antibiotic resistance mechanism
DESCRIPTION
hivTRANSCRIPT
FIRST SEMINAR
ON
Molecular Mechanisms of Antibiotic Resistance in Microbes
GANAPATI BHAT
PGS 04 AGR 3672
INTRODUCTION
MECHANISM OF ANTIBIOTIC RESISTANCE
MOVEMENT OF ANTIBIOTIC RESISTANCE GENE
CASE STUDY
CONCLUSION
OUTLINE
Antibiotic : Substances produced by one organism
kills or inhibit the growth of other organism.
1928-Alexander Fleming discovered Penicillin
from Penicillium notatum.
1939-Commercially exploited by Flory and Chain.
INTRODUCTION
In 1946, Staphylococcus aureus is first bug to resist penicillin.
Aminoglycoside - Kanamycin,Neomycin
Streptomycin
β- lactum ring antibiotics - Penicillin ,Ampicillin
Polykatids
Tetracycline - Oxy tetracycline,
Doxycycline
Cephalosporin and cephamycine - Cephataxine
Glycopeptides antibiotics - Vencomycin
Polymyxine - Polymyxine B
Quinolones - Nalidixic acid
Others - Chloremphenicol,
Fucidic acid
CLASSIFICATION OF ANTIBIOTICS
Antibiotic Year
marketed
Year Resistance
first observed
No.of
years
Sulfonamides 1930 1940 10
Penicillin 1943 1946 3
Streptomycin 1943 1959 10
Chloramphenicol 1947 1959 12
Tetracycline 1948 1959 11
Erythromycin 1952 1988 36
Methicillin 1960 1961 1
Ampicillin 1961 1973 12
Antibiotic Resistance
Mode of action of antibiotics
Movement of Antibiotic Resistance Gene.
Mechanisms of Antibiotic Resistance.
ANTIBIOTIC RESISTANCE
Alteration of Antibiotic Target
Protein synthesis
DNA Replication
RNA Synthesis
Alteration in Cell wall Synthesis
Antibiotic Inactivation
MECHANISMS OF ANTIBIOTIC RESISTANCE
Antibiotic Efflux
Selection
• Inherent capacity
• Selection
• Multiplication of
resistance type
• “Survival of the
fittest”
SELECTION
Antibiotic
mRNA
DEFECTIVE
PROTEIN
WRONG
AMINO ACID
RIGHT
AMIO ACID
Eg: Streptomycin,
Kanamycin,Tetracycline,Ge
ntamycin.
30s
50s
PRTEIN SYNTHESIS
Action of Antibiotics
Point mutation in rrs locus of agene which codes for
16s subunit of 30s ribosome complex.
Point mutation in rspL gene codes for another protein
portion i.e.,S12.
These mutations cause decreased binding affinity of
antibiotics.
Antibiotic resistance
Antibiotics attacks to 50s ribosomal subunit
Eg:Erythromycin,chloremphenicol etc..
These antibiotics attacks 50s sub unit,specifically at
Peptidyl transferase which is centered in 23s part.
This result in to lack of peptide bond formation between
amino acids.
Ultimately defective protein.
Antibiotic action
Erythromycin resistance gene(erm ).
This gene methylates “adenine 2058” in peptidyl
transferase loop of rRNA.
OR
Point mutation that involves the replacement of
“adenine”at 2058 position with either G or C or U.
Both mechanism reduces the antibiotic action on
50s subunit.
Resistance mechanism
These proteins have homology to elongation factor
EF-Tu and EF-G.
Greater homology at N-terminal end.
Eg: Tet(M),Tet(O),Otr(A) in tetracycline resistance.
Ribosomal protection proteins
It is more affinity than the antibiotics.
Antibiotic action
Antibiotics attacks
on DNA gyrase.
Eg:Fluoroqunolones
like Ciprofloxacin
DNA Replication
Quinolone resistance determining region(QRDR)located
on N-terminal of the A subunit of DNA gyrase.
A single or several different point mutation between
“67-106”Residues can result in to resistance.
Mutation in QRDR region cause 4 to 8 fold increase
in the fluoroquinolone resistance.
Resistance mechanism
Antibiotics targets the β-subunit of RNA polymerase.
Mutation in 505 and 534 residues of β-sub unit leads
to antibiotic resistance.
These regions are highly conserved in β-sub unit.
RNA synthesis
Eg:Penicillin
CELL WALL SYNTHESIS
cell
cell
Penicillin binding protein (PBP)
Inhibit cell wall synthesis
Osmotic pressure
Cell dies
β-lactum
Specific point mutation in transpeptidase domain at
“thr 338” present immediately adjacent to catalytic
“serine337”.
Mutation of “thr 338”to glycine, alanine, proline or valine
lowers the acylation efficiency of PBP for β-lactum.
Mutation of “Gln 552” in to glutamate reduces the affinity
towards the cefotaxime and penicillin G.
PBP resistance is governed by “mec A” gene present
on the chromosomal DNA.
