Transcript beta-lactamases

Mechanisms of antimicrobial
action directed against the
bacterial cell wall and
corresponding resistance
mechanisms
M-4 Advanced
Therapeutics
Course
Mechanisms of antimicrobial resistance
Drug-modifying
enzymes
(e.g., - lactamases,
aminoglycosidemodifying enzymes)
Altered
drug
targets
(e.g., PBPs
ribosomes,
DNA gyrase)
Altered
uptake or
accumulation of
drug
(e.g., altered porins,
membrane
efflux pumps)
Subunits for cell wall construction
N-acetylglucosamine
N-acetylmuramic acid
pentapeptide
D-ala-D-ala
Cell Wall Assembly
Second layer of cell
wall cross-linked to
the lower layer
Layer of cell wall
with cross links
of 5 glycines
(gray)
A subunit is added
to the growing chain
Transpeptidase (PBP) forms a 5-glycine bridge between peptides
Transpeptidase, or PBP
(orange sunburst)
is bound by beta-lactam antibiotic (light blue)
and its activity is inhibited
(turns gray)
5-glycine crosslinking bridges cannot form in the presence of a
beta-lactam, and the cell wall is deformed and weakened
Mechanisms of beta-lactam resistance
• Drug-modifying enzymes (beta-lactamases)
– Gram-positives(e.g., S. aureus) excrete the enzyme
– Gram-negative (e.g., E. coli) retain the enzyme in the
periplasm
• Overexpression of
•
cell wall synthetic enzymes
– e.g., vancomycin-intermediate S. aureus (VISA)
Alteration of the PBPs so antibiotic cannot bind
– e.g., S. pneumoniae, gonococcus
• Exclusion from the site of cell wall synthesis
– Porin mutations in the outer membrane of Gramnegative bacteria only (e.g., Ps. aeruginosa)
Beta-lactamases
Beta-lactamases
(dark orange)
bind to the antibiotics
(light blue)
and cleave the beta-lactam ring.
The antibiotic is no longer able to inhibit the
function of PBP
(orange sunburst)
Beta-lactamase activity
Altered drug targets
Vancomycin-intermediate S. aureus
Production of excessive cell wall; the antibiotic cannot keep up
MRSA
vancomycin MIC = 2 µg/ml
VISA
vancomycin MIC =8 µg/ml
MRSA
VISA
Mechanism of vancomycin action
V
D-ala-D-ala
Mechanism of vancomycin resistance
Vancomycin is
unable to bind
to the D-ala-Dlactate structure
D-ala-D-lactate
V
·June 2002: isolated from the catheter exit site in a chronic
dialysis patient
·The patient had received multiple courses of abx since
April 2001; toe amputation in April 2002 --> MRSA
bacteremia
·VRSA also found at amputation stump wound (with VRE
and Klebsiella); not in the patient’s nose
·Vancomycin MIC >128mcg/ml!! (contains vanA)
·Sensitive to trim/sulfa, chloro, tetracyclines, Synercid,
linezolid
MRSA and penicillin-resistant S.
pneumoniae
• These bacteria are both resistant because they
•
•
have altered bacterial targets -- penicillin-binding
proteins (PBPs or transpeptidases)
In MRSA, the altered PBP2 (mecA) gene is acquired
by gene transfer from another bacterium.
In pneumococci, the alteration in PBP is generated
by uptake of DNA released by dead oral
streptococci and recombination at the
pneumococcal pbp gene to create a new, chimeric
protein that does not bind penicillin.
– depicted on the next slide . . .
DNA
alpha-strep pbp
Alpha-strep
S. pneumoniae
transformation
alpha-strep pbp
S. pneumoniae chromosomal pbp; penicillin-sensitive
Chimeric pbp (resistant to penicillin)
Outer membrane permeability in
Gram-negative bacteria
Beta-lactam (blue)
enters through an outer
membrane porin channel
Altered porin channel
prevents access of the
antibiotic to the cell wall
Outer membrane
Cell wall
(peptidoglycan)
Inner membrane
Cytoplasm
Bacterium