FIRST SEMINAR

ON

Molecular Mechanisms of Antibiotic Resistance in Microbes

GANAPATI BHAT
PGS 04 AGR 3672
 OUTLINE

INTRODUCTION

MECHANISM OF ANTIBIOTIC RESISTANCE

MOVEMENT OF ANTIBIOTIC RESISTANCE GENE

CASE STUDY

CONCLUSION
 INTRODUCTION

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.

In 1946, Staphylococcus aureus is first bug to resist penicillin.

 CLASSIFICATION OF ANTIBIOTICS

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
 Antibiotic Resistance

Antibiotic Year Year Resistance No.of

marketed first observed 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

Mode of action of antibiotics
 ANTIBIOTIC RESISTANCE

Mechanisms of Antibiotic Resistance.

Movement of Antibiotic Resistance Gene.

 MECHANISMS OF ANTIBIOTIC RESISTANCE

Selection

Alteration of Antibiotic Target

Protein synthesis
DNA Replication
RNA Synthesis

Alteration in Cell wall Synthesis

Antibiotic Inactivation

Antibiotic Efflux
 SELECTION

• Inherent capacity

• Selection

• Multiplication of
resistance type

• “Survival of the
fittest”
 PRTEIN SYNTHESIS

Action of Antibiotics
Eg: Streptomycin,
RIGHT Kanamycin,Tetracycline,Ge
AMIO ACID
WRONG
ntamycin.
AMINO ACID

50s
DEFECTIVE
PROTEIN
mRNA
30s

Antibiotic
Antibiotic resistance

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 action

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.

Resistance mechanism

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.
Ribosomal protection proteins

These proteins have homology to elongation factor

EF-Tu and EF-G.

Greater homology at N-terminal end.

It is more affinity than the antibiotics.

Eg: Tet(M),Tet(O),Otr(A) in tetracycline resistance.

 DNA Replication

Antibiotic action

Antibiotics attacks
on DNA gyrase.

Eg:Fluoroqunolones
like Ciprofloxacin
Resistance mechanism

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.
 RNA synthesis

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.

 CELL WALL SYNTHESIS

β-lactum
Eg:Penicillin

Penicillin binding protein (PBP)

cell Inhibit cell wall synthesis

cell Osmotic pressure

Cell dies
 Protection for cell wall synthesis

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.
 Cell wall synthesis(contd….)

Transglycosylation

Transpeptidation

Normal
cell wall
Vancomycin
D-alanine
D-glu
L-lysine
No cell wall
L-alanine synthesis
NAM
NAG
 Cell wall synthesis (contd..)

Van H Van A
Puruvate D-Lactate D-alanine-D-lactate

UDP- Muranyl
D-Alanine tripeptidase

D-Ala-D-Ala D-ala
Van X

D-lactate

Normal cell
Vancomycine wall synthesis
 Mycobacterium has special mechanism

Mycobacterium has mycolic acid(MA) layer.

Antibiotic disrupt biosynthesis of MA through

inhibition of “inhA”i.e.,enoyl ACP reductase.

Resistance is due to single point mutation in

“inhA”gene.

Mutation changes “serine 94” to “alanine” imparts

the antibiotic resistance.
 ANTIBIOTIC INACTIVATION

Microorganisms produces enzymes which degrades

the antibiotics.

O-phosphotransferase(APHS) Adds phosphate group

N-Acetyl trnsferase(AAC’s) Acytelate amino group

Nucleotidyl transferase(ANT) Adds AMP molecule

ANTIBIOTIC INACTIVATION (CONTD...)
 ANTIBIOTIC INACTIVATION (CONTD...)

O
H
S CH3

_
R C N
CH3
N COOH
O
Penicillin
β- lactamase

O H H
S CH3
R C N C
CH3
COOH
N COOH
H Penicilloic acid (inactive)
 ANTIBIOTIC EFFLUX

Primary active transport

Cytoplasm + ADP ATP

Antibiotic
Secondary active transport

+ H+ H+
H

Cytoplasm
H+
Antibiotic Antiport

H+
H+ +
H

+ Cytoplasm
H +
H
H+
Antibiotics H+ Symoprt
 ANTIBIOTIC EFFLUX (contd..)

ABC transporters

Major facilitator super

family(MFS)

Small drug resistance(SMR)

Resistance nodulation
division(RND)
 Movement of resistance gene

R-plasmid Transduction

Transformation Transposon

Conjugation Integrons
 R-plasmid

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.
 TRANSFORMATION

Up take of “naked DNA”

from surrounding
environment.

Integration of DNA into

chromosome or plasmid.
 CONJUGATION

Transfer of genes from

F+ to F-

Direct contact is necessary

High frequency recombination

(Hfr)
 TRANSDUCTION

Transfer is mediated by
virus.

Temperate phage –no

cell lysis

DNA integrate in to
chromosome
 TRANSPOSON

Discrete movable DNA segment having insertional

sequence(IS) on either side.

One or more resistance gene in middle of the transposon.

R-gene
IS IS Plasmid

Transposon

Chromosomal
DNA
 INTEGRONS

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.
 SOCIOECONOMIC REASONS FOR
ANTIBIOTIC RESISTANCE

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.
 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
 USE OF ANTIBIOTIC RESISTANCE IN
MOLECULAR BIOLOGY

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.
 FUTURE ASPECTS

Clavulanic acid inhibits the β-lctamase and it is

combined with amoxicillin.

Strict quarantine measures.

Relaxation in rules for new drug approval.