Departamento de Patología Animal I, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain
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Abstract |
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Introduction |
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The original quinolone drugs and the fluoroquinolones have been shown to have excellent in vitro activity against clinical E. coli isolates of human and animal origin.2 However, the number of reports of fluoroquinolone-resistant E. coli strains isolated from humans and animals seems to be increasing.2 To our knowledge, no fluoroquinolone-resistant VTEC or eae-positive E. coli have been reported. Several authors have studied the resistance of VTEC strains, principally of E. coli O157, to fluoroquinolones and have observed that none of these strains was resistant to these antimicrobials.2 Moreover, our group has studied the quinolone susceptibility of E. coli strains isolated from diarrhoeic dairy calves3 and from diarrhoeic lambs.4 Some of the strains investigated in these studies3,4 were NTEC, VTEC and/or eae-positive and, although relatively high percentages of fluoroquinolone-resistant strains were found, all VTEC and eae-positive strains were susceptible to fluoroquinolones and only one NTEC strain was resistant to these antimicrobials. Thus, the results of our studies indicate that the ruminant E. coli strains which possess some potential virulence factors are more susceptible to quinolones than those that do not possess these factors.
The aims of this study were to evaluate the quinolone susceptibilities of E. coli strains isolated from healthy ruminants and the relationships between potential virulence factors of E. coli and susceptiblity to these antimicrobials.
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Materials and methods |
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The study investigated 557 strains of E. coli isolated from 416 healthy ruminants (204 strains from 146 cows, 210 strains from 151 sheep and 143 strains from 119 goats). The bacterial strains were isolated from faecal samples in our laboratory between 1997 and 1998 from 24 farms (seven cattle farms, nine sheep farms and eight goat farms) located in central Spain. Precise information about fluoroquinolone use on the farms sampled for this study was not available. The isolates were tested for cytotoxic necrotizing factors (CNF) and verotoxins (VT) by cytotoxicity assays and PCR, and for the eae gene by colony blot hybridization.3 Four strains per animal were analysed but only one strain with different characteristics (presence or absence of potential virulence factors) per animal was included in the study. Two hundred and twelve of the strains were selected because they possessed the following potential virulence factors (potentially pathogenic strains): 37 were NTEC (22 from cattle, 13 from sheep and two from goats); 98 were VTEC (24 from cattle, 46 from sheep and 28 from goats); and 98 possessed the eae gene (29 from cattle, 47 from sheep and 22 from goats). Six of the potentially pathogenic strains from cattle were positive for both VT and eae gene, and 15 of the potentially pathogenic strains from sheep and goats were positive for both CNF and eae gene (13 from sheep and two from goats). The remaining potentially pathogenic strains possessed only one potential virulence factor. Moreover, 345 strains that did not produce any of the toxins studied and were eae-negative (non-pathogenic strains) were also studied. Two hundred and sixteen of these non-pathogenic strains were from animals from which no potentially pathogenic strains were isolated (81 from cattle, 62 from sheep and 73 from goats), and 129 were from animals from which potentially pathogenic strains were isolated [28 from animals from which NTEC strains were isolated (21 from cattle and seven from sheep); 61 from animals from which VTEC strains were isolated (17 from cattle, 31 from sheep and 13 from goats); and 52 from animals from which eae-positive strains were isolated (20 from cattle, 25 from sheep and seven from goats)].
Quinolones
The following quinolones were studied and were provided by the manufacturers: nalidixic acid (Hipra, Amer, Girona, Spain), enrofloxacin (Bayer, Barcelona, Spain) and ciprofloxacin (Bayer).
Antimicrobial susceptibility testing
In vitro susceptibility tests were performed by the agar dilution method, according to the recommendations of the NCCLS,5 with MuellerHinton agar (Difco, Detroit, MI, USA). The range of interpretative categories of susceptibility for nalidixic acid, enrofloxacin and ciprofloxacin were those recommended by the NCCLS.5,6 The NCCLS breakpoint for ciprofloxacin is based on human clinical isolates.
Statistical analysis
Significant differences in the frequencies of resistance to the tested quinolones among the potentially pathogenic strains and the non-pathogenic strains were determined by the 2 test. A P value of <0.05 was considered significant.
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Results |
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In sheep and goats, only three strains (one non-pathogenic from a sheep from which no potentially pathogenic strains were isolated, one VTEC from goats and one non-pathogenic from a goat from which eae-positive strains were isolated) were resistant to nalidixic acid and none was resistant to fluoroquinolones.
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Discussion |
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The percentages of strains resistant to nalidixic acid found in this study in healthy cattle and sheep (5.9% and 0.5%, respectively) were lower than those found previously by us in E. coli strains isolated from diarrhoeic dairy calves (17.9%)3 and diarrhoeic lambs (26.3%).4 These results may be explained because antimicrobials are used frequently by animal owners and veterinarians in the treatment of diarrhoea.
The fluoroquinolones are an exceptionally important and rapidly developing group of antimicrobial drugs, and are being introduced into human and veterinary medicine for a wide variety of antimicrobial purposes.2 In early reports on fluoroquinolones, resistance of human and ruminant E. coli strains to these antibiotics was rarely observed.2 However, several investigators have described increases in the levels of resistance to these antimicrobials among E. coli strains isolated from humans and animals.2 In this study the percentages of strains resistant to enrofloxacin (4.9% in cattle and 0% in sheep) were lower than those found in previous studies performed by our group with diarrhoeic calves (11.8%)3 and diarrhoeic lambs (14%)4 from the same geographical area as the animals in this study. As mentioned previously, this may be because herds with diarrhoea often have an increased consumption of antimicrobials.
