Vol. 37, No. 11ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1993, P. 2379-23840066-4804/93/112379-06$02.00/0Copyright X 1993, American Society for Microbiology

Antibiotic Preparations Contain DNA: aSource of Drug Resistance Genes?

VERA WEBB AND JULIAN DAVIES*Department ofMicrobiology, University of British Columbia, Vancouver,

British Columbia, Canada V6T 1Z3

Received 11 June 1993/Returned for modification 5 August 1993/Accepted 9 September 1993

Fluorescence measurements and polymerase chain reaction amplification of streptomycete 16S ribosomalDNA sequences were used to show that a number of antibiotic preparations employed for human and animaluse are contaminated with chromosomal DNA of the antibiotic-producing organism. The DNA containsidentifiable antibiotic resistance gene sequences; the uptake of this DNA by bacteria and its functionalincorporation into bacterial replicons would lead to the generation of antibiotic resistance determinants. Wepropose that the presence of DNA encoding drug resistance in antibiotic preparations has been a factor in therapid development of multiple antibiotic resistance in bacteria.

Antibiotics, which have been used worldwide for morethan 40 years, are the foundation of modem infectiousdisease treatment and have led to the effective control ofmany common microbial infections. However, the use ofantibiotics has failed to eradicate any form of bacterialpathogen. On the contrary, the number of organisms recal-citrant to treatment has increased (8) and there are numerousreports of epidemics due to multiple-antibiotic-resistant bac-teria. This situation has arisen largely because the extensiveuse of antibiotics has been accompanied by the widespreadappearance of drug resistance genes in bacteria (27, 30). Theresistance determinants are commonly carried by plasmidswhich are present in an unlimited range of microbial genera;the determinants are often transposable and exist in bothplasmid and chromosomal locations (24). Antibiotic resis-tance mechanisms of at least seven different biochemicaltypes have been characterized (11). Mutations in chromo-somal genes for the bacterial target have played only a minorrole in this process, such as resistance to fluoroquinolones(4) and antituberculosis drugs (34).

In addition to their use in human therapy, antibiotics havebeen employed extensively as growth promotants in animalhusbandry and therapeutic agents in agriculture (as spraysfor fruit trees, for example) with substantial economic im-pacts in the industry. There is evidence that such nonthera-peutic use has contributed to the development of a largenatural reservoir of antibiotic-resistant microbes; a numberof studies have linked this reservoir to the development ofresistance in the human population (14).

Analysis of bacterial collections from the preantibiotic eraindicates that although plasmids were present in some of thestrains, they did not harbor antibiotic resistance genes (21).The conclusion is that the development of antibiotic-resis-tant microbial populations occurred after the introduction ofantibiotics into clinical use. In addition, epidemiologicalstudies have suggested that, in most cases, antibiotic resis-tance plasmids emerge in microbial populations within 5years after introduction of the antibiotic for therapeutic use(8). Since most resistance determinants are extrachromo-somal and are believed to have been inherited from a

* Corresponding author.

microbial pool, important questions are: from where andhow?A number of reports have suggested that the antibiotic

resistance genes found in human and animal isolates couldhave originated in the very industrial microbes that are usedfor production of antibiotics (3, 33). Analyses of the bio-chemical mechanisms of resistance and the molecular char-acterization of antibiotic resistance genes from actino-mycetes and clinical isolates have been consistent with thisnotion (11). However, if antibiotic-producing actinomycetes,which are ubiquitous soil microorganisms, were the sourceof many of the resistance genes, what is a plausible scenariofor contact between them and microbes that are responsiblefor disease in humans and animals?We show that a number of antibiotic preparations are

contaminated with DNA from the organism used in thefermentative production of the active secondary metabo-lites; thus, administration of antibiotics is accompanied bysmall amounts of chromosomal DNA containing resistancegenes from the producing organism. We propose that underthe simultaneous selection pressure of the antibiotic, uptakeof one or more resistance genes by unidentified members ofthe microbial population in the host would lead to antibiotic-resistant organisms being constructed by natural geneticengineering. Subsequent inter- and intraspecific genetictransfers would permit other microbes to become resistant tothe antibiotics. Our proposal provides an explanation for therapid appearance, following extensive use of antibiotic prep-arations, of resistance mechanisms directed specificallyagainst the therapeutic agents being used.

