BIOL 62 Antibiotic Resistance Consequences & Mechanisms

1. BIOL 62Antibiotic ResistanceConsequences & Mechanisms Scott F. Singleton, PhD UNC Eshelman School of Pharmacy scott_singleton@unc.edu

2. “…it is time to close the book on infectious diseases.” • US Surgeon General, 1967 • “During the last 150 years the Western world has virtually eliminated death due to infectious disease.” • US Surgeon General, 1975

3. 4 decades later

4. Just how bad is it? CONSIDER THE FOLLOWING STORY…

5. Day 1 • A 34-year-old New Hampshire expectant mother visits her doctor’s office complaining of severe stomach pain, vomiting, diarrhea, fever, and chills. She is diagnosed with an intestinal infection, given intravenous fluids and a prescription for a fluoroquinolone and is sent home.

6. Day 2 • At a Massachusetts hospital’s emergency room, a 2-year-old boy with a severe case of diarrhea, vomiting, dehydration, and fever is given fluids and administered a cephalosporin and is admitted to the hospital.

7. Day 3 • The infection of the 34-year-old pregnant woman results in a miscarriage of an otherwise normal baby, followed by the woman’s death.

8. Day 4 • The boy’s lab results come back identifying the cause of his illness as Salmonella, a common foodborne bacterial infection, but, in this instance, the “bug” is highly resistant to the antibiotics commonly used to treat such infections, including cephalosporins and fluoroquinolones. • The baby boy dies of dehydration and bloodstream infection.

9. Day 5 • 325 people are dead. • Thousands — many of them children, the elderly, and other vulnerable individuals — jam emergency rooms across the Northeast complaining of similar symptoms. • Cases have been reported in 15 states along the East Coast and in the Mid-Atlantic region. Isolated cases are reported in other states, including Texas and California. • 14 cases are reported in Mexico and 27 cases in Canada.

10. Day 6 • 1,730 deaths and 220,000 illnesses have occurred in the United States. The epidemic expands in other countries. • Canada, Mexico, and Europe close their borders to U.S. food imports, and travel initiated from the United States is banned around the globe. • Economic losses to the U.S. and global economies soon reach tens of billions of dollars. • The Food and Drug Administration and Centers for Disease Control and Prevention identifiy the source of the infections as a milk distribution facility located in New York state. They confirm that the Salmonella not only causes severe illness, but also is resistant to all available antibiotics. • Doctors can only provide supportive care, not specific, antibiotic treatment.

11. Day 7 • The number of deaths and illnesses continues to climb...

12. That can’t happen!Can it? Think it can’t happen? Think again.

13. A true story. • In 1985, milk contaminated with Salmonella typhimurium infected 200,000 people across the Midwest. • What distinguishes that case from our scenario is the development of a fully antibiotic-resistant strain of the bacteria as compared to the one that is only partially drug-resistant. • Such “bad bugs” are evolving. Some are already here.

14. ...some are already here. • On April 13, 2000, the Pennsylvania State Department of Health notified the Centers for Disease Control and Prevention (CDC) of an increase in Salmonella enterica (Typhimurium). • 100% of the isolates tested for antimicrobial resistance were resistant to at least 3 drugs. • 75% were resistant to Ampicillin, Kanamycin, Streptomycin, Sulfamethoxazole, and Tetracycline (AKSSuT) • 19% were AKSSu resistant • 6% were ASSu resistant S.J. Olsen et al., Emerg. Infect. Dis. 10: 932-935 (2004).

15. What if bioterrorists got involved? • Even relative to our fictional scenario, infection rates could have been significantly higher, as several sources could have been intentionally contaminated. • The toll on human lives and the U.S. economy would have been substantially worse.

