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Antimicrobial Agents and Chemotherapy, June 2005, p. 2490-2494, Vol. 49, No. 6
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.6.2490-2494.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

DNA in Antibiotic Preparations: Absence of Intact Resistance Genes

Markus Woegerbauer,1,2* Heimo Lagler,1 Wolfgang Graninger,1 and Heinz Burgmann1

Division of Infectious Diseases, Department of Internal Medicine I, University of Vienna, 1090 Vienna, Austria,1 Birkmayer Laboratories, 1090 Vienna, Austria2

Received 4 August 2004/ Returned for modification 10 October 2004/ Accepted 1 February 2005


   ABSTRACT
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Fragments of erm(E2), otrA, and aph(6) shorter than 400 bp and producer strain-specific rRNA genes were amplified from various antibiotics. The amount of genetic material and the sizes of amplicons recovered from murine feces after oral administration of a ß-lactamase-encoding plasmid indicated substantial DNA degradation in the mammalian gastrointestinal tract. These observations imply that antibiotics are no major source for horizontal resistance gene transfer in clinical settings.


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Antimicrobial chemotherapy is severely troubled by the emergence and rapid spread of multiresistant bacteria (3, 16). Resistance genes per se either originate from spontaneous mutations of cellular genes or are newly introduced into a bacterial population by acquisition from already resistant microbes (13, 14, 17). Antibiotic producer strains, which possess genetically encoded functions for protection, are thus an important potential source for resistance genes (12). Similarities between resistance genes found in pathogenic clinical isolates and the resistance determinants in the respective antibiotic producer strains have been reported (1). Producer strain-specific DNA and fragments of resistance genes in semisynthetic and biotechnologically produced antibiotics for livestock and animal husbandry were detected (2, 22).

Our aims were (i) to screen antimicrobials currently used to treat infections in humans for producer strain-specific ribosomal RNA and resistance genes and (ii) to monitor the fate of orally applied resistance determinants in the mammalian gastrointestinal tract to assess the risk for resistance gene transfer by contaminated antibiotic preparations.

Film tablets or capsules of synthetic or semisynthetic origin and antibiotics of microbial origin (Table 1) were ground in a mortar and analyzed with the double-stranded DNA-specific fluorescent dye Picogreen (Molecular Probes). Quantitation was standardized with lambda phage DNA; fluorescence inhibition was detected by spiking the samples with linear pUC18 DNA. DNA amounts found in preparations of microbial and semisynthetic origin were between 8 ng (kanamycin sulfate) and 1,500 ng per gram of antibiotic (vancomycin; Table 1). The synthetic fosfomycin was also positive for DNA. Fungal contaminations or DNA from additives may be a reason. DNase I digestion prevented fluorescence emission in treated antibiotic samples. Picogreen was extremely sensitive to contaminations leading to inhibition of fluorescence (e.g., tetracyclines; Table 1).


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  		TABLE 1. DNA contaminations in commercially available antibiotic preparations

 
For characterization of the DNA load, 2 grams of erythromycin, oxytetracycline, streptomycin, teicoplanin, or fosfomycin was resuspended in 20 ml of 1x Tris-EDTA buffer supplemented with sodium dodecyl sulfate and proteinase K. After extraction with cetyltrimethylammonium bromide (18), the supernatant was dialyzed with a CELLUSep H1 dialysis bag (Membrane Filtration Products; cutoff, 15,000 Da). The sample volume was reduced to 2 ml (dialysis bag on hygroscopic silica; Sigma). The DNA was isolated with the Prep A Gene DNA purification kit (Bio-Rad). Producer strain DNA was isolated with cetyltrimethylammonium bromide (Table 2).


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  		TABLE 2. Strains and target sequences used for PCR amplification

 
Different amplicon sizes were checked by the primer pairs described in Table 3. A HotStar Taq PCR protocol (QIAGEN, Germany) was applied with the following modifications: 5 µl of template, 10 µl of 5x Q-solution, and 0.1 µM concentrations of the primers were used in a total volume of 50 µl. The annealing temperature was optimized for each target in increments of 1°C from 72°C to the primer-specific melting temperature (5). Sixty amplification cycles were used. The amplicons were analyzed on 1.5% agarose gels, purified with the PCR gel extraction kit (QIAGEN), and sequenced (VBC Genomics, Austria).


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  		TABLE 3. Amplicons from antibiotic preparations and from murine feces

 
Ribosomal RNA genes and fragments of erm(E2) were found in erythromycin (Table 3). The 222-bp erm(E2) amplicon and the 178-bp ribosomal fragment showed 100% and 98.9% homology, respectively, to Saccharopolyspora erythraea sequences. All other sequenced amplicons, which were specific for ribosomal RNA genes of Streptomyces rimosus, Streptomyces griseus, and "Actinoplanes teichomyceticus," as well as otrA and aph(6) sequences, were identical to the corresponding database sequences. TecR resistance (21) in teicoplanin and Streptomyces fradiae-specific sequences in fosfomycin were not detectable (Table 3). The amplicon sizes were between 87 bp [aph(6)] and 378 bp (otrA). The largest proportion of a resistance determinant was obtained with erm(E2), comprising approximately 20% of the intact gene (Table 3). Amplicons longer than 400 bp could not be amplified from antibiotic preparations (≥6 PCRs under various amplification conditions). No full-length resistance gene could be detected (Table 3). The DNA isolation procedure did not lead to DNA fragmentation (successful amplification of a 1,103-bp amplicon from antibiotics spiked with 1 µg/ml pGFP).

