Mechanism of Reduced Susceptibility to Fosfomycin inEscherichia coliClinical Isolates

Abstract

In recent years, multidrug resistance of Escherichia coli has become a serious problem. However, resistance to fosfomycin (FOM) has been low. We screened E. coli clinical isolates with reduced susceptibility to FOM and characterized molecular mechanisms of resistance and reduced susceptibility of these strains. Ten strains showing reduced FOM susceptibility (MIC ≥ 8 μg/mL) in 211 clinical isolates were found and examined. Acquisition of genes encoding FOM-modifying enzyme genes (fos genes) and mutations in murA that underlie high resistance to FOM were not observed. We examined ability of FOM incorporation via glucose-6-phosphate (G6P) transporter and sn-glycerol-3-phosphate transporter. In ten strains, nine showed lack of growth on M9 minimum salt agar supplemented with G6P. Eight of the ten strains showed fluctuated induction by G6P of uhpT that encodes G6P transporter expression. Nucleotide sequences of the uhpT, uhpA, glpT, ptsI, and cyaA shared several deletions and amino acid mutations in the nine strains with lack of growth on G6P-supplemented M9 agar. In conclusion, reduction of uhpT function is largely responsible for the reduced sensitivity to FOM in clinical isolates that have not acquired FOM-modifying genes or mutations in murA. However, there are a few strains whose mechanisms of reduced susceptibility to FOM are still unclear.

Growth medium-dependent antimicrobial activity of early stage MEP pathway inhibitors

“…Fosmidomycin is known to enter cells by the glycerol-3-phosphate transporter, GlpT [ 16 , 34 , 71 – 75 ]. However, studies of fosmidomycin entry mechanism are largely conducted in rich growth medium, where the effects of GlpT deletion are dramatic ( Fig 6A , [ 16 , 71 , 74 , 76 , 77 ]). Pertinent to this study, GlpT is tightly regulated by catabolite repression machinery [ 78 – 80 ] in M9-glucose, and is therefore unlikely to be a major mechanism for fosmidomycin uptake.…”

Frequency and Mechanisms of Spontaneous Fosfomycin Nonsusceptibility Observed upon Disk Diffusion Testing of Escherichia coli

“…Fosfomycin resistance is rare in E. coli but can occur from decreased transport across the outer membrane, from a reduced affinity of the target enzyme MurA, or from the production of FosA-family enzymes that inactivate fosfomycin (31). Of these mechanisms, the loss of function of hexose-6-phosphate transporter UhpT or glycerol-3phosphate transporter GlpT, through changes either in the transporter genes themselves or in their regulatory genes, is the most common pathway to fosfomycin resistance (7,14,21,(32)(33)(34). Changes in the phosphoenolpyruvate protein phosphotransferase I gene (ptsI) and adenylyl cyclase gene (cyaA) are also reported to impact the expression of UhpT and GlpT through the modulation of cAMP levels (31).…”

“…qRT-PCR with the induction of uhpT was performed as previously reported (7). Briefly, the cells of overnight cultures were collected and washed twice with saline.…”

“…This often results from defects in either the GlpT (glycerol-3-phosphate) or UhpT (hexose-6-phosphate) transporter systems utilized by fosfomycin for cell entry, which may occur thorough loss-of-function mutations in glpT or uhpT and their regulatory genes themselves or sometimes in genes that maintain cAMP levels that positively regulate glpT, such as cyaA and ptsI (5). While data regarding resistance mechanisms underlying fosfomycin-resistant clinical strains remain relatively scarce, a defective GlpT or UhpT system and, in some instances, specific substitutions in MurA have been implicated (5)(6)(7). The overall resistance rates of fosfomycin remain very low among E. coli clinical strains, likely due to the fitness costs incurred by these GlpT or UhpT transporter mutations, which have been further described to result in decreased growth rates in both laboratory medium and urine (5,8).…”

Urinary Tract Conditions Affect Fosfomycin Activity against Escherichia coli Strains Harboring Chromosomal Mutations Involved in Fosfomycin Uptake

“…The effect of FOS resistance mutations on fitness is particularly interesting in the case of uropathogenic E. coli (UPEC), because if the cost is high, the resistant bacteria will not grow at the minimal rate needed to establish infection (30,31). However, FOS-resistant clinical isolates containing mutations in the above-cited chromosomal genes have been described (30,32,33). Moreover, because fitness costs can easily be ameliorated by compensatory mutations, as shown for other antibiotic resistance genes (34), additional explanations for the low prevalence of FOS-resistant strains can be invoked.…”