16. AR : The Economic Burden • Antibiotics: $36 Billion worldwide sales • second-largest therapeutic category (sales) • four antibiotics > $1 Billion each • Treatment is longer, more expensive, and increasingly relies on new antibiotics • AR costs US health system $28-45 Billion annually Source: National Institutes of Allergies & Infectious Disease (www.niaid.nih.gov)

17. AR : The Human Toll • 1.7 Million U.S. patients acquired nosocomial infections • 70% resistant to ≥ 1 antibiotic • 99,000 deaths • Millions more worldwide • inadequate sanitation • lack of potable water • Rx not required in all countries • international travel – rapid, frequent, inexpensive Mariana Bridi (1988 – 2009) “Brazilian model who lost hands and feet dies” – Associated Press Source: National Institutes of Allergies & Infectious Disease (www.niaid.nih.gov)

18. The problem is worsening…The pace may be quickening… • U.S. Deaths (nosocomial infections) • 1992: 13,300 • 2002: 99,000 • U.S. cases of sepsis • tripled since 1980 • U.S. hospital MRSA • 1970: < 5% • 2004: > 50% • Canadian hospital VRE • 1995: 2 cases • 2002: 101 cases • resistance traits in nursing homes, athletic facilities, the community Source: National Institutes of Allergies & Infectious Disease (www.niaid.nih.gov)

19. Resistant Bugs are Spreading Rapidly Source: Centers for Disease Control and Protection (CDC)

20. Leading Causes of Death in the U.S. • 1900 Infectious Disease • Pneumonia and Influenza • Tuberculosis • Diarrhea and enteritis • Heart disease • Intracranial Lesions • Kidney disease • Accidents • Cancer • Senility • Diptheria • 2010 - Return of I.D.? • Pneumonia • Tuberculosis • Diarrhea and enteritis • Diptheria • Scarlet Fever • Bronchitis • 1998 Chronic Diseases • Heart disease • Malignant neoplasms • Cerebrovascular diseases • Pulmonary disease • Accidents and adverse effects • Pneumonia and influenza • Diabetes mellitus • Suicide • Kidney disease • Liver disease Source: CDC/NCHS, National Vital Statistics System, Mortality, NVSR vol 48 (11).

21. Leading Causes of Death in the U.S. • 1900 Infectious Disease • Pneumonia and Influenza • Tuberculosis • Diarrhea and enteritis • Heart disease • Intracranial Lesions • Kidney disease • Accidents • Cancer • Senility • Diptheria • 2010 - Return of I.D.? • Pneumonia • Tuberculosis • Diarrhea and enteritis • Diptheria • Scarlet Fever • Bronchitis • Can you imagine a world • where invasive surgery can’t be risked? • where retirement & nursing homes are dangerous places? • where nurseries & daycare facilities must be avoided? • where children can’t play outside? • where you can’t swim in the lakes or hike in the mountains?

22. No problemo.“Yeah, that’s scary, but...” We have the best & most innovative pharmaceutical industry in the world. They’ll save us, won’t they?

23. The Antibiotic Pipeline is Drying UpAntibacterial Agents Approved 1983-2007 Source: B. Spellberg et al., Clin. Infect. Dis. 38: 1279-86 (2004) (modified).

24. The Antibiotic Pipeline15 largest pharmaceuitcal companies7 major biotechnology companies • Since 1998, 10 new antibiotics approved • only 2 were “novel” (new target, no resistance) • In 2002, 89 new medicines, 0 new antibiotics • 506 drugs in phase 2 or 3 clinical trials • 5 are new anitbacterials (< 1%) • 4 in big pharma • 1 in biotech • all are derivatives of known antibiotics • 67 for cancer, 33 for inflammation, 3 for ED Source: B. Spellberg et al., Clin. Infect. Dis. 38: 1279-86 (2004).

