ABSTRACT
Despite the first-line recommendation 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 scientific error. 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 scientific error 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 scientific error should not be the main detractor from BMD use.IMPORTANCEDespite the recommendation of fosfomycin for uncomplicated urinary tract infections (UTIs), there are barriers for optimizing its use. There are no approved breakpoints for oral use against other Enterobacterales, and the recommended agar dilution (AD) reference method for MIC determination is largely impractical. The use of broth microdilution (BMD) for fosfomycin testing is not recommended by the Clinical and Laboratory Standards Institute due to unsatisfactory precision and skipped wells-occurrence of no-growth in a single well before the minimal inhibitory concentration (MIC)-and trailing endpoints. We sought to understand rates of skipped wells and growth at concentrations above measured MICs in BMD and how that compares to scientific error using AD. No-growth wells at concentrations below the MIC occurred in up to 10.9% of wells for BMD and single colonies at or beyond measured MICs for AD were also common. Frequent scientific error in both methods should prompt re-evaluation of both AD and BMD for fosfomycin susceptibility testing.
ABSTRACT
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 EUCASTIV AD as the reference method. MIC values ranged from 1 to >256 µg/mL with an MIC50/90 of 32/256 µg/mL. Extrapolating EUCASToral and CLSI AD Escherichia coli breakpoints, 12.5% and 83.8% of isolates were susceptible, respectively, whereas 66.3% were susceptible by EUCASTIV 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 EUCASTIV AD was greatest for CLSI AD (65.0%) and poorest for EUCASToral 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. IMPORTANCE Fosfomycin susceptibility testing recommendations are complex. Both the Clinical and Laboratory Standards Institute and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recognize agar dilution as the reference method, but they also support disk diffusion as an approved method for Escherichia coli. However, these two organizations have conflicting recommendations for the interpretation of inner colonies that arise during disk diffusion testing which can lead to varying zone diameters and interpretations despite isolates having identical MIC values. Using a collection of 80 Klebsiella pneumoniae isolates, we found that a large (82.5%) portion produced discrete inner colonies during disk diffusion and isolates were frequently classified into different interpretive categories. The more conservative breakpoints of EUCAST resulted in more isolates categorized as resistant despite frequent inner colonies. 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.