Drug Resistance mechanisms in bacteria.ppt

Mechanism of drug resistance

1. Definition
Definition:-Relative or complete lack of effect of
antibiotic against a previously susceptible microbe

2. 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

3. Molecular genetics of antibiotic
resistance
Genetic variability is essential for microbial
evolution. It may occur in a variety of ways :-
oMicro evolutionary changes by point mutations-in
nucleotide base pair
oMicro evolutionary changes [whole scale changes] like
-Inversions
Duplications
Deletions
Transposition
oAcquisition of foreign Dna –plasmid
mediated/bacteriophages/transposons

4. MECHANISMS OF RESISTANCE

5. 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
Many beta lactamases are plasmid mediated all are produced
constitutively there are 6 main groups
1.Those that hydrolyze benzylpenicillin

6. Beta lactamases
1.Those that hydrolyze oxacillin and related penicillins
2.Carbenicillinases
3.Those that break extended spectrum beta lactams
like aztreonam
4.Those that break down oxyimino B lactams
5.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

7. Beta lactamases site of action

8. Modifying enzymes
Reactions are-
N-acetylation
O nuleotidylation
O 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.

9. 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. -lactamases


10. 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

11. Alteration of bacterial membranes
continued
Promotion of antibiotic efflux-major mechanism
for tetracycline resistance in gram
neg-plasmid/chromosomal/transposone
mediated

12. .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

13. 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.

14. Efflux pump
The efflux pump is a
membrane bound protein
that "pumps" the
antibiotic out of the
bacterial cell.

15. 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,lincosa
mides.

16. 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

17. 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

18. 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

19. Modification of AB
target sites:
disruption in
protein synthesis

20. 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 -lactamase producing

K. pneumoniae
. PRSP penicillin-resistant S. pneumoniae

21. Antibiotic resistance

22. 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

23. Antibiotics and mechanisms of
resistance
ANTIBIOTIC TARGET MOA MECHANIS
M
OF
RESISTANC
E
CELL WALL
b-Lactams Transpeptida
ses/
transglycosyl
ases (PBPs
Blockade of
cross linking
enzymes in
peptidoglyca
n layer
b-
Lactamases,
PBP mutants
Vancomycin D-Ala-D-Ala
termini of
peptidoglyca
n and of lipid
II
Sequestratio
n of
substrate
required for
cross linking
Reprogramm
ing of D-Ala-
D-Ala to D-
Ala-D-Lac od
D-Ala –D-ser
PROTEIN
SYNTHESIS
Macrolides
of the
erythromyci
n class
Peptidyl
transferase,
centre of the
ribosome
Blockade of
protein
synthesis
rRNA
methylation,
drug efflux
TetracyclinesPeptidyl
transferase
Blockade of
protein
synthesis
Drug efflux
Aminoglycos
ides
Peptidyl
transferase
Blockade of
protein
synthesis
Enzymatic
modification
of drug
Oxazolidinon
es
Peptidyl
transferase
Blockade of
protein
synthesis
unknown
DNA
replication/
repair
Fluoroquinol
ones
DNA gyrase Blockade of
DNA
replication
Gyrase
mutations to
drug
resistance