Novel Chromosomal Mutations Responsible for Fosfomycin Resistance in .

Fosfomycin resistance in results from chromosomal mutations or acquisition of plasmid-mediated genes. Because these mechanisms may be absent in some resistant isolates, we aimed at decipher the genetic basis of fosfomycin resistance in . Different groups of isolates were studied: fosfomycin-resistant mutants selected from CFT073 (MIC = 1 mg/L) and two groups (wildtype and non-wildtype) of clinical isolates. Single-nucleotide allelic replacement was performed to confirm the implication of novel mutations into resistance. Induction of expression by glucose-6-phosphate (G6P) was assessed by RT-qPCR. The genome of all clinical isolates was sequenced by MiSeq (Illumina). Two first-step mutants were obtained from CFT073 (MICs, 128 mg/L) with single mutations: G469R in (M3); F384L in (M4). Second-step mutants (MICs, 256 mg/L) presented additional mutations: R282V in (M7 from M3); Q558 in (M8 from M4). Introduction of or mutations by site-directed mutagenesis conferred a 128-fold increase in fosfomycin MICs, whereas single mutations in or were only responsible for a 2-fold increase. Also, these mutations abolished the induction of expression by G6P. All 14 fosfomycin-susceptible clinical isolates (MICs, 0.5-8 mg/L) were devoid of any mutation. At least one genetic change was detected in all but one fosfomycin-resistant clinical isolates (MICs, 32 - >256 mg/L) including 8, 17, 18, 5, and 8 in , , , , and genes, respectively. In conclusion, novel mutations in and are associated with fosfomycin resistance in clinical isolates.