In the present study, the level of resistance to enrofloxacin (a fluoroquinolone used in domestic animals and approved in Spain for the treatment of colibacillosis and diarrhoea in ruminants) was the same as the level of resistance to ciprofloxacin (a fluoroquinolone available for human clinical use). This is owing to the fact that resistance to one fluoroquinolone generally confers resistance to the entire class of fluoroquinolone agents.2 The development of cross-resistance among the fluoroquinolones used in veterinary and human medicine is a source of debate on the use of these antibiotics for the treatment of infections in animals.2 Threlfall et al.7 have suggested that the emergence and spread in the UK of Salmonella typhimurium DT 104, a human pathogen with reduced susceptibility to ciprofloxacin, has followed the licensing of the related fluoroquinolone enrofloxacin for veterinary use in the UK in 1993. Because of this, Threlfall et al.7 have recommended a restriction of the veterinary use of fluoroquinolones; however, there is considerable controversy over the apparent association between enrofloxacin usage in food animals and the emergence of strains with decreased susceptibility to ciprofloxacin in humans. Without scientific evidence, such an association cannot be substantiated.8 However, Walker et al.9 recently traced clearly an outbreak of infection in humans due to S. typhimurium DT 104 from dairy cattle. These investigators demonstrated a distinct mutation in the gyrA gene (a gene implicated in resistance to quinolones) in S. typhimurium DT 104 isolated from dairy cattle, a milk filter and humans during an outbreak.
In this study, most organisms resistant to quinolones were non-pathogenic strains from cattle from which no potentially pathogenic strains were isolated; only one of the potentially pathogenic strains studied (VTEC and eae-positive from cattle) was resistant to these antimicrobials. To the authors' knowledge, this is the first report of fluoroquinolone-resistant VTEC and/or eae-positive E. coli. Moreover, the non-pathogenic strains isolated from cattle from which no potentially pathogenic strains were isolated were significantly more resistant to the quinolones tested than the potentially pathogenic strains isolated from cattle. However, the potentially pathogenic strains from cattle were not significantly more susceptible to quinolones than the non-pathogenic strains from cattle from which potentially pathogenic strains were isolated. On the other hand, in sheep and goats only three strains were resistant to nalidixic acid and none to fluoroquinolones. Thus, these results are not consistent with, although not contradictory to, our previous results in diarrhoeic calves3 and diarrhoeic lambs,4 in which the bovine and ovine E. coli strains that possessed some potential virulence factors were more susceptible to quinolones than those that did not possess these factors. This may be owing to the fact that in previous studies3,4 all the non-pathogenic strains were isolated from animals other than those carrying potentially pathogenic strains.
The percentages of fluoroquinolone-resistant E. coli strains isolated from ruminants found by our group (relatively high in isolates from diarrhoeic calves and lambs but low in isolates from healthy ruminants) show that the resistance to quinolones in E. coli isolates from ruminants should be seen as a resistance reservoir rather than an imminent threat to public health. However, if fluoroquinolones are not used properly, the emergence of fluoroquinolone resistance in pathogenic E. coli may present a risk to public health because these strains may cause human disease,1 and because resistance to the fluoroquinolones used in veterinary medicine may confer resistance to the fluoroquinolones used in human medicine.2,10
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Acknowledgements |
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Notes |
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References |
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2 . Orden, J. A., Ruiz-Santa-Quiteria, J. A., Cid, D. & de la Fuente, R. (2000). Quinolone resistance in bacteria of animal origin and implications on human health. Research Advances in Antimicrobial Agents and Chemotherapy 1, 3548.
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Orden, J. A., Ruiz-Santa-Quiteria, J. A., García, S., Cid, D. & de la Fuente, R. (1999). In vitro activities of cephalosporins and quinolones against Escherichia coli strains isolated from diarrheic dairy calves. Antimicrobial Agents and Chemotherapy 43, 5103.
4 . Orden, J. A., Ruiz-Santa-Quiteria, J. A., García, S., Cid, D. & de la Fuente, R. (2000). Quinolone resistance in Escherichia coli strains isolated from diarrhoeic lambs in Spain. Veterinary Record 147, 5768.[ISI][Medline]
5 . National Committee for Clinical Laboratory Standards. (1999). Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals: Approved Standard M31-A. NCCLS, Wayne, PA.
6 . National Committee for Clinical Laboratory Standards. (1993). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-A3. NCCLS, Wayne, PA.
7 . Threlfall, E. J., Angulo, F. J. & Wall, P. G. (1998). Ciprofloxacin-resistant Salmonella typhimurium DT104. Veterinary Record 142, 255.
8 . Jones, P. G. H. (1998). The authorisation of antimicrobials in the EU for veterinary use. In Use of Quinolones in Food Animals and Potential Impact on Human Health, pp. 10313. World Health Organization, Geneva.
9 . Walker, R. A., Lawson, A. J., Lindsay, E. A., Ward, L. R., Wright, P. A., Bolton, F. J. et al. (2000). Decreased susceptibility to ciprofloxacin in outbreak-associated multiresistant Salmonella typhimurium DT104. Veterinary Record 147, 3956.[ISI][Medline]
10 . Wegener, H. C., Aarestrup, F. M., Gerner-Smidt, P. & Bager, F. (1999). Transfer of antibiotic resistant bacteria from animals to man. Acta Veterinaria Scandinavica, Suppl. 92, 517.
Received 8 February 2001; returned 27 April 2001; revised 16 May 2001; accepted 31 May 2001