MATERIALS AND METHODS

Bacterial strains and culture conditions. Streptomyces fra-diae NRRL 2338 (22), S. nimosus M4018 (6), and S. griseusATCC 10971 were obtained from culture collections. Esch-erichia coli JM109 served as the host in the cloning ofpolymerase chain reaction (PCR) products. For preparationof chromosomal DNAs of S. fradiae and S. nimosus, cultureswere grown in YEME (19), and for S. griseus, cultures weregrown in tryptic soy broth (Difco).DNA manipulations. Chromosomal DNA was prepared

from Streptomyces spp. as described by Hopwood et al.(19). The 378-bp PCR product obtained from an extract of

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TABLE 1. Oligonucleotide primers used in PCR amplification assays.'

Name 5' primer 3' primer Product size

S16S1 5'-CTGCGAGGTTCGAAAGCTCCGG-3' 5'-CACCAGGAATTCCGATCTCCCC-3' 470S16S2 5'-GTGGGGATTAGTGGCGAACGGGTG-3' 5'-CACCAGGAATTCCGATCTCCCC-3' 577S16S3 5'-GTGGGGATTAGTGGCGAACGGGTG-3' 5'-CTCACCAAGGCGACGACGGGTAGC-3' 201ermSF 5'-GAGCTCTCGCAGAACTTCCTC-3' 5'-TCCATAGTCGCCGGTCCGCTT-3' 415otrA 5'-GAACAAGCTGAATCTGGGCATCC-3' 5'-GACGAAGACCAGCGTGGGAATG-3' 378gaph 5'-GGCCGTCACCGCGGGCGAATCG-3' 5'-GCAGAGATCACCGTGGCAGACG-3' 510

a The primer sequences shown are homologous to regions in the following genes: S16S1, 16S RNA genes of S. lividans TK21 (32) and S. coelicolor A3(2) (2);S16S2 and S16S3, 16S RNA genes of S. lividans TK21 (32), S. coelicolor A3(2) (2), and S. gnseus (23); ermSF, macrolide-lincosamide-streptogramin amethylation gene, ermSF, of S. fradiae NRRL 2338 (22); otrA, oxytetracycline resistance gene, otrA, of S. rinmosus (13); gaph, streptomycin-3'-phosphotrans-ferase gene, aphE, of S. griseus (17).

terramycin was eluted with Qiaex (Qiagen); the purifiedproduct was cloned into pGEM-T (Promega) and sequencedwith an automated sequencer (Applied Biosystems Inc.).Sequencing employed double-stranded DNA templates andfluorescence-labeled dideoxynucleotides in accordance withApplied Biosystems protocols.

Detection of DNA with nucleic acid-specific dyes. Equalvolumes of DNA solution or antibiotic preparation andYO-PRO-1 (benzoxazolium-4-quinolinium dye [Y-3603];Molecular Probes) diluted in 10 mM Tris (pH 7.4)-2 mMNaCl-1 mM EDTA (TNE) in accordance with the manufac-turer's specifications were mixed, and the resulting fluores-cence was measured with excitation and emission filterscentered at 485 and 530 nm, respectively, in a Perkin-ElmerLS50 luminescence spectrometer. Blanks were run withTNE alone.PCR assays and primers. The primers used in this study

(Table 1) were synthesized with an oligonucleotide synthe-sizer (Applied Biosystems).PCR assays were carried out in 50-pl reaction mixtures

containing 20 mM Tris (pH 8.3 at 22°C), 1.5 mM MgCl2, 25mM KCl, 0.05% Tween 20, 100 ,g of gelatin per ml, 10%glycerol, 5% formamide, 1 mM deoxyribonucleoside triphos-phates (0.25 mM each), and 2 ,uM primers (1 ,uM 5' primer,1 ,uM 3' primer). Five to fifteen microliters of DNA solutionor antibiotic solution was added to the reaction. PCR wasperformed with an automated thermal cycler (EppendorfMicroCycler E5465). Taq polymerase (1.25 U) was addedafter initial denaturation at 95°C for 2 min. The followingtemperature profile was used: 25 cycles of denaturation(95°C for 15 s), annealing (55°C for 30 s), and extension (72°Cfor 90 s); 1.25 U of Taq polymerase was added, and thetemperature profile was repeated for a further 25 cycles.Finally, 1.25 U of Taq polymerase was added and thetemperature profile was repeated for a further 12 cycles(total, 62 cycles). Reactions were stopped by temperaturereduction to 15 or -20°C. A 12-,ul volume of each reactionmixture was loaded onto a 1.5% agarose gel, electrophore-sed, and visualized by staining with ethidium bromide. Allpreparations were carried out in a laminar-flow hood withfiltered solutions. Primers were synthesized by the NucleicAcid and Protein Synthesis Unit of the Biotechnology Lab-oratory, University of British Columbia.Premix extraction. Premix extract was prepared as fol-

