Abstract
The antibiotic susceptibilities of 43 strains of Escherichia coli O157:H7 identified in the summer of 1996 in Japan were investigated. Growth of 90% of O157 strains was inhibited at a concentration of ≤0.5 μg/ml by several agents including fosfomycin with glucose-6-phosphate.
In 1996, enterohemorrhagic Escherichia coli (EHEC) O157:H7 was the cause of serious outbreaks of diarrheal illness, with sporadic cases of hemorrhagic colitis and hemolytic-uremic syndrome in Japan. EHEC O157 is the organism frequently isolated from patients with these diseases in the United States, Canada, and Europe (1, 3, 6, 13).
During this epidemic of EHEC infection caused by O157, the disease spread throughout Japan, and more than 9,000 people were affected, resulting in 11 deaths. The main route by which this infection spread may have been food and drink; however, the specific route of infection remains to be clarified. In routine bacterial infections, the use of antibiotics during the initial stage is considered a part of basic treatment; however, there are only a few studies that have strictly tested various antibiotics for O157 strains. In addition, there are many different opinions regarding selection of an antibiotic, as it is unknown whether hemorrhagic colitis and hemolytic-uremic syndrome can be treated with antibiotics. Treatment has not yet been fully accepted, as some studies have not recommended the use of antibiotics (10, 11). The Japanese Ministry of Health and Welfare recommended that fosfomycin, norfloxacin, or kanamycin be administered orally to children and that fosfomycin or a new quinolone be given to adults orally during the initial stage of infection (4). Basic studies supporting this opinion have not yet been reported.
We compared the antibiotic susceptibilities of 43 strains of O157 from 97,160 stool specimens collected in Osaka, Sizuoka, and Tokyo districts during July and August 1996.
Antibiotics and reagents used in our study were purchased as follows: ampicillin, arbekacin, dibekacin, cefditoren, and fosfomycin, Meiji Seika Kaisha, Ltd.; cefmetazole, cefpodoxime, and piperacillin, Sankyo Co., Ltd.; ceftazidime, Tanabe Pharmaceutical Industrial Co., Ltd.; sulbactam-cefoperazone, Pfizer International Inc.; flomoxef, Shionogi Pharmaceutical Co., Ltd.; clavulanic acid-amoxicillin (Smith Kline and Beecham Co., Ltd.; cefotiam, Takeda Chemical Industries, Ltd.; cefaclor and kanamycin, Sigma Chemical Co., St. Louis, Mo.; cefdinir, Fujisawa Pharmaceutical Co., Ltd.; tosufloxacin and cefteram, Toyama Chemical Co., Ltd.; ciprofloxacin, Bayer Inc.; levofloxacin and ofloxacin, Daiichi Seiyaku Co., Ltd.; norfloxacin, Kyorin Pharmaceutical Co., Ltd.; vancomycin, Shionogi Pharmaceutical Co., Ltd.; minocycline, Lederle-Japan; glucose-6-phosphate (G6P), Oriental Yeast Co., Ltd.; G6P-dehydrogenase and NADP, Asahi Kasei Industry Co., Ltd.
More than 97,000 stool specimens were collected in Osaka, Sizuoka, and Tokyo districts during July and August 1996. A single fresh stool sample from each patient was analyzed. O157 was isolated by the conventional sorbitol IPA bile salts agar plating method (8) and then confirmed with specific antisera to the O157 serotype (Denkaseiken Co., Ltd.). Forty-three strains of O157 from patients whose gender and age could be identified were used. MICs were determined by the broth microdilution method (5, 12).
G6P levels in human serum were measured with a Hitachi 7150 autoanalyzer (2). Three hundred microliters of 68.5 mM phosphate buffer (pH 7.5) containing 2.6 mM NADP and 3.8 mM magnesium sulfate was added to 20 μl of serum at 37°C. After 5 min, 50 μl of 68.5 mM phosphate buffer (pH 7.5) containing 14.0 U of G6P-dehydrogenase per ml and 3.8 mM magnesium sulfate was mixed at 37°C. After another 5 min, absorbance of the mixture was measured at 340 nm.
