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. You now know how antibiotics work

10. evolution.berkeley.edu And how drug resistant bugs arise

11. evolution.berkeley.edu And how drug resistant bugs arise

12. evolution.berkeley.edu And how drug resistant bugs arise

13. evolution.berkeley.edu And how drug resistant bugs arise

14. How did that 1 st streptomysin 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

20. And plasmids are?

21. 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

22. 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.

24. Bacterial conjugation Driven by conjugative plasmids; 1 st example =the fertility factor F 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

25. F Plasmid A 100 kb (single copy) with ~ 100 genes – Replicates using host machinery – Partitions to daughter cells William Hayes

26. A selfish genetic element! Encodes pillin which assembles into pili allowing cell contact Only F+ cells have pili F+ inhibited from contacting other F+ cells

27. Here’s how it happens F + donates single-stranded copy of F to F – cell (rolling circle) F - cell converted to F + by replication of ssDNA F plasmid rapidly spreads through bacterial population

28. Bacterial conjugation is the primary mechanism which spreads antibiotic resistance among bacterial populations

29. Pumping ssDNA

30. Let’s look at the machine 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 = pillus

31. This machine can be a drug target

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

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

34. Plasmid transfer provides a drug target

35. 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

36. 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

37. Transposable Genetic Elements are also key to antibiotic resistance

38. What is a transposon?

39. Transposable Genetic Elements are also key to antibiotic resistance Transposable genetic elements (transposons) = DNA segments that can insert themselves at one or more sites in a genome. Remarkably, almost 50% of our chromosomes consist of transposable elements

40. Composite versus simple Tns

41. Transposons can carry drug resistance genes onto “R plasmids”

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

43. How does transposition occur?

44. Our genome is filled with transposons and their “fossils”

45. R plasmids can become increasingly complex through natural selection

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

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