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1. Mechanisms of acquired resistance (Continued)
1. Prevention of Access to Target
2. Modification (& Protection) of Target
3. Direct Modification of Antibiotics
• Bacteria can destroy or modify antibiotics, thus resisting their
action
• The enzyme-catalyzed modification of antibiotics is a major
mechanism of antibiotic resistance
• Thousands of such enzymes has been identified that can
degrade & modify antibiotics of different classes

2. a) Inactivation of antibiotics by enzymatic hydrolysis
• The classes include β–lactams, aminoglycosides & macrolides
• There are also subclasses of enzymes that can degrade different
antibiotics within the same class
• For example the β–lactams: penicillins, cephalosporins,
carbapenems & monobactams are hydrolyzed by diverse range
of β–lactamases

3. b) Inactivation of Antibiotics by Transfer of a Chemical Group
• Bacterial enzymes can add chemical groups to vulnerable sites
on the antibiotic molecule
• This causes antibiotic resistance by preventing the antibiotic
from binding to the target protein as a result of steric hindrance
• Various different chemical groups can be transferred including
acyl & phosphate groups
• Aminoglycoside antibiotics are particularly susceptible to
modification as they tend to be large molecules with many
exposed hydroxyl & amino groups

4. • Aminoglycosides like kanamycins & neomycins function by
binding to the organism’s rRNA & inhibit protein synthesis
Kanamycin
O
O
O
O
HO
HO
NH2
OH
HO
H2N
NH2
HO
NH2
OH
OH

5. • Resistance develops for aminoglycosides because the bacteria
acquire enzymes that catalyze the modification of hydroxyl &
amino groups of these antibiotics by acetylation, adenylylation
& phosphorylation
• The modification block binding to rRNA
• There are three main classes of aminoglycosides modifying
enzymes:
■ Acetyltransferases
■ Phosphotransferases
■ Nucleotidyltransferases

6. Inactivation of Antibiotics by hyrolysis or Transfer of a Chemical
group

7. Resistance to aminoglycosides e.g. Kanamycin by
phosphotransferase
• This takes place through the enzyme-catalyzed phosphorylation
of the 3’-OH group
O
O
O
O
HO
HO
NH2
OH
HO
H2N
NH2
HO
NH2
OH
OH
O
O
O
O
HO
O OH
HO
H2N
NH2
HO
NH2
OH
OH
P
-O
-O O
NH2
Phosphotransferase
3' 3'

8. • To avoid this type of resistance, analogs of kanamycin can be
designed so that:
◙ It has low affinity to the drug-modifying enzyme
◙ It is less susceptible to modification such as topramycin which
lacks 3’-OH group which is phosphorylated by resistant organisms
◙ Inhibit the enzyme responsible for destroying the drug
O
O
O
O
HO
NH2
NH2
HO
H2N
NH2
HO
NH2
OH
OH
3'

9. • A clever approach was reported in literature which involves the
design of an analog that is less susceptible to modification
• The 3’-OH of kanamycin was converted to a carbonyl group
• The keto compound is in equilibrium with the corresponding
hydrate
O
O
O
O
HO
NH2
OH
HO
H2N
NH2
HO
NH2
OH
OH
O

10. • The resistant organism can phosphorylates one of the 3’-OH groups of the
hydrate in a reaction catalyzed by the ATP-dependent aminoglycoside 3’-
phosphotransferase
• The phosphorylated product spontaneously decomposes back to the active
carbonyl compound
O
O
O
O
HO
HO
NH2
OH
HO
H2N
NH2
HO
NH2
OH
OH
O
O
O
O
HO
NH2
OH
HO
H2N
NH2
HO
NH2
OH
OH
O
OH
O
O
O
O
HO
O
NH2
OH
HO
H2N
NH2
HO
NH2
OH
OH
O
P
-O
-O O
P
OH
HO
O O
H
ATP
ADP

11. • An interesting mechanism of resistance was elucidated for the
antibiotic vancomycin
• The antibiotic is the drug of last defense against resistant
streptococcal & staphylococcal organisms

12. • The mechanism of action of vancomycin is by formation of a
complex with the terminal D-alanyl-D-alanine of the substrate
peptidoglycan
• It thus inhibits the cross-linking step in the cell wall biosynthesis

13. • A surprising resistance to vancomycin arises in bacteria by
induction of genes used to construct an altered peptidoglycan in
which the terminal D-alanyl-D-alanine is replaced by D-alanyl-D-
lactate
• During the cross-linking of the two peptidoglycan strands in
antibiotic-sensitive bacteria the terminal D-alanine residue is
released

14. Mechanism of cross-linking reaction catalyze by transpeptidase
NAM NAG
L-Ala
D-Glu
L-Lys Gly
D-Ala
D-Ala
Gly Gly Gly Gly
D-Ala
L-Lys
D-Glu
(Gly)4
L-Ala
NAM
Gly
NAG
NAM NAG
L-Ala
D-Glu
L-Lys Gly
D-Ala
D-Ala
Gly Gly Gly Gly
D-Ala
L-Lys
D-Glu
(Gly)4
L-Ala
NAM
Gly
NAG
D-Ala
Transpeptidase
+ D-Ala

15. • In the antibiotic-resistant bacteria, however, D-lactate is released
instead of D-alanine, but the same cross-linked product is formed
• Thus the terminal D-alanine & D-lactate are not involved in cross-
linking
N
H
O C
CH3
Peptide
O
O NH3
+
SERINE LYSINE
_
O
O
N
H
O
CH3
Peptide
O NH3
+
SERINE LYSINE
+
HO C
OH
O
CH3
NH2
O
Peptide
O-
NH3
H
N
N
H
CH3 O
O
Peptide
Peptide
+
+
CH3

16. • Substitution of D-alanine by D–lactate results in:
◙ Deletion of a hydrogen bond to vancomycin
◙ Production of a nonbonded electron repulsion between the
lactate ester oxygen & the amide carbonyl oxygen of the
vancomycin preventing complex formatioon
• This in effect produces a 1000-fold reduction in binding of
vancomycin to the peptidoglycan substrate
• In this case, therefore, the resistant organism has not mutated
an essential enzyme but has mutate the substrate for the
enzyme which forms the complex with vancomycin

17. Mechanism of bacterial resistance to vancomycin by substrate
peptidoglycan modification: