Effect of polysorbate 80 on oritavancin binding to plastic surfaces – implications for susceptibility testing

Abstract number: 1733_609

Arhin F.F., Sarmiento I., Parr Jr T.J., Moeck G.

Objectives: Oritavancin (ORI) is a lipoglycopeptide (LG) with activity against most Gram-positive bacteria including drug-resistant pathogens. Broth microdilution (BMD) assays with dalbavancin, another LG, require addition of 0.002% polysorbate 80 (P80). To investigate whether P80 affects ORI susceptibility test results, we performed BMD assays for ORI with and without P80 and quantitated 14C-ORI recovery from assay plates containing cation-adjusted Mueller Hinton Broth (CAMHB) ± P80.

Methods: For recovery assays, 14C-ORI and 14C-ciprofloxacin (14C-CIP) were dissolved in water ± P80, diluted in CAMHB or CAMHB plus 2% lysed horse blood (LHB) ± P80, and dispensed into 96-well polystyrene plates (Sensititre) without cells. Recovery of radiolabeled agents was assessed by scintillation counting of supernatant over time to yield residual counts relative to input label at time 0. BMD assays with Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212 and Streptococcus pneumoniae ATCC 49619 followed CLSI guidelines. P80, where present, was at 0.002%.

Results: In the absence of P80, 14C-ORI was rapidly lost from solution: at 1 mg/L, <10% of input ORI was recovered at 1 h. ORI loss was concentration dependent: proportionately greater losses were observed at lower ORI concentrations, suggesting saturable binding of ORI to surfaces. Inclusion of P80 or LHB promoted recovery of 80–100% of 14C-ORI at all concentrations tested up to 24 h. Quantitative recovery of input 14C-CIP +/- P80 was observed for all concentrations and time points tested. Concordantly, minimal inhibitory concentrations (MICs) of ORI for S. aureus and E. faecalis were 16- to 32-fold lower in the presence of P80 whereas P80 had no impact on MICs of vancomycin, teicoplanin or CIP. Assay plates with low-binding surfaces afforded 4- to 8-fold reductions in ORI MICs without P80, suggesting that ORI binding to plastic in the absence of P80 may underestimate in vitro potency. ORI MICs for S. pneumoniae tested in CLSI-recommended CAMHB + 2% LHB were identical ± P80.

Conclusions: ORI exhibits rapid, saturable binding to susceptibility test plates. P80 minimises binding of ORI to microtitre plate surfaces, thereby maintaining ORI available in solution for growth inhibition. 2% LHB in CAMHB promoted ORI recovery, helping to explain the lack of ORI MIC shifts for S. pneumoniae with P80. Current literature MIC data for ORI significantly underestimates ORI in vitro potency.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Location: ICC, Munich, Germany
Presentation type:
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