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Antibiotic resistance is ancient

Antibiotic resistance is ancient Vanessa M. D'Costa1,2*, Christine E. King3,4*, Lindsay Kalan1,2, Mariya Morar1,2, Wilson W. L. Sung4, Carsten Schwarz3,Duane Froese5, Grant Zazula6, Fabrice Calmels5, Regis Debruyne7, G. Brian Golding4, Hendrik N. Poinar1,3,4 & Gerard D. Wright1,2 The discovery of antibiotics more than 70 years ago initiated a with high concentrations of Escherichia coli harbouring the gfp (green period of drug innovation and implementation in human and fluorescent protein) gene from Aequorea victoria (Supplementary animal health and agriculture. These discoveries were tempered in all cases by the emergence of resistant microbes1,2. This history After fracturing of the samples (Supplementary Fig. 3), total DNA has been interpreted to mean that antibiotic resistance in patho- was extracted from a series of five subsamples taken along the radius of genic bacteria is a modern phenomenon; this view is reinforced by each core (Supplementary Information). Quantitative polymerase the fact that collections of microbes that predate the antibiotic eraare highly susceptible to antibiotics3. Here we report targeted metagenomic analyses of rigorously authenticated ancient DNAfrom 30,000-year-old Beringian permafrost sediments and the Dawson tephra: 25,300 14C yr BP identification of a highly diverse collection of genes encoding res- (30,430–30,030 calendar yr BP) istance to b-lactam, tetracycline and glycopeptide antibiotics.
Structure and function studies on the complete vancomycin resist- Microtus & ance element VanA confirmed its similarity to modern variants.
These results show conclusively that antibiotic resistance is anatural phenomenon that predates the modern selective pressure of clinical antibiotic use.
Recent studies of modern environmental and human commensal microbial genomes have a much larger concentration of antibioticresistance genes than has been previously recognized4–6. In addition, metagenomic studies have revealed diverse homologues of knownresistance genes broadly distributed across environmental locales.
Elevation above datum (m) This widespread dissemination of antibiotic resistance elements isinconsistent with a hypothesis of contemporary emergence and Loess (primary and instead suggests a richer natural history of resistance2. Indeed, estimates of the origin of natural product antibiotics range from2 Gyr to 40 Myr ago7,8, suggesting that resistance should be similarly old. Previous publications claim to have cultured resistant bacteria from Siberian permafrost (for example ref. 9), but these results remaincontentious (see Supplementary Information).
To determine whether contemporary resistance elements are modern or whether they originated before our use of antibiotics, we analysed DNA sequences recovered from Late Pleistocene permafrost sediments.
The samples were collected east of Dawson City, Yukon, at the BearCreek (BC) site (Fig. 1); prominent forms of ground ice (ice wedges and surface icings) are preserved in the exposure, immediately overlain by a distinctive volcanic ash layer, the Dawson tephra10,11 (SupplementaryTable 1 and Supplementary Figs 1 and 2). The tephra has been dated at several sites in the area to about 25,300 radiocarbon (14C) years BP, or about 30,000 calendar years10,12. The cryostratigraphic context is similarto other sites in the area preserving relict permafrost and indicates thatthe permafrost has not thawed since the time of deposition (Sup- plementary Information). In the absence of fluid leaching, the site repre-sents an ideal source of uncontaminated and securely dated ancient Figure 1 Stratigraphic profile and location of Bear Creek site. Elevation isgiven in metres above base of exposure. Permafrost samples from below Dawson tephra were dated to about 30 kyr BP. Preservation of the ice below and Two frozen sediment cores (BC1 and BC4), 10 cm apart, were above the sample indicates that the sediments have not thawed since obtained 50 cm below the tephra. In accordance with appropriate deposition. Silhouettes represent mammals and birds identified from ancient protocols13, we monitored contamination introduced during coring DNA sequences that are typical of the regional Late Pleistocene environment.
by spraying the drilling equipment and the outer surface of the cores aDNA, ancient DNA.
1Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada, L8N 3Z5. 2Department of Biochemistry and Biomedical Sciences, McMaster University,Hamilton, Ontario, Canada, L8N 3Z5. 3McMaster Ancient DNA Centre, Department of Anthropology, McMaster University, Hamilton, Ontario, Canada, L8S 4L9. 4Department of Biology, McMaster University,Hamilton, Ontario, Canada, L8S 4K1. 5Department of Earth and Atmospheric Sciences, University of Alberta, Edmonton, Alberta, Canada, T6G 2E3. 6Yukon Palaeontology Program, Department of Tourismand Culture, Yukon Government, PO Box 2703, Whitehorse, Yukon, Canada, Y1A 2C6. 7Muse´um National d'Histoire Naturelle, UMR 7206 Eco-anthropologie, 57 rue Cuvier, CP139, 75231 Paris cedex 05,France.
*These authors contributed equally to this work.
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chain reaction (qPCR) analysis confirmed extremely high yields of gfp Supplementary Table 8). Querying the permafrost sequences against on both core exteriors, with 0.1% or less of this amount at the centre the contaminant data set with the use of BLAST further confirmed (Supplementary Information and Supplementary Fig. 4). This sup- their disparity: only 1% of the reads had 95–100% identity to a con- ports negligible leaching or cross-contamination during subsampling.
taminant sequence, with a single sequence showing 100% identity.
A crucial step lending support for the authenticity of the ancient We next developed a series of assays to detect genes encoding resist- DNA was to confirm the presence of DNA derived from flora and fauna ance to several major classes of antibiotic and representing diverse characteristic of a late Pleistocene age, and the absence of common strategies of drug evasion (for example target modification, target pro- modern or Holocene floral and faunal sources. To explore the vertebrate tection and enzymatic drug inactivation) (Supplementary Information).
and plant diversity, we amplified fragments of the mitochondrial 12S Determinants included the ribosomal protection protein TetM, which rRNA and chloroplast trnL and rbcL genes (Supplementary Table 3).
confers resistance to tetracycline antibiotics by weakening the inter- Amplicons were sequenced with the 454 GS-FLX platform and iden- action between the drug and the ribosome; the D-Ala-D-Ala dipeptide tified by BLAST analysis of GenBank sequences (Supplementary hydrolase VanX, which is a component of the vancomycin resistance operon; the aminoglycoside-antibiotic-modifying acetyltransferase The vertebrate sequences included abundant Late Pleistocene AAC(3); a penicillin-inactivating b-lactamase Bla (a member of the megafauna such as Bison, Equus and Ovis, as well as rodents (Microtus TEM group of b-lactamases); and the ribosome methyltransferase and Ellobius) and the rock ptarmigan, Lagopus mutus (Supplementary Erm, which blocks the binding of macrolide, lincosamide and type B Fig. 6 and Supplementary Table 5). Mammuthus was detectable at low streptogramin antibiotics. Amplification of vanX, tetM and bla frag- copy numbers with the use of a mammoth-specific qPCR assay, which is ments was successful, and triplicate PCR products from multiple consistent with the low ratio of these fossils relative to bison and horse in extracts were cloned and multiple clones were sequenced.
the region11,14. The rbcL and trnL sequences revealed many plant groups The b-lactamase sequences demonstrated amino-acid identities that are also well documented in Beringia, including the grasses Poa and between 53% and 84% with known determinants and clustered with Festuca, sage (Artemisia) and willow (Salix)15 (Supplementary Figs 7 and one of two groups of enzymes: characterized b-lactamases from strepto- 8, and Supplementary Tables 6 and 7). No sequences of common mycetes and uncharacterized b-lactamase-like hydrolytic proteins Holocene vertebrates (for example elk or moose) or plants (for example (Fig. 2a and Supplementary Fig. 14). We identified several tetM-related spruce) were identified despite sequence conservation across the primer- genes in the permafrost, most of which were most closely related to the binding sites; these results are consistent with other reports16 that have actinomycete subset of ribosomal protection proteins, including the argued against DNA leaching in permafrost sediments.
biochemically characterized self-resistance element OtrA from the We focused our investigation of bacterial 16S rRNA sequences on oxytetracycline producer Streptomyces rimosus18 (Fig. 2b). Most intri- the Actinobacteria, known for their ability to synthesize diverse guing was the identification of vanX gene fragments, which spanned secondary metabolites and for harbouring antibiotic resistance genes4.
the entire phylogenetic space of characterized vancomycin resistance Deep sequencing of 16S amplicons (Supplementary Information) determinants found in the clinic and in the environment. These branch revealed genera commonly found in soil and permafrost microbial away from the cellular dipeptidases that are the likely progenitors the communities17, including Aeromicrobium, Arthrobacter and Frankia vanX family (Supplementary Fig. 10).