Resistance gene recovery from murine feces. Four female BALB/c mice (Himberg, Austria) were housed separately in single cages. A metal lattice serving as the floor allowed for the collection of feces without any further contact between the animal and its excretions. Different amounts of the ß-lactamase-containing plasmid pGFP (100 pg, 100 ng, and 100 µg in 300 µl of Tris buffer, pH 8) were administered to three animals by gavage to avoid plasmid contamination. One animal served as a control. The pGFP-encoded sequence of the green fluorescent protein from Aequorea victoria allowed the exclusion of PCR-positive signals from silent ß-lactamase genes, which may be present in the fecal flora.

A 343-bp fragment could be amplified from murine feces between 3 and 6 h after gavage application of 100 µg of DNA (Fig. 1). Quantification was performed on the LightCycler by using 1 µl template (10 µg/ml DNA, purified from feces), the primer pair pGFP13/14, and the Faststart DNA SYBR Green master mix (Roche, Austria) in a total volume of 10 µl. After an oral administration of 2.8 x 1013 copies of pGFP, a total of 1.8 x 109 copies could be recovered. A 572-bp fragment retrieved from the same DNA isolation could be amplified only at 3 h after application (data not shown). Fragments corresponding to approximately 49% of the intact ß-lactamase gene and 80% of the coding region for green fluorescent protein could be recovered from feces after the administration of 100 µg DNA (Table 3). Fragments comprising 17% of the intact plasmid were found after passing the murine gastrointestinal tract. The amplification of 735-bp, 851-bp, and 1,103-bp fragments failed. No amplification was achieved with animals treated with 100 pg DNA. The administration of 100 ng resulted in amplicon sizes of 86 bp and 174 bp 3 h after onset (Table 3).



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  		FIG. 1. Quantitative recovery of resistance gene fragments from feces. Feces were collected before and after oral application of 100 µg of the resistance-encoding plasmid pGFP. DNA recovery was quantified by real-time PCR with primers specific for a 343-bp fragment of the plasmid. (A) Feces were collected before (curve 1) and each hour for 7 h after (curves 2 to 8) oral inoculation of plasmid DNA. Excrements were also tested 24 h (curve 9) and 48 h (curve 10) later. Melting peak analysis shows a positive signal for the amplicon 3 to 6 h after oral administration (curves 4 through 7) but not before or afterwards (curves 1 through 3 and 8 through 10). Curves 11 (24 h + 1 µg/ml pGFP) and 12 (pGFP, 10 ng/assay) represent positive controls. (B) Quantification of the positive samples is shown: 5,992 pg, 263 pg, 138 pg, and 41 pg (dotted lines, curves 4 through 7 from left to right) totally recovered plasmid DNA. Solid lines represent logarithmic dilutions (10 to 0.0001 ng DNA/assay) of pGFP in water, used as standards for the generation of the quantification standard curve (C).

 
In vivo transformation. Two mice with an ampicillin resistance-negative gut flora were pipette fed with 100 µg pGFP for 5 days. A third animal was inoculated with a competent Escherichia coli DH5{alpha} strain until growth and then treated as described above. No ampicillin-resistant transformants in the feces of the animals were observed within 4 weeks after pGFP administration.

Natural transformation of competent bacteria may be below the detection limit under usually applied laboratory conditions (9, 15). A low probability for transformation may require higher numbers of bacteria/test animals or extremely long observation periods for a positive result under simulated conditions, but natural microbial ecosystems eventually provide such requirements (4, 9, 17). Long-time exposure to resistance genes by contaminated antibiotics used as feed additives in animal husbandry may support horizontal transfer of free DNA (22, 23). Recently, a report specified the detection of a vancomycin resistance gene cluster in the growth promoter avoparcin (10). This cluster may have been acquired by enterococci residing in the animal gut from the preparations and transferred to human pathogenic strains (7, 10, 20). Mosaic genes found in Neisseria meningitidis and Streptococcus pneumoniae (11) imply a potential for functional recombination of resistance gene fragments in an evolutionary time scale and genetic elements like integrons may provide promoter and recombination sites (6, 19). However, a phylogenetic comparison of resistance genes in producer strains and bacterial pathogens did not provide evidence for a producer strain-specific origin of clinically relevant resistance functions (8).

Conclusions. The analyzed semisynthetic and microbial antibiotic preparations contain small amounts of producer strain-specific DNA but no full-length resistance genes. Resistance determinants entering the mammalian gastrointestinal tract are lost in large scale, but units up to 49% of the entire gene are transiently and size-dependently detectable in mammalian feces. These results indicate that antibiotic preparations are no major source for horizontal resistance gene transfer during therapy.


   ACKNOWLEDGMENTS
 
This work was supported in part by grants of the Austrian Federal Ministry of Health and Consumer Protection (Bundeskanzleramt) the Oesterreichische Nationalbank, and the Austrian Society for Chemotherapy.

We would like to thank Seth Hallström for rewarding discussions, Irene Lechner for excellent technical assistance and carefully reviewing the manuscript, and Bernhard Sautner for his patience with some of the PCR experiments.

Markus Woegerbauer, Heimo Lagler, Wolfgang Graninger, and Heinz Burgmann have no commercial or other associations that might pose a conflict of interest.


   FOOTNOTES
 
* Corresponding author. Mailing address: Birkmayer Laboratories, Department of Research and Development, Schwarzspanierstrasse 15, A-1090 Vienna, Austria. Phone: 0043/1/403 17 55-11. Fax: 0043/1/403 17 55-19. E-mail: m.woegerbauer{at}birkmayer.com. Back


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Antimicrobial Agents and Chemotherapy, June 2005, p. 2490-2494, Vol. 49, No. 6
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.6.2490-2494.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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