Of 24 antibiotics tested, the growth of 90% of the O157 strains used was inhibited at a concentration of ≤0.5 μg/ml by several agents (Table 1). These included cephalosporins except cefaclor, new quinolones, and fosfomycin with 3 μg of G6P per ml. G6P promotes the uptake of fosfomycin into bacteria (12). Fifty-six strains of E. coli other than O157 were isolated at the same time, and their susceptibilities to fosfomycin, norfloxacin, and kanamycin were compared (Table 1). Fosfomycin with G6P was more active on O157 isolates than non-O157 strains.
TABLE 1.
Susceptibilities of 43 clinical isolates of E. coli O157 and 56 clinical isolates of non-O157 E. coli to antimicrobial agentsa
| Antimicrobial agent | MIC (μg/ml)
|
||
|---|---|---|---|
| Range (μg/ml) | 50% | 90% | |
| Ampicillin | 2 to >128 | 2 | 4 |
| Arbekacin | 0.5 to 1 | 1 | 1 |
| Cefaclor | 2 to 4 | 2 | 2 |
| Cefdinir | ≤0.13 to 0.5 | 0.25 | 0.25 |
| Cefditoren | ≤0.13 to 0.5 | 0.25 | 0.25 |
| Cefmetazole | 0.25 to 1 | 0.50 | 0.50 |
| Cefotetiam | ≤0.13 | ≤0.13 | ≤0.13 |
| Cefpodoxime | 0.25 to 0.5 | 0.50 | 0.50 |
| Ceftazidime | ≤0.13 to 0.25 | ≤0.13 | ≤0.13 |
| Cefteram | ≤0.13 to 0.25 | 0.25 | 0.25 |
| Ciprofloxacin | ≤0.13 | ≤0.13 | ≤0.13 |
| Clavulanic acid-amoxicillin | 4 to 8 | 4 | 4 |
| Dibekacin | 1 to 2 | 1 | 1 |
| Flomoxef | ≤0.13 | ≤0.13 | ≤0.13 |
| Levofloxacin | ≤0.13 | ≤0.13 | ≤0.13 |
| Minocycline | 1 to 16 | 1 | 2 |
| Ofloxacin | ≤0.13 | ≤0.13 | ≤0.13 |
| Piperacillin | 0.50 to 8 | 1 | 1 |
| Sulbactam-cefoperazone | ≤0.13 to 0.25 | ≤0.13 | ≤0.13 |
| Tosufloxacin | ≤0.13 | ≤0.13 | ≤0.13 |
| Vancomycin | 64 to >128 | 128 | 128 |
| Fosfomycin | |||
| O157 | 16 to 32 | 16 | 32 |
| Non-O157 | 2 to 128 | 16 | 128 |
| Fosfomycin-G6Pb | |||
| O157 | ≤0.13 to 0.5 | 0.5 | 0.5 |
| Non-O157 | 1 to 64 | 4 | 16 |
| Kanamycin | |||
| O157 | 2 to 4 | 2 | 4 |
| Non-O157 | 2 to 64 | 4 | 32 |
| Norfloxacin | |||
| O157 | ≤0.13 | ≤0.13 | ≤0.13 |
| Non-O157 | ≤0.13 to 2 | ≤0.13 | ≤0.13 |
The data are for O157 unless indicated otherwise. 50% and 90%, concentrations of antimicrobial agents which inhibited growth in 50 and 90% of the strains, respectively.
3 μg of G6P per ml.
Fosfomycin acts as a phosphoenolpyruvate analog, irrevers- ibly inhibiting phosphoenolpyruvate UDP-N-acetylglucos- amine-3-O-enolpyruvyltransferase, an enzyme which catalyzes the first step of peptidoglycan biosynthesis (7, 9). The active transport of this antibiotic is associated with a transport system induced by G6P. We found that the mean G6P level in sera from 40 healthy adults was 4.0 ±1.0 μg/ml. Accordingly, the release of G6P-containing serum into the intestinal area immediately surrounding the inflammatory tissues of patients with O157 strains may potentiate the activity of fosfomycin.
Acknowledgments
Our sincere gratitude is extended to Y. Iwai, H. Ogawa, and K. Hoshino.
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