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 g/ml glucose-6-phosphate (G6P). When we retrospectively tested 12 fosfomycin-resistant (MIC, >256 g/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 diskbased 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.

Evaluation of Disk Potentiation Test Using Kirby-Bauer Disks Containing High-Dosage Fosfomycin and Glucose-6-Phosphate To Detect Production of Glutathione S -Transferase Responsible for Fosfomycin Resistance

“…When the FR-GST-positive strains were grown on MH-G6P plates, the growth inhibition zone was larger than that on MH plates. Thus, the addition of G6P to MH plates resulted in a larger inhibitory zone, confirming the results of our previous study (2). However, MH plates without the addition of G6P resulted in enough growth inhibition zone expansion to enable the detection of FR-GST production (Fig.…”

“…2). The FR-GST-negative strains showed no growth inhibition zone expansion by PPF, although a slight reduction in the size of the zone was observed, as shown by our previous study (2). Therefore, the use of high-dosage fosfomycin-G6P disks containing 200 g fosfomycin and 50 g G6P could eliminate the need for additional G6P supplementation in the potentiation test for identifying FR-GST producers.…”

“…negative E. coli isolates were used for the evaluation (2). The strains tested were prepared according to the CLSI guidelines (9) and spread on MH plates and MH-G6P plates.…”

Characterization of fosA5 , a new plasmid-mediated fosfomycin resistance gene in Escherichia coli

“…; Nakamura et al . ). Primer pair bla CTX‐M‐9 ‐F2/ bla CTX‐M‐9 ‐R2 (Table ) was used for PCR amplification of bla CTX‐M‐9 gene from E265, and the product was sequenced.…”

“…; Nakamura et al . ) . fosA encodes a glutathione S ‐transferase catalyzing the addition of glutathione to C1 of the epoxide ring of the antibiotic (Karageorgopoulos et al .…”