1. Antimicrobial resistance arises through genetic mutations and the acquisition of resistance genes from other bacteria. 2. Resistance genes can be acquired horizontally via mobile genetic elements such as plasmids, leading to rapid spread. 3. Common resistance mechanisms include enzymatic inactivation of antibiotics, modification or protection of antibiotic targets, and efflux pumps that pump out antibiotics.

1. 1
Antimicrobial Resistance
Mechanisms

2. Outline
• Antibiotic selection pressure
• Genetic element transfer mechanisms
• Antibiotic resistance mechanisms
• Resistance in major class of antibiotics
2

3. Antibiotic Resistance – A Global
Problem
MRSA MBL
VISA
VRSA
PRP
ESBL
VRE
1961 1967 1983 1986 1988 1996 2002
All
-lactams
Penicillin
3rd gen
cephalosporin
Carbapenem
Vancomycin
Vancomycin
and
teicoplanin
Vancomycin
and
teicoplanin
Emergence → Spread

4. Genetic Basis of Resistance
• Spontaneous mutations in endogenous genes
– Structural genes: expanded spectrum of enzymatic activity,
target-site modification, transport defect
– Regulatory genes: increased expression
• Acquisition of exogenous genes
– Usually genes that encode inactivating enzymes or modified
targets, regulatory genes
– Mechanisms of DNA transfer: conjugation (cell–cell contact);
transformation (uptake of DNA in solution); transduction
(transfer of DNA in bacteriophages)
• Expression of resistance genes
– Reversible induction/repression systems can affect
resistance phenotypes

5. evolution.berkeley.edu
How do drug resistant bugs arise?

6. evolution.berkeley.edu
How do drug resistant bugs arise?

7. evolution.berkeley.edu
How do drug resistant bugs arise?

8. evolution.berkeley.edu
How do drug resistant bugs arise?

9. How did that 1st drug 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

10. A couple of pieces of information
• 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

11. Can you think of other
mechanisms of drug resistance?
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

13. Horizontal Gene Transfer-Another
Mechanism For Resistance
http://www.bioteach.ubc.ca/Biodiversity/AttackOf
TheSuperbugs

14. 14

15. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
The insertion sequence (IS)
transposable element, IS1

16. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Schematic of the integration of an
IS element into chromosomal DNA

17. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Structure of the composite
transposon Tn10

18. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Structure of the noncomposite
transposon Tn3

19. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
DNA sequence of a target site of
Tn3

20. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Cointegration model for transposition of a
transposable element by
replicative transposition

21. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Organizational maps of bacterial
plasmids with transposable elements

22. Mechanisms of Transposition
Transposition is catalyzed by an enzyme, transposase, encoded by
the transposon
The ends of the transposon are critical for transposition

23. R plasmids

24. F Plasmid
• F+ cells conjugate with F– cells
– F+ donates single-stranded copy of F to F– cell (rolling circle)
– F+ retains copy of plasmid, F- cell converted to F+ by replication of
ssDNA donated to the F- cell
– Allows F plasmid to rapidly spread through a bacterial population

25. R Plasmid

26. Pumping ssDNA

27. Mechanisms of Resistance
Eliopoulos. Infectious Diseases. 1992.

28. 28

29. 29

30. 30

31. Resistance Mechanisms

32. TEM1 beta-lactamase Structure

33. TEM1 beta-lactamase Structure

34. 34

35. Resistance Mechanisms

36. Altered Target
• Antimicrobics act by binding and inactivating their
target, which is typically a crucial enzyme or
ribosomal site.
• Substitutions of one amino acid in a protein can alter
its’ binding.
• First generation aminoglycosides and quinolones
only bind one site.
• Newer agents bind at multiple sites on their target
making resistance improbable.

37. In widely divergent gram-pos and gram-
neg species changes in one or more of
peptidoglycan transpeptidase penicillin-
binding proteins (PBP) have been
correlated with decreased susceptibility to
multiple -lactams
Causes: point mutations, substitutions of
amino acid sequence, and
synthesis of a new enzyme.
An important example : -lactam
family

38. Target Site Alteration
• MRSA: the essential function of PBP2 is replaced
by PBP2A, the protein product of the resistance gene
mecA
• Penicillin resistance streptococcus
pneumoniae: alteration of: PBP 1A, 2B, 2X
• Macrolide resistance: Streptococcus pyogenes,
strept pneumo, staphylococcus
– MLSB phenotype
• Macrolide, Lincomycin and Streptogramin B
(quinupristin/dalfopristin)
• Methylation of a single adenine on the 23S rRNA of the 50S
ribosomal subunit
• Encoded by the erm gene (erythromycin ribosomal methylase)

39. Mechanism Of Resistance
http://www.jci.org/cgi/content/full/114/12/1693/F1

45. Mechanism of Action of Vancomycin
Vancomycin binds to the D-alanyl-D-alanine dipeptide on the peptide side chain of newly synthesized
peptidoglycan subunits, preventing them from being incorporated into the cell wall by penicillin-binding
proteins (PBPs). In many vancomycin-resistant strains of enterococci, the D-alanyl-D-alanine dipeptide is
replaced with D-alanyl-D-lactate, which is not recognized by vancomycin. Thus, the peptidoglycan subunit
is appropriately incorporated into the cell wall.

