Microbial Resistance to Antibiotics

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Microbial Resistance to Antibiotics
Henry F. Chambers, M.D. Professor of Medicine
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Most Important Infectious Agents on the Planet

• Mycobacterium tuberculosis
• Plasmodium falciparum
• Human immunodeficiency virus

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Most Common Bacterial Pathogens on the Planet

• Mycobacterium tuberculosis
• Enterics (Salmonella enterica, Shigella sp., E.
  coli)
• Staphylococcus aureus
• Streptococcus sp. (S. pneumoniae, S. pyogenes)
• Neisseria sp. (N. gonorrhoeae, N. meningitidis)
• Hemophilus sp.

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Resistance is relative, depending on .

• The host and the site of infection
• The intrinsic activity of the drug and its
  achievable concentration in the host
• The level of susceptibility exhibited by the
  microorganism at the site of infection

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Selective Pressure
Low fitness cost or compensated
Resistance
High fitness cost
Ton-Days of Antibiotics
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Compensatory Mutations

• Wild-type is best adapted, most fit
• Mutation (or acquisition of resistance genes)
  comes at a cost
• Cost can be compensated by
• loss of resistance (by reversion or
  recombination)
• by compensatory mutations
• Compensatory mutations are
• environmentally determined
• a consequence of a low recombination rate

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Example of Compensatory Mutations

• Salmonella typhimurium LT2
• SmR resistant to streptomycin (ribosomal
  inhibitor)
• Cost slow growth because of reduced rate of
  protein synthesis
• SmR passaged in broth (LB) and mice
• Mutants recovering fitness (fast growth) analyzed
  for acquisition of compensatory mutations

Bjorkman, Science 287479 (2000)
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Compensatory Mutations
Bjorkman, Science 287479 (2000)
Salmonella typhimurium LT2
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Relative Fitness of SmR Mutants
Bjorkman, Science 287479 (2000)
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Resistant Strain Passaged w/o Antibiotic Pressure
Types of Mutations
Bjorkman, Science 287479 (2000)
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Effect of Bottleneck on Emergence of Compensatory
Mutations
Levin, Genetics 154985, 2000
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Compensatory Mutations

• Favored when the rate of their generation exceeds
  reversion
• Bottlenecks play an important role
• Function to maintain resistance (implications for
  antibiotic control)
• Less likely to occur if gene exchange or
  recombination is common

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General Types of Resistance

• Extrinsic resistance
• Gram-neg. cocci
• Staphylococci
• Streptococci
• E. coli

• Intrinsic resistance
• M. tuberculosis
• Pseudomonas
• Enterococci

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Biochemistry of Resistance

• Antibiotic is not in an active (e.g., hydrolyzed,
  modified)
• Antibiotic does not reach its target (fails to
  penetrate a membrane, efflux pump)
• Target is changed, modified

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Genetics of Resistance

• Vertical, mutation and selection
• Fluoroquinolone resistance in all bacterial
  species
• All drug resistance in Mtb
• Comparatively less advantageous
• Horizontal, transfer of mobile element and
  selection
• Transducing phage (e.g., transfer of S. aureus
  bla plasmid)
• Transformation, homologous recombination (e.g.,
  penicillin-resistance in pneumococci)
• Conjugation (e.g., vanA resistance of
  enterococci)
• Comparatively more advantageous

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Mycobacterium tuberculosis A Paradigm of
Mutation-selection Mediated Resistance
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Drug Resistance in Mycobacterium tuberculosis

• MTB infects 1/3 of the worlds population
• About 10-25 of strains are resistant to at least
  one drug depending on region of the world
• Up to 10-20 resistant to isoniazid (INH) and
  rifampin (RIF), i.e., MDR
• Important cause of treatment failure

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Emergence of Resistance with Single Drug Therapy
of Active TB
Start INH alone
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Resistant Subpopulations in MTB
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Pathogenesis of Secondary Drug Resistance
Meanwhile, in the lab...
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Molecular Basis of MDR TB

