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.

Background: Daptomycin is approved by Health Canada for the treatment of Staphylococcus aureus bacteremia and complicated skin and soft tissue infections caused by gram-positive organisms, but is often used for other indications. We aimed to understand the indications, dosing, and safety profile of daptomycin use in a Canadian inpatient setting. Methods: We included consecutive adult patients who received intravenous daptomycin as inpatients from January 1, 2016, to December 31, 2016, at two tertiary care teaching hospitals in Hamilton, Ontario. Results: We identified 86 courses in 77 unique patients. S. aureus was the most common pathogen ( n = 38, 44%) of which 87% ( n = 33) were methicillin-resistant. The most common indications were bloodstream infections ( n = 31, 36%). The average treatment duration was 10 days, at an average dose of 7.4 mg/kg. The infectious diseases service was consulted in all but two courses. Less than half of treatment courses were given for an indication approved by Health Canada ( n = 41, 48%). Almost half of the unapproved indications ( n = 21, 47%) followed Infectious Diseases Society of America (IDSA) recommendations. Creatine kinase elevation of 3 × the upper limit of normal or higher occurred in a small number of courses ( n = 7, 8%), with only one instance requiring discontinuation of the drug. Conclusions: Daptomycin is being used to treat inpatients for a variety of unapproved indications. Importantly, a sizable portion of these are within IDSA guideline recommendations. Most patients are treated with doses higher than the approved 6 mg/kg without major safety concerns.