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BIOL 62 Antibiotic Resistance Consequences & Mechanisms Scott F. Singleton, PhD UNC Eshelman School of Pharmacy [email protected] • “…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 4 decades later Just how bad is it? CONSIDER THE FOLLOWING STORY… 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. 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. 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. 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. 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. 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. Day 7 • The number of deaths and illnesses continues to climb... That can’t happen! Can it? Think it can’t happen? Think again. 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. ...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). 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. 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 $2845 Billion annually Source: National Institutes of Allergies & Infectious Disease (www.niaid.nih.gov) 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) 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) Resistant Bugs are Spreading Rapidly Source: Centers for Disease Control and Protection (CDC) Leading Causes of Death in the U.S. 1900 2010 Infectious - Return ofDisease I.D.? 1998 Chronic Diseases •Pneumonia •Pneumoniaand Influenza •Tuberculosis •Tuberculosis •Diarrhea •Diarrheaand andenteritis enteritis •Heart •Diptheria disease •Intracranial •Scarlet Fever Lesions •Kidney •Bronchitis disease •Accidents •Cancer •Senility •Diptheria •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). Leading Causes of Death in the U.S. 1900 2010 Infectious - Return ofDisease I.D.? Can you imagine a world •Pneumonia •Pneumoniaand Influenza •Tuberculosis •Tuberculosis •Diarrhea •Diarrheaand andenteritis enteritis •Heart •Diptheria disease •Intracranial •Scarlet Fever Lesions •Kidney •Bronchitis disease •Accidents •Cancer •Senility •Diptheria • 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? 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? The Antibiotic Pipeline is Drying Up Antibacterial Agents Approved 1983-2007 Source: B. Spellberg et al., Clin. Infect. Dis. 38: 1279-86 (2004) (modified). The Antibiotic Pipeline 15 largest pharmaceuitcal companies 7 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). Where have all the drug companies gone? R.P. Wenzel, N. Engl. J. Med. 351: 523-526 (2004). Drug Discovery & Development Timeline Discovery Exploratory Development Full Development Pre-Clinical Early-stage research & discovery Clinical Preclinical Pharmacology studies in animal models 4 – 6 years 0 2 – 3 yr Phase I safety 20-80 volunteers Phase II efficacy & AE 100-300 patients 1 – 2 yr 1 – 2 yr 10 5 Idea 12 - 15 years Phase III efficacy & safety 1000-3000 patients 2 – 3 yr FDA Review & Approval Post-approval surveillance & marketing 1 – 2 yr 15 Drug Discovery & Development Attrition isolation & screening 5,000 - 5,000,000 compounds further screening 500 preclinical studies 250 clinical trials 12 – 15 years $802 M – 1.7 B 5 1 or 2 new drugs (may or may not be profitable) 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). Industrial pharmaceutical development is risk-averse antibiotic discovery 10 classes only 2 in last 40 yrs Discovery risk ??? Biotech FDA hurdles Abx Tx 3 – 14 days fragmented markets pressure not to use Big Pharma Market risk R.L. Stein, Drug Disc. Today 8: 245-248 (2003). 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? 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) Bad news: Promising hospital antiinfection 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). Any other bright ideas? How do we understand the mechanisms for the development and transmission of antibiotic resistance genes? Development of antibiotic resistance Biochemical Bases of Antibiotic Resistance • 1. 2. 3. 4. 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/ Development of antibiotic resistance Origins 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 a) virus b) plasmid vector c) free DNA uptake • selective pressure Levy, SB. The challenge of antibiotic resistance. Sci. Am. March 1998:46-53. Origins of antibiotic resistance Mutation and Recombination growth DNA damage antibiotics “stress” ssDNA external DNA source SOS response stress bypass? recombination mutation RecA Antibiotic Resistance Two important facts. • Bacteria can quickly become less susceptible (or resistant) to any drug • Lots of antibiotic resistance genes are “out there” 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. From Microbial Warfare to 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 Penicillin Alexander Fleming, Ernst Chain & Howard Florey Nobel Prize for Medicine, 1945 Chain & Florey, 1938 read Fleming’s work Pasteur, 1877 “life hinders life” “Penicillin as a Chemotherapeutic Agent” Chain & Florey, 1940 first animal tests Heatley & Florey, 1939 produce Penicillin Fleming, 1929 Penicillium supernatant The Lancet, 1940 The Lancet, 1941 “Further Observations on Penicillin” 1941 first human treated