MECHANISMS OF ANTIBIOTIC RESISTANCE

Dr T. Aswani NdongaMsc TID I

April 2010

Definition .Definition:-Relative or complete lack of effect

of antibiotic against a previously susceptible microbe

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

Molecular genetics of antibiotic resistanceGenetic variability is essential for microbial

evolution. It may occur in a variety of ways :-o Micro evolutionary changes by point mutations-

in nucleotide base pairo Micro evolutionary changes [whole scale

changes] like-InversionsDuplicationsDeletionsTranspositiono Acquisition of foreign Dna –plasmid

mediated/bacteriophages/transposons

MECHANISMS OF RESISTANCE

1.Enzymatic inhibition Enzymes inactivating antibioticsBeta-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 enzymesMany beta lactamases are plasmid mediated all are

produced constitutively there are 6 main groups1. Those that hydrolyze benzylpenicillin

Beta lactamases1. Those that hydrolyze oxacillin and related

penicillins2. Carbenicillinases3. Those that break extended spectrum beta lactams

like aztreonam4. Those that break down oxyimino B lactams5. 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

Beta lactamases site of action

Modifying enzymesReactions are-N-acetylationO nuleotidylationO phosphorylationChloramphenicol 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.

ENZYMES

Degrading enzymes will bind tothe antibiotic and essentially degrade itor make the antibiotic inactive

Blocking enzymes attach side chains tothe antibiotic that inhibit its function.E.g. -lactamases

Alteration of bacterial membranesOuter 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

Alteration of bacterial membranes continuedPromotion of antibiotic efflux-major

mechanism for tetracycline resistance in gram neg-plasmid/chromosomal/transposone mediated

.Efflux /influx mechanismBacterial 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

Influx/effluxWhen 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.

Efflux pumpThe efflux pump is a membrane bound protein that "pumps" theantibiotic out of the bacterial cell.

3. Modification of target sitesAlteration of ribosomal target sites-hence

failure to inhibit protein synthesis and cell growth.

Affected antibiotics are aminoglycosides ,tetracylines,macrolides,lincosamides.

Altered cell wall precursor targetsGlycopeptides 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

Alteration of target enzymesAlteration of PBPs in B lactamsSMX/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

By pass inhibitionDevelopment 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

Modification of AB target sites:disruption in protein synthesis

Some terminologies. VRE . vancomycin-resistant enterococci. 70% of E. faecium strains in USA. GISA . glycopeptide intermediately susceptibleS.aureus. VISA . vancomycin intermediately susceptibleS.aureus. VRSA & VRSE . vancomycin-resistant S.aureus andS.epidermidis. (MIC> 32 mcg/ml; 1st clinical case described in 2002

in USA). ESBL producing K.pneumoniae . extendedspectrum -lactamase producing K. pneumoniae. PRSP penicillin-resistant S. pneumoniae

Antibiotic resistance

Factors promoting drug resistanceExposure to sub-optimal levels of antimicrobial Exposure to microbes carrying resistance genesInappropriate drug use- Lack of quality control inmanufacture or outdated antimicrobial Inadequate surveillance ordefective 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

Antibiotics and mechanisms of resistanceANTIBIOTIC

TARGET MOA MECHANISMOF RESISTANCE

CELL WALL

b-Lactams Transpeptidases/transglycosylases (PBPs

Blockade of cross linking enzymes in peptidoglycan layer

b-Lactamases, PBP mutants

Vancomycin D-Ala-D-Ala termini of peptidoglycan and of lipid II

Sequestration of substrate required for cross linking

Reprogramming of D-Ala-D-Ala to D-Ala-D-Lac od D-Ala –D-ser

PROTEIN SYNTHESIS

Macrolides of the erythromycin class

Peptidyl transferase, centre of the ribosome

Blockade of protein synthesis

rRNA methylation, drug efflux

Tetracyclines

Peptidyl transferase

Blockade of protein synthesis

Drug efflux

Aminoglycosides

Peptidyl transferase

Blockade of protein synthesis

Enzymatic modification of drug

Oxazolidinones

Peptidyl transferase

Blockade of protein synthesis

unknown

DNA replication/repair

Fluoroquinolones

DNA gyrase Blockade of DNA replication

Gyrase mutations to drug resistance

END