lows. A 700-mg premix sample was extracted in 6 ml of 50/10buffer (0.05 M Tris, 0.01 M EDTA [pH 8]) and centrifuged at5,900 x g for 5 min. The supematant liquid was extractedtwice with phenol-chloroform, ethanol precipitated, andresuspended in 0.6 ml of 50/10 buffer. Three volumes ofBio-Rad Prep-A-Gene binding buffer were added, and the

diluted solution was absorbed onto a 30-,ul volume of Prep-A-Gene matrix in 0.6-ml aliquots as follows: the mixture wasshaken at 250 rpm and 25°C for 10 min and centrifuged, andthe supernatant liquid was discarded. After absorption, thematrix was washed three times with 0.25 ml of 4 M sodiumperchlorate and four times with Prep-A-Gene wash buffer.The matrix was eluted in a final volume of 70 ,ul.Chromatography of streptomycin sulfate. A solution of

streptomycin sulfate (Sigma) (300 mg) in 2 ml of TE (10 mMTris [pH 8], 1 mM EDTA) was divided into two, andpancreatic DNase at a final concentration of 4 U/ml wasadded to one half. The two samples were allowed to stand at4°C for a minimum of 24 h and then loaded onto a column (5ml) of the cationic resin AGSOWX8 (Bio-Rad) and elutedwith water; fractions (1 ml) were collected and assayed forantibiotic activity, optical density at 260 nm, and DNaseactivity and subjected to PCR amplification as describedabove. Chromatography was carried out in a laminar-flowhood.

RESULTS

Detection of DNA with nucleic acid-specific dyes. By usingfluorescent enhancement with the dye YO-PRO-1, a numberof antibiotics were tested for the presence of contaminatingnucleic acid. This dye has a high binding specificity for DNAand RNA and can be used to detect nucleic acids at thenanogram level (15). The results indicated that some antibi-otic preparations (reagent or animal feed grade) were con-taminated with nucleic acids in excess of 65 ,ug g-1 (Fig. 1).In other experiments, we found high levels of nucleic acidcontamination (>10 jig g-1) in clinical-grade preparations oferythromycin, vancomycin, and tetracycline. RNase diges-tion of the preparations indicated that this was principallyDNA (results not shown). Nucleic acid was detected inantibiotic preparations that were produced by both actino-mycetes and fungi. Semisynthetic antibiotic preparationswere contaminated at much lower levels than their naturallyoccurring precursors, and totally synthetic compounds, suchas chloramphenicol and isoniazid, were nucleic acid free(results not shown).

Analysis of antibiotic solutions by PCR. As a preliminaryexamination of the nature of nucleic acid found in antibioticpreparations, DNA isolated from an antibiotic preparation(streptomycin) was 5' end labeled with T4 polynucleotidekinase; electrophoresis of the labeled material showed frag-ments that ranged in molecular size from 0.5 to 10 kb (resultsnot shown); this result indicates that the nucleic acid wasprobably double stranded. To identify rigorously the natureof the nucleic acid contamination in antibiotic preparations,

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3 s

3 -

I.-~~~~~~~~~~~~~~~~

7.

reagent veterinary

FIG. 1. Detection of DNA content in antibiotic preparations. (A)

Standard curve of S. griseus DNA. (B) Histogram of corrected

fluorescence values of antibiotic preparations. (The values were

corrected by subtracting the value of a no-dye control.) The num-

bers above the bars are micrograms of DNA per gram of antibiotic

preparation. The following antibiotics were tested: kan, kanamycin;

siso, sisomicin. The following were animal or fish feed grade

samples: asp, aureomycin, sulfonamide, and penicillin; bac, baci-

tracin; lin, lincomycin; tin, terramycin; ty, tylosin.

