resistance

1. MECHANISMS OF ANTIBIOTIC
RESISTANCE

2. DEFINITION
• Relative or complete lack of
effect of antibiotic against a
previously susceptible
microbe

3. MECHANISMS OF RESISTANCE
 Enzymatic inhibition
 Alteration of bacterial membranes
 Outer membrane permeability
 Inner membrane permeability
 Rapid ejection of the drug [efflux] or reduced drug influx.
 By pass of antibiotic inhibition.
 Alteration of target sites
 Altered ribosomal target sites
 Altered cell wall precursor targets
 Altered target enzymes

4. MOLECULAR GENETICS OF
ANTIBIOTIC RESISTANCE
 Genetic variability is essential for microbial evolution. It
may occur in a variety of ways :-
o Micro evolutionary changes by point mutations-in nucleotide
base pair
o Micro evolutionary changes [whole scale changes] like
• Inversions
• Duplications
• Deletions
• Transposition
• Acquisition of foreign DNA - plasmid
mediated/bacteriophages/transposons

5. INVERSION
A chromosomal defect in which a segment of the chromosome
breaks off and reattaches in the reverse direction.

6. DUPLICATIONS
one or more copies of a DNA segment (which can be as small as a
few bases or as large as a major chromosomal region) is produced

7. DELETIONS
Involves the loss of one or more nucleotides from a
segment of DNA

8. TRANSPOSITION
A biological process that allows genes to be transferred to a host organism's
chromosome, interrupting or modifying the function of an extant gene on the
chromosome and causing mutation

9. ACQUISITION OF FOREIGN DNA-PLASMID
MEDIATED/BACTERIOPHAGES/TRANSPOSONS
The transfer of antibiotic resistance genes which are carried on
plasmids

10. MECHANISMS OF RESISTANCE

11. 1.ENZYMATIC INHIBITION
Enzymes inactivating antibiotics
 Beta-lactamases-split amide bond of the beta lactam ring.
 There are many types - characterised by amino acid and nucleotide
sequencing
 Class A MWT 29000-Preferentially hydrolyze penicillins e.g. TEM-1 prevalent in
many gram neg
 Class B-metalloenzymes have a zinc –binding thiol group required for beta
lactamase activity
 Class C mwt 39000 –mainly cephalosporinases
 Class D-oxacillin –hydrolyzing enzymes

12. BETA LACTAMASES
Many beta lactamases are plasmid mediated all are produced constitutively:
there are 6 main groups
1. Those that hydrolyze oxacillin and related penicillins
2. Those that hydrolyze benzylpenicillin
3. Carbenicillinases
4. Those that break extended spectrum beta lactams like aztreonam
5. Those that break down oxyimino B lactams
6. Carbapenemases-found in pseudomonas
Most cephalosporinases are inhibited by clavulanate,sulbactam or tazobactam.
Carbapenamases are metalloenzymes inhibited by EDTA but not clavulanate or
sulbactam
Production of enzymes modifying antibiotics
Aminoglycosides, chloramphenicol-coded by plasmids or chromosomal genes

13. BETA LACTAMASES SITE OF
ACTION

14. MODIFYING ENZYMES
 Reactions are
 N-acetylation
 nuleotidylation
 phosphorylation
 Chloramphenicol acetyltransferase-inactivates the drug by 3-o-
acetylation: plasmid mediated/chromosomal
 Erythromycin esterase: seen in E coli-hydrolyze lactone ring thus
deactivating it - limits utility of oral erythromycin in reducing the
aerobic gram neg flora of the GIT prior to Gi surgery.

15. ENZYMES
Degrading enzymes will bind to
the antibiotic and essentially degrade it
or make the antibiotic inactive
Blocking enzymes attach side chains to
the antibiotic that inhibit its function.
E.g. beta-lactamases

16. ALTERATION OF BACTERIAL
MEMBRANES
 Outer membrane permeability—outer
membrane of gram neg acts as a barrier to
antibiotics esp hydrophobic ones.
 Inner membrane permeability- rate of entry
of aminoglycosides into bacterial cells is a
function of them binding to a non saturable
anionic transporter,where they retain their
positive charge and are pulled across the
cytoplasmic membrane by the internal
charge of the cell.This is an energy
dependent process. The energy generation
or proton motive force may be altered
through mutation

17. ALTERATION OF BACTERIAL
MEMBRANES CONTINUED
• Promotion of antibiotic efflux-major mechanism for
tetracycline resistance in gram neg-
plasmid/chromosomal/transposone mediated

