Determinants of intrinsic aminoglycoside resistance in Pseudomonas aeruginosa.
Screening of a transposon insertion mutant library of Pseudomonas aeruginosa for increased susceptibility to paromomycin identified a number of genes whose disruption enhanced susceptibility of this organism to multiple aminoglycosides, including tobramycin, amikacin, and gentamicin. These included genes associated with lipid biosynthesis or metabolism (lptA, faoA), phosphate uptake (pstB), and two-component regulators (amgRS, PA2797-PA2798) and a gene of unknown function (PA0392). Deletion mutants lacking these showed enhanced panaminoglycoside susceptibility that was reversed by the cloned genes, confirming their contribution to intrinsic panaminoglycoside resistance. None of these mutants showed increased aminoglycoside permeation of the cell envelope, indicating that increased susceptibility was not related to enhanced aminoglycoside uptake owing to a reduced envelope barrier function. Several mutants (pstB, faoA, PA0392, amgR) did, however, show increased cytoplasmic membrane depolarization relative to wild type following gentamicin exposure, consistent with the membranes of these mutants being more prone to perturbation, likely by gentamicin-generated mistranslated polypeptides. Mutants lacking any two of these resistance genes in various combinations invariably showed increased aminoglycoside susceptibility relative to single-deletion mutants, confirming their independent contribution to resistance and highlighting the complexity of the intrinsic aminoglycoside resistome in P. aeruginosa. Deletion of these genes also compromised the high-level panaminoglycoside resistance of clinical isolates, emphasizing their important contribution to acquired resistance.
Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada.
Thomas
T
Krahn
Christie
C
Gilmour
Justin
J
Tilak
Sebastien
S
Fraud
Nicholas
N
Kerr
Calvin Ho-Fung
CH
Lau
Keith
K
Poole
D000583:Amikacin / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects; D004251:DNA Transposable Elements; D024881:Drug Resistance, Bacterial / Q000187:drug effects / Q000235:genetics*; D017353:Gene Deletion; D015723:Gene Library; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005839:Gentamicins / Q000494:pharmacology; D006801:Humans; D008826:Microbial Sensitivity Tests; D016254:Mutagenesis, Insertional; D010303:Paromomycin / Q000494:pharmacology; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D014031:Tobramycin / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D004251:DNA Transposable Elements; D005839:Gentamicins; D010303:Paromomycin; D000583:Amikacin; D014031:Tobramycin