Investigation of Klebsiella pneumoniae inner colonies during fosfomycin susceptibility testing

Abstract

Fosfomycin is a broad-spectrum antibiotic indicated for first-line treatment of uncomplicated urinary tract infection (UTI) caused by Escherichia coli. However, it is often used for non-E. coli Enterobacterales when limited oral antibiotics are available to treat UTI caused by increasingly resistant urinary pathogens. Both agar dilution (AD) and disk diffusion (DD) are approved methods for fosfomycin susceptibility testing, though DD is used more often in clinical settings. During DD testing, visually distinct inner colony (IC) sub-populations frequently develop. A collection of K. pneumoniae (n = 262), obtained from five US locations in 2013-2023 and their corresponding IC (n = 116; 44.3%) underwent susceptibility testing. Median minimal inhibitory concentration (MIC) values increased three, 2-fold dilutions from IC-producers to IC. IC-producers along with IC never producers (NP) underwent heteroresistance screening. IC-producers had greater odds (odds ratio: 1.71) of screening positive for heteroresistance. Whole genome sequencing among selected IC-producer/IC pairs revealed variations in several known fosfomycin resistance genes within IC isolates not found in their IC-producers or NP. Further in vitro fitness testing including growth curve and generation time analysis demonstrated no loss of fitness for IC in monoculture. Among a 72-hour competition time growth between IC-producers and IC, prolonged generation times were observed across all IC-producers and IC. In competition, some IC entered death phase as early 24-36 hours post inoculation where all IC-producers maintained logarithmic growth for all 72 hours. Finally, in vivo fitness testing using an ascending UTI mouse model, showed varying colonizing abilities of IC isolates when in competition—up to 50% for IC isolates. This work reveals that specific genetic variations did not alter in vivo fitness of K. pneumoniae when colonizing the mouse urinary tract. This indicates that fosfomycin IC should be considered relevant, and not ignored during DD testing.