Heteroresistance associated with the production of fosfomycin-resistant inner colonies during disk diffusion testing among a geographically diverse collection of Klebsiella pneumoniae clinical isolates

Background: Fosfomycin susceptibility breakpoints apply only to Escherichia coli despite clinical use against Klebsiella pneumoniae. EUCAST and CLSI have different breakpoints and guidelines for disk diffusion (DD) interpretation that are frequently extrapolated to K. pneumoniae. Guidelines differ in interpreting inner colonies (IC) that grow within the zone of inhibition, but specificity to E. coli leaves knowledge gaps when extrapolating to other uropathogens. Objectives: To examine the frequency and MIC of K. pneumoniae IC during fosfomycin DD testing and to determine potential relationships between IC production, heteroresistance and fosA presence. Methods: A collection of K. pneumoniae clinical isolates (n = 262) and their IC (n = 116) underwent broth microdilution testing. Heteroresistance screening and PCR for fosA was performed on susceptible isolates that either never produced (NP) IC (n = 14) or produced ≥5 resistant IC (n = 43). Results: The MIC range (≤2 to >256 mg/L) of clinical isolates increased to 32 to >1024 mg/L for the IC collection with a median MIC increase of three, 2-fold dilutions. IC producers had 1.71 greater odds (P < 0.01) of a positive heteroresistance screen compared to NP isolates. No relationship was found between fosA presence and either IC production or heteroresistance. Conclusions: Production of ≥5 IC among clinical K. pneumoniae isolates was frequent and often resulted in an increased IC isolate MIC. Significantly greater odds of heteroresistance among IC producers were found when compared to NP isolates. Thus, presence of IC during fosfomycin DD testing should prompt avoidance of fosfomycin treatment.