title: Influence of transcriptional activator RamA on expression of multidrug efflux pump AcrAB and tigecycline susceptibility in Klebsiella pneumoniae.
abstract: Tigecycline is an expanded broad-spectrum antibacterial agent that is active against many clinically relevant species of bacterial pathogens, including Klebsiella pneumoniae. The majority of K. pneumoniae isolates are fully susceptible to tigecycline; however, a few strains that have decreased susceptibility have been isolated. One isolate, G340 (for which the tigecycline MIC is 4 microg/ml and which displays a multidrug resistance [MDR] phenotype), was selected for analysis of the mechanism for this decreased susceptibility by use of transposon mutagenesis with IS903phikan. A tigecycline-susceptible mutant of G340, GC7535, was obtained (tigecycline MIC, 0.25 microg/ml). Analysis of the transposon insertion mapped it to ramA, a gene that was previously identified to be involved in MDR in K. pneumoniae. For GC7535, the disruption of ramA led to a 16-fold decrease in the MIC of tigecycline and also a suppression of MDR. Trans-complementation with plasmid-borne ramA restored the original parental phenotype of decreased susceptibility to tigecycline. Northern blot analysis revealed a constitutive overexpression of ramA that correlated with an increased expression of the AcrAB transporter in G340 compared to that in tigecycline-susceptible strains. Laboratory mutants of K. pneumoniae with decreased susceptibility to tigecycline could be selected at a frequency of approximately 4 x 10(-8). These results suggest that ramA is associated with decreased tigecycline susceptibility in K. pneumoniae due to its role in the expression of the AcrAB multidrug efflux pump.
authors.affiliation: Wyeth Research, Department of Infectious Disease, 401 North Middletown Rd., Bldg. 200, Rm. 3219, Pearl River, NY 10965, USA. ruzina@wyeth.com
authors.forename: Alexey
authors.initials: A
authors.lastname: Ruzin
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Visalli
authors.forename: David
authors.initials: D
authors.lastname: Keeney
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Bradford
mesh_terms: D001426:Bacterial Proteins / Q000502:physiology*; D004251:DNA Transposable Elements; D024901:Drug Resistance, Multiple, Bacterial; D007711:Klebsiella pneumoniae / Q000187:drug effects* / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D008911:Minocycline / Q000031:analogs & derivatives* / Q000494:pharmacology*; D009154:Mutation; D000078304:Tigecycline
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D026901:Membrane Transport Proteins; C095947:RamA protein, Klebsiella; D000078304:Tigecycline; D008911:Minocycline
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