from 1997 to 2000 , fecal samples were obtained from woodmice ( apodemus sylvaticus ) and bank voles ( clethrionomys glareolus ) in woodland and on small islands in a lake near farmland in the wirral , northwest england . we collected 177 fecal samples from bank voles and 13 from woodmice captured on the islands and 211 from bank voles and 118 from woodmice captured in the woodland . fecal samples from badgers ( meles meles ) were obtained near their burrows in a 10x10-km area in cheshire , england . the 165 samples from the badgers were collected in 2000 . isolation was attempted by using both enterococcosel agar and broth , neither incorporating vancomycin ( bbl microbiology systems , cockeysville , md ) . on thawing , fecal samples were emulsified in an equal volume of sterile saline , and 10 l was spread onto enterococcosel agar and incubated at 37c for 48 hours . in addition , approximately 500 l of each sample was added into enterococcosel broth and incubated at 37c for 48 hours , after which samples were subcultured onto blood agar plates with a vancomycin disk ( 5 g ) on the main inoculum . suspect colonies ( esculin positive or from around the vancomycin disk ) were further tested to confirm their identity as enterococci ( streptococcal grouping kit : oxoid , basingstoke , uk ; api 20 strep : biomerieux , basingstoke , uk ) . provisionally identified vre were tested for mics of vancomycin and teicoplanin ( e - test , ab biodisk , solna , sweden ) . whole bacterial cell dna was extracted by suspending 1 l of pure culture in 250 l of chelex-100 ( bio - rad laboratories , hercules , ca ) in water slurry ( 5% wt / vol ) and boiling for 10 minutes . cell debris was removed by centrifugation ( 13,000 rpm for 15 minutes ) and dna extract stored at -70c . genetic identification of enterococci to species level and detection of vana , vanb , vanc1 , or vanc2/c3 and vand ligase genes were performed by using a multiplex polymerase chain reaction amplification ( 7 ) . this amplification detects both structural genes encoding d - alanine  d - alanine ligases and glycopeptide resistance genes . as controls , strains of e. faecalis ( nctc 775 ) and e. faecium ( nctc 12202 ) and previously characterized strains of e. faecium encoding vana and vanb were included . the primers used ( 7,9 ) were vanaa1 : 5-ggg aaa acg aca att gc-3  ; vanaa2 : 5-gta caa tgc ggc cgt ta-3  ; vanbb1 : 5-atg gga agc cga tag tc-3  ; vanbb2 : 5-gat ttc gtt cct cga cc-3  ; vanc1/c1 : 5-ggt atc aag gaa acc tc-3  ; vanc1/c2 : 5-ctt ccg cca tca tag ct-3  ; vanc23/c1 : 5-ctc cta cga ttc tct tg-3  ; vanc23/c2 : 5-cga gca aga cct tta ag-3  ; vandd1 : 5-taa ggc gct tgc ata tac cg-3  ; vandd2 : 5-tgc agc caa gta tcc ggt aa-3  ; ddl faecalis - e1 : 5-atc aag tac agt tag tctt-3  ; ddl faecalis - e2 : 5-acg att caa agc taa ctg-3  ; ddl faecium - f1 : 5-gca agg ctt ctt aga ga-3  ; and ddl faecium - f2 : 5-cat cgt gta agc taa cttc-3  . amplicons were separated by electrophoresis through agarose ( 1% wt / vol ) gels ( sigma - aldrich , poole , uk ) . pulsed - field gel electrophoresis ( pfge ) of macrorestricted chromosomal dna was performed to establish the genetic relatedness of the vre isolates ( 10 ) . bacteria were harvested from solid media and embedded in lysozyme ( 25 mg / ml)agarose blocks . released nucleases were neutralized with 25 mg / ml proteinase k ( sigma - aldrich ) at 50c overnight . chromosomal dna was digested with smal ( 40 u / block ) ; gel slices were embedded in 1% ( wt / vol ) pfge agarose gels ( bio - rad laboratories ) and separated by electrophoresis on a chef 3d system ( bio - rad laboratories ) in x 0.5 tris - borate - edta buffer . electrophoresis conditions were an initial switch time of 1 second with a linear increase to 20 seconds after 20 hours at a buffer temperature