1 National Center for Antimicrobials and Infection Control, Statens Serum Institut, 5 Artillerivej, DK-2300 Copenhagen S; 2 Danish Zoonosis Centre and 3 Section for Antibiotic Resistance and Environmental Microbiology, Danish Veterinary Institute, 27 Bülowsvej, DK-1790 Copenhagen V, Denmark
Keywords: vanA, PFGE, gene transfer
Sir,
The occurrence of vancomycin-resistant Enterococcus faecium (VREF) in faeces from food animals and from meat has been associated with the use of the glycopeptide, avoparcin, for growth promotion. Tn1546, the vanA gene cluster encoding vancomycin resistance, has been detected both in VREF of human and animal origin. The use of avoparcin was banned in Denmark in May 1995, and in all of the European Union in 1997. Between 1995 and 2002 the occurrence of VREF isolated from Danish pigs declined from 20% to 2%.1 The prevalence of vancomycin-resistant enterococci (VRE) in healthy humans was not monitored before the ban of avoparcin in Denmark. The aim of this study was to investigate if Danish healthy humans were carrying VREF 7 years after the ban of avoparcin.
A programme to monitor antibiotic resistance in faecal bacteria from healthy human volunteers was initiated in Denmark in 2002. All volunteers were screened for VRE. Candidates for the programme were randomly selected from the Danish Civil Register System, which is a continuously updated register of all residents in Denmark. The selection algorithm was based on a gender and age distribution resembling the total Danish population.
The Scientific Ethics Committee for Copenhagen and Frederiksberg municipalities has approved the protocol [(KF) 01-006/02].
Between May 2002 and May 2003, 149 faecal samples were obtained from healthy volunteers. High-level vancomycin-resistant Enterococcus spp. were isolated using pre-enrichment in enterococcosel broth overnight at 35°C followed by spread on bile aesculin agar with 16 mg/L vancomycin and incubation for 2 days at 35°C. One vanA VREF isolate, 841V03, was obtained from a 35-year-old female office worker. She had not received antibiotics, was not a vegetarian and had eaten pork, broiler meat and beef. During the last month before isolation of faeces she had consumed Idoform (Ferrosan A/S, Denmark), an E. faecium containing probiotic product, and the yogurt product Gaio (Arla Foods, Denmark), containing high concentrations of E. faecium.
Tn1546 from 841V03 was transferable in a filter mating. An in vitro transfer rate of 9 x 105 transconjugants/donor was obtained from 841V03 to E. faecium BM4105-RF.
This characteristic of E. faecium has given concern regarding the safety of probiotics containing this bacterium. Previously, in vitro transfer of Tn1546 from a clinical isolate to the E. faecium strain from Gaio has been detected by Lund & Edlund.2 A possible in vivo transfer of Tn1546 could have taken place in the intestine of the woman in our study, since she had consumed high concentrations of enterococci from both Gaio and Idoform. Two E. faecium isolates were obtained from Gaio and Idoform, respectively. Both isolates were susceptible to vancomycin. 841V03 was PFGE-typed with the Gaio and Idoform isolates (Figure 1). The PFGE profile of 841V03 differed by more than six bands from the E. faecium strains obtained from Gaio and Idoform, indicating that these strains were not likely to be related according to the criteria of Tenover et al.3 Therefore 841V03 was not a transconjugant of one of the E. faecium strains from Gaio and Idoform. The criteria of Tenover et al. have never been suggested for the assessment of relatedness of independently collected isolates from humans, animals or food, but were used in our study because no other criteria exist.
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Earlier studies have shown that the occurrence of VREF in the Danish pig population was primarily caused by the presence of a single E. faecium clone.6,7 E8sv3 was chosen as a representative of the vanA pig clone.7 841V03, E8sv3 and four vanA positive VREF isolates (S163-1, S166-3, S328-2 and S658-3) obtained from pigs in 20021 were typed by PFGE using SmaI (Figure 1). The aim was to investigate if 841V03 was related to the VREF pig isolates obtained in 2002 and to see if the porcine VREF clone persisted. The PFGE profile of S163-1 was unrelated to the other strains. The PFGE profiles of S166-3 and S658-3 were identical to the PFGE profile of E8sv3, whereas the PFGE profile of S328-2 differed by one band, indicating a close relation. The PFGE profile of 841V03 differed from the PFGE profiles of E8sv3, S166-3 and S658-3 by less than six bands, indicating possible pig origin of the strain.3
After banning of avoparcin, tetracycline and tylosin were still extensively used therapeutically in pigs in 2002.1 Aarestrup found that the persistence of the porcine VREF clone during the period from 1995 to 1998 was associated with co-resistance to macrolides and selection by tylosin used for growth promotion and therapy.6 The use of the macrolide, tylosin, as a growth promoter was banned in all of the European Union in July 1999. A decrease in the occurrence of VREF in the pig herds has been detected following the termination of the use of tylosin as a growth promoter and it reached 2% in 2002.1 All the vanA VREF isolates in this study were resistant to tetracycline, encoded by tet(M); additionally three of the four pig isolates were resistant to erythromycin, encoded by erm(B). Although 841V03 and 166-3 were not resistant to erythromycin, they share the same mutation in vanX and have related PFGE patterns. This could indicate a possible pig origin of 841V03.
We suggest that the persistence of VREF in the pig herds at a low level 7 years after the ban of avoparcin, may, in part, be related to the therapeutic use of tetracycline and tylosin.
Acknowledgements
At SSI, Karin S. Pedersen, Frank Hansen, Stine Frese-Madsen, Mette Skafte Thomsen and at DVI, Charlotte Simonsen are thanked for their technical assistance. This work is part of The Danish Integrated Antimicrobial Resistance and Research Programme (DANMAP).
Footnotes
* Corresponding author. Tel: +45-32-68-33-99; Fax: +45-32-68-38-87; E-mail: ama{at}ssi.dk
References
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2 . Lund, B. & Edlund, C. (2001). Probiotic Enterococcus faecium strain is a possible recipient of the vanA gene cluster. Clinical Infectious Diseases 32, 13845.[CrossRef][ISI][Medline]
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