• INH
• High level resistance katG (catalase) mutations
  and deletions
• Low level resistance inhA (mycolic acid
  synthetic enzyme) overexpression
• Rifampin, rifabutin, rifapentine rpoB RNA
  polymerase target mutation

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Molecular Basis of MDR TB

• PZA pncA (pyrazinamidase/nicotinamidase), which
  activates PZA
• Fluoroquinolones gyrA (gyrase) target
  alteration
• Streptomycin rpsL (ribosomal protein S12) or
  rrs (16S rRNA), altered binding to the ribosome
• Ethambutol point mutations in embCAB gene
  cluster (role in arabinoglycan synthesis)

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What about Fitness?

• High-level INH resistance is mediated by
  catalase-gene deletion or mutation at the cost of
  susceptibility to oxidative stress
• Rifampin resistance is mediated by rpoB mutation,
  which is relatively cost neutral
• MDR strains generate fewer secondary cases
• But what about the W strain (Beijing)?

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Resistance Mediated by Horizontal Transfer
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Toxins and Mobile Elements
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Mobile Elements Involved in Resistance
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Resistance Integrons
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Integrons

• An integrase, gene cassette(s), and a cassette
  integration site (att), a strong promoter
• Not themselves mobile, but excise and integrate
  cassettes that are
• Found on transposons, plasmids, chromosome
• Two major groups resistance integrons and
  super-integrons (presumed ancestor, 100 kb)

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Integron Structural Features
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Gene Cassettes

• Open reading frame, usually promoterless
  (provided by integron)
• A repeat sequence, attC, that is recognized by
  integrase
• Cassettes for b-lactamases, aminoglycoside
  resistance, chloramphenicol resistance,
  trimethoprim resistance, ciprofloxacin, etc

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Integrons - Epidemiology

• Mainly found in gram-negatives, especially
  enterobacteriacae (but also M. fortuitum and E.
  faecalis)
• Resistance integrons are interspecies
  transferrable
• Link between integrons in agriculatural isolates
  and human isolates

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Methicillin Resistance in Staphylococcus aureus
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Beta-lactam Antibiotic Resistance of
Staphylococcus aureus

• Inactivation penicillinase (blaZ)
• Four types, relatively narrow spectrum
• Usually plasmid encoded, inducible
• Methicillin resistance target alteration
• Low affinity transpeptidase, PBP 2a (mecA)
• Inducible, chromosomal, found only in MRSA,
  foreign
• Can substitute for other synthetic PBPs

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Characteristics of Community MRSA

• Associated with outbreaks, often in new
  populations (e.g., children, football players,
  MSM)
• Specific clones, different from those typically
  found in hospitals
• Susceptible to many antibiotics
• SCCmec type IV
• Toxin production (Panton-Valentine leukocidin,
  PVL)

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Common MRSA Clones
Enright, PNAS 997687, 2002
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Clonal Complexes of MRSA strains
Enright Fey Okuma
MN, MS, ND, TN Europe, Oceania, Australia
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Molecular Epidemiology of CA-MRSAEmergence of
Common Clone USA300 (ST8)

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80
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Football Team X MRSA, Abscess
Football Team Y MRSA, Abscess
California College Football
Pennsylvania HS Football
Colorado Fencers
California MSM
Mississippi Prison
Texas Jail
Georgia Prison
Tennessee Children
Texas Children
Missouri Children
Football Team X MSSA, Nasal
USA300 Community
USA100 Hospital strain
USA200 Hospital strain
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MRSA from Bay Area Patients, 2004-5
1 2 3 4 5 6 7 8
origin clone 1 ST
FRANCIS 1 ST8S1 2 ST FRANCIS 2
ST8S1 3 SFGH blood ST8S7 4 SFGH eye
ST8S7 5 SFGH eye ST8S1 6 UCSF eye
(HA) ST5D 7 UCSF eye ST8S1 8 MARIN CAP
ST8S1
Merl 261/2005
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PVL as a Putative Virulence Factor