of 14c , 6 v / cm with a pulsing angle of 120c ( 10 ) . were detected in feces from 10 ( 76.9% ) of 13 woodmice and 135 ( 76.3% ) of 177 bank voles on the islands and 110 ( 93.2% ) of 118 woodmice and 185 ( 87.7% ) of 211 bank voles from the woodland site . vre were isolated from 6 ( 5.1% ) of 118 woodmice samples from the woodland area only . the remaining vre ( from a woodmouse ) was obtained from broth culture , as were each of the other vre also isolated by direct culture . each of the vres had vancomycin mics > 64 mg / l and teicoplanin mics 0.756.0 mg / l ( table ) . each vre had the vana gene but none of the other resistance genes , and each was confirmed as e. faecium by polymerase chain reaction ( figure ) . pfge analysis showed that six of the isolates had band patterns differing by > 10 bands , but two woodmice isolates were closely related , differing by only two bands . all isolates in this table are enterococcus faecium , vana ge ne ( polymerase chain reaction ) . abbreviations used : wm , woodmouse , b , badger .  genetically similar by pulsed - field gel electrophoresis of macrorestricted chromosomal dna ( differing by two bands ) . polymerase chain reaction analysis of vancomycin - resistant enterococci isolates for glycopeptide resistance genotypes and species identification . this first study of enterococcal and specifically vre excretion by wild mammals showed that enterococci appear to be part of the normal flora of most badgers ( 74.5% ) , woodmice ( 92% ) , and bank voles ( 82% ) , like many other animal species ( 1 ) . we also found vre in a small number of woodmice ( 5.1% ) and badgers ( 1.2% ) . each of our isolates was e. faecium , and each encoded the vana but not vanb , vanc , or vand genes . although all had mics for vancomycin > 64 mg / l , only one had an mic for teicoplanin of 6 mg / l , which does not correspond to the generally accepted vana phenotype . however , an isolate of e. raffinosus has been described with a vanb phenotype yet vana genotype ( 11 ) . on pfge of macrorestricted chromosomal dna , only two isolates ( both from woodmice ) were genotypically related . the two related isolates were from different woodmice ( tagged on capture with transponders ) , trapped 6 months apart . how the mammals acquired vre and whether they are long - term carriers are less clear . the woodmice and bank voles may have been exposed in the woodland to either avoparcin ( a glycopeptide antibiotic related to vancomycin ) or fecal material from farm animals ; however , avoparcin had not been used for any livestock raised in proximity to the sites , and the samples ( including the badger samples ) were taken after the use of avoparcin as a growth promoter had been banned . none of the bank voles were excreting vre , in spite of the fact that they can be reservoirs for enterococci . in general , bank voles tend to be herbivorous and have a limited territory , beyond which they rarely stray . woodmice and badgers are omnivorous and will travel distances in search of food and territory ( 12 ) . in conclusion , we demonstrated that woodmice and badgers can excrete vre in their feces and may be an unexpected reservoir for such bacteria . however , bank voles occupying the same habitat as the woodmice did not excrete vre . how long such reservoirs may persist and if the bacteria can be transmitted to other animals , including humans , remain to be determined .
a survey of fecal samples found enterococcal excretion in 82% of 388 bank voles ( clethrionomys glareolus ) , 92% of 131 woodmice ( apodemus sylvaticus ) , and 75% of 165 badgers ( meles meles ) . 
 vancomycin - resistant enterococci , all enterococcus faecium of vana genotype , were excreted by 4.6% of the woodmice and 1.2% of the badgers , but by none of the bank voles .