Vancomycin-resistant Enterococcus faecium clone in swine, Europe.

To the Editor: The use of antimicrobial agents for growth promotion (AGP) in food-producing animals has been extensively debated because of the risk of establishing a reservoir of antimicrobial resistance genes or antimicrobial-resistant organisms of potential relevance for human health. This concern has motivated the progressive ban of the use of different AGP in the European Union, which began in 1997 with avoparcin and will end in 2006 (1). Worldwide trade of living animals for food production or breeding and of meat products enables multidrug-resistant pathogens to spread across national borders.

Intercontinental dissemination of antimicrobial-resistant bacteria associated with food animals has been described for particular clones such as Salmonella enterica Typhimurium DT104 or Escherichia coli O157:H7 and for transferable genetic elements such as the genomic island SG1 or the streptococcal plasmid pRE25 (2). Vancomycin-resistant enterococci (VRE) in European farms were initially associated with the intensive use of avoparcin; however, the persistence of VRE in food animal environments after years of avoparcin withdrawal indicates that coselection by further antimicrobial or other agents, increased fitness of strains, and mobile genetic elements cannot be ruled out (1-3).

A specific clone was recently detected among vancomycin-resistant E. faecium (VREF) isolated from different swine farms in Denmark and Switzerland and from a healthy Danish woman without antimicrobial drug exposure who ate pork, chicken, and beef (4,5). Since Portugal and Spain maintain commercial trade of food-producing swine (living or meat products) between them and with other European countries, including Denmark (http//:www.dgv.min-agricultura.pt/dgv.nsf), we investigated a possible relationship among VREF swine fecal isolates from Portugal and Spain and compared these isolates with the Swiss/Danish clone. We studied 3 VREF from a Figueira da Foz slaughterhouse in central Portugal (1997-1998) and 3 VREF isolates from 3 Spanish slaughterhouses in Valencia, Lugo, and Murcia in eastern, northern, and southern Spain, respectively (1998-2000). These isolates were recovered in the course of previous surveillance studies (C. Novais/I. Herrero, unpub data). Antimicrobial susceptibility was tested for 13 antimicrobial agents by using the agar dilution method (6). Clonal relationships were analyzed by pulsed-field gel electrophoresis (PFGE) and characterization of pur-K alleles by amplification and further sequencing (6,7; http://efaecium. mlst.net). Species identification, genes coding for antimicrobial resistance genes or for putative virulence traits, and the backbone structure of Tn1546 were analyzed by polymerase chain reaction followed by sequencing when necessary (6,8). Broth and filter mating were performed by using E. faecium GE1 as recipient strain (6).

Following criteria published elsewhere (6), the VREF isolates studied were considered a single clone (0-4 bands difference by PFGE). Some vancomycin-susceptible E. faecium swine isolates (VSEF) from Spain and Switzerland showed an SmaI-PFGE pattern closely related to that of VREF isolates (data not shown; [4]).

Representative VREF of each country harbored the allele 9 of the housekeeping gene purK, previously found among E. faecium isolates from swine and healthy persons (7). All VREF isolates were resistant to glycopeptides (vanA), erythromycin [erm(B)], and tetracycline. Two Spanish isolates were also highly resistant to streptomycin and kanamycin [aph(3')-IIIa] (Table). All VREF isolates tested carried a Tn1546 type D, previously found in isolates from food-producing animals (8). This element showed alterations in orf1 and a G-T point mutation in the position 8234 at vanX. Transfer of vancomycin resistance was detected for the Swiss (4), Spanish, and Portuguese isolates and was associated with erythromycin resistance in all cases. Tetracycline resistance was also transferable in the Spanish strains. No virulence traits were detected.

We describe the simultaneous occurrence of a VREF strain among swine in 4 distant European countries for at least a 4-year period. Tn1546 type D has been largely described in European swine isolates, which indicates stability of this particular type among the high diversity of Tn1546 described to date (8). The finding of a group of genetically closely related strains, which include both VSEF and VREF isolates and which harbor a particular purK allele previously associated with E. faecium swine strains, might mirror wide dissemination of a host-specific clone more prone than others to acquire and spread different antimicrobial resistance, as reported for human clinical E. faecium isolates (9). Since enterococci from swine are able to colonize in the human gut (5,7) and isolates harboring purK-9 can be recovered from hospitalized patients with severe infections (10), specific swine enterococcal strains might represent a risk for antimicrobial resistance spread in the clinical setting. Further analyses need to be performed to understand the role of international animal movements, animal feed, and colonized farmers in the spread of this particular strain and to assess whether this clone shows an increased fitness in the porcine intestine when compared to other E. faecium strains.

C. Novais was supported by a fellowship from Fundacao para a Ciencia e Tecnologia (SFRH/BD/3372/2000).

References

(1.) Phillips I, Casewell M, Cox T, Groot B, Friis C, Jones R, et al. Does the use of antibiotics in food animals pose a risk to human health? A critical review of published data. J Antimicrob Chemother. 2004;53(Suppl 1):28-52.

(2.) Teuber M. Veterinary use and antibiotic resistance. Curr Opin Microbiol. 2001;4:493-9.

(3.) Johnsen PJ, Osterhus JI, Sletvold H, Sorum M, Kruse H, Nielsen K, et al. Persistence of animal and human glycopeptide-resistant enterococci on two Norwegian poultry farms formerly exposed to avoparcin is associated with a widespread plasmid-mediated vanA element within a polyclonal Enterococcus faecium population. Appl Environ Microbiol. 2005;71:159-68.

(4.) Boerlin P, Wissing A, Aarestrup F, Frey J, Nicolet J. Antimicrobial growth promoter ban and resistance to macrolides and vancomycin in enterococci from pigs. J Clin Microbiol. 2001;39:4193-5.

