1. Bacteria Subsisting on Antibiotics Gautam Dantas Church Lab CEGS Annual Grantee Meeting - 2008

2. Agency for healthcare research and quality, 2006 US reported MRSA infections [1000’s] in US: MRSA kills more people than AIDS 2006: 18,964 2006: 14,627

3. Mechanisms of antibiotic resistance antibiotic modificationeffluxtarget modification

4. resistance gene origin hypothesis: antibiotic resistance genes originated as producer self immunity and was transferred to human pathogens Proc. Nat. Acad. Sci. 70:2276 (1973)

5. Science 312:374 (2006)

6. many antibiotics are produced in the environment, but it is likely that they undergo further biological transformations

7. G. Dantas., M. Sommer et al. Science 320:100 (2008) GROWTH NO GROWTH Bacteria subsisting on antibiotics are widely distributed in the environment

8. Antibiotic eaters are phylogenetically diverse G. Dantas., M. Sommer et al. Science 320:100 (2008)

9. microbes subsisting on antibiotics are phylogenetically diverse they comprise of 3 out the 4 major phyla of human microbiomes 27% of the phylogenetic nearest named isolates are pathogens compared to antibiotic producer organisms they constitute a more accessible reservoir of resistance

10. Bacteria subsisting on antibiotics are extremely resistant G. Dantas., M. Sommer et al. Science 320:100 (2008)

12. using functional genomic selections we have identified more than 150 unique antibiotic resistance genes from antibiotic eaters

13. Gentamicin resistance from eater vs. Salmonella enterica subsp. enterica serovar Typhimurium genomic island I Experiments by: G. Dantas, M. Sommer. Unpublished data

14. resistance genes from some antibiotic catabolizers are identical to resistance genes of pathogens at the nucleotide level

15. best genetic support for producer hypothesis: >gi|27461214|gb|AY082011.1| Enterococcus faecium strain 10/96A vanD gene cluster, complete sequence; and ISEfa4 insertion sequence element >gi|4104707:67-693 Streptomyces toyocaensis D-ala- D-ala dipeptidase (vanXst) gene, complete cds; and unknown gene ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 42: 2215 (1998), Science 312:374 (2006), Nature Reviews Microbiology 5:175 (2007)

16. Producer vs. Pathogen

17. best homology between resistance gene from producer and pathogen: 66% identity @ nucleotide level homology between resistance gene from eater and pathogen: 98.5%, 99.9%, 99.9%, 100% identity @ nucleotide level

18. some antibiotic resistance genes acquired by pathogens by HGT may have originated from antibiotic eaters

19. Acknowledgements Morten Sommer George Church John Aach Duhee Bang Jay Lee Church Lab Rantimi Oluwasegun (Cornell) Jay Shendure (U.W.) Lynn Bry (BWH Pathology) Roberto Kolter (HMS) Peter Girguis, Hellen White (Harvard) Will Haas (HMS) Wayne Curtis (Penn State) David Ellison

21. publications about antibiotic resistance

22. Experimental Design resuspend in minimal media 4x filter 500uL Innoculum 11 soils X 18 antibiotics toothpick passage 1 toothpick passage 2 plate on solid (100,1,0.01uL, no carbon) liquid passage of clones restreak Experiments by: G. Dantas, M. Sommer, R. Oluwasegun.

23. 11 soils: pristine, farm, urban 18 antibiotics: chemical class: beta-lactams quinolones sulfonamides amphenicols aminoglycosides glycopeptides amino-acid deriv origin: natural products semi-synthetic synthetic compounds

24. Penicillin Catabolism in Action G. Dantas., M. Sommer et al. Science 320:100 (2008)

25. Penicillin Catabolism in Action

26. some antibiotic resistance genes acquired by pathogens by HGT may have originated from antibiotic eaters

27. >gi|94960156|gb|DQ519091.1| Acinetobacter baumannii strain DB60079/01 class I integron Experiments by: G. Dantas, M. Sommer. Unpublished data