we used PCR to amplify and identify specific DNA se-

quences. We screened first for the presence of 16S ribosomal

DNA (rDNA) by using primers corresponding to known

streptomycete sequences (Table 1). As shown in Fig. 2, 16S

rDNA sequences could be detected by direct PCR analysis

of a variety of antibiotic preparations; however, in some

cases the contaminating DNA was isolated by means of

solvent extraction, cation-exchange chromatography, or af-

finity bead purification prior to PCR analysis. A summary of

the results from the PCR amplification studies with a diverse

FIG. 2. PCR amplification of antibiotic samples with S16S2primers. Lanes contained 12 pl of a 50-pl assay reaction in which thefollowing were used as substrates: lane +, S. nmosus chromosomalDNA-positive control; lane C, cefoxitin; lane K, kanamycin; lane V,vancomycin; lane A, adriamycin; lane T, tobramycin; lane -,

no-DNA control. Lanes M contained 1 ,ug of a 1-kb ladder (BethesdaResearch Laboratories).

TABLE 2. Direct DNA amplification from antibiotic preparationswith rDNA and resistance gene primersa

Antibiotic Primer used(grade[s])b 16S rRNA Resistance

Tylosm (A) +Erythromycin (R, H) + NDStreptomycin (R) + +Tobramycin (R) + NDParomomycin (R) + NDOxytetracycline (A, R) + +Vancomycin (H) + NDCefoxitin (R) + ND

a For 16S rDNA amplification, the S16S1 and S16S2 primer sets were usedwith tylosin, the S16S2 and S16S3 primer sets were used with oxytetracyclineand erythromycin, and all other compounds were assayed with the S16Sprimer set. For detection of antibiotic resistance genes, the following primerswere used: tylosin, ermSF; streptomycin, gaph; oxytetracycline, otrA. Allcompounds were tested at least twice in independent assays.

b A, animal feed; R, reagent; H, human.c ND, not done.

group of antibiotics is presented in Table 2. The experimentswere reproducible, and preparations of the same antibioticmanufactured by different pharmaceutical companies werepositive for the same 16S rDNA sequences.As confirmation of the presence of chromosomal DNA

sequences from the producing organisms in antibiotic prep-arations, PCR primers corresponding to known resistancegene sequences were employed (Table 1). A 378-bp fragmentof the oxytetracycline resistance gene (otrA) of S. nmosuswas amplified from reagent grade and animal feed prepara-tions. The assay results for the animal feed preparation areshown in Fig. 3. The 378-bp PCR amplification productisolated from samples of oxytetracycline was subsequentlycloned into pGEM-T (Promega); nucleotide sequence deter-

Ml 2 34 5

378...

FIG. 3. PCR amplification assay of extracts of a feed gradeantibiotic premix with otrA primers. Assays were performed asdescribed in the legend to Fig. 2. The lanes contained 12 ,ul of a 50-,ulreaction in which the following were employed as substrates in thePCR assay: lane 1, 1.15 pg of S. rimosus DNA; lane 2, 10 ,ul of aTylan 10 (tylosin) extract; lane 3, 10 ,ul of a TmllO (oxytetracycline)extract; lane 4, 10 pl of Terramycin Aqua (oxytetracycline); lane 5,buffer control. Lane M contained 1 pg of a 1-kb ladder (BethesdaResearch Laboratories). The position of a 378-bp PCR amplificationproduct is shown.

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FIG. 4. PCR amplification of chromatographic fractions of a

streptomycin preparation following DNase I treatment. The S16S2primer set was employed for the PCR assay. The lanes contained 12pl of a reaction mixture with the column fractions indicated. Lane +contained 12 jul of the PCR assay that contained 2 pg of S. gm'seusDNA. Lanes M contained 0.5 pg of a 1-kb ladder (BethesdaResearch Laboratories). Lane - was a no-DNA control.

