Fosfomycin Disk Diffusion Testing among Klebsiella pneumoniae Results in Frequent Inner Colonies and Categorical Disagreement Based on Conflicting Breakpoint Organization Recommendations

Recent studies indicate that discrete inner colonies (ICs) arise during fosfomycin disk diffusion (DD) testing. CLSI and EUCAST have contradicting recommendations on the interpretation of ICs; CLSI recommends considering them while EUCAST recommends ignoring them when interpreting DD results. We sought to compare the categorical agreement of DD and agar dilution (AD) MIC and to assess the implications of ICs interpretation on zone diameter readings. A convenience sample of 80 Klebsiella pneumoniae clinical isolates with varied phenotypic profiles collected from 3 United States locations was included. Susceptibility was determined in duplicate, using both organization recommendations and interpretations for Enterobacterales. Correlations between methods were calculated using EUCAST_IV AD as the reference method. MIC values ranged from 1 to >256 μg/mL with an MIC_50/90 of 32/256 μg/mL. Extrapolating EUCAST_oral and CLSI AD Escherichia coli breakpoints, 12.5% and 83.8% of isolates were susceptible, respectively, whereas 66.3% were susceptible by EUCAST_IV AD—which applies to K. pneumoniae. CLSI DD measurements were 2 to 13 mm smaller than EUCAST measurements due to 66 (82.5%) isolates producing discrete ICs. Categorical agreement with EUCAST_IV AD was greatest for CLSI AD (65.0%) and poorest for EUCAST_oral DD (6.3%). Isolates among this collection were frequently classified into different interpretive categories based on varying breakpoint organization recommendations. The more conservative oral breakpoints of EUCAST resulted in more isolates categorized as resistant despite frequent ICs. Differing zone diameter distributions and poor categorical agreement highlight issues of extrapolating E. coli breakpoints and methods to other Enterobacterales, and the clinical relevance of this issue warrants further investigation.