Role of inoculum and mutant frequency on fosfomycin MIC discrepancies by agar dilution and broth microdilution methods in Enterobacteriaceae.

Clin Microbiol Infect

Unidad intercentros de Enfermedades Infecciosas, Microbiología y Medicina Preventiva, Hospital Universitario Virgen Macarena, Seville, Spain; Instituto de Biomedicina de Sevilla IBIS, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Seville, Spain; Departmento de Microbiología, Universidad de Sevilla, Seville, Spain; Red Española de Investigación en Patología Infecciosa (REIPI), Instituto de Salud Carlos III, Madrid, Spain.

Published: May 2017

Objectives: Fosfomycin is re-evaluated as a treatment of multidrug-resistant Enterobacteriaceae infections. However, MIC differences have been described among the different susceptibility testing. The aim was to study the role of the different inoculum size used in agar dilution with respect to broth microdilution, according to CLSI, in the fosfomycin MIC discrepancies.

Methods: Fosfomycin MICs were determined using agar dilution (reference) and broth microdilution in 220 Escherichia coli (n=81) and Klebsiella pneumoniae (n=139) clinical isolates. Fosfomycin mutant frequencies were determined in 21 E. coli (MIC=1mg/L) and 21 K. pneumoniae (MIC=16mg/L). The emergence of resistant subpopulations of five E. coli strains (MIC=1mg/L) was monitored over the time by microdilution assay using 0, 4 and 8 mg/L of fosfomycin, and eight different inocula (5×10-3.91×10 CFU/well, 1 : 2 dilutions).

Results: For E. coli, 86.4% of categorical agreement (CA), 9.1% very major errors (VME), 3.3% major errors (ME) and 9.9% minor errors (mE) were found. For K. pneumoniae, CA was 51.1%, VME 15.7%, ME 28.4% and mE 25.2%. Essential agreement (±1-log) was observed in 55.45%. By microdilution, 35.9% of the MICs showed discrepancies of ≥2 dilutions. Initial inoculum used was 5.63 times higher in the microdilution method, in range with CLSI methodology for both techniques. Fosfomycin mutant frequencies were 6.05×10 (4×MIC) to 5.59×10 (256×MIC) for E. coli, and 1.49×10 (4×MIC) to 1.58×10 (16×MIC) for K. pneumoniae. Resistant subpopulations arose mainly after 8 h of incubation with inocula >3.13×10 CFU/well.

Conclusions: The higher inoculum used in the microdilution method enriched the initial inoculum with resistant subpopulations and could partially explain the fosfomycin MIC discrepancies with respect to the agar dilution method.

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http://dx.doi.org/10.1016/j.cmi.2016.12.022DOI Listing

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