baumannii pumps. For instance, derivatives of the MDR clinical isolate BM4454 in which adeABC was inactivated had increased susceptibility to the same antibiotics (fluoroquinolones, chloramphenicol, tetracycline, tigecycline and erythromycin) as inactivation of adeIJK in the same isolate [6]. When both adeABC and adeIJK were inactivated in BM4454, increased susceptibility to ticarcillin, previously not observed in the ΔadeABC mutant or the ΔadeIJK mutant, was seen [6]. Furthermore, overexpression of

a pump gene did not always result in an increase in the MIC of the same antibiotics that had increased activity in the pump inactivated mutants. For example, inactivation RSL3 molecular weight of adeABC in the MDR clinical isolate BM4454 did not affect Cell Cycle inhibitor its susceptibility

to imipenem, amikacin and cotrimoxazole, but overexpressing adeABC in a non-MDR clinical isolate BM4587 increased the MIC of these antibiotics [4]. Therefore, it is possible that inactivation of a gene by inserting an antibiotic-resistance gene may affect the antimicrobial susceptibility of the pump gene-inactivated mutants, thus complicating the interpretation of the results. To address this possibility and to define clearly the impact of each efflux pump on antibiotic resistance, we propose that genes encoding efflux pumps be deleted using a marker-less strategy first described by Hamad et al (2009) for Burkholderia spp. [8]. The suicide vector, pMo130 was modified to carry a tellurite resistance cassette, a non-antibiotic selection marker [9]. The A. baumannii isolates we have tested, including MDR isolates, were

sensitive to tellurite and can be counter-selected in LB medium containing 30-60 mg/L tellurite. Gene deletion by allelic replacement was selected using a modification of the two-step process described by Hamad et al (2009) [8]. In this study, the adeFGH and adeIJK operons were deleted separately and together in two MDR A. baumannii strains, DB and R2. The adeIJK deletion mutant showed increased susceptibility to nalidixic crotamiton acid, chloramphenicol, trimethoprim, tetracycline, tigecycline, minocycline and clindamycin, but the deletion of adeL-adeFGH operon had no impact on antimicrobial susceptibility in the two MDR isolates. Genetic and gene expression analyses revealed that the allelic replacement in both MDR strains had occurred. The marker-less gene deletion method we describe is robust and, unlike the creation of mutants by inserting an antibiotic resistance gene, is suitable for deleting multiple genes in MDR A. baumannii. Results Deletion of the A. baumannii adeFGH and adeIJK operons To ensure reproducibility of the method, gene deletions were created for the adeFGH and adeIJK operons, separately and together, in two clinical MDR A. baumannii isolates, DB and R2. A suicide vector harboring a tellurite-resistance marker was first created by inserting a 3.