18. EFFLUX /INFLUX MECHANISM
• Bacterial cells have an intrinsic capacity to restrict the
entry of small molecules especially gram neg-outer
membrane is protective,gram pos no outer membrane
hence more antibiotic sensitive
• Restriction of influx is a physiological way to reduce
toxixity to bacterial cell.
• The most wellstudied efflux system in E. coli is the
AcrAB/TolC system this system comprises of an inner
membrane proteinAcr B, and an outer membrane
protein, Tol C, linked by a periplasmic protein, Acr A

19. INFLUX/EFFLUX
• When activated, the linker protein is
believed to fold on itself, bringing the
AcrB and Tol C proteins in close contact,
thus providing an exit path from the
inside to the outside of the cell.
Antibiotics are pumped out through this
channel.

20. EFFLUX PUMP
The efflux pump is a
membrane bound protein
that "pumps" the
antibiotic out of the
bacterial cell.

21. 3. MODIFICATION OF TARGET
SITES
• Alteration of ribosomal target sites-
hence failure to inhibit protein synthesis
and cell growth.
• Affected antibiotics are aminoglycosides,
tetracylines,macrolides, lincosamides.

22. ALTERED CELL WALL PRECURSOR
TARGETS
• Glycopeptides like vancomycin-bind D-
alanine-D-alanine which is present at the
termini of peptidoglycan precursors.
• The large glycopeptide molecules
prevent the incorporation of the pre
cursors into the cell wall

23. ALTERATION OF TARGET
ENZYMES
• Alteration of PBPs in B lactams
• SMX/TMP-production of a
dihdropteroate synthetase that is
resistant to binding by sulphonamides-
plasmid mediated
• Quinolones-DNA gyrase is made up of
gyr Aand gyr B genes-mutations in gyr A
result in resistance

24. BY-PASS INHIBITION
• Development of auxotrophs-have
growth factor requirements different
from those of wild strain these
mutants require subtrates that
normally are synthesized by the
target enzymes and if these are
present in the environment the
organisms grow despite inhibition by
synthetic enzymes

25. MODIFICATION OF
AB TARGET SITES:
DISRUPTION IN
PROTEIN SYNTHESIS

26. SOME TERMINOLOGIES
 VRE - vancomycin-resistant enterococci
 70% of E. faecium strains in USA
 GISA glycopeptide intermediately susceptible S.aureus
 VISA - vancomycin intermediately susceptible S.aureus
 VRSA & VRSE - vancomycin-resistant S.aureus and
S.epidermidis
 (MIC> 32 mcg/ml; 1st clinical case described in 2002 in USA)
 ESBL producing K. pneumoniae - Extended spectrum beta-
lactamase producing K. pneumoniae
 PRSP - penicillin-resistant S. pneumoniae

27. ANTIBIOTIC RESISTANCE

28. FACTORS PROMOTING DRUG
RESISTANCE
 Exposure to sub-optimal levels of antimicrobial
 Exposure to microbes carrying resistance genes
 Inappropriate drug use-
 Lack of quality control in manufacture or outdated
antimicrobial
 Inadequate surveillance or
defective susceptibility assays
 Poverty or war
 Use of antibiotics in foods-Antibiotics are used in animal
feeds and sprayed on plants to prevent infection and
promote growth Multi drug-resistant Salmonella typhi has
been found in 4 states in 18 people who ate beef fed
antibiotics

29. ANTIBIOTICS AND MECHANISMS
OF RESISTANCE
ANTIBIOTIC TARGET MOA MECHANISM
OF RESISTANCE
CELL WALL
b-Lactams Transpeptidases/transglycosylases
(PBPs
Blockade of cross-linking
enzymes in peptidoglycan
layer
b-Lactamases, PBP mutants
Vancomycin D-Ala-D-Ala termini of
peptidoglycan and of lipid II
Sequestration of substrate
required for cross linking
Reprogramming of D-Ala-D-
Ala to D-Ala-D-Lac od D-Ala –
D-ser
PROTEIN SYNTHESIS
Macrolides of the
erythromycin class
Peptidyl transferase, centre of the
ribosome
Blockade of protein synthesis rRNA methylation, drug efflux
Tetracyclines Peptidyl transferase Blockade of protein synthesis Drug efflux
Aminoglycosides Peptidyl transferase Blockade of protein synthesis Enzymatic modification of
drug
Oxazolidinones Peptidyl transferase Blockade of protein synthesis unknown
DNA replication/repair
Fluoroquinolones DNA gyrase Blockade of DNA replication Gyrase mutations to drug
resistance