• Hygiene and predisposing host conditions appear
  to influence CA-MRSA colonization
• PVL implicated in skin and soft-tissue infections
• May enhance spread by creating an easily
  contacted reservoir shedding large numbers of
  bacteria
• Synergism with SCCmec ineffective antimicrobial
  therapy may prolong time of shedding

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Vancomycin Resistance
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Vancomycin

• A glycopeptide, 1450 daltons
• Inhibitor of cell wall synthesis
• Target D-alanyl-D-alanine
• Interferes with transglycosylation and
  transpeptidation (crosslinking) reactions
• Bactericidal

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Vancomycin Use United States
VISA
VRE
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Outside cell
TG
TG transglycosylase
Inside cell
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Players in vanA resistance

• Enterococcus faecium and E. faecalis donors
• A broad-host range conjugative plasmid
• Suitable recipient cell (pheromone producer)
• A transposon, Tn1546, resident on the plasmid
• The D-ala-D-ala target

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Map of Transposon Tn1546
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racemase
Inside cell
L-ala
D-ala D-ala
D-ala D-ala L-lys D-glu L-ala NAM
D-ala D-ala L-lys D-glu L-ala NAM
L-lys D-glu L-ala NAM
ligase
D-ala D-ala
NAG-
NAG-
Pi
Pi
NAG
Outside cell
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Outside cell
VanY
D-ala
TG
TG transglycosylase
Inside cell
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O
O C
R__D-ala
C O
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Enterococcus faecalis Conjugative Plasmid
Clewell, Plasmid 48193 (2002)
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Some General Features of Conjugative Plasmids

• Tend to be relatively large gt 50kb
• A DNA transport system that combines elements of
• A DNA rolling circle replication system
• Type IV secretion system
• Plasmid encoded features
• Origin of transfer (oriT)
• DNA replicating enzymes (e.g., helicase,
  relaxase, replicase)
• Coupling proteins, transport proteins,
  aggregating proteins
• Regulators of the pheromone response
  anti-pheromone

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Model of DNA Transport by a Conjugative Plasmid
Llosa, Mol Microbiol 451, 2002
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Vancomycin Resistant Staphylococcus aureus

• Intermediate presumably due to false target
  overproduction MIC 8 mg/ml
• vanA type three strains reported (MICs 16-1024
  mg/ml)
• Presumed donor E. faecalis with a vanA encoding
  conjugative plasmid
• Tn1546 present on a staphylococcal conjugative
  plasmid, which is transferable
• A residual, nontransferable presumed enterococcal
  conjugative plasmid relic

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Closing Comments

• Given enough time and pressure, resistance will
  eventually occur
• When possible acquisition of resistance (and
  probably other traits as well) occurs by
  horizontal gene transfer
• Compensatory mutations are a way to mitigate the
  cost of resistance and maintain the trait in a
  fit pool (and there may be no way back)
• Hopefully, a better understanding of microbial
  pathogenesis can lead to reduce antibiotic
  pressure
• Have a happy day!

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Additional References

• Chang S, et al. Infection with
  vancomycin-resistant Staphylococcus aureus
  containing the vanA resistance gene. NEJM
  3481342, 2003.
• De la Cruz F, and Davies J. Horizontal gene
  transfer and the origin of species lessons from
  bacteria. Trends Microbiol 8128, 2000.
• Fluit AC and Schmitz F-J. Resistance integrons
  and superintegrons. Clin Microbiol Infect
  10272, 2004
• Kazakova SV, et al. A clone of methicillin-resista
  nt Staphylococcus aureus among professional
  football players. NEJM 352468, 2005.
• Levin B and Bergstrom C. Bacteria are different
  Observations, interpretations, speculations, and
  opinions about the mechanisms of adaptive
  evolution in prokaryotes. PNAS 976981, 2000.
• Paulsen I, et al. Role of mobile DNA in the
  evolution of vancomycin-resistant Enterococcus
  faecalis. Science 2992071, 2003
• Tenaillon O, et al. Mutators and sex in bacteria
  conflict between adaptive strategies. PNAS
  9710465, 2000.