Practical agar-based disk potentiation test for detection of fosfomycin-nonsusceptible Escherichia coli clinical isolates producing glutathione S-transferases

Abstract

The number of reports concerning Escherichia coli clinical isolates that produce glutathione S-transferases responsible for fosfomycin resistance (FR-GSTs) has been increasing. We have developed a disk-based potentiation test in which FR-GST producers expand the growth inhibition zone around a Kirby-Bauer disk containing fosfomycin in combination with sodium phosphonoformate (PPF). PPF, an analog of fosfomycin, is a transition-state inhibitor of FosA(PA), a type of FR-GST from Pseudomonas aeruginosa. Considering its mechanism of action, PPF was expected to inhibit a variety of FR-GSTs. In the presence of PPF, zone enlargement around the disk containing fosfomycin was observed for FosA3-, FosA4-, and FosC2-producing E. coli clinical isolates. Moreover, the growth inhibition zone was remarkably enlarged when the Mueller-Hinton (MH) agar plate contained 25 μg/ml glucose-6-phosphate (G6P). When we retrospectively tested 12 fosfomycin-resistant (MIC, ≥256 μg/ml) E. coli clinical isolates from our hospital with the potentiation test, 6 FR-GST producers were positive phenotypically by potentiation disk and were positive for FR-GST genes: 5 harbored fosA3 and 1 harbored fosA4. To identify the production of FR-GSTs, we set the provisional cutoff value, 5-mm enlargement, by adding PPF to a fosfomycin disk on the MH agar plates containing G6P. Our disk-based potentiation test reliably identifies FR-GST producers and can be performed easily; therefore, it will be advantageous in epidemiological surveys and infection control of fosfomycin-resistant bacteria in clinical settings.

FIG 1

Predicted amino acid sequence of fosfomycin resistance determinants. *, amino acid residues conserved among the eight fosfomycin resistance determinants; colons and dots, amino acid substitutions that result in homologous amino acid residues. The resistance determinants (GenBank accession no.) are FosA^PA (AAT49669), FosA^TN (AAA98399), FosA2 (ACC85616), FosA3 (BAJ10054), FosA4 (AB908992), open reading frame 1 (ORF1) (AAP50248), FosC (AAZ14834), and FosC2 (BAJ10053). The box indicates the conserved Thr9 residue in the fosfomycin resistance glutathione S-transferases.

FIG 2

(A) Potentiation by PPF in growth inhibition zone diameters around a fosfomycin disk. PPF was added to a fosfomycin disk (right side in each panel). Shown are a fosA3-positive strain on an MH plate (upper left) and MH-G6P plate (upper right), and an FR-GST-negative strain on an MH plate (lower left) and MH-G6P plate (lower right). (B) The results of fosC2-positive (left) and fosA4-positive strains on MH-G6P plates (right). FR-GST, fosfomycin resistance-mediating glutathione S-transferase; PPF, phosphonoformate; MH, Mueller-Hinton; G6P, glucose-6-phosphate.

FIG 3

Summary of changes in growth inhibition zone diameter with PPF. The y axis represents the enlargement of the growth inhibition zone (mm) by PPF. FR-GST, fosfomycin resistance-mediating glutathione S-transferase; PPF, phosphonoformate; MH, Mueller-Hinton; G6P, glucose-6-phosphate.