1. Conjugative DNA transfer, antibiotic resistance and MDR bacteria With thanks to Steve Matson Who first created this lecture

2. Antibiotics – a medical miracle The discovery of antibiotics changed the medical landscape http://www.nature.com/nature/journal/v406/n6797

3. Life expectancy increased by 8 years between 1944 and 1972 Bacterial infection as cause of death plummeted www.gro-scotland.gov.uk Deaths in Scotland due to infectious disease per 100,0000

4. Life expectancy increased by 8 years between 1944 and 1972 Bacterial infection as cause of death plummeted www.gro-scotland.gov.uk Deaths in Scotland due to TB per 100,0000

5. The antibiotic resistance problem Drug resistant bacteria are very wide spread occurring throughout the world

6. The antibiotic resistance problem Drug resistance happens quickly One study observed an increase from 0% to 28% drug resistant E. coli in less than 5 years

7. The antibiotic resistance problem In 2005 there were more deaths in the US from Methicillin resistant Staphylococcus aureus than from AIDS MRSA Staph aureus 19,000 deaths HIV 17.011 deaths Stats from CDC

8. The antibiotic resistance problem 85% of the cases of MRSA Staph were acquired in hospitals or other health care settings MRSA Staph aureus 19,000 deaths HIV 17.011 deaths

9. How antibiotics work

10. evolution.berkeley.edu How do drug resistant bugs arise?

11. evolution.berkeley.edu How do drug resistant bugs arise?

12. evolution.berkeley.edu How do drug resistant bugs arise?

13. evolution.berkeley.edu How do drug resistant bugs arise?

14. How did that 1 st drug resistant bug arise? A simple error in DNA replication that produced a mutation Occurs at low frequency Mutation is on the chromosome Mutation affects either ribosomal protein S12 or 16S rRNA to produce streptomycin resistance Does not explain MDR bugs or high rate of spread

15. How do we solve this puzzle? We know that drug resistance spreads at an alarming rate Far too fast to be the result of single mutations in the chromosome that arise independently

16. How do we solve this puzzle? We know that drug resistance spreads at an alarming rate Far too fast to be the result of single mutations in the chromosome that arise independently We also know that bacteria become resistant to more than a single drug If this were the result of point mutations in the chromosome the rate would be even slower

17. Vancomycin resistant The four waves of antibiotic resistance in Staph. aureus

18. There are many ways of becoming drug resistant

19. Plasmids are a key to combining them together in one bacterium A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms

20. Two questions 1– how are plasmids rapidly transferred in a bacterial population? 2 – how do plasmids encode resistance to multiple drugs? To understand the rapid increase in multiple drug resistant strains of bacteria there are two questions we must answer.

22. Bacterial conjugation Driven by conjugative plasmids; 1 st example called the fertility factor or F found in some but not all E. coli one of several different types of conjugative plasmid Mating only between cell with F (F + ) and cell without F (F – ) Transfer of information is one- way from donor to recipient Cells must be in close cell-cell contact for DNA transfer to occur

23. F Plasmid A 100 kbp plasmid (single copy) with ~ 100 genes – Replicates inside host cell using host machinery for replication – Partitions to daughter cells in a manner similar to chromosome William Hayes

24. F Plasmid Contains genes encoding synthesis of pillin which is assembled into pili that allow cell contact F+ cells have pili and F- cells lack pilli F+ inhibited from making contact with other F+ cells

25. F Plasmid F + cells conjugate with F – cells – F + donates single-stranded copy of F to F – cell (rolling circle) – F + retains copy of plasmid, F - cell converted to F + by replication of ssDNA donated to the F- cell – Allows F plasmid to rapidly spread through a bacterial population

26. Bacterial Conjugation Bacterial conjugation is the primary mechanism used to spread antibiotic resistance among bacterial populations There will be several million infections involving antibiotic resistant bacteria this year This is now a very significant health problem

27. Pumping ssDNA

28. Tra I (H) = helicase Tra Y (R)= nicks donor DNA at oriT and remains covalently linked during transfer Tra D = links TraY to Type 4 secretion machine

29. This machine can be a drug target

30. Look among existing drugs for small molecules that inhibit the Relaxase 1 nM 10 nM Proc Natl Acad Sci U S A. 2007 Jul 24;104(30):12282-7

31. These inhibit DNA transfer! Proc Natl Acad Sci U S A. 2007 Jul 24;104(30):12282-7

32. Plasmid transfer provides a drug target

33. Plasmid transfer provides other drug targets Plasmids that replicate in similar ways (top, red and blue) compete for resources, and the losing plasmid is lost from the bacterial cell. J. Am. Chem. Soc., 2004, 126 (47), pp 15402–15404

34. Plasmid transfer provides a drug target An aminoglycoside that binds the small RNA causing plasmid incompatibility can mimic this natural process, Causing elimination of a drug-resistance plasmid (bottom, green). J. Am. Chem. Soc., 2004, 126 (47), pp 15402–15404

35. Transposable Genetic Elements are also key to antibiotic resistance A variety of colorful names have been used to describe these genetic elements Controlling elements Jumping genes Roving genes Mobile genetic elements Transposons Definition: Transposable genetic elements (transposons) are DNA segments that can insert themselves at one or more sites in a genome. They are ubiquitous among organisms and play an important role in genome evolution. Remarkably, almost 50% of our chromosomes consist of transposable elements We are still unsure of the normal genetic role, if any, of these elements

36. Composite versus simple Tns Composite Tns contain a variety of genes between two IS elements Transposase is encoded by one of the elements Individual IS elements cannot move Simple Tn contains short IRs at each end Encode their own transposase and other genes

37. Transposons carry drug resistance genes onto plasmids called R plasmids

38. The plasmid can then be transferred to another bacterium by conjugation

39. How does transposition occur? Transposition is catalyzed by an enzyme, transposase, encoded by the transposon The ends of the transposon are critical for transposition

40. Our genome is filled with transposons and their “fossils” Human genome is typical in terms of abundance and distribution of mobile elements How do we survive? Elements inserted into introns Vast majority of elements cannot move There are instances of mutations caused by mobile elements

41. R plasmids can become increasingly complex through natural selection

42. http://www.fbs.leeds.ac.uk/staff/profile.php?tag=ONeill_AJ

43. Integrases can move DNA flanked by direct repeats From plasmids to chromosome and back

44. CDC Research into this area is key to combating TB and other bacterial infections!