Abraham, 1940 in vitro Staph AR Fleming, 1945 Nobel lecture 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 Trimethoprim George H. Hitchings & Gertrude B. Elion Nobel Prize for Medicine, 1988 1969 TMP + Sulfonamides Tmp introduced to the universe, 1957 1972 Tmp first used alone 1971 (Bristol) Coliform bacteria 2.5% 1962 first human treated 1972 R plasmids reported Hitchings, 1988 Nobel prize 1972 (Paris) Enteric bacteria 18% Bacteria can quickly become less susceptible (or resistant) to any drug A.E. Chatworthy et al. (2007) Nature Chem. Biol. “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). Antibiotic Use in the United States • 35 – 50 Million lbs annually • 13 - 60% in human medicine enrofloxacin - used in chickens (Baytril®) • remainder in agriculture/pets – 32 - 78% nontherapeutic uses in agriculture – 6 - 8% therapeutic uses in agriculture ciprofloxacin - used in humans – 0 - 3% in pets (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). Lots of antibiotic resistance genes are “out there” 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... 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 The Road to Resistance: It’s A Bug’s Life 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 Development of antibiotic resistance Stationary Phase Mutation: An Escape Pod • Mutations occurring in aged, stressed, or growthstunted cells which may allow relief from these stressors • Program of genomic hypermutation – RecA & induction of SOS – low-fidelity polymerases (Pol IV & Pol V) G.J. McKenzie et al, PNAS 97:6646-51 (2000). J.D. Tompkins et al. J Bacteriol. 185:3469-72 (2003). I. Bjedov et al. Science 300:1404-9 (2003). Development of antibiotic resistance Neo-Darwinian Model 100 90 80 70 60 50 40 30 20 10 0 1 2 Phenotype • pre-existing mutation • selective pressure Development of antibiotic resistance Stationary-Phase Mutation Model 100 90 80 70 60 50 40 30 20 10 0 1 2 3 4 Phenotype • pre-existing mutation • stress induces MORE mutations • selective pressure How does this happen? Mutation & Recombination • bacteria have evolved mutagenic program to respond to physiological stress – not Darwinian • antibiotics induce stress that results in mutagenesis • bacteria acquire resistance genes from other bacteria via recombination Horizontal transfer of resistance & virulence genes Transmission by Recombination • Recombination involved in transformation, integrative conjugal elements (I.C.E.’s), and integrated phages/ transduced genes • These transferable genetic elements can be pathogenicity islands (Yersinia, Salmonella) http://www.bioteach.ubc.ca/Biodiversity/AttackOfTheSuperbugs/ Horizontal transfer of resistance & virulence genes The Griffith (1928) & Avery (1944) Experiments • Naturally competent S. pneumoniae demonstrate the ability of RecA-dependent recombination in the transfer of virulence factors fig.cox.miami.edu/~cmallery/150/gene/mol_gen.htm Origins of antibiotic resistance Mutation and Recombination growth DNA damage antibiotics “stress” ssDNA external DNA source SOS response stress bypass? recombination mutation RecA Antibiotic Resistance SOS Mediates a Dynamic Balance Preservation of Genetic Information Diversification of Genetic Information SOS SOS response = DNA repair enzymes activation of error-prone DNA synthesis recombinational DNA repair homologous genetic recombination Two Likely Successful Outcomes leftward shift in MIC increase therapeutic index increase effectiveness of antibacterials Novel. Needed. Now. Synereca Pharmaceuticals, Inc will develop products that restore or increase the effectiveness of existing (and future) antibiotics. + antibiotic www.synereca.com + Synereca’s inhibitor increased effectiveness http://endeavors.unc.edu/fall2009/learning_to_ bust_drug-resistant_bugs.php Two Likely Successful Outcomes leftward shift in MIC prevent SOS mutagenesis increase therapeutic index inhibit homologous recombination increase effectiveness of antibacterials attenuate development of resistance The Antibiotic Paradox the more you use an antibiotic to kill human pathogens, the more rapidly the antibiotic loses its efficacy with every use of antibiotics, resistant bacteria survive while susceptible strains are annihilated, thus increasing the prevalence of resistance in the environment every major class of antibiotic has lost varying degrees of efficacy against a number of pathogenic bacteria because of resistance Contributing Factors • inappropriate use of antibiotics in both medicine and agriculture (cf FDA & Baytril) • expanding population of immuno-compromised patients • increased use of invasive medical procedures • inadequate sanitation and lack of clean drinking water • rapid, frequent, and inexpensive international travel allows diseases to leap from continent to continent What can I do? • stay out of the hospital • finish your prescriptions • use antibiotics only when they might actually do some good • support funding of fundamental biomedical research aimed toward understanding mechanisms of bacterial resistance – BioShield II Act of 2005 (Hatch et al.) – Biodefense & Pandemic Vaccine & Drug Development Act of 2005 (Burr, Dole et al.)