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J Clin Microbiol. 2014 Sep;52(9):3175-9. doi: 10.1128/JCM.01094-14. Epub 2014 Jun 20.

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

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Department of Bacteriology, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Aichi, Japan.
Department of Bacteriology, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Aichi, Japan
Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Higashi-ku, Nagoya, Aichi, Japan.
Department of Bacteriology II, National Institute of Infectious Diseases, Musashi-Murayama, Tokyo, Japan.
Department of Infectious Diseases, Nagoya University Hospital, Showa-ku, Nagoya, Aichi, Japan.


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.

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