25. Where have all the drug companies gone? R.P. Wenzel, N. Engl. J. Med. 351: 523-526 (2004).

26. Early-stage research & discovery Preclinical Pharmacology studies in animal models Phase I safety 20-80 volunteers Phase II efficacy & AE 100-300 patients Phase III efficacy & safety 1000-3000 patients FDA Review & Approval Post-approval surveillance & marketing 0 15 10 5 Drug Discovery & Development Timeline Discovery Exploratory Development Full Development Pre-Clinical Clinical 4 – 6 years 2 – 3 yr 1 – 2 yr 1 – 2 yr 2 – 3 yr 1 – 2 yr Idea 12 - 15 years

27. 5,000 - 5,000,000 compounds isolation & screening 12 – 15 years $802 M – 1.7 B further screening 500 preclinical studies 250 clinical trials 1 or 2 new drugs (may or may not be profitable) 5 Drug Discovery & Development Attrition Sources: C. O’Driscoll, Horizon Symposia 2004: Charting Chemical Space. J. Gilbert et al. IN VIVO: The Business & Medicine Report, 21: 73-82 (2003). J.A. DiMasi et al. J. Health Econ. 835, 1-35 (2003).

28. ??? Biotech Discovery risk Big Pharma Market risk Industrial pharmaceutical developmentis risk-averse antibiotic discovery 10 classes only 2 in last 40 yrs FDA hurdles Abx Tx 3 – 14 days fragmented markets pressure not to use R.L. Stein, Drug Disc. Today 8: 245-248 (2003).

29. OK – so we’re not gonna be “saved”(at least not by Big Pharma). But aren’t there lots of possible new drugs and clinical strategies to cope with this issue?

30. Bad news: Antimicrobial Peptides • Cationic antimicrobial peptides are natural products used as “defensins” by vertebrates • CAPs exploit fundamental “physical” features of the bacterial cell wall • resistance is much less likely to evolve than in the case of convential antibiotics... • or NOT! • G.G. Perron et al., Proc. R. Soc. B. (2006)

31. Bad news: Promising hospital anti-infection strategy probably won’t work • resistant microbes are passed from old hospital patients to new patients • cycling -- alternate between two or more classes of antibiotics every few months • mixing -- might work Source: University of Washington News & Information (Aug. 9, 2004).

32. Any other bright ideas? How do we understand the mechanisms for the development and transmission of antibiotic resistance genes?

33. Development of antibiotic resistanceBiochemical Bases of Antibiotic Resistance • mutant genes carried either on the bacterial chromosome or on plasmids • degrade or chemically modify (inactivate) antibiotics • eliminate the entry ports into the cell • export the antibiotic from the cell before it has met its target • modify or replace target molecules that are normally bound by an antibiotic Levy, SB. The challenge of antibiotic resistance. Sci. Am. March 1998:46-53. http://www.bioteach.ubc.ca/Biodiversity/AttackOfTheSuperbugs/

34. Development of antibiotic resistanceOrigins of Antibiotic Resistance Genes • spontaneous mutations occur readily in bacteria • mutations can • produce new resistance • strengthen existing resistance • bacteria may acquire resistance genes • inherit the genes from their resistant forerunners • Integrating Conjugative Element (ICE) transfer • virus • plasmid vector • free DNA uptake • selective pressure Levy, SB. The challenge of antibiotic resistance. Sci. Am. March 1998:46-53.

35. Origins of antibiotic resistanceMutation and Recombination growth DNA damage antibiotics “stress” stress bypass? SOS response ssDNA RecA external DNA source recombination mutation Antibiotic Resistance

36. Two important facts. • Bacteria can quickly become less susceptible (or resistant) to any drug • Lots of antibiotic resistance genes are “out there”

37. Whence Antibiotic Resistance? “In the inferior organisms, still more than in the great animal and vegetable species, life hinders life. Louis Pasteur & Jules-François Joubert, 1877 “Antibiosis” first used by Jean-Paul Vuillemin, in 1889, to describe the fight for survival between two living things.

