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.