(Supplementary Fig. 9 and Supplementary Table 8). Analysis of con- Vancomycin resistance took the clinical community by surprise taminant 16S sequences derived from extraction and PCR control re- when it emerged in pathogenic enterococci in the late 1980s19. In both actions (Supplementary Table 4) suggested that these do not clinical pathogens19 and contemporary soil environments4, resistance contribute to the ancient DNA data set; in fact not only were the copy results from the acquisition of a three-gene operon vanH–vanA–vanX numbers 1,000–30,000-fold lower than from the permafrost extracts, (vanHAX). These enzymes collectively reconstruct bacterial peptido- but with the exception of unclassified bacteria there was also very glycan to terminate in D-alanine-D-lactate in place of the canonical little overlap in the genera identified (Supplementary Fig. 9 and D-alanine-D-alanine, which is required for vancomycin binding and Kinetococcus radiotolerans St. griseus cellulosae St. rimosus* St. scabies St. aver BC4 St. sp. AA#4 St. coelicolor St. scabies St. griseusGTP-binding protein Figure 2 Genetic diversity of ancient antibiotic resistance elements.
activity has been biochemically verified are noted with a single asterisk a, b, Unrooted Bayesian phylogenies of translated b-lactamase (bla) (a) and (Supplementary Information). The scale bar represents 0.1 substitutions per tetracycline resistance (tetM) (b). Blue denotes predicted resistance enzymes, site. Posterior probabilities are shown for a, and those of 0.7 or more are and green those associated with other functions; permafrost-derived sequences indicated for b. All unlabelled tips derive from ancient sequences. BC1, Bear are labelled with the originating core name. Sequences in which resistance Creek sample 1; BC4, Bear Creek sample 4.
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subsequent antibiotic action. Although most forms of resistance are With few exceptions, the vanHAX operon is invariant in genetic attributed to a single gene, this complex mechanism is exclusively organization; it therefore offers a matchless template for confirming its associated with resistance and thus its presence provides unambiguous presence with PCR assays that span the vanHA and vanAX boundaries.
confirmation of its role as a resistance determinant.
Two short qPCR assays were designed to confirm this contiguity a vanHAX operon orientation
Amplicons and primer sites Figure 3 Ancient vancomycin resistance elements. a, vanHAX amplicons overlaid with modern VanA (green), where the V-loop is coloured red; right: used in this study, with primer names noted above each arrow. b, Unrooted ball-and-stick representation of ATP binding. The electron density shown is an Bayesian phylogeny of translated vanA sequences; blue denotes strains with Fo 2 Fc map contoured at 3s. d, Comparison of modern and ancient VanA vanHAX clusters confirmed to confer resistance; sequences containing stop monomer structures. The V-loop is coloured red and detailed in the ball-and- codons but homology throughout are noted with a single asterisk stick figures. Ligands are shown in grey. Dashed lines represent hydrogen (Supplementary Information). BC1, Bear Creek sample 1; BC4, Bear Creek sample 4. c, VanAA2 structure. Left: ribbon diagram of the VanAA2 dimer (blue) 2011 Macmillan Publishers Limited. All rights reserved
Table 1 vanHAX permutation tests Length (base pairs) Probability of similarity by chance alone to Streptomyces coelicolor genes * Clones from independent replication in France. {Includes both H1AX and H2AX.
(Fig. 3a and Supplementary Information). Positive results, including resistance is an ancient, naturally occurring phenomenon widespread particularly high yields of the smallest amplicon, A6X (Supplemen- in the environment. This is consistent with the rapid emergence of tary Table 9), encouraged us to attempt amplification across both resistance in the clinic and predicts that new antibiotics will select for boundaries (that is, the complete vanA gene) in a single 1.2-kilobase pre-existing resistance determinants that have been circulating within amplicon. We also targeted fragments anchored on either boundary the microbial pangenome for millennia. This reality must be a guiding and extending as far as possible into vanA. None of the sequences from principle in our stewardship of existing and new antibiotics.
these products, or those generated by an independent laboratory (Sup-plementary Information), were present in GenBank. No contaminants were detected in more than 300 control reactions.
