1 Department of Pharmacy, Yamaguchi University Hospital, 1-1-1 Minamikogushi, Ube 755-8505; 2 Faculty of Pharmaceutical Science, Hiroshima International University, 5-1-1 Hiro-koshingai, Kure 737-0112; 3 Yamaguchi Prefectural Research Institute of Health, 2-5-67 Aoi, Yamaguchi 753-0821, Japan
Received 23 February 2004; returned 11 March 2004; revised 24 June 2004; accepted 2 September 2004
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Abstract |
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Methods: Pulsed-field gel electrophoresis was carried out with the Bio-Rad Gene Path system. Each antimicrobial agent was added to logarithmic phase of enterohaemorrhagic E. coli (four strains of E. coli O157:H7, two of E. coli O26, two of E. coli O111, and one of E. coli O165) in broth to obtain a concentration of 10 or 50 mg/L, and viable cells were counted after 1, 2, 6 and 24 h.
Results: All nine strains were confirmed to differ in their DNA pattern by pulsed-field gel electrophoresis. Norfloxacin at concentrations of 10 and 50 mg/L had bactericidal effects on all nine strains of enterohaemorrhagic E. coli. However, cefoperazone, kanamycin and fosfomycin had no bactericidal effects on some strains. In particular, after addition of 10 mg/L fosfomycin or kanamycin, four of the nine strains showed proliferation.
Conclusions: Norfloxacin had marked bactericidal effects on enterohaemorrhagic E. coli. This information could be of value in planning randomized clinical trials of antimicrobial agents as treatment for enterohaemorrhagic E. coli infection.
Keywords: killing kinetics , norfloxacin , cefoperazone , kanamycin , fosfomycin
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Introduction |
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Materials and methods |
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Four strains of E. coli O157:H7, two of E. coli O26, two of E. coli O111, and one of E. coli O165 were used. The four E. coli O157:H7 strains had been isolated in outbreaks in Japan: one strain in an outbreak in Osaka Prefecture (12 680 patients) in 1996 (E. coli O157 Osaka),15,23 one in an outbreak in Iwate Prefecture (220 patients) in 1996 (E. coli O157 Iwate), one in an outbreak in Saitama Prefecture (67 patients) in 2001 (E. coli O157 Saitama), and one in an outbreak in Yamaguchi Prefecture (26 patients) in 2001 (E. coli O157 Yamaguchi). The two strains of E. coli O26 (E. coli O26-1 and E. coli O26-2), two strains of E. coli O111 (E. coli O111-1 and E. coli O111-2), and one strain of E. coli O165 had been isolated from five patients with sporadic infection in Yamaguchi Prefecture, Japan in 2001.
Killing test
Each drug was added to 5 mL of Sensitivity Test broth (Nissui Pharm., Tokyo) containing log-phase inocula (final concentration, approximately 5 x 105 cfu/mL) to obtain a drug concentration of 10 or 50 mg/L. Viable cells were counted after incubation at 37°C for 1, 2, 6 and 24 h. The evaluated agents were norfloxacin (Kyorin Pharm., Tokyo), cefoperazone sodium (Pfizer Pharm. Inc., USA), kanamycin sulphate (Meiji Seika Co., Tokyo), and fosfomycin sodium (Meiji Seika Co., Tokyo). In fosfomycin killing tests, 25 mg/L of glucose-6-phosphate (Oriental Yeast Co., Ltd., Tokyo) was also used. The two agents were combined because in vitro susceptibility tests for fosfomycin have been standardized by adding 25 mg/L of glucose-6-phosphate, and glucose-6-phosphate is present physiologically wherever glycolysis takes place.24 The three agents excluding norfloxacin were provided in the form of a freeze-dried amorphous powder.
Viable cells were counted by a membrane filter filtration method to prevent antimicrobial agent carry-over.17,25 The broth (0.5 mL) was filtered through a 0.22 µm membrane filter (diameter, 5 cm; Nippon Becton Dickinson Co., Japan), and sterile saline (100 mL) was filtered a total of four times. Subsequently, this membrane filter was placed in a bottle containing 10 mL sterile physiological saline, and this bottle was ultrasonicated (Sine Sonic 100, Ikemoto Rikagaku Co., Japan) at 37 kHz for 10 min. Each sample of physiological saline from this bottle was diluted 101- to 107-fold in sterile saline; two aliquots (0.5 mL each) of 101- to 107-fold dilution and of an undiluted sample were plated on two trypticase soy agar plates (Nippon Becton Dickinson Co., Japan). Viable cells were counted after 24 h incubation at 37°C. The experiments were carried out twice, and the mean values were calculated.
The MICs were also determined for the nine strains of enterohaemorrhagic E. coli after 18 h incubation at 37°C by dilution on Sensitivity Test agar (Nissui Pharm., Tokyo). Twofold serial dilutions ranging from 128 to 0.008 mg/L were employed. The inocula (104 cfu/spot) were plated using a multipoint inoculator (Sakuma Co., Japan). MICs were defined as the lowest concentration of agent inhibiting visible growth.
Pulsed-field gel electrophoresis
The preparation of high molecular weight chromosomal DNA was carried out according to Murray et al.26 Digestion was carried out by placing a small slice of an agarose plug in 200 µL of reaction buffer with 30 U of XbaI. Pulsed-field gel electrophoresis was carried out with the Bio-Rad Gene Path system in a 1% agarose gel in 0.5x TBE buffer at 14°C with a linear ramp time of 4 to 50 s over a period of 20 h. Thereafter, the gels were stained with ethidium bromide and photographed.
Data analysis
The effects on the viability of nine strains of enterohaemorrhagic E. coli were compared among control (without drugs), norfloxacin, cefoperazone, kanamycin and fosfomycin by the KruskalWallis test.
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Results |
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Discussion |
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Norfloxacin had bactericidal effects on all the nine strains, showing the highest bactericidal activity among the four agents evaluated. Some studies have shown a significant decrease in the incidence of haemolytic uraemic syndrome (HUS) after administration of appropriate antimicrobial agents such as fluoroquinolones for E. coli O157:H7 infection, or a significant decrease in the mortality rate after administration of a fluoroquinolone for this infection in a mouse model.913 Therefore, fluoroquinolones such as norfloxacin may be useful for treating enterohaemorrhagic E. coli infection. On the other hand, cefoperazone, kanamycin and fosfomycin had no bactericidal effects on some strains. In particular, fosfomycin's low bactericidal activity suggests that it may not be the optimal agent for treatment of enterohaemorrhagic E. coli infection.
Only a small number of strains of enterohaemorrhagic E. coli were studied, but they were chosen to include representative strains from large outbreaks. In addition, pulsed-field gel electrophoresis confirmed that the study strains were all distinct. Our preliminary study has shown that the MICs for these nine strains of enterohaemorrhagic E. coli were low (less than or equal to 8 mg/L) for all antimicrobial agents studied, however the bactericidal activity differed markedly, with norfloxacin showing the highest level of activity. This possible activity of norfloxacin against enterohaemorrhagic E. coli may be of value in planning future clinical trials of antimicrobial agents for the management of enterohaemorrhagic E. coli infection.
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Acknowledgements |
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Footnotes |
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References |
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