a suspension ( 0.7 ml ) of avoparcin ( roche , sydney , australia ) in h2o ( 100 g / ml ) was centrifuged in a 1.5-ml eppendorf tube for 6 min and the supernatant , after being shaken with 1 volume of phenol : chloroform : isoamyl alcohol ( 25:24:1 ) , was centrifuged at 16,000 x g for 3 min . the aqueous phase was subjected to two additional phenol - chloroform extractions . the nucleic acid in the pooled aqueous fractions was precipitated with ethanol ; the pellet was recovered by centrifugation and further purified by using a geneclean spin kit ( bio101 ) and resuspended in 100 l of double - distilled h2o . the dna concentration was measured with a fluorometer ( model tko100 , hoefer scientific instruments , san francisco , ca ) . primers 16s 440f and 16s 1491r ( table ) were designed to amplify partial 16s rdna sequences . the polymerase chain reaction ( ( pcr ) contained 2 mm mgcl2 , 0.16 mm dntp , 0.4 l of each primer , taq polymerase ( 1 u ) , 315 ng template , and 5% dimethyl sulfoxide ( dmso ) . pcr was done in a minicycler ( mj research , waltham , ma ) by using the following program : 96c , 3 min ; 96c , 30 s ; 60c , 45 s ; 72c , 1 min 30 s ; 35 cycles ; and 72c , 10 min . different combinations of pcr primers ( 9 ) were used to amplify the entire van cluster ( table ) . pcr products were cloned by using vector pcr 2.1-topo ( invitrogen , burling , ontario , canada ) according to the manufacturer s instructions , and the insertion size was confirmed by a second pcr . plasmid dna was extracted by using the concert rapid plasmid miniprep system ( invitrogen ) . cycle sequence reactions were carried out with a bigdye terminator dna sequencing kit ( applied biosystems , foster city , ca ) with plasmid dna templates . the cycle sequence program was as follows : 96c , 1 min ; 96c , 30 s ; 5060c ( dependent on different primers and fragments ) , 15 s ; 60c , 4 min , for 25 cycles . excess oligos and dyes were removed by using centrisep spin columns ( princeton separations , aldelphia , nj ) . reaction products were sequenced by the nucleic acid and protein service , university of british columbia , using an abi prism 377 sequencer . sequences were analyzed by using the standard nucleotide - nucleotide blast program ( national center for biotechnology information , bethesda , md ) . and comparisons were carried out by using clustal w ( european bioinformatics institute , cambridge , uk ) . a suspension ( 0.7 ml ) of avoparcin ( roche , sydney , australia ) in h2o ( 100 g / ml ) was centrifuged in a 1.5-ml eppendorf tube for 6 min and the supernatant , after being shaken with 1 volume of phenol : chloroform : isoamyl alcohol ( 25:24:1 ) , was centrifuged at 16,000 x g for 3 min . the aqueous phase was subjected to two additional phenol - chloroform extractions . the nucleic acid in the pooled aqueous fractions was precipitated with ethanol ; the pellet was recovered by centrifugation and further purified by using a geneclean spin kit ( bio101 ) and resuspended in 100 l of double - distilled h2o . the dna concentration was measured with a fluorometer ( model tko100 , hoefer scientific instruments , san francisco , ca ) . primers 16s 440f and 16s 1491r ( table ) were designed to amplify partial 16s rdna sequences . the polymerase chain reaction ( ( pcr ) contained 2 mm mgcl2 , 0.16 mm dntp , 0.4 l of each primer , taq polymerase ( 1 u ) , 315 ng template , and 5% dimethyl sulfoxide ( dmso ) . pcr was done in a minicycler ( mj research , waltham , ma ) by using the following program : 96c , 3 min ; 96c , 30 s ; 60c , 45 s ; 72c , 1 min 30 s ; 35 cycles ; and 72c , 10 min . different combinations of pcr primers ( 9 ) were used to amplify the entire van cluster ( table ) . pcr products were cloned by using vector pcr 2.1-topo ( invitrogen , burling , ontario , canada ) according to the manufacturer s instructions , and the insertion size was confirmed by a second pcr . plasmid dna was extracted by using the concert rapid plasmid miniprep system ( invitrogen ) . cycle sequence reactions were carried out with a bigdye terminator dna sequencing kit ( applied biosystems , foster city , ca ) with plasmid dna templates . the cycle sequence program was as follows : 96c , 1 min ; 96c , 30 s ; 5060c ( dependent on different primers and fragments ) , 15 s ; 60c , 4 min , for 25 cycles . excess oligos and dyes were removed by using centrisep spin columns ( princeton separations , aldelphia , nj ) . reaction products were sequenced by the nucleic acid and protein service , university of british columbia , using an abi prism 377 sequencer . sequences were analyzed by using the standard nucleotide - nucleotide blast program ( national center for biotechnology information , bethesda , md ) . and comparisons were carried out by using clustal w ( european bioinformatics institute , cambridge , uk ) . direct extraction of avoparcin powder with phenol / chloroform / isoamyl alcohol provided substantial amounts of dna ( 30.5 g / g of avoparcin ) ( figure 3a ) . pcr amplification of the dna with oligonucleotide primers specific for a region of streptomycete 16s rrna gave a single amplicon ( figure 3b ) , which was sequenced and shown to be 16s closely related to that of amycolatopsis coloradensis , the producer of avoparcin . figure 3b shows similarities between the 16s rrna of species that produce glycopeptide antimicrobial agents . a , molecular mass of total dna extracted from animal feed grade avoparcin . m : 1 kb plus dna ladder ( invitrogen , burlington , ontario , canada ) . b , polymerase chain reaction amplification of partial 16s rdna ( 1051 bp ) with primers 16s 440f and 16s 1491r . lane 2 : dna of the avoparcin producer amycolatopsis coloradensis nrrl 3218 . to examine for the presence of genes involved in glycopeptide resistance from the antimicrobial agent  derived dna , we used the dna primers described by marshall et al . the amplicons ( figure 4 ) were cloned , sequenced , and assembled , indicating a van - like cluster closely related to that found in a. orientalis and streptomyces toyocaensis . polymerase chain reaction amplification of the partial vanhax clusters from dna extracted from animal feed  grade avoparcin and the