1. MECHANISMS OF ANTIBIOTIC RESISTANCE Dr T. Aswani Ndonga Msc TID I April 2010

2. Definition. Definition:-Relative or complete lack of effect of antibiotic against a previously susceptible microbe

3. 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

4. Molecular genetics of antibiotic resistance Genetic variability is essential for microbial evolution. It may occur in a variety of ways :- o Micro evolutionary changes by point mutations-in nucleotide base pair o Micro evolutionary changes [whole scale changes] like -Inversions Duplications Deletions Transposition o Acquisition of foreign Dna –plasmid mediated/bacteriophages/transposons

5. MECHANISMS OF RESISTANCE

6. 1.Enzymatic inhibition Enzymes inactivating antibiotics Beta-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 enzymes Many beta lactamases are plasmid mediated all are produced constitutively there are 6 main groups 1. Those that hydrolyze benzylpenicillin

7. Beta lactamases 1. Those that hydrolyze oxacillin and related penicillins 2. Carbenicillinases 3. Those that break extended spectrum beta lactams like aztreonam 4. Those that break down oxyimino B lactams 5. 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

8. Beta lactamases site of action

9. Modifying enzymes Reactions are-  N-acetylation  O nuleotidylation  O phosphorylation Chloramphenicol 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.

10. ENZYMES Degrading enzymes will bind to the antibiotic and essentially degrade it or make the antibiotic inactive Blocking enzymes attach side chains to the antibiotic that inhibit its function. E.g.  -lactamases

11. Alteration of bacterial membranes Outer 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

12. Alteration of bacterial membranes continued Promotion of antibiotic efflux-major mechanism for tetracycline resistance in gram neg- plasmid/chromosomal/transposone mediated

13. .Efflux /influx mechanism Bacterial 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

14. Influx/efflux When 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.

15. Efflux pump The efflux pump is a membrane bound protein that "pumps" the antibiotic out of the bacterial cell.

16. 3. Modification of target sites Alteration of ribosomal target sites-hence failure to inhibit protein synthesis and cell growth. Affected antibiotics are aminoglycosides,tetracylines,macrolides,lincosamides.

17. Altered cell wall precursor targets Glycopeptides 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

18. Alteration of target enzymes Alteration of PBPs in B lactams SMX/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

19. By pass inhibition Development 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

20. Modification of AB target sites: disruption in protein synthesis

21. Some terminologies. VRE. vancomycin-resistant enterococci. 70% of E. faecium strains in USA. GISA. glycopeptide intermediately susceptible S.aureus. VISA. vancomycin intermediately susceptible S.aureus. VRSA & VRSE. vancomycin-resistant S.aureus and S.epidermidis. (MIC> 32 mcg/ml; 1st clinical case described in 2002 in USA). ESBL producing K.pneumoniae. extended spectrum  -lactamase producing K. pneumoniae. PRSP penicillin-resistant S. pneumoniae

22. Antibiotic resistance

23. Factors promoting drug resistance Exposure to sub-optimal levels of antimicrobial Exposure to microbes carrying resistance genes Inappropriate drug use- Lack of quality control in manufacture or outdated antimicrobial Inadequate surveillance or defective 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

24. Antibiotics and mechanisms of resistance ANTIBIOTICTARGETMOAMECHANISM OF RESISTANCE CELL WALL b-LactamsTranspeptidas es/transglycos ylases (PBPs Blockade of cross linking enzymes in peptidoglycan layer b-Lactamases, PBP mutants VancomycinD-Ala-D-Ala termini of peptidoglycan and of lipid II Sequestration of substrate required for cross linking Reprogrammi ng 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 TetracyclinesPeptidyl transferase Blockade of protein synthesis Drug efflux Aminoglycosi des Peptidyl transferase Blockade of protein synthesis Enzymatic modification of drug Oxazolidinone s Peptidyl transferase Blockade of protein synthesis unknown DNA replication/r epair Fluoroquinolo nes DNA gyraseBlockade of DNA replication Gyrase mutations to drug resistance

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