Mechanisms of fosfomycin resistance in clinical isolates of carbapenem-resistant Klebsiella pneumoniae

Older antimicrobials such as fosfomycin are being considered as alternative agents in the treatment of drug-resistant organisms. However, there are limited data on the usefulness of fosfomycin against carbapenem-resistant Klebsiella pneumoniae (CRKP). This study aimed to investigate the prevalence of fosfomycin resistance and associated mechanisms in CRKP. A total of 99 clinical CRKP isolates were collected in the Second Affiliated Hospital of Wenzhou Medical University (Wenzhou, China) between January 2017 and June 2018. Fosfomycin susceptibility testing was performed by the agar dilution method. Carbapenemase and fosfomycin resistance genes were detected by PCR. Analysis of the murA, glpT, uhpT, uhpA, ptsI and cyaA genes was performed by PCR and sequencing of four fosfomycin-resistant fosA3-negative CRKP strains. Conjugation experiments were employed to determine the mobility of the fosA3 gene. Of the 99 CRKP isolates, fosfomycin-non-susceptibility was detected in 48 (48.5%) isolates, among which the fosA3 gene was detected in 44. Among the four fosfomycin-resistant fosA3-negative CRKP isolates, one isolate possessed a single nucleotide insertion and deletion mutations as well as 219 nucleotide substitution mutations in murA, two isolates possessed deletion or mutation of large DNA fragments in glpT, and one isolate possessed a fragment insertion sequence in glpT. Transfection into Escherichia coli J53 via plasmid conjugation was successful for 19 (43.2%) of the 44 fosA3-positive CRKP isolates. The fosA3 gene is the primary mechanism of fosfomycin resistance in CRKP and can be transmitted widely by plasmid in hospitals. Mutations in murA and glpT were found in fosfomycin-resistant fosA3-negative CRKP.