Abstract
With multidrug-resistant (MDR) Enterobacterales on the rise, a nontoxic antimicrobial agent with a unique mechanism of action such as fosfomycin seems attractive. However, establishing accurate fosfomycin susceptibility testing for non-Escherichia coli isolates in a clinical microbiology laboratory remains problematic. We evaluated fosfomycin susceptibility by multiple methods with 96 KPC-producing clinical isolates of multiple strains and species collected at a single center between 2008 and 2016. In addition, we assessed the presence of fosfomycin resistance genes from whole-genome sequencing (WGS) data using NCBI's AMRFinder and custom HMM search. Susceptibility testing was performed using a glucose-6-phosphate-supplemented fosfomycin Etest and Kirby-Bauer disk diffusion (DD) assays, and the results were compared to those obtained by agar dilution. Clinical Laboratory and Standards Institute (CLSI) breakpoints for E. coli were applied for interpretation. Overall, 63% (60/96) of isolates were susceptible by Etest, 70% (67/96) by DD, and 88% (84/96) by agar dilution. fosA was detected in 80% (70/88) of previously sequenced isolates, with species-specific associations and alleles, and fosA-positive isolates were associated with higher MIC distributions. Disk potentiation testing was performed using sodium phosphonoformate to inhibit fosA and showed significant increases in the zone diameter of DD testing for isolates that were fosA positive compared to those that were fosA negative. The addition of sodium phosphonoformate (PPF) corrected 10/14 (71%) major errors in categorical agreement with agar dilution. Our results indicate that fosA influences the inaccuracy of susceptibility testing by methods readily available in a clinical laboratory compared to agar dilution. Further research is needed to determine the impact of fosA on clinical outcomes.
Highlights
Antimicrobial resistance among Gram-negative organisms continues to increase and presents a serious threat to modern medicine, with carbapenemase-producing Enterobacteriaceae (CPE) considered one of the most pressing issues [1]
Both the Clinical Laboratory and Standards Institute (CLSI) and EUCAST recommend against the use of broth microdilution methods, which likely impacts the inaccuracies with most automated susceptibility testing platforms for E. coli or Klebsiella pneumoniae [17, 18]
We demonstrate that fosfomycin susceptibility testing by routinely used laboratory diffusion-based methods (Etest and disk diffusion (DD)) largely overcalls resistance compared to agar dilution as a gold standard (Table 1)
Summary
Antimicrobial resistance among Gram-negative organisms continues to increase and presents a serious threat to modern medicine, with carbapenemase-producing Enterobacteriaceae (CPE) considered one of the most pressing issues [1]. Fosfomycin resistance in Enterobacterales has been primarily driven by mutations in the glpT and uhpT genes, preventing active transport of fosfomycin into the cell [7] These mutations are, thought to be associated with a fitness cost in E. coli and are unstable [8, 9]. Susceptibility testing of fosfomycin for non-E. coli Enterobacterales is difficult for clinical microbiology labs [14,15,16]. With agar dilution as the only accurate method for non-E. coli Enterobacterales, we aimed to characterize some of the molecular mechanisms (by whole-genome sequencing [WGS] for a subset of isolates) that may be contributing to the inaccuracies with these diffusion methods, using a diverse set of clinical, carbapenemase-producing strains and agar dilution-based reference phenotyping
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