title: A method for generating marker-less gene deletions in multidrug-resistant Acinetobacter baumannii.
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 (second-crossover) 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.
authors.affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, Singapore 117597, Singapore.
authors.forename: Ismawati Mohammad
authors.initials: IM
authors.lastname: Amin
authors.forename: Grace E
authors.initials: GE
authors.lastname: Richmond
authors.forename: Paromita
authors.initials: P
authors.lastname: Sen
authors.forename: Tse Hsien
authors.initials: TH
authors.lastname: Koh
authors.forename: Laura J V
authors.initials: LJ
authors.lastname: Piddock
authors.forename: Kim Lee
authors.initials: KL
authors.lastname: Chua
mesh_terms: D040981:Acinetobacter baumannii / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D024901:Drug Resistance, Multiple, Bacterial; D017353:Gene Deletion; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D012641:Selection, Genetic; D013691:Tellurium / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; C026660:tellurous acid; D013691:Tellurium