antibiotic producer amycolatopsis coloradensis nrrl 3218 , and genes vanh , ddln , and vanx from dna extracted from animal feed grade avoparcin . lanes 12 : the partial van cluster ( 2.32.4 kb ) amplified with primers vanh1 and vanx4 ; lane 1 : dna extracted from animal feed grade avoparcin ; lane 2 : dna of the avoparcin producer a. coloradensis nrrl 3218 . the genes encoded three putative proteins showing > 50% amino acid identity to the van h , a , and x proteins of vre ( figure 2 ) . all of the clusters have translational overlaps between the vana and vanx genes and their homologs , suggesting cotranslational regulation of expression . this finding clearly implies that the van cluster must be transferred and acquired in toto from any source organism . we suggest that the use of crude avoparcin preparations in animal feeds from 1975 to 1996 was the origin of the vanhax cluster in the genesis of vre ( and possibly that found in vrsa ) ( 22,23 ) . large amounts of avoparcin were used in animal feed ; in denmark , for example , total vancomycin use in 1994 amounted to 24 kg , whereas avoparcin use in animals was 24,000 kg ( 24 ) . during their entire lives , broiler chickens received 15 mg / kg and pigs 2040 mg / kg of antimicrobial agentin their feed . each pig was fed 510 g of the crude drug for its life span and , consequently , received a steady dose of dna encoding vancomycin resistance . in europe , an estimated 100 mg of antimicrobial agents are used in animal feed for the production of 1 kg of meat for human consumption . we believe that this regimen would have favored the selection and maintenance of rare bacterial transformants carrying the resistance genes . if one bears in mind that large numbers of pigs and chickens were exposed to the antimicrobial agent , the probability of gene pick - up by bacterial commensals in the animal gastrointestinal tract would be favored , and once incorporated into a gut commensal genome , further dissemination would have followed under antimicrobial selection . the finding that organization of the van cluster in contaminating dna of the feed is identical to that in vre , with overlapping reading frames typical of translational coupling of gene expression between the vana and vanx homologs ( 9 ) , reinforces this supposition . the mechanism by which a van cluster becomes functionally integrated into bacteria is not known . we propose that intestinal bacteria were the original recipients of the dna ; many of the resident strains are known to be competent for dna uptake ( 25,26 ) . however , mere uptake is not sufficient for function , and the actinomycete genes differ from vre genes in their g+c content ( approximately 65% vs. 50% ) and codon usage . given the enormous complexity of bacterial populations in the mammalian gastrointestinal tract ( 27,28 ) , we assume that a variety of intestinal species may have incorporated the resistance - encoding dna ; expression ( at low levels ) would have been rare , depending on the compatibility of the van genes with the transcription and translation system of the host . under constant antimicrobial selection pressure , translationally competent sequences nonetheless , the conversion ( evolution ) of the actinomycete genes into functional enterococcal genes likely would have required many generations of growth under constant selection , and any intermediate stages in this process are a matter of speculation ; however , once established on conjugative transposons , the genes would be readily disseminated ( 17,29 ) . a number of similar van clusters have been identified in different bacterial species , and whether these evolved independently or by divergent evolution is unknown . the finding of resistance genes in crude antimicrobial products intended to be fed to animals adds to the already strongly voiced opinion that use of antimicrobial agents in this way constitutes a serious public health concern and further emphasizes the need for prohibiting the use in animal feed of all antimicrobial agents that are employed in human therapy . this ban should include structurally or biologically related antimicrobial agents and the use of any compound with the potential to select for cross - resistance to another antimicrobial agent ( 15,30 ) . the use of avoparcin in denmark was prohibited in 1995 and in the european union in 1997 . , the united states and canada permit the use of many such products , including penicillin , tetracycline , macrolides , and sulfonamides . nonhuman applications of antimicrobial agents , such as in agriculture and aquaculture , should employ only chemically and biologically distinct classes of compounds developed specifically for that purpose . clearly such measures should be combined with a requirement for rational and prudent measures for antimicrobial use in the human population . many antimicrobial agents ( or their close structural relatives ) have been used extensively as animal - feed additives . in almost all cases , crude antimicrobial preparations are used , and thus the antimicrobial agent acts as a carrier for its cognate resistance genes . these delivery systems provide the opportunity for resistant strains of bacteria to evolve and so create an enormous gene pool for antimicrobial resistance determinants in the environment .
avoparcin , a glycopeptide antimicrobial agent related to vancomycin , has been used extensively as a growth promoter in animal feeds for more than 2 decades , and evidence has shown that such use contributed to the development of vancomycin - resistant enterococci . a cluster that includes three genes , vanh , vana , and vanx , 
 is required for high - level resistance to glycopeptides . in the vancomycin producer amycolatopsis orientalis c329.2 , 
 homologs of these genes are present , suggesting an origin for the cluster . 
 we found substantial bacterial dna contamination in animal - feed - grade avoparcin . 
 furthermore , nucleotide sequences related to the cluster vanhax are present in this dna , suggesting that the prolonged use of avoparcin in agriculture led to the uptake of glycopeptide resistance genes by animal commensal bacteria , which were subsequently transferred to humans .