Assessment of heteroresistance to vancomycin and other antibiotics by broth macrodilution in Staphylococcus aureus strains
Abstract number: P1031
Arhin F.F., Sarmiento I., Moeck G.
Objective: Heteroresistance (HR) to vancomycin (VAN), defined as a small subpopulation with elevated VAN MIC (>2 mg/L) in a culture, is becoming increasingly prevalent and has been linked to clinical therapeutic failure. The most reliable procedure to detect VAN HR is the agar-based population analysis profile-area under curve (PAP-AUC) method. This procedure is labour-intensive and cannot be applied to agents for which agar-based assays are unsuitable. We developed a broth macrodilution assay (Bmac) to assess HR to vancomycin. The Bmac, with an increased number of cells relative to broth microdilution assay (Bmic), increases the probability of identifying rare resistant subpopulations while respecting the inoculum density recommended by CLSI. This assay was used to assess HR to daptomycin (DAP), teicoplanin (TEI), telavancin (TEL) and oritavancin (ORI) in heterogeneous VAN-intermediate Staphylococcus aureus (SA) (hVISA) strains.
Methods: 5 SA clinical strains (from NARSA or Eurofins Medinet) used in this study exhibited VAN HR by PAP-AUC. MICs were determined by Bmac and broth microdilution assay (Bmic) following CLSI M7-A8. SA strains ATCC 29213 (VAN-susceptible) ATCC 700699 (VAN intermediate SA [VISA]) and NRS2 (Mu3; hVISA) were used as reference strains. Bmac was performed in 50 mL polyproplylene screw cap tubes containing 5×105 CFU/mL in a final assay volume of 20 mL (total of 107 CFU). Bmac was read after 24 h at 37°C. All experiments were performed at least twice.
Results: By PAP-AUC, the 5 S. aureus test strains were VAN HR. These isolates, along with NRS2 (hVISA reference strain), had VAN MICs of 1 or 2 mg/L by Bmic and 4 mg/L by Bmac, indicating growth of subpopulations at a VAN concentration of 2 mg/mL in Bmac. In contrast, the VSSA and VISA control strains showed MIC values that were identical for both Bmic and Bmac. Two of the five VAN HR isolates showed ORI and TEI Bmac MICs that were higher (2 doubling dilutions) than the Bmic MICs, suggestive of HR to ORI and TEI. Bmac and Bmic MICs for DAP and TEL were identical, suggesting lack of HR in this set of isolates.
Conclusions: The Bmac assay described here provides a simple means to identify the presence of rare HR isolates. The procedure is useful for agents such as ORI for which agar-based assays are not possible. Two of the 5 VAN HR strains of SA were identified by Bmac assay as possibly HR to TEL and ORI. Clinical significance of HR as identified by Bmac remains to be determined.
|Session name:||Abstracts of 21st ECCMID / 27th ICC|
|Location:||Milan, Italy, 7 - 10 May 2011|
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