Protection for cell wall synthesis
No cell wall
synthesis
Normal
cell wall
Vancomycin
D-alanine
L-alanine
D-glu
L-lysine
NAM
Transpeptidation
Transglycosylation
NAG
Cell wall synthesis(contd….)
Puruvate D-Lactate
D-Alanine
D-alanine-D-lactate
D-Ala-D-Ala D-ala
Van H Van A
Van X
UDP- Muranyl
tripeptidase
Normal cell
wall synthesisVancomycine
Cell wall synthesis (contd..)
D-lactate
Mycobacterium has mycolic acid(MA) layer.
Resistance is due to single point mutation in
“inhA”gene.
Mutation changes “serine 94” to “alanine” imparts
the antibiotic resistance.
Antibiotic disrupt biosynthesis of MA through
inhibition of “inhA”i.e.,enoyl ACP reductase.
Mycobacterium has special mechanism
Microorganisms produces enzymes which degrades
the antibiotics.
O-phosphotransferase(APHS)
N-Acetyl trnsferase(AAC’s)
Nucleotidyl transferase(ANT)
Adds phosphate group
Acytelate amino group
Adds AMP molecule
ANTIBIOTIC INACTIVATION
ANTIBIOTIC INACTIVATION (CONTD...)
N
S
O
R N
H
CH3
COOH
C
CH3
N
S
O
R NCH3
COOH
C
CH3C
_
COOH
H HO
H
Penicillin
Penicilloic acid (inactive)
β- lactamase
ANTIBIOTIC INACTIVATION (CONTD...)
ADP+Cytoplasm ATP
ANTIBIOTIC EFFLUX
Primary active transport
Antibiotic
Secondary active transport
H +
H +Cytoplasm
Antibiotic Antiport
H +
H +
Cytoplasm
Antibiotics
H +
H +
H +
H +
H +
Symoprt
H +H +
ANTIBIOTIC EFFLUX (contd..)
ABC transporters
Major facilitator super
family(MFS)
Small drug resistance(SMR)
Resistance nodulation
division(RND)
Conjugation
Transformation
Transduction
Movement of resistance gene
R-plasmid
Transposon
Integrons
Contains resistance gene for many antibiotics.
It can be transmissible through conjugation.
R751 plasmid easily cross inter-specific barrier.
Acinetobacter calcaceticus and E.coli confer
Resistance to ampicillin,chlorephenicol, kanamycin
and streptomycin.
R-plasmid
Up take of “naked DNA”
from surrounding
environment.
Integration of DNA into
chromosome or plasmid.
TRANSFORMATION
Transfer of genes from
F+ to F-
Direct contact is necessary
High frequency recombination
(Hfr)
CONJUGATION
Transfer is mediated by
virus.
Temperate phage –no
cell lysis
DNA integrate in to
chromosome
TRANSDUCTION
Discrete movable DNA segment having insertional
sequence(IS) on either side.
One or more resistance gene in middle of the transposon.
IS IS
R-gene
Plasmid
Transposon
Chromosomal
DNA
TRANSPOSON
Tn 21 type of transposon.
5’ segment encodes a site specific recombinase.
Often found in R-plasmid.
Most of the integrons have sul I gene codes for
sulfonamide resistance.
INTEGRONS
Indiscriminate use of antibiotics.
Less interest in drug companies, because
“antibiotics cure the disease”.
Since,1962,only two new classes of antibiotics were
discovered i.e., oxazolidinone(in 2000) and
daptomycin (in 2003).
All other new antibiotics are merely the modification
of pre-existing antibiotics.
SOCIOECONOMIC REASONS FOR
ANTIBIOTIC RESISTANCE
ANTIBIOTIC RESISTANT MICROBES
Staphylococcus aureus Chloramphenicol, Rifampin,
Methicillin, Ciprofloxacin, Clindamycin,
Erythromycin, Beta-lactams,
Tetracycline, Trimethoprim
Streptococcus pneumoniae Aminoglycosides, Penicillin,
Chloramphenicol, Erythromycin,
Trimethoprim- Sulfamethoxazole
Mycobacterium tuberculosis Aminoglycosides, Ethambutol,
Isoniazid, Pyrazinamide, Rifampin
Pseudomonas aeruginosa Aminoglycosides, Beta-lactams,
Ciprofloxacin, Tetracycline,
Sulfonamides
Shigella dysenteriae Ampicillin, Trimethoprim-
Sulfamethoxazole, Tetracycline,
Chloramphenicol
Almost all plasmid vectors contains antibiotic
resistance gene used as a selectable marker.
Antibiotic resistance genes helps in identification of
recombinants by insertional inactivation.
Earliest vector pSC101 contains tetracycline
resistance gene.
USE OF ANTIBIOTIC RESISTANCE IN
MOLECULAR BIOLOGY
FUTURE ASPECTS
Clavulanic acid inhibits the β-lctamase and it is
combined with amoxicillin.
Strict quarantine measures.
Relaxation in rules for new drug approval.