mination of the fragment indicated better than 95% identitywith the published otrA sequence (6) (results not shown).Using primers to amplify part of the aminoglycoside phos-photransferase resistance gene (aph3") of S. griseus (17)permitted detection of a 750-bp fragment (not the expected510-bp product; Table 1) from an S. griseus control and a

streptomycin sample. Nucleotide sequence determination ofthe fragment indicated 60% identity with the published aph3"sequence.Removal of DNA in antibiotic preparations by DNase treat-

ment. To confirm that the template for the PCR product waspresent in the antibiotic preparation, one half of a sample ofstreptomycin sulfate was treated with DNase I. Both treatedand untreated samples were fractionated by ion-exchangechromatography as described in Materials and Methods, andthe fractions were analyzed by PCR with the S16S2 primerset. DNase I treatment eliminated the DNA contaminationfrom the streptomycin preparation (Fig. 4). The columnfractions were tested for the presence of DNase I andshowed no activity (results not shown).

DISCUSSIONWe have shown, by fluorescence spectroscopy and PCR

amplification, that a number of antibiotic preparations arecontaminated with significant amounts of DNA from theproducing organism. The fluorescence tests, although ex-tremely sensitive, do not identify the source of the nucleicacid, and they neither discriminate between DNA and RNAnor indicate whether it is single or double stranded, nicked,or even functional. Chakrabarty et al. (7) examined a numberof antibiotic preparations and detected DNA contaminationby chemical tests but did not identify the nature or thesource of the nucleic acid.rDNA sequences could not be detected in all cases (e.g.,

kanamycin), even in samples whose fluorescence analysisindicated the presence of considerable amounts of contami-nating nucleic acid. In all probability, the appropriate primersequences were not used. An amplification product was

obtained with primer set S16S2 (Table 1) from the chromo-somal DNA control of the laboratory strain of S. nmosus butnot from the DNA present in oxytetracycline preparations;however, an amplification product was seen in both caseswith primer set S16S3. There is a paucity of available rRNAsequence information about actinomycetes. In addition,there is considerable species variability among the rRNAsequences of actinomycetes (31). It is also possible thatcontaminating DNA came from another source, for example,actinophage released during growth of the producing strep-tomycete.The demonstration that antibiotic preparations contain

DNA encoding known antibiotic resistance genes is, webelieve, of considerable significance. For example, the otrAgene is closely related to tetM and tetO, which are widelydisseminated in both gram-positive and gram-negative clini-cal isolates. Since streptomycete 16S rDNA gene sequenceswere detected in a vancomycin preparation (Fig. 2 and Table2),DNA encoding an associated resistance gene(s) may bepresent in the drug. The biochemical mechanism of resis-tance to vancomycin in clinical isolates involves formationof a novel cell wall component refractory to vancomycininhibition (1); it is not known whether the producing strep-tomycete possesses a biochemically similar resistance mech-anism.We conclude that since the 1950s (9) microbes are likely to

have been exposed to DNA encoding resistance to many ofthe antibiotics being used to eliminate them. Presumably thisDNA comes from the antibiotic-producing organism duringfermentation. Our results suggest that the DNA contamina-tion level in some antibiotic preparations is at least 1 part in107. Thus, a therapeutic dose may contain a considerablenumber of copies of genes that encode resistance to theantibiotic.

If DNA contamination of antibiotic preparations repre-sents a source for the development of clinically relevantresistance genes, a first and key step in the generation ofantibiotic-resistant bacteria is DNA transformation of thebacteria in the environment. It may not be possible toidentify the initial recipient species; for example, gut bacte-ria consist of a large and diverse number of species. Inaddition, nonliving cells may be implicated in gene transfer(18). It is not known whether actinomycete DNA can betaken up by bacterial species in the gastrointestinal tract orwhether the DNA can survive in such an environment;however, ingested DNA can be detected in animal excre-ment (20). Transfer of resistance genes between unrelatedbacterial genera occurs frequently in nature, although theactual mechanisms and the donors and recipients cannot beidentified (5, 26). In all probability, pickup, integration, andexpression of a resistance gene occurs extremely rarely andthe event may be undetectable under laboratory conditions.For example, Tetr-encoding genes are widespread in clinicalisolates, but many of the apparent transfer events cannot bereproduced in the laboratory. One single gene incorporationwould be sufficient to elicit a microbial population refractoryto an antibiotic, since the event is likely to be followed by aseries of transfers involving different microbial genera and avariety of gene transfer mechanisms.

