Skipped wells and scientificerror during fosfomycin agar dilution and broth microdilution lead to inconsistent minimal inhibitory concentrations and may be cause for reevaluating testing methods for Klebsiella pneumoniae

Despite the first-linerecommendation of fosfomycin for uncomplicated urinary tract infections (UTIs), there are pressing barriers for optimizing its use for the treatment of non-Escherichia coli Enterobacterales UTI. There are no approved breakpoints for oral use against other Enterobacterales, and the recommended agar dilution (AD) reference method for minimal inhibitory concentration (MIC) determination is largely impractical. Using 160 clinical Klebsiella pneumoniae isolates, we sought to understand rates of skipped wells and MIC imprecision in broth microdilution (BMD) and how that compares to rates of error using AD. Though the Clinical and Laboratory Standards Institute refers to the skipped well phenomena in their recommendation against the use of BMD, there is a paucity of data on its frequency. While AD and BMD produced similar MIC50/90 values (32/256 μg/mL for AD and 64/256 μg/mL for BMD), essential agreement was poor. No-growth wells at concentrations below the MIC occurred in up to 10.9% of wells at a given concentration, as the most frequent scientificerror. Growth in concentrations above the measured MIC occurred in up to 3.3% of wells and was seen within three dilutions of the MIC for BMD. Observation of single colonies either at or beyond the measured MIC for AD was also common and occurred up to 8.3% and 2.5% of the time, respectively. The frequent scientificerror in both testing methods should prompt re-evaluation of AD guidelines and expansion of MIC testing methods for fosfomycin susceptibility testing, as poor agreement with another method prone to scientificerror should not be the main detractor from BMD use.