AUTHOR=Cattoir Vincent , Pourbaix Annabelle , Magnan Mélanie , Chau Françoise , de Lastours Victoire , Felden Brice , Fantin Bruno , Guérin François 

TITLE=Novel Chromosomal Mutations Responsible for Fosfomycin Resistance in Escherichia coli

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.575031

DOI=10.3389/fmicb.2020.575031

ISSN=1664-302X

ABSTRACT=<p>Fosfomycin resistance in <italic>Escherichia coli</italic> 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 <italic>E. coli</italic>. Different groups of isolates were studied: fosfomycin-resistant mutants selected <italic>in vitro</italic> from <italic>E. coli</italic> CFT073 (MIC = 1 mg/L) and two groups (wildtype and non-wildtype) of <italic>E. coli</italic> clinical isolates. Single-nucleotide allelic replacement was performed to confirm the implication of novel mutations into resistance. Induction of <italic>uhpT</italic> 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 <italic>in vitro</italic> from CFT073 (MICs, 128 mg/L) with single mutations: G469R in <italic>uhpB</italic> (M3); F384L in <italic>uhpC</italic> (M4). Second-step mutants (MICs, 256 mg/L) presented additional mutations: R282V in <italic>galU</italic> (M7 from M3); Q558<sup>∗</sup> in <italic>lon</italic> (M8 from M4). Introduction of <italic>uhpB</italic> or <italic>uhpC</italic> mutations by site-directed mutagenesis conferred a 128-fold increase in fosfomycin MICs, whereas single mutations in <italic>galU</italic> or <italic>lon</italic> were only responsible for a 2-fold increase. Also, these mutations abolished the induction of <italic>uhpT</italic> 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 – &gt;256 mg/L) including 8, 17, 18, 5, and 8 in <italic>uhpA</italic>, <italic>uhpB</italic>, <italic>uhpC</italic>, <italic>uhpT</italic>, and <italic>glpT</italic> genes, respectively. In conclusion, novel mutations in <italic>uhpB</italic> and <italic>uhpC</italic> are associated with fosfomycin resistance in <italic>E. coli</italic> clinical isolates.</p>