Novel Chromosomal Mutations Responsible for Fosfomycin Resistance in Escherichia coli

Abstract

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

Strategies of organic phosphorus recycling by soil bacteria: acquisition, metabolism, and regulation

“…The UhpT transporter, which is encoded by the uhpT gene, mediates the uptake of hexose‐6‐phosphate (Sonna et al ., 1988 ; Cattoir et al ., 2020 ). The synthesis of UhpT transporter is tightly regulated by the products of the uhpABC regulatory genes: a response regulator UhpA, a histidine kinase UhpB and a receptor/transporter protein UhpC (Islandt and Kadner, 1993 ; Schwöppe et al ., 2002 ; Västermark and Saier, 2014 ; Cattoir et al ., 2020 ). The UhpB protein functions as an activator of UhpA, which regulates uhpT transcription (Weston and Kadner, 1988 ; Wright and Kadner, 2001 ).…”

“…The UhpC protein has a high sequence similarity to the UhpT transporter (Islandt and Kadner, 1993 ) and can serve as a G6P‐specific transporter with a relatively low transport rate compared to UhpT (Västermark and Saier, 2014 ). The UhpC protein functions as a high‐affinity G6P receptor that signals UhpB to undergo autophosphorylation and activate the UhpA regulator in the presence of external G6P (Wright and Kadner, 2001 ; Västermark and Saier, 2014 ; Cattoir et al ., 2020 ). Thus, the expression of the UhpT transporter is induced by extracellular G6P (Dietz and Heppel, 1971 ; Hoffer et al ., 2001b; Castañeda‐García et al ., 2013 , Cattoir et al ., 2020 ).…”

“…The UhpC protein functions as a high‐affinity G6P receptor that signals UhpB to undergo autophosphorylation and activate the UhpA regulator in the presence of external G6P (Wright and Kadner, 2001 ; Västermark and Saier, 2014 ; Cattoir et al ., 2020 ). Thus, the expression of the UhpT transporter is induced by extracellular G6P (Dietz and Heppel, 1971 ; Hoffer et al ., 2001b; Castañeda‐García et al ., 2013 , Cattoir et al ., 2020 ). Similar to the GlpT system, the expression of the UhpT transporter requires the cAMP‐CRP complex to bind to the promoter site of the uhpT gene (Castañeda‐García et al ., 2013 ; Cattoir et al ., 2020 ).…”

Identification of Multiple Low-Level Resistance Determinants and Coselection of Motility Impairment upon Sub-MIC Ceftriaxone Exposure in Escherichia coli

“…Porins also mediate transport of other antibiotics across the outer membrane, increasing the risk of multidrug resistance ( 55 ). Indeed, OmpF mutants are resistant to multiple antibiotics ( 56 ), and a galU deletion and point mutant had a 2-fold increase in MIC to fosfomycin ( 57 ). One study found that 42.9% of clinically ceftriaxone-resistant uropathogenic E. coli (UPEC) isolates were multidrug resistant.…”

Clinical effectiveness and bacteriological eradication of three different Short-COurse antibiotic regimens and single-dose fosfomycin for uncomplicated lower Urinary Tract infections in adult women (SCOUT study): study protocol for a randomised clinical trial

“…19 Fosfomycin resistance is rare in areas with limited use but is on the rise in countries with higher usage, although the susceptibility rates are variable and do not exceed 10% of the isolates. [20][21][22] However, the use of a single dose is associated with a higher percentage of relapses, mainly in patients with recurrent LUTIs. 23 A single dose of fosfomycin-tromethamine and short-courses of nitrofurantoin and pivmecillinam are now recommended by the latest guideline of the European Association of Urology for empirical therapy of uncomplicated LUTI, 24 but no study has compared more than two short-course antimicrobial agents for uncomplicated LUTIs.…”