a Danish Veterinary Laboratory, Bülowsvej 27, DK-1790 Copenhagen V; b Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark
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Abstract |
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Introduction |
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Materials and methods |
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A total of 83 Danish E. faecium isolates from humans (22 isolates from 22 patients), pigs (27 isolates from 16 herds) and chickens (34 isolates from 17 flocks) were selected from a previously described isolate collection.2 The human vanA reference isolate BM4147 was also included.3 The animal isolates were collected during March to June 1995, from the faeces of chickens and pigs in Denmark. The human isolates were obtained in 1995 from the faeces of hospitalized patients with diarrhoea, except for one isolate obtained from a urinary tract infection at the same hospital in Denmark. None of the patients were treated with glycopeptides. Two of the human isolates, 31 of the chicken isolates and 15 of the pig isolates were high-level resistant to vancomycin (MIC 64 mg/L) and had the vanA genotype.2 From each flock or herd, isolates were obtained from one to three animals. Isolates were presumed to be epidemiologically unrelated if they originated from different herds at different geographical locations. Isolates originating from the same flock or herd were presumed to be potentially epidemiologically related.
RiboPrinting
RiboPrinting was performed using the RiboPrinter, as recommended by the manufacturer (Qualicon, Wilmington, DE, USA). Analysis, including EcoRI restriction of DNA, was carried out automatically. The resultant RiboPrint patterns were aligned according to the position of a molecular size standard and compared with patterns obtained previously, including a database consisting of 750 validated ribogroups supplied by the manufacturer.
PFGE
Whole cell DNA in agarose plugs was prepared as previously described4 and digested with 20 U of SmaI (Amersham Pharmacia Biotech, Uppsala, Sweden) for a minimum of 4 h. DNA fragments were separated on a 1.4% agarose gel (pulsed-field certified agarose; Bio-Rad, Hercules, CA, USA) by use of a CHEF DRIII apparatus (Bio-Rad) with pulse times 28 s for 20 h followed by 822 s for 21 h (temperature, 12°C; voltage, 6 V/cm; angle, 120°).
PFGE types were differentiated assuming that a single genetic event (introduction or loss of a single restriction endonuclease cutting site) could introduce a maximum of three fragment differences in the restriction pattern.5 Isolates showing variation fewer than three fragments were assigned to subtypes of the major types.
Analysis of PFGE and RiboPrints
PFGE profiles and RiboPrints were analysed in GelCompar (Applied Marths, Kortrijk, Belgium). The levels of similarity between fingerprints were expressed as Dice coefficients, which were calculated by determining the ratio of twice the number of bands shared by two patterns to the total number of bands in both patterns.6 Isolates were clustered by using the unweighted pair group method with arithmetic averages (UPGMA).6
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Results and discussion |
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Eleven VRE isolates obtained from six pig herds belonged to a single ribogroup and PFGE type (R20/45), which could indicate spread of the same clone between these pig herds. Furthermore, the finding of this type in a human VRE isolate indicated animal to human transmission.
Our results showed that neither VRE nor VSE isolates could be grouped in discrete clusters by ribotyping or PFGE typing; rather, VRE and VSE isolates showed a great diversity of types, which were evenly distributed within the dendrograms. These data are consistent with the highly transmissible nature of Tn1546.
In conclusion, our results show that both clonal spread of isolates as well as horizontal transfer of Tn1546 occurred in Danish pig and chicken production, as has been shown in previous studies of the epidemiology of VRE in and between hospitals.1 The isolates obtained from different reservoirs (pigs, chickens and humans) did not group into separate clusters by either of the typing methods, indicating a non-host-specific preference of isolates and suggesting that the population of E. faecium spreads freely between the animal and human reservoirs.
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Acknowledgments |
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Notes |
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References |
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2 . Aarestrup, F. M., Ahrens, P., Madsen, M., Pallesen, L. V., Poulsen, R. L. & Westh, H. (1996). Glycopeptide susceptibility among Danish Enterococcus faecium and Enterococcus faecalis isolates of animal and human origin and PCR identification of genes within the VanA cluster. Antimicrobial Agents and Chemotherapy 40, 193840.[Abstract]
3 . Leclercq, R., Derlot, E., Duval, J. & Courvalin, P. (1988). Plasmid-mediated resistance to vancomycin and teicoplanin in Enterococcus faecium. New England Journal of Medicine 319, 15761.[ISI][Medline]
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Jensen, L. B., Ahrens, P., Dons, L., Jones, R. N., Hammerum, A. M. & Aarestrup, F. M. (1998). Molecular analysis of Tn1546 in Enterococcus faecium isolated from animals and humans. Journal of Clinical Microbiology 36, 43742.
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Tenover, F. C., Arbeit, R. D., Goering, R. V., Mickelsen, P. A., Murray, B. E., Persing, D. H. et al. (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. Journal of Clinical Microbiology 33, 22339.
6 . Sokal, R. R. & Sneath, P. H. A. (1963). Principles of Numerical Taxonomy. W. H. Freeman, San Francisco, CA.
7 . Bates, J., Jordens, J. Z. & Griffiths, D. T. (1994). Farm animals as a putative reservoir for vancomycin-resistant enterococcal infection in man. Journal of Antimicrobial Chemotherapy 34, 50714.[Abstract]
8 . Klare, I., Heier, H., Claus, H., Böhme, G., Marin, S., Seltmann, G. et al. (1995). Enterococcus faecium strains with vanA-mediated high-level glycopeptide resistance isolated from animal foodstuffs and fecal samples of humans in the community. Microbial Drug Resistance 1, 26572.[ISI][Medline]
9 . Gordillo, M. E., Singh, K. V. & Murray, B. E. (1993). Comparison of ribotyping and pulsed-field gel electrophoresis for subspecies differentiation of strains of Enterococcus faecalis. Journal of Clinical Microbiology 31, 15704.[Abstract]
10 . Plessis, P., Lamy, T., Donnio, P. Y., Autuly, F., Grulois, I., Le Prisé, P. Y. et al. (1995). Epidemiologic analysis of glycopeptide-resistant Enterococcus strains in neutropenic patients receiving prolonged vancomycin administration. European Journal of Clinical Microbiology and Infectious Diseases 14, 95963.[ISI][Medline]
Received 23 June 1999; returned 31 October 1999; revised 14 December 1999; accepted 24 January 2000