We examined the prevalence and mechanism of fosfomycin resistance in CTX-M-producing Escherichia coli isolates from healthy Japanese individuals. One hundred thirty-eight CTX-M-producing E. coli isolates were subjected to fosfomycin susceptibility testing. The presence of acquired fosfomycin resistance genes such as fosA, fosA3, and fosC2 was explored, and the transmissibility of fosfomycin resistance, replicon type of plasmid, and genetic environment of fosA3 were investigated. Eight isolates (5.8%) showed resistance to fosfomycin, five of which harbored fosA3, which was in genetic linkage with blaCTX-M. The replicon types of the five transferred fosA3-carrying plasmids were as follows: IncI1 (n=2), IncN (n=1), and IncFII (n=2). Each fosA3 gene was located close to the blaCTX-M gene and was flanked by IS26 elements. These genetic environments of fosA3 in E. coli from healthy individuals were quite similar to those observed in the clinical and veterinary settings. Our results indicate that fosA3 genes possibly inserted by small mobile genetic elements flanked by two IS26 elements have already spread throughout the plasmids along with the blaCTX-M genes of commensal E. coli colonizing in healthy Japanese people.

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.

To develop a novel low-temperature plasma sterilizer using pure N 2 gas as a plasma source, we evaluated bactericidal ability of a prototype apparatus provided by NGK Insulators. After determination of the sterilizing conditions without the cold spots, the D value of the BI of Geobacillus stearothermophilus endospores on the filter paper was determined as 1.9 min. However, the inactivation efficiency of BI carrying the same endospores on SUS varied to some extent, suggesting that the bactericidal effect might vary by materials of sterilized instruments. Staphylococcus aureus and Escherichia coli were also exposed to the N 2 gas plasma and confirmed to be inactivated within 30 min. Through the evaluation of bactericidal efficiency in a sterilization bag, we concluded that the UV photons in the plasma and the high-voltage pulse to generate the gas plasma were not concerned with the bactericidal effect of the N 2 gas plasma. Bactericidal effect might be exhibited by activated nitrogen atoms or molecular radicals. Key words bactericidal effect, low-temperature sterilization, N 2 gas plasma. Sterilization is an essential and important technology not only for the modern medical services but for food and pharmaceutical industries. Many techniques and apparatuses for the sterilization were invented and improved. The selection of the sterilizing procedure depends on the materials and the shapes of medical instruments. Auto-clave, the representative technique to kill pathogenic microorganisms by heat, is of good cost performance and versatile application for many medical materials, therefore , is applied mainly in the hospital. However, because of the high temperatures such as 121 C, the autoclave cannot be applied for instruments and implants made from thermolabile polymers. Dry-heat sterilization is also used for the glassware and the metal instruments but the ap-Correspondence plication is also restricted because of its high temperature. In addition, both procedures take several hours to complete the sterilization, because heating takes a lot of time to raise the temperature and to cool the materials after sterilization. Drying of the instruments before use is also necessary in some cases. The sterilization methods using γ-ray or electron beam is not applicable for daily use in hospitals, because the exceptional facility is required for the applications and the management of the system is complicated. Meanwhile, EOG sterilization is applicable to thermolabile and fine structural instruments, because of the low handling temperature and the penetrative property of EOG. However, EOG is toxic and carcinogenic and release of EOG into the environment is severely restricted

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