title: Role of the AcrAB-TolC efflux pump in determining susceptibility of Haemophilus influenzae to the novel peptide deformylase inhibitor LBM415.
abstract: Haemophilus influenzae isolates vary widely in their susceptibilities to the peptide deformylase inhibitor LBM415 (MIC range, 0.06 to 32 microg/ml); however, on average, they are less susceptible than gram-positive organisms, such as Staphylococcus aureus and Streptococcus pneumoniae. Insertional inactivation of the H. influenzae acrB or tolC gene in strain NB65044 (Rd strain KW20) increased susceptibility to LBM415, confirming a role for the AcrAB-TolC pump in determining resistance. Consistent with this, sequencing of a PCR fragment generated with primers flanking the acrRA region from an LBM415-hypersusceptible H. influenzae clinical isolate revealed a genetic deletion of acrA. Inactivation of acrB or tolC in several clinical isolates with atypically reduced susceptibility to LBM415 (MIC of 16 microg/ml or greater) significantly increased susceptibility, confirming that the pump is also a determinant of decreased susceptibility in these clinical isolates. Examination of acrR, encoding the putative repressor of pump gene expression, from several of these strains revealed mutations introducing frameshifts, stop codons, and amino acid changes relative to the published sequence, suggesting that loss of pump repression leads to decreased susceptibility. Supporting this, NB65044 acrR mutants selected by exposure to LBM415 at 8 microg/ml had susceptibilities to LBM415 and other pump substrates comparable to the least sensitive clinical isolates and showed increased expression of pump genes.
authors.affiliation: Infectious Diseases, Novartis Institute for Biomedical Research, Inc., 100 Technology Square, Cambridge, MA 02139, USA. charlesr.dean@novartis.com
authors.forename: Charles R
authors.initials: CR
authors.lastname: Dean
authors.forename: Shubha
authors.initials: S
authors.lastname: Narayan
authors.forename: Denis M
authors.initials: DM
authors.lastname: Daigle
authors.forename: JoAnn L
authors.initials: JL
authors.lastname: Dzink-Fox
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Puyang
authors.forename: Kathryn R
authors.initials: KR
authors.lastname: Bracken
authors.forename: Karl E
authors.initials: KE
authors.lastname: Dean
authors.forename: Beat
authors.initials: B
authors.lastname: Weidmann
authors.forename: Zhengyu
authors.initials: Z
authors.lastname: Yuan
authors.forename: Rakesh
authors.initials: R
authors.lastname: Jain
authors.forename: Neil S
authors.initials: NS
authors.lastname: Ryder
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D006193:Haemophilus influenzae / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D016254:Mutagenesis, Insertional; D020411:Oligonucleotide Array Sequence Analysis; D010455:Peptides / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C474926:AcrB protein, Salmonella enterica; C477884:AcrR protein, Klebsiella pneumoniae; D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C487638:NVP PDF 713; D010455:Peptides; C073126:tolC protein, E coli; D000581:Amidohydrolases; C084507:peptide deformylase