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

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  • 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 membranesOuter membrane permeabilityInner membrane permeability

    Rapid ejection of the drug [efflux] or reduced drug influx.By pass of antibiotic inhibition.Alteration of target sitesAltered ribosomal target sitesAltered cell wall precursor targetsAltered target enzymes

  • Molecular genetics of antibiotic resistanceGenetic variability is essential for microbial evolution. It may occur in a variety of ways :-Micro evolutionary changes by point mutations-in nucleotide base pairMicro evolutionary changes [whole scale changes] like-InversionsDuplicationsDeletionsTranspositionAcquisition 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 sequencingClass A MWT 29000-Preferentially hydrolyze penicillins e.g. TEM-1 prevalent in many gram negClass B-metalloenzymes have a zinc binding thiol group required for beta lactamase activityClass C mwt 39000 mainly cephalosporinasesClass D-oxacillin hydrolyzing enzymesMany beta lactamases are plasmid mediated all are produced constitutively there are 6 main groupsThose that hydrolyze benzylpenicillin

  • Beta lactamasesThose that hydrolyze oxacillin and related penicillinsCarbenicillinasesThose that break extended spectrum beta lactams like aztreonamThose that break down oxyimino B lactamsCarbapenemases-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/chromosomalErythromycin 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.

  • ENZYMESDegrading enzymes will bind tothe antibiotic and essentially degrade itor make the antibiotic inactiveBlocking enzymes attach side chains tothe antibiotic that inhibit its function.E.g. -lactamases

  • Alteration of bacterial membranesOuter membrane permeabilityouter 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 sensitiveRestriction 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 mediatedQuinolones-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 resistance

    ANTIBIOTICTARGETMOAMECHANISMOF RESISTANCECELL WALLb-LactamsTranspeptidases/transglycosylases (PBPsBlockade of cross linking enzymes in peptidoglycan layerb-Lactamases, PBP mutantsVancomycinD-Ala-D-Ala termini of peptidoglycan and of lipid IISequestration of substrate required for cross linkingReprogramming of D-Ala-D-Ala to D-Ala-D-Lac od D-Ala D-serPROTEIN SYNTHESISMacrolides of the erythromycin classPeptidyl transferase, centre of the ribosomeBlockade of protein synthesisrRNA methylation, drug effluxTetracyclinesPeptidyl transferaseBlockade of protein synthesisDrug effluxAminoglycosidesPeptidyl transferaseBlockade of protein synthesisEnzymatic modification of drugOxazolidinonesPeptidyl transferaseBlockade of protein synthesisunknownDNA replication/repairFluoroquinolonesDNA gyraseBlockade of DNA replicationGyrase mutations to drug resistance

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