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Background   Acinetobacter baumannii  is an important nosocomial pathogen that has become increasingly resistant to multiple antibiotics. Genetic manipulation of MDR  A. baumannii  is useful especially for defining the contribution of each active efflux mechanism in multidrug resistance. Existing methods rely on the use of an antibiotic selection marker and are not suited for multiple gene deletions.    Results  A tellurite-resistant ( sacB  + ,  xylE  + ) suicide vector, pMo130-Tel R , was created for deleting the  adeFGH  and  adeIJK  operons in two clinical MDR  A. baumannii,  DB and R2 from Singapore .  Using a two-step selection, plasmid insertion recombinants (first-crossover) were selected for tellurite resistance and the deletion mutants (second-crossover) were then selected for loss of  sacB . The DNA deletions were verified by PCR while loss of gene expression in the Δ adeFGH , Δ adeIJK  and Δ adeFGH Δ adeIJK  deletion mutants was confirmed using qRT-PCR. The contribution of AdeFGH and AdeIJK pumps to MDR was defined by comparing antimicrobial susceptibilities of the isogenic mutants and the parental strains. The deletion of  adeIJK  produced no more than eight-fold increase in susceptibility to nalidixic acid, tetracycline, minocycline, tigecycline, clindamycin, trimethoprim and chloramphenicol, while the deletion of  adeL - adeFGH  operon alone had no impact on antimicrobial susceptibility. Dye accumulation assays using H33342 revealed increased dye retention in all deletion mutants, except for the R2Δ adeFGH  mutant, where a decrease was observed. Increased accumulation of ethidium bromide was observed in the parental strains and all pump deletion mutants in the presence of efflux inhibitors. The efflux pump deletion mutants in this study revealed that only the AdeIJK, but not the AdeFGH RND pump, contributes to antimicrobial resistance and dye accumulation in MDR  A. baumannii  DB and R2.    Conclusions  The marker-less gene deletion method using pMo130-Tel R  is applicable for creating single and multiple gene deletions in MDR  A. baumannii . The  adeFGH  and  adeIJK  operons were successfully deleted separately and together using this method and the impact of each efflux pump on antimicrobial resistance could be defined clearly.

A Method for generating marker-less gene deletions in multidrug-resistant Acinetobacter baumannii