimicrobials, such as tellurite and Zeo, can be used for in vitro selection (39,40). Zeo is not intended for clinical use, though it has already been employed as a selectable marker for XDR A. baumannii strains (40). In this study, replacement of the aacC1 cassette of pVRL1 and pVRL2 with the Zeo resistance gene (i.e., ble) led to the generation of pVRL1Z and pVLR2Z, respectively ( Table 1).…”

“…The Zeo concentrations used for E. coli and A. baumannii were 25 g/ml and 250 g/ml, respectively. Zeo selection was performed on low-salt LA (40).…”

Next Generation of Tn 7 -Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii

“…However, antibiotics/antimicrobials that are not widely used clinically can be utilized for selection purposes, since incidences of resistance against them are likely to be very low due to the lack of selection pressure. Zeocin (Zeo) resistance markers have recently been shown to be useful for selection purposes in MDR clinical isolates of A. baumannii (13,14). Similarly, apramycin (Apr) has also been shown to have promising activity against MDR A. baumannii strains (15).…”

“…Similarly, apramycin (Apr) has also been shown to have promising activity against MDR A. baumannii strains (15). Although apramycin is used in veterinary medicine, both apramycin and zeocin are antibiotics that are not used in human clinical settings (14,16), and therefore they are less prone to already-acquired resistance due to lack of selective pressure.…”