38. From Microbial Warfareto Wonder Drugs “A liquid invaded by an organized ferment, or by an aerobe, makes it difficult for an inferior organism to multiply. … These facts may, perhaps, justify the greatest hope from the therapeutic point of view.” Louis Pasteur & Jules-François Joubert, 1877 “If the study of the mutual antagonisms of bacteria were sufficiently far advanced a disease caused by one bacterium could probably be treated by another bacterium.” –1885 “Medicinal properties attributed by tradition to certain fungi may possibly represent an untapped source of therapeutic virtue.” Lancet editorial, 1925

40. PenicillinAlexander Fleming, Ernst Chain & Howard FloreyNobel Prize for Medicine, 1945 The Lancet, 1940 “Penicillin as a Chemotherapeutic Agent” Chain & Florey, 1938 read Fleming’s work The Lancet, 1941 “Further Observations on Penicillin” Pasteur, 1877 “life hinders life” Chain & Florey, 1940 first animal tests Heatley & Florey, 1939 produce Penicillin 1941 first human treated Fleming, 1929 Penicillium supernatant Abraham, 1940 in vitro Staph AR Fleming, 1945 Nobel lecture

41. What doesn’t kill me makes me stronger. “The time may come when penicillin can be bought by anyone in the shops. Then there is the danger that the ignorant man may easily underdose himself and by exposing his microbes to non-lethal quantities of the drug make them resistant. Here is a hypothetical illustration. Mr. X. has a sore throat. He buys some penicillin and gives himself, not enough to kill the streptococci but enough to educate them to resist penicillin. He then infects his wife. Mrs. X gets pneumonia and is treated with penicillin. As the streptococci are now resistant to penicillin the treatment fails. Mrs. X dies. Who is primarily responsible for Mrs. X’s death? Why Mr. X whose negligent use of penicillin changed the nature of the microbe. Moral: If you use penicillin, use enough.” http://nobelprize.org/medicine/laureates/1945/fleming-lecture.pdf

42. TrimethoprimGeorge H. Hitchings & Gertrude B. ElionNobel Prize for Medicine, 1988 1972 R plasmids reported 1969 TMP + Sulfonamides Tmp introduced to the universe, 1957 1972 Tmp first used alone 1971 (Bristol) Coliform bacteria 2.5% Hitchings, 1988 Nobel prize 1962 first human treated 1972 (Paris) Enteric bacteria 18%

43. Bacteria can quickly become less susceptible (or resistant) to any drug A.E. Chatworthy et al. (2007) Nature Chem. Biol.

44. “They’re out there.” • antibiotic producing organisms harbor resistance elements for self-protection • soil-dwelling bacteria produce and encounter a myriad of antibiotics • potential reservoir of resistance determinants? • 480 strains screened against 21 antibiotics Source: VM D’Costa et al., Science 311: 374-377 (2006).

45. Antibiotic Use in the United States • 35 – 50 Million lbs annually • 13 - 60% in human medicine • remainder in agriculture/pets • 32 - 78% nontherapeutic uses in agriculture • 6 - 8% therapeutic uses in agriculture • 0 - 3% in pets enrofloxacin - used in chickens (Baytril®) ciprofloxacin - used in humans (Cipro®) Sources: M.N Swartz, Human Health Risks with the Subtherapeutic Use of Penicllin or Tetracycline in Animal Feed. National Academy Press: 1989. M. Mellon et al., Hogging It! Estimates of Antimicrobial Abuse in Livestock. Union of Concerned Scientists, 2001. K.M. Shea, Pediatrics 112: 253-258 (2003).

46. Lots of antibiotic resistance genes are “out there”

47. Two important facts. • Bacteria can quickly become less susceptible (or resistant) to any drug • Lots of antibiotic resistance genes are “out there” It’s only a matter of time...

48. How does this happen?Mutation & Recombination • bacteria have evolved mutagenic program to respond to physiological stress • not simply Darwinian • antibiotics induce stress that results in mutagenesis • bacteria acquire resistance genes from other bacteria via recombination

49. The Road to Resistance: It’s A Bug’s Life

50. How does this happen?Mutation & Recombination • bacteria have evolved mutagenic program to respond to physiological stress • not simply Darwinian • antibiotics induce stress that results in mutagenesis • bacteria acquire resistance genes from other bacteria via recombination