title: Role of inoculum and mutant frequency on fosfomycin MIC discrepancies by agar dilution and broth microdilution methods in Enterobacteriaceae.
abstract: OBJECTIVE
  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 (5×105-3.91×103CFU/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-log2) 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 6.05×10-5(4×MIC) to 5.59×10-7(256×MIC) for E. coli, and 1.49×10-4(4×MIC) to 1.58×10-5(16×MIC) for K. pneumoniae. Resistant subpopulations arose mainly after 8 h of incubation with inocula >3.13×104CFU/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.
authors.affiliation: 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.
authors.forename: M
authors.initials: M
authors.lastname: Ballestero-Téllez
authors.affiliation: 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. Electronic address: fdocobop@yahoo.es.
authors.forename: F
authors.initials: F
authors.lastname: Docobo-Pérez
authors.affiliation: 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.
authors.forename: J M
authors.initials: JM
authors.lastname: Rodríguez-Martínez
authors.affiliation: Departmento de Microbiología, Universidad de Sevilla, Seville, Spain.
authors.forename: M C
authors.initials: MC
authors.lastname: Conejo
authors.affiliation: Unidad intercentros de Enfermedades Infecciosas, Microbiología y Medicina Preventiva, Hospital Universitario Virgen Macarena, Seville, Spain.
authors.forename: M S
authors.initials: MS
authors.lastname: Ramos-Guelfo
authors.affiliation: Instituto de Biomedicina de Sevilla IBIS, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Seville, Spain; Red Española de Investigación en Patología Infecciosa (REIPI), Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Blázquez
authors.affiliation: 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; Red Española de Investigación en Patología Infecciosa (REIPI), Instituto de Salud Carlos III, Madrid, Spain; Departmento de Medicina, Universidad de Sevilla, Seville, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Rodríguez-Baño
authors.affiliation: 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.
authors.forename: A
authors.initials: A
authors.lastname: Pascual
mesh_terms: D000362:Agar / Q000737:chemistry; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D003470:Culture Media / Q000737:chemistry; D024901:Drug Resistance, Multiple, Bacterial; D004755:Enterobacteriaceae / Q000187:drug effects*; D004926:Escherichia coli / Q000187:drug effects; D005578:Fosfomycin / Q000494:pharmacology*; D007711:Klebsiella pneumoniae / Q000187:drug effects; D008826:Microbial Sensitivity Tests
chemical_list: D000900:Anti-Bacterial Agents; D003470:Culture Media; D005578:Fosfomycin; D000362:Agar
keywords: Bacterial subpopulation; Fosfomycin; Heteroresistance; Mutant frequency; Susceptibility test