Permafrost cores were collected at Bear Creek, Yukon, then shipped frozen to the Phylogenetic analyses showed that many of the ancient vanHAX McMaster Ancient DNA Centre and stored at 240 uC. All subsequent procedures sequences cluster with characterized glycopeptide-resistant strains of before PCR/qPCR amplification were performed in dedicated clean rooms, physicallyseparated from laboratories containing modern DNA, bacterial cultures and amp- Actinobacteria containing vanHAX cassettes (for example streptomy- lification products. Contaminant leaching into the centre of cores after sampling was cetes, glycopeptide-producing Amycolatopsis species and the nitrogen- monitored by qPCR assays designed to detect E. coli DNA encoding the jellyfish green fixing Frankia sp. EAN1pec) (Fig. 3b and Supplementary Figs 11 and fluorescent protein sprayed onto coring equipment and the external surfaces of all 12). Another group falls between the actinobacterial sequences and the collected cores. DNA was extracted from the centre of subsampled permafrost cores.
Firmicutes-derived cluster, which includes environmental Paenibacillus PCR assays were designed to target vertebrates, plants, bacteria and specific antibiotic isolates and the pathogenic Enterococci, and may reflect an intermediate resistance elements. All products were sequenced with either the 454 GS-FLX platform or by standard cloning and sequencing procedures (GenBank accession Permutation tests were performed with the PRSS algorithm20 (1,000 numbers JN316287–JN366376). The ancient vanA gene identified from thepermafrost was synthesized and expressed in E. coli, and the His permutations each) to confirm that the sequences were statistically was purified by immobilized metal-affinity chromatography. This protein was similar to those of vancomycin resistance genes (vanHAX) present used in enzymatic studies to determine steady-state kinetics and was also studied in modern Streptomyces. As shown in Table 1, all vanHA-spanning by crystallography using the vapour-diffusion hanging-drop method. Data were clones have significant similarity to vanH and vanA, and all vanAX- collected at the National Synchrotron Light Source, Brookhaven National spanning clones have significant similarity to vanA and vanX.
Laboratory, beamline X25 (PDB 1E4E).
To ascertain whether the complete vanA sequences are indeed func- tional and do not represent PCR artefacts or pseudogenes, we synthe- Received 28 March; accepted 22 July 2011.
sized four open reading frames from the 40 H1AX/H2AX sequences Published online 31 August 2011.
(Supplementary Information). Two of these generated soluble proteins Livermore, D. M. Has the era of untreatable infections arrived? J. Antimicrob.
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the clinic and the environment (Supplementary Table 10). These D'Costa, V. M., McGrann, K. M., Hughes, D. W. & Wright, G. D. Sampling theantibiotic resistome. Science 311, 374–377 (2006).
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on antibiotics. Science 320, 100–103 (2008).
We further confirmed the link between 30,000-year-old VanA and Sommer, M. O. A., Dantas, G. & Church, G. M. Functional characterization of theantibiotic resistance reservoir in the human microflora. Science 325, 1128–1131 contemporary enzymes by determining the three-dimensional struc- ture of VanAA2 by X-ray crystallography (Supplementary Table 11 and Baltz, R. H. Antibiotic discovery from actinomycetes: will a renaissance follow the Supplementary Information). The quaternary and tertiary structures decline and fall? SIM News 55, 186–196 (2005).
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of ancient and modern VanA (Fig. 3c, d) reveals conservation of 10. Froese, D. G., Zazula, G. D. & Reyes, A. V. Seasonality of the late Pleistocene Dawson quaternary and tertiary structure with minor differences in Mg21 tephra and exceptional preservation of a buried riparian surface in central Yukon and ATP c-phosphate coordination. The V-loop comprises the biggest Territory, Canada. Quat. Sci. Rev. 25, 1542–1551 (2006).
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from the electron density map of VanAA2, including His 241 (His 244 12. Brock, F., Froese, D. G. & Roberts, R. G. Low temperature (LT) combustion of in modern VanA), responsible for the lactate selectivity. The last seven sediments does not necessarily provide accurate radiocarbon ages for sitechronology. Quat. Geochronol. 5, 625–630 (2010).
