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 - lactum ring antibiotics Polykatids Tetracycline - Kanamycin,Neomycin Streptomycin - Penicillin ,Ampicillin

Cephalosporin and cephamycine Glycopeptides antibiotics Polymyxine Quinolones Others

- Oxy tetracycline, Doxycycline - Cephataxine - Vencomycin - Polymyxine B - Nalidixic acid - Chloremphenicol, Fucidic acid

Antibiotic Resistance

Antibiotic

Year marketed 1930 1943

Year Resistance first observed 1940 1946

No.of years 10 3

Sulfonamides Penicillin

Streptomycin
Chloramphenicol Tetracycline Erythromycin Methicillin

1943
1947 1948 1952 1960

1959
1959 1959 1988 1961

10
12 11 36 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 AMIO ACID WRONG AMINO ACID

Kanamycin,Tetracycline,Ge ntamycin.

50s mRNA
DEFECTIVE PROTEIN

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 adenineat 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 67-106Residues can result in to resistance. Mutation in QRDR region cause 4 to 8 fold increase in the fluoroquinolone resistance.

RNA synthesis Antibiotics targets the -subunit of RNA polymerase.

Mutation in 505 and 534 residues of -sub unit leads to antibiotic resistance. These regions are highly conserved in -sub unit.

CELL WALL SYNTHESIS

-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 338to glycine, alanine, proline or valine lowers the acylation efficiency of PBP for -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 L-alanine NAM NAG

No cell wall synthesis

Cell wall synthesis (contd..)

Van H
Puruvate D-Lactate

Van A

D-alanine-D-lactate UDP- Muranyl tripeptidase

D-Alanine

D-Ala-D-Ala

Van X

D-ala

D-lactate

Vancomycine

Normal cell wall synthesis

Mycobacterium has special mechanism

Mycobacterium has mycolic acid(MA) layer.

Antibiotic disrupt biosynthesis of MA through inhibition of inhAi.e.,enoyl ACP reductase.

Resistance is due to single point mutation in inhAgene.

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(AACs)
Nucleotidyl transferase(ANT)

Acytelate amino group

Adds AMP molecule

ANTIBIOTIC INACTIVATION (CONTD...)

ANTIBIOTIC INACTIVATION (CONTD...)

O H R C N N O S

CH3
CH3 COOH

- lactamase

Penicillin

O
R C

H
N

H
C
COOH

CH3
CH3

N H

COOH

Penicilloic acid (inactive)

ANTIBIOTIC EFFLUX

Primary active transport

Cytoplasm

+ ADP

ATP

Antibiotic

Secondary active transport H

+

H+

H+

Cytoplasm H+ Antibiotic H+ H+ + H Antiport

Antibiotics

Cytoplasm H+ H+ 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.

Resistance to ampicillin,chlorephenicol, kanamycin and streptomycin.

Acinetobacter calcaceticus and E.coli confer

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 Transposon
Chromosomal DNA

IS

Plasmid

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

Aminoglycosides, Ethambutol, Isoniazid, Pyrazinamide, Rifampin Aminoglycosides, Beta-lactams, Ciprofloxacin, Tetracycline, Sulfonamides

Mycobacterium tuberculosis Pseudomonas aeruginosa

Shigella dysenteriae

Ampicillin, TrimethoprimSulfamethoxazole, 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 -lctamase and it is combined with amoxicillin.

Strict quarantine measures. Relaxation in rules for new drug approval.