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

Amin et al. BMC Microbiology 2013, 13:158 http://www.biomedcentral.com/1471-2180/13/158 METHODOLOGY ARTICLE Open Access A Method for generating marker-less gene deletions in multidrug-resistant Acinetobacter baumannii Ismawati Mohammad Amin1, Grace E Richmond3, Paromita Sen1, Tse Hsien Koh2, Laura JV Piddock3 and Kim Lee Chua1* Abstract 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-TelR, 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 (secondcrossover) were then selected for loss of sacB. The DNA deletions were verified by PCR while loss of gene expression in the ΔadeFGH, ΔadeIJK and ΔadeFGHΔadeIJK 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 R2ΔadeFGH 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-TelR 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. Keywords: Acinetobacter baumannii, Multidrug-resistant, AdeFGH, AdeIJK, RND pumps, Allelic replacement Background Acinetobacter baumannii, a non-fementing Gramnegative cocco-bacillus, is a frequent cause of nosocomial bloodstream infections and is associated with considerable morbidity and mortality, especially among patients in intensive care or with burns [1]. A. baumannii has become increasingly resistant to multiple * Correspondence: 1 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, Singapore 117597, Singapore Full list of author information is available at the end of the article antibiotics, including imipenem and meropenem, the carbapenems of choice for treating multidrug resistant (MDR) A. baumannii infections. The incidence of carbapenem-resistant A. baumannii in the United States and Europe is around 54% and 16%, respectively, while the incidence in the Asia/Pacific rim is about 80% [2]. A. baumannii possesses a variety of intrinsic and acquired resistance determinants, including β-lactamases, class D oxacillinases, aminoglycoside-modifying enzymes, outer membrane proteins and active efflux systems [3]. Among its intrinsic resistance determinants, overexpression of © 2013 Amin et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Amin et al. BMC Microbiology 2013, 13:158 http://www.biomedcentral.com/1471-2180/13/158 the chromosomally encoded active efflux systems of the resistance-nodulation and division (RND) family, such as AdeABC, AdeFGH and AdeIJK pumps, are a mechanism of resistance to a number of antibiotics [4]. The impact of RND pumps to antibiotic resistance in A. baumannii has been demonstrated by inactivating the genes that encode the efflux pumps and the method for gene inactivation involves insertion of an antibiotic resistance gene to select mutants [5-7]. Studies using mutants in which RND efflux pump genes have been inactivated have suggested significant overlap in antibiotics that are substrates of the A. 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 of adeABC in the MDR clinical isolate BM4454 did not affect its susceptibility to imipenem, amikacin and cotrimoxazole, but overexpressing adeABC in a nonMDR 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 geneinactivated 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 counterselected 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 acid, chloramphenicol, trimethoprim, tetracycline, tigecycline, minocycline and clindamycin, but the deletion of adeL-adeFGH opero (...truncated)