Three separate experiments

showed consistent results and representative examples are shown. Standard deviation represents variation between biological replicates. Selleck PP2 Asterisks indicate significant differences (P ≤ 0.05) in accumulation compared with the parental isolate or with addition of an EI. Panel A, Fold-change in level of ethidium bromide accumulated by R2 and mutants. Panel B, Fold-change in level of ethidium bromide accumulated by R2 and mutants with addition of EIs. Panel C, Fold-change in level of ethidium bromide accumulated by DB and mutants. Panel D, Fold-change in level of ethidium bromide accumulated by DB and mutants with addition of EIs. Dark grey, selleck chemicals llc no EI; light grey, CCCP; white, PAβN. Discussion The two-step deletion strategy we have described was used for creating unmarked deletions in the adeFGH and adeIJK efflux pump operons, separately and together, in two clinical MDR A. baumannii isolates. It is an improvement from the simple method for gene replacement in A. baumannii described by Aranda et al (2010) that uses an antibiotic resistance cassette [12]. To adapt the method first described for use in MDR A. baumannii, we introduced a tellurite resistance cassette into the pMo130 suicide vector created by Hamad et al (2009) to facilitate the selection of MDR A. baumannii transconjugants with the suicide plasmid inserted

into the genome, i.e. first crossover products [8]. It was helpful to first ascertain the growth inhibitory concentration of tellurite for the parental A. baumannii strain so the number of transconjugants (first crossover) that are false positives can be minimized by using a suitable tellurite concentration. Passaging the first crossover recombinants in media containing sucrose provided the selection pressure for loss of the plasmid by a second crossover, leading to the formation of white colonies when sprayed with pyrocathechol. The main advantage of this method, which does not use antibiotic selection for the gene deletion mutants, Vasopressin Receptor is its application for generating multiple gene deletions in a single strain as we have

demonstrated by creating DBΔadeFGHΔadeIJK and R2ΔadeFGHΔadeIJK mutants. This is particularly important because the majority of A. baumannii strains are MDR or extensively drug-resistant (XDR). Other than the MDR strains described in this study, we have also tested this method in a carbapenem-susceptible A. baumannii strain (data not shown). Un-marked deletion mutants are especially useful for ascertaining the contribution of each efflux pump to MDR as the presence of antibiotic resistance cassettes in the mutants may complicate the interpretation of antimicrobial susceptibility. We believe that the marker-less method would allow the impact of each efflux system on antimicrobial resistance to be clearly defined.