(5.) Hammerum A, Lester C, Neimann J, Porsbo L, Olsen K, Jensen L, et al. A vancomycin-resistant Enterococcus faecium isolate from a Danish healthy volunteer, detected 7 years after the ban of avoparcin, is possibly related to pig isolates. J Antimicrob Chemother. 2004;53(Suppl 3):5474-9.

(6.) Novais C, Coque TM, Sousa JC, Baquero F, Peixe L. Local genetic patterns within a vancomycin-resistant Enterococcus faecalis clone isolated in three hospitals in Portugal. Antimicrob Agents Chemother. 2004;48:3613-7.

(7.) Homan WL, Tribe D, Poznanski S, Li M, Hogg G. Spalburg E, et al. Multilocus sequence typing scheme for Enterococcus faecium. J Clin Microbiol. 2002;40: 1963-71.

(8.) Woodford N, Adebiy AMA, Palepou MFI, Cookson B. Diversity of VanA glycopeptide resistance elements in enterococci from humans and animals. Antimicrob Agents Chemother. 1998;42:502-8.

(9.) Willems RJL, Top J, van Santen M, Robinson A, Coque TM, Baquero F, et al. Global spread of vancomycin-resistant Enterococcus faecium from distinct nosocomial genetic complex. Emerg Infect Dis. 2005;11:821-8.

(10.) Coque TM, Willems RJ, Fortun J, Top J, Diz S, Canton R, et al. Population structure of Enterococcus faecium causing bacteremia in a Spanish university hospital: setting the scene for a future increase in vancomycin resistance? Antimicrob Agents Chemother. 2005;49:2693-700.

Carla Novais, * Teresa M. Coque, ([dagger]) Patrick Boerlin, ([double dagger]) Inmaculada Herrero, ([section]) Miguel A. Moreno, ([section]) Lucas Dominguez, ([section]) and Luisa Peixe *

* REQUIMTE at Universidade do Porto, Porto, Portugal; ([dagger]) Hospital Universitario Ram6n y Cajal, Madrid, Spain; ([double dagger]) University of Guelph, Ontario, Canada; and ([section]) Universidad Complutense de Madrid, Madrid, Spain

Address for correspondence: Luisa Peixe, REQUIMTE, Laboratario de Microbiologia. Faculdade de Farmacia,Universidade do Porto, Rua Anibal Cunha, 4050-030 Porto, Portugal; fax: [phone]; email: [e-mail]

Table. Features of vancomycin-resistant Enterococcus faecium swine
isolates from European countries **

                                     Antimicrobial drug
                                  susceptibility (mg/L) **
                                         ([dagger])

Isolate             PFGE   purK     VC       TC    AMP   TET

Portugal
    ([section])
  7S4                 A      9    >256 *   256 *    <2    64
  8S1                A3     ND    >256 *   256 *    <2    32
  35S2               A4     ND    >256 *    256     <2    64

Spain
    ([paragraph])
  S1                 A1      9    >256 *   128 *    <2   >64
  S2                 A2     ND    >256 *   128 *    <2    64
  S8                 A1'    ND    >256 *   128 *    <2   >64

Switzerland (#)
  4D                  A      9    >256 *    64 *    <2    64

                        Antimicrobial drug susceptibility
                              (mg/L) ** ([dagger])

Isolate               ER      CP    CL    GM      KM       SM

Portugal
    ([section])
  7S4                32 *    <0,5    8   <256    1,000    <256
  8S1               >32 *    <0,5   16   <256    <256     <256
  35S2               >32     <0,5    8   <256    <256     <256

Spain
    ([paragraph])
  S1                >32 *   <0,25    8   <256   >2,000   >2,000
  S2                >32 *   <0,25    8   <256    <256     <256
  S8                >32 *   <0,25    8   <256   >2,000    2,000

Switzerland (#)
  4D                >32 *   <0,25   <4   <256    <256     <256

                     Antimicrobial
                         drug
                    susceptibility
                       (mg/L) **
                      ([dagger])

                                      Resistance      Mating
                                         genes       ([double-
Isolate             LIN    DA   NIT   ([dagger])     dagger])

Portugal
    ([section])
  7S4                2     1     64   vanA *       [10.sup.-4]
                                      erm B *
  8S1                2     1     64   vanA *       [10.sup.-8]
                                      erm B *
  35S2               2    0.5    32   vanA              ND
                                      erm(B)
Spain
    ([paragraph])
  S1                 2     1     64   vanA *       [10.sup.-5]
                                      erm(B) *
                                      aphIII
  S2                 2     2     64   vanA  *      [10.sup.-4]
                                      erm B *
  S8                 2    0.5    64   vanA *       [10.sup.-5]
                                      erm B *
                                      aphIII
Switzerland (#)
  4D                 2     4     64   vanA *       [10.sup.-5]
                                      erm(B) *

** PFGE, pulsed-field gel electrophoresis; VC, vancomycin; TC,
teicoplanin; AMP, ampicillin; TET, tetracycline; ER, erythromycin;
CP, ciprofloxacin; CL, chloramphenicol; GM, high level of resistance
to gentamicin; KM, high-level resistance to kanamycin; SM, high-level
resistance to streptomycin; LIN, linezolid, DA, daptomycin; NIT,
nitrofurantoin, ND, not done. All isolates were TN 1546 type D.

([dagger]) Antimicrobial resistance or resistance genes detected in
transconjugants appear underlined.

([double dagger]) Conjugation frequency is expressed as
transconjugants per donors.

([section]) First 2 isolates were collected in 1997; third in 1998.

([paragraph]) S1 was isolated in 1998; S2, 1999; and S8, 2000.

(#) Isolate was collected in 1999.

Note: Antimicrobial resistance or resistance genes detected in
transconjugants is indicated with *.