Journal of Antimicrobial Chemotherapy (2000) 45, 677-680
© 2000 The British Society for Antimicrobial Chemotherapy
Brief reports |
Characterization of vancomycin-resistant and vancomycin-susceptible Enterococcus faecium isolates from humans, chickens and pigs by RiboPrinting and pulsed-field gel electrophoresis
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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Forty-eight vancomycin-resistant and 35 vancomycin-sensitive Danish Enterococcus faecium isolates obtained from pigs, chickens and humans, as well as the human vanA reference isolate BM4147, were characterized by EcoRI RiboPrinting and SmaI pulsed-field gel electrophoresis. RiboPrinting of the 84 isolates yielded 40 types whereas PFGE-typing yielded 57 types discriminated by differences in more than three bands. By molecular typing, both clonal spread of E. faecium as well as horizontal transmission of Tn1546 between animals and humans was supported. Furthermore, it was found that the population of E. faecium spreads freely between the animal and human reservoir.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Infections with enterococci are a major cause of nosocomial infections, and these bacteria have been reported to have a high potential for acquiring resistance to vancomycin. Vancomycin-resistant enterococci (VRE) can be isolated from healthy people, foodstuffs, domestic animals and sewage.1 The epidemiology of VRE has been studied in hospital environments and it has been shown that vancomycin resistance is transmitted through both clonal and horizontal spread.1 Avoparcin, a related glycopeptide, was until May 1995 widely used as a growth promoter for animal production in Denmark.2 A previous study has shown that avoparcin induces resistance to vancomycin as well.2 Little is know about the epidemiology of both VRE and vancomycin-sensitive enterococci (VSE) outside hospital environments. The aim of the present study was therefore to investigate the relatedness of VRE and VSE isolated from animals and humans by RiboPrinting and pulsed-field gel electrophoresis (PFGE), and to study the spread of Tn1546 (the vanA gene cluster encoding vancomycin resistance).
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Bacterial isolates
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 2–8 s for 20 h followed by 8–22 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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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The diversity and distribution of VRE types outside the hospital environment have been investigated in only a few studies.7,8 In the present study, a population of E. faecium isolated from humans, chickens and pigs, as well as the vanA reference isolate BM4147, was investigated by EcoRI RiboPrinting and SmaI PFGE typing. Forty-nine of the 84 isolates were high-level resistant to vancomycin and had the vanA genotype. RiboPrinting is comparable with ribotyping, with the difference that most of the process is automated. RiboPrinting divided the population of 84 isolates into 40 ribogroups (Figure 1
). PFGE-typing differentiated the population into 57 PFGE types, of which eight types consisted of two to three subtypes (Figure 2). In our study, the better discrimination of isolates by SmaI PFGE-typing than EcoRI RiboPrinting was in agreement with previous studies comparing SmaI PFGE-typing with HindIII, EcoRI and PvuII ribotyping of E. faecalis.9,10
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From nine of the chicken flocks and six of the pig herds in this study, more than one isolate could be obtained. In five of these cases, the isolates were shown to be identical by both RiboPrinting and PFGE-typing, indicating clonal spread between animals within these flocks or herds.
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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We gratefully acknowledge Réne Hendriksen (Danish Veterinary Laboratory) and Jane Andersen (Statens Serum Institut) for their technical assistance. We are grateful to Professor P. Courvalin for providing E. faecium BM4147. This study is part of the Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP), and was funded jointly by the Danish Ministry of Health and the Danish Ministry of Food, Agriculture and Fisheries.
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Notes |
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* Corresponding author. Tel: +45-35-30-01-00; Fax: +45-35-30-01-20; E-mail: aha{at}svs.dk
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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1 . Woodford, N. (1998). Glycopeptide-resistant enterococci: a decade of experience. Journal of Medical Microbiology 47, 849–62.
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, 1938–40.[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, 157–61.[Web of Science][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, 437–42.
5 . 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, 2233–9.[Web of Science][Medline]
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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, 265–72.
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Received 23 June 1999; returned 31 October 1999; revised 14 December 1999; accepted 24 January 2000
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