46. N-acetylglucosamine
N-acetylmuramic acid
d-ala
L-glu
Lys
d-ala
d-ala
N-acetylglucosamine
N-acetylmuramic acid
d-ala
L-glu
Lys
d-ala
d-ala
Mechanism of action
of Vancomycin
Vancomycin blocks cell wall synthesis
By binding to the d-alanyl-d-alanine site
on the growing peptidoglycan chain

47. N-acetylglucosamine
N-acetylmuramic acid
d-ala
L-glu
Lys
d-ala
d-ala
Cell membrane
Cell
wall
Transpeptidases (PBP’s)
d-lactate

48. N-acetylglucosamine
N-acetylmuramic acid
d-ala
L-glu
Lys
d-ala
d-lactate
Mechanism of resistance
to Vancomycin

49. Resistance Mechanisms

50. Multi Drug Efflux Pump
50

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52. 52

53. Efflux Pump System in Gram
Positives and Gram Negatives
53

54. Efflux Pump
– Macrolide resistance in S. aureus, S.
pneumoniae and S. pyogenes
• Macrolides not lincosamides or streptogramin B
• encoded by the mef gene (macrolide efflux
pump)
• Pumps out the 14 (erythromycin and
clarithromycin) and 15 member (azithromycin)
rings of macrolides but not 16 member ring
macrolides (miocamycin), lincosamides,
streptogramins B or ketolides (telithromycin)
– ß-lactam resistance in pseudomonas
aeruginosa

55. 55

56. 56

57. Resistance Mechanisms

58. Major Classes of Antibiotics
Antibiotic Mechanism of action
Major resistance
mechanisms
β-Lactams Inactivate PBPs
(peptidoglycan synthesis)
• β-lactamases
• Low affinity PBPs
• Efflux pumps
Glycopeptides Bind to precursor of
peptidoglycan
• Modification of precursor
Aminoglycosides Inhibit protein synthesis
(bind to 30S subunit)
• Modifying enzymes (add
adenyl or Phosphate)
Macrolides Inhibit protein synthesis
(bind to 50S subunit)
• Methylation of rRNA
• Efflux pumps
(Fluoro)Quinolones Inhibit topoisomerases
(DNA synthesis)
• Altered target enzyme
• Efflux pumps
PBPs penicillin-binding proteins

59. Major Bacterial Beta-Lactam
Resistance Mechanisms
MSSA
Haemophilus
influenzae
M. catarrhalis
N. gonorrhoeae
E. coli
Klebsiella pneumoniae
Proteus vulgaris
Proteus mirabilis
Bacteroides fragilis
MRSA
S. pneumoniae
p. aeruginosa

60. Mechanisms of Resistance:
Pseudomonas and Efflux Pumps
Adapted with permission from
Livermore DM. Clin Infect Dis 2002;34:634-640.
Imipenem
and
meropenem
enter here
Meropenem
is pumped
out while
imipenem
is not
Efflux System
Exit Portal
(OprM)
Outer
Membrane
Periplasm
Linker
Lipoprotein
(Mex A)
Cytoplasmic
Membrane
Efflux System Pump (Mex B)
Porin

61. DNA
Staphylococcus aureus
MRSA
mecA gene
Cell Membrane Enzymes:
Abnormal Penicillin Binding Protein (PBP2a)
Β-Lactam
Antibiotics
Penicillins,
(Methicillin)
Cephalosporins,
Monobactams,
Carbapenems,Staphylococcal Cassette Chromosome
(SCC)
MSSA

62. Mechanisms of penicillin
resistance in N. Gonorrhoeae (1)
62
Présentation des activités de
l’Institut Pasteur
Plasmid mediated
-lactamases
Acquired, Transferable
(e.g. TEM)
Chromosomally- mediated
alterations
• Altered penicillin binding proteins
• Porins
• Efflux pumps

63. Mechanisms of penicillin
resistance in N.gonorrhoeae (2)
63
Présentation des activités de l’Institut
Pasteur

64. 64

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