Coadministration of antibiotics and DNA could be ofconsequence, since the antibiotics may, indeed, play a roleas effectors of DNA survival and uptake. Some antibiotics(aminoglycosides, for example) bind tightly to DNA, whichmay protect the nucleic acid from the action of nucleases oreven enhance DNA uptake into bacteria. Other antibioticscould promote the entry of contaminating DNA by perme-

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abilizing or weakening the bacterial cell surface. In addition,the activity of antibiotics as agents active in promotingbacterial gene transfer has been noted (26).There are other aspects of DNA contamination in antibi-

otic preparations. Might it be possible to isolate an entirebiosynthetic gene cluster (25) from an antibiotic? Mutatedgenes coming from industrial production (improved) strainscould be isolated by amplifying DNA obtained from a vialpurchased at the local pharmacy and used to pirate a highlyproductive industrial strain; limited DNA libraries of strep-tomycete sequences can be prepared directly from antibioticpreparation extracts (unpublished results). Alternatively, theidentification and confirmation of a proprietary source or-ganism could be accomplished by direct DNA fingerprintingof the antibiotic product.The amount of DNA contamination that can be present in

protein pharmaceuticals produced by recombinant organ-isms is strictly regulated (less than 10 pg per dose); however,the regulatory requirements for antibiotic purity do notinclude contamination with nucleic acids. Given the poten-tial consequences of DNA contamination, newly introducedpharmaceuticals may have to be monitored for the presenceof DNA which encodes potential resistance determinants.Nevertheless, such a requirement will not change the situa-tion for antibiotics that have been in use for some time andfor which a substantial resistance pool already exists innature. Improved quality control measures, such as inclu-sion of a DNA-removing step (nuclease treatment or chro-matography) during antibiotic processing might be one wayto eliminate the problem of contamination. We found thattreatment of streptomycin with DNase led to elimination ofthe PCR product from the S16S2 primer set (Fig. 4).These studies do not exclude the possibility that there are

other sources of antibiotic resistance determinants leading tothe development of clinically significant resistance (12).There is a large pool of antibiotic resistance genes in nature.Many actinomycetes contain resistance genes that are notassociated with antibiotic production; often these genes arecryptic-that is, not associated with a resistance phenotype(28). For example, chloramphenicol resistance in clinicalisolates is due mainly to the presence of chloramphenicolacetyltransferase, but the gene is not found in the producingorganism, S. venezuelae (29). Since a number of otheractinomycetes carry a gene for chloramphenicol resistance,the determinant may have been derived from DNA contam-ination in a preparation of another antibiotic. The presenceof more than one drug resistance gene in the same species ofactinomycete could lead to multiple resistance. A similarargument may apply to resistance to 1-lactam antibiotics,which are produced largely (but not exclusively) by fungi. AP-lactamase gene has been shown to be part of the biosyn-thetic gene cluster for the antibiotic cephamycin C in Nor-cardia lactamdurans (10); in addition, a large number ofactinomycetes produce 1-lactamases and may be the sourceof this form of resistance. Is it conceivable that resistance toantitumor agents might arise in the same way? S. peucetius,the organism producing adriamycin, has a multiple drugresistance determinant that is similar to those of mammaliancells (16).We submit that the presence of specific DNA sequences in

certain antibiotic agents used for growth promotion in ani-mals or therapy and prophylaxis in humans has contributedto the development of antibiotic resistance in clinicallyimportant bacteria. More detailed examination of this prob-lem should shed light on important general questions ofnatural gene transfer in bacterial evolution and speciation.

Attention has recently focused on the (apparent) risks atten-dant on the release of genetically engineered organisms;there is a need for detailed studies of gene pickup andtransfer with the release of any kind of DNA into theenvironment, especially when there is strong selection for anacquired phenotype.

ACKNOWLEDGMENTS

We appreciate the encouragement and helpful suggestions ofCharles Thompson, George Spiegelman, and Bob Hancock. Fund-ing was provided by the Canadian Bacterial Diseases Network andthe National Science and Engineering Council of Canada. We aregrateful to Carl Douglas for use of a fluorescence spectrometer andthank Tom Yagisawa for technical assistance. Dorothy Daviesassisted in the preparation of the manuscript. Many of the antibiot-ics were provided generously by friends in the pharmaceuticalindustry.

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