28. >dbj|AB207867.1| Escherichia coli transposon Tn2610, complete sequence gi|83627312|dbj|AB207867.1|_Es TCCGGTGCGGGGACGGCTGCTCAGTGTTGCGCCTGTGTTCGCACGTTCGTGE_AB95Da04_01_mgRes1_R.trim.f ---------------------------------TCTGTTCGCACGTTCGT * ***************gi|83627312|dbj|AB207867.1|_Es ATCGGTGCGTTCTGATCTTCGCGTCAGACATTGCCGCGGCGCGGGCACAAGE_AB95Da04_01_mgRes1_R.trim.f ATCGGTGCGTTCTGATCTTCGCGTCAGACATTGCCGCGGCGCGGGCACAA **************************************************gi|83627312|dbj|AB207867.1|_Es CAAAAAGCCCGGCATCGCTGCCGGGCTCCGGCCCCGTCCTTGGGGCCTTGGE_AB95Da04_01_mgRes1_R.trim.f CAAAAAGCCCGGCATCGCTGCCGGGCTCCGGCCCCGTCCTTGGGGCCTTG **************************************************gi|83627312|dbj|AB207867.1|_Es ATGTCGGGTCGTTGCCGGGATCGGACCGCGCTGGCGCGGTCCGGTTCCCTGE_AB95Da04_01_mgRes1_R.trim.f ATGTCGGGTCGTTGCCGGGATCGGACCGCGCTGGCGCGGTCCGGTTCCCT **************************************************gi|83627312|dbj|AB207867.1|_Es GACGACCGGGCCAACCGGATCAGAAATCCATGCCGCCCATGCCGCCCATAGE_AB95Da04_01_mgRes1_R.trim.f GACGACCGGGCCAACCGGATCAGAAATCCATGCCGCCCATGCCGCCCATA **************************************************gi|83627312|dbj|AB207867.1|_Es CCGCCAGCACCCGGCATGGCCGGCTCTTCCTTCTTCGGCACTTCGGCCACGE_AB95Da04_01_mgRes1_R.trim.f CCGCCAGCACCCGGCATGGCCGGCTCTTCCTTCTTCGGCACTTCGGCCAC **************************************************gi|83627312|dbj|AB207867.1|_Es GACCACTTCGGTCGTGATCGCAAGGCCGGCGACGGAAGCGGCGTGCTGCAGE_AB95Da04_01_mgRes1_R.trim.f GACCACTTCGGTCGTGATCGCAAGGCCGGCGACGGAAGCGGCGTGCTGCA **************************************************gi|83627312|dbj|AB207867.1|_Es GGGCCGAGCGGGTCACCTTGGTCGGGTCCAGGATGCCCATGGCGATCATGGE_AB95Da04_01_mgRes1_R.trim.f GGGCCGAGCGGGTCACCTTGGTCGGGTCCAGGATGCCCATGGCGATCATG **************************************************gi|83627312|dbj|AB207867.1|_Es TCGCCGAACTCGCCGGTGGCGGCGTTGTAGCCGTAGCTGCCTTCGCCGGCGE_AB95Da04_01_mgRes1_R.trim.f TCGCCGAACTCGCCGGTGGCGGCGTTGTAGCCGTAGCTGCCTTCGCCGGC **************************************************gi|83627312|dbj|AB207867.1|_Es CTTGACGTTGGCCACGATCACGCTCGGTTCTTCACCGGCGTTGGCCACGAGE_AB95Da04_01_mgRes1_R.trim.f CTTGACGTTGGCCACGATCACGCTCGGTTCTTCACCGGCGTTGGCCACGA **************************************************gi|83627312|dbj|AB207867.1|_Es TGGCGCGCAGCGGGGCTTCCAGCGCACGGCGGGTGATGGCGATGCCCAGGGE_AB95Da04_01_mgRes1_R.trim.f TGGCGCGCAGCGGGGCTTCCAGCGCACGGCGGGTGATGGCGATGCCCAGG **************************************************gi|83627312|dbj|AB207867.1|_Es TTCTGGTCTTCGTTGATGCCCTGCCAGGGCAGATCCGTGCACAGCACCTTGE_AB95Da04_01_mgRes1_R.trim.f TTCTGGTCTTCGTTGATGCCCTGCCAGGGCAGATCCGTGCACAGCACCTT **************************************************gi|83627312|dbj|AB207867.1|_Es GCCGTAGAAGAACAGCAAGGCCGCCAATGCCTGACGATGCGTGGAGACCGGE_AB95Da04_01_mgRes1_R.trim.f GCCGTAGAAGAACAGCAAGGCCGCCAATGCCTGACGATGCGTGGAGACCG **************************************************gi|83627312|dbj|AB207867.1|_Es AAACCTTGCGCTCGTTCGCCAGCCAGGACAGAAATGCCTCGACTTCGCTGGE_AB95Da04_01_mgRes1_R.trim.f AAACCTTGCGCTCGTTCGCCAGCCAGGACAGAAATGCCTCGACTTCGCTG **************************************************gi|83627312|dbj|AB207867.1|_Es CTGCCCAAGGTTGCCGGGTGACGCACACCGTGGAAACGGATGAAGGCACGGE_AB95Da04_01_mgRes1_R.trim.f CTGCCCAAGGTTGCCGGGTGACGCACACCGTGGAAACGGATGAAGGCACG **************************************************gi|83627312|dbj|AB207867.1|_Es AACCCAGTGGACATAAGCCTGTTCGGTTGGTAAGCTGTAATGCAAGTAGCGE_AB95Da04_01_mgRes1_R.trim.f AACCCAGTGGACATAAGCCT------------------------------ ******************** Experiments by: G. Dantas, M. Sommer. Unpublished data

29. >gi|4583495|gb|AF133140.1| Pseudomonas sp. plasmid pPSTG2 Experiments by: G. Dantas, M. Sommer. Unpublished data