Mechanisms of antimicrobial action directed against the bacterial cell wall and corresponding resistance mechanisms M-4 Advanced Therapeutics Course.

Mechanisms of antimicrobial action directed against the

bacterial cell wall and corresponding resistance

mechanisms

Mechanisms of antimicrobial action directed against the

bacterial cell wall and corresponding resistance

mechanisms

M-4 Advanced Therapeutics Course

Mechanisms of antimicrobial resistanceMechanisms of antimicrobial resistance

Drug-modifying enzymes(e.g., - lactamases, aminoglycoside- modifying enzymes)

Altered drug targets (e.g., PBPs ribosomes, DNA gyrase)

Altered uptake oraccumulation of drug(e.g., altered porins, membrane efflux pumps)

Subunits for cell wall construction

D-ala-D-ala

pentapeptide

N-acetylmuramic acid N-acetylglucosamine

Cell Wall AssemblyCell Wall Assembly

Layer of cell wall with cross links of 5 glycines (gray)

Second layer of cell wall cross-linked to the lower layer

Transpeptidase (PBP) forms a 5-glycine bridge between peptides

A subunit is added to the growing chain

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 Gram-

negative bacteria only (e.g., Ps. aeruginosa)

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-lactamases

Beta-lactamase activityBeta-lactamase activity

Altered drug targets

Vancomycin-intermediate S. aureus

vancomycin MIC = 2 µg/ml vancomycin MIC =8 µg/ml

MRSA VISA

Production of excessive cell wall; the antibiotic cannot keep up

MRSA VISA

Mechanism of vancomycin actionMechanism of vancomycin action

D-ala-D-ala

V

Mechanism of vancomycin resistanceMechanism of vancomycin resistance

V

D-ala-D-lactate

Vancomycin is unable to bind to the D-ala-D-lactate structure

·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 . . .

S. pneumoniae chromosomal pbp; penicillin-sensitive

alpha-strep pbp

alpha-strep pbp

Chimeric pbp (resistant to penicillin)

Alpha-strep

transformationS. pneumoniae

DNA

Outer membrane permeability in Gram-negative bacteria

Inner membrane

Outer membrane

Cell wall(peptidoglycan)

Cytoplasm

Beta-lactam (blue) enters through an outer membrane porin channel

Altered porin channel prevents access of the antibiotic to the cell wall

Bacterium