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This work firmly establishes that antibiotic resistance genes predate 15. Zazula, G. D. et al. Ice-age steppe vegetation in east Beringia. Nature 423, 603 our use of antibiotics and offers the first direct evidence that antibiotic 2011 Macmillan Publishers Limited. All rights reserved
16. Haile, J. et al. Ancient DNA reveals late survival of mammoth and horse in interior Author Contributions D.F., G.Z. and F.C. collected permafrost cores and performed Alaska. Proc. Natl Acad. Sci. USA 106, 22352–22357 (2009).
geochemical analyses followed by subsampling by C.S., V.M.D. and C.E.K. C.E.K 17. Gilichinsky, D. et al. in Psychrophiles: From Biodiversity to Biotechnology performed ancient DNA laboratory work and 454 sequencing. V.M.D. designed primers (eds Margesin, R., Schinner, F., Marx, J.-C. & Gerday, C.) 83–102 (Springer, 2008).
for resistance genes, 16S and gfp. V.M.D. and C.E.K. designed and optimized the qPCR 18. Doyle, D., McDowall, K. J., Butler, M. J. & Hunter, I. S. Characterization of an assays, and cloned and sequenced the resistance gene products. R.D. independently oxytetracycline-resistance gene, otrA, of Streptomyces rimosus. Mol. Microbiol. 5, confirmed the results in France. L.K. purified and characterized VanA, and M.M.
2923–2933 (1991).
crystallized VanA and determined the three-dimensional structure. W.S., G.B.G., C.E.K.
19. Courvalin, P. Vancomycin resistance in gram-positive cocci. Clin. Infect. Dis. 42, and H.N.P. processed and analysed the floral/faunal data; V.M.D. and G.B.G. performed S25–S34 (2006).
phylogenetic and bioinformatic analyses of the resistance gene sequences. H.N.P. and 20. Pearson, W. R. & Lipman, D. J. Improved tools for biological sequence comparison.
G.D.W. conceived the project, and V.M.D., C.E.K., D.F., H.N.P. and G.D.W. wrote the Proc. Natl Acad. Sci. USA 85, 2444–2448 (1988).
manuscript. All authors edited the final draft.
Supplementary Information is linked to the online version of the paper at Author Information The metagenomic sequences determined from permafrost are deposited in GenBank under accession numbers JN316287–JN366376. Reprints and Acknowledgements We thank A. Guarne´ for assistance in X-ray data collection. This permissions information is available at The authors declare work was supported by Canada Research Chairs to D.F., H.N.P. and G.D.W., a Canadian no competing financial interests. Readers are welcome to comment on the online Institutes of Health Research operating grant to G.D.W. (MOP-79488) and a scholarship version of this article at Correspondence and requests for to V.M.D., and by grants from the Natural Sciences and Engineering Research Council of materials should be addressed to G.D.W. or H.N.P.
Canada to D.F. and H.N.P. and scholarship to C.E.K.
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Tlo13244 297.305

Volume 6 Number 3 pp. 297–304 297 RASSF1A Promoter Methylation Viera Kajabova*, Bozena Smolkova*, Levels Positively Correlate with Iveta Zmetakova*, Katarina Sebova*,Tomas Krivulcik*, Vladimir Bella†, Karol Kajo‡, Estrogen Receptor Expression Katarina Machalekova‡ and Ivana Fridrichova* in Breast Cancer Patients1,2 *Laboratory of Cancer Genetics, Cancer ResearchInstitute of Slovak Academy of Sciences, Bratislava,Slovakia; †Department of Senology, St Elizabeth CancerInstitute, Bratislava, Slovakia; ‡Department of Pathology,Slovak Medical University and St Elizabeth CancerInstitute, Bratislava, Slovakia

Page 1 of 11 Jeff Gudin, Abel Gonzalez, Joon Lee Pain Management and Palliative Care, Englewood Hospital and Medical Center, Englewood, New Jersey, USACorrespondence to: Jeff Gudin, MD. Clinical Instructor, Anesthesiology, Icahn School of Medicine at Mount Sinai, Board Certified Pain Management, Anesthesiology, Palliative Care and Addiction Medicine; Director. Pain Management and Palliative Care, Englewood Hospital and Medical Center, 350 Engle St. Englewood, New Jersey 07631, USA. Email:; Abel Gonzalez, MD. Department of Internal Medicine, Englewood Hospital and Medical Center, 350 Engle St, Englewood NJ 07631, USA. Email:; Joon Lee, MD. Pain Management and Palliative Care, Englewood Hospital and Medical Center, 350 Engle St, Englewood NJ 07631, USA. Email: