National Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries, 1-15-1 Tokura, Kokubunji, Tokyo 185-8511, Japan
Received 30 May 2002; returned 2 August 2002; revised 15 September 2002; accepted 25 September 2002
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Abstract |
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Keywords: Escherichia coli, antimicrobial resistance, cattle, pig, broiler
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Introduction |
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Many studies on bacterial resistance have been published to date, but most have focused on human and animal pathogens, and have been based on data generated to support therapy.2 However, the selective pressure of antimicrobial use for therapy should be taken into account when interpreting these studies. Escherichia coli is the most common enterobacterium, and can serve as an indicator bacterium that easily acquires antimicrobial resistances commonly found in different animal species.2,3 The use of E. coli as an indicator bacterium allows a representative stratified random sampling strategy from healthy animals, enabling direct comparison of resistance between different animals and analysis of trends over time.3 The use of E. coli as an indicator bacterium is also important because changes in the resistance of this species may serve as an early warning system for resistance in potentially pathogenic bacteria.2
The Japanese Veterinary Antimicrobial Resistance Monitoring System (JVARM) was formed in 1999 in response to international concern about the impact of antimicrobial resistance on public health. This paper deals with the antimicrobial susceptibility of E. coli isolates from groups of the major food-producing animals throughout Japan, determined under the JVARM.
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Materials and methods |
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Sampling was carried out by the Livestock Hygiene Service Centers of all 47 prefectures across Japan from June to December 1999. Fresh faecal samples were collected from healthy beef cattle, pigs and broilers at the farm. In general, four samples per animal species were collected from different farms in each prefecture. Faecal samples were placed in sterile plastic specimen tubes on ice and transported to our laboratory for bacterial isolation within 3 days.
Culture procedures for isolation of E. coli
Faeces were plated on to desoxycholatehydrogen sulphatelactose agar (Eiken Co. Ltd, Japan) directly or, occasionally, after enrichment in trypticase soy broth (BBL; Becton-Dickinson, Cockeysville, MD, USA) containing vancomycin (Sigma Chemical Co.). Candidate colonies were then plated on to nutrient agar (Eiken), identified biochemically and kept at 4°C in Dorset egg medium (Nissui Pharmaceuticals Co. Ltd, Japan) until use. For individual samples, two E. coli isolates were selected randomly for the purpose of determining susceptibility.
Antimicrobial susceptibility testing
Eighteen widely used antimicrobial agents approved in Japan as veterinary medicines were tested. The MIC for each isolate was determined by a standardized agar dilution method, as described by the Japanese Society of Chemotherapy,4 using MuellerHinton agar (Difco). E. coli ATCC 25922, E. coli ATCC 35218, E. coli NIHJ, Staphylococcus aureus ATCC 25923, S. aureus 209P and Pseudomonas aeruginosa ATCC 27853 were used as quality controls. Breakpoint was defined from a population of the isolates tested in this study.
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Results and discussion |
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The use of chloramphenicol was banned in all animals except pets in Japan in 1998, so that the emergence of resistance due to its use would no longer take place. However, our study has shown that chloramphenicol resistance levels are still comparable to those of previous Japanese studies,57 indicating that continuous monitoring is clearly necessary.
Resistance rates were found to be <10% for the other antimicrobials, with the exception of quinolones. Gentamicin and apramycin resistance levels were found to be comparable to previous levels in Japan7 and other countries.2,3 Neither antimicrobial is approved for use in cattle or broilers in Japan, accordingly, apramycin resistance was only observed in seven pig isolates. Although gentamicin resistance was detected in broiler isolates, it may have been caused by off-label use or the clonal spread of resistant isolates, as suggested by Aarestrup et al.3 Simultaneous resistance to gentamicin and apramycin, which has been reported in other countries,3 was observed in our study for all apramycin-resistant isolates. Colistin showed substantial activity against E. coli isolates from all animal origins, with an MIC50 of 0.39 mg/L and an MIC90 of 0.78 mg/L, after three decades of use of this antimicrobial. Resistance to bicozamycin was low for all animal isolates, although the breakpoint was high, at 100 mg/L. Regardless of the animal origin, only a limited number of isolates were resistant to cephalosporins, including the third-generation cephalosporin, ceftiofur, which is an important human medicine. The use of cephalosporins is restricted in Japan and oral formulations for mass administration of these antimicrobials have not been available since their introduction to the veterinary market around 1986. Such restricted use may have resulted in lower levels of resistance against cephalosporins.
Resistance levels against quinolones were markedly higher in broiler isolates. Table 2 shows the distribution of MICs of the fluoroquinolones and a comparison of the resistance rates observed in this study with those of a previous survey. In the survey of 19921993,7 only a limited number of isolates were resistant to fluoroquinolones, regardless of the animal origin. However, the resistance rates increased among the broiler isolates from 0% in 1992 to 9.9% for enrofloxacin and 1.3% in 1993 to 10.5% for ofloxacin, whereas resistance rates were comparable or decreased in isolates from other animals. Among the broiler isolates, extremely high MICs of fluoroquinolones (100 mg/L) were also determined, although the MIC50s of enrofloxacin and ofloxacin were 0.05 and 0.1 mg/L, respectively, demonstrating that E. coli are still extremely sensitive to fluoroquinolones. Regionally, fluoroquinolone-resistant E. coli were isolated on 24 farms, located in 16 prefectures across Japan, indicating that fluoroquinolone resistance is widely dispersed in Japan.
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In conclusion, antimicrobial resistance in E. coli as an indicator bacterium has generally decreased in Japan, with the exception of fluoroquinolone resistance among broiler isolates, which has increased.
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Acknowledgements |
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Footnotes |
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References |
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2
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