Transcript The Recombination Molecular Motor of Escherichia coli

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.)