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Antimicrobial agents and mechanisms of action 2
 

Antimicrobial agents and mechanisms of action 2

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    Antimicrobial agents and mechanisms of action 2 Antimicrobial agents and mechanisms of action 2 Presentation Transcript

    •  Relative or complete lack of effect of antimicrobial against a previously susceptible microbe Increase in MIC
    • Figure 20.20
    • Horizontal Gene TransferA = Transformation; B = Conjugation; C = Transduction
    • • Enzymatic destruction of drug• Prevention of penetration of drug• Alteration of drugs target site• Rapid ejection of the drug
    •  Clinical resistance vs actual resistance Resistance can arise by mutation or by gene transfer (e.g. acquisition of a plasmid) Resistance provides a selective advantage Resistance can result from single or multiple steps Cross resistance vs multiple resistance › Cross resistance -- Single mechanism-- closely related antibiotics › Multiple resistance -- Multiple mechanisms -- unrelated antibiotics
    • TerminologiesResistant organism MICs of organism are higher than achieved drug concentrations in tissuesIntermediately resistant the antibiotic may still be effective but higher doses should be usedHighly resistant the antibiotic tissue concentrations are likely not to exceed MICs of the microorganisms
    • Types of resistanceIntrinsic or natural resistanceG-neg bacteria are resistant to vancomycin (largemolecule)Tetracyclines are hydrophobic, G-neg bacilli areresistantAcquired resistanceMutations (PBP)Disseminated by plasmids and transposonsSpontaneous mutations
    • Mechanisms of antibiotic resistance 1. Production of enzymes destroying and modifying AB ß-lactamases AG modifying enzymes 2. Decrease of cell membrane permeability 3. Active efflux of AB from cell 4. Modification of AB target sites
    • Genetics and spread of drug resistance Viridans Streptococci S.pneumoniae S.Epidermidis S.aureus E.faecium S.aureus
    • Transposon .genes moving from one point to another (jumping genes)Bacteriophagevirus, infecting bacteria (virus of bacteria)Integronslice(s) of DNA, cassette of gene that may be entered intoother cellPlasmidcircular double stranded DNA molecule, located separatelyof the chromosomal RNA
    • (1) Mechanisms of resistanceProduction of enzymes inactivating (destroying)antibioticsß-lactamasesMain mechanism of resistance in ß-lactamantibioticsPenicillin-resistant S.aureusAmpicillin-resistant E.coliProduction of enzymes modifying antibioticsAminoglycosides, chloramphenicol
    • Resistance mechanisms: inactivating enzymes (2)Degrading enzymes will bind to the Blocking enzymes attach side chainsantibiotic and essentially degrade it to the antibiotic that inhibit its function.or make the antibiotic inactive E.g. ß-lactamases
    • PBP & ß-lactamaseSerine proteases (PBP) a metalloenzymes (Zn-binding thiole group ascoenzyme)200 different enzymes e.g. penicillinases, cephalosporinases, ESBL,AmpCESBL - extended spectrum ß-lactamases (broad spectrum of activity);encoded in plasmids, can be transferred from organism to organism
    • Production of ß-lactamases: mechanism of action Examples TEM-1 is a widespread ß- lactamase of Enterobacteriaciae that attacks Penicillin G and narrow spectrum cephalosporins >50% AmpR E.coli isolates are caused by TEM-1
    •  Altered permeability › Altered influx  Gram negative bacteria
    • Efflux Mechanisms of resistanceAntibiotics are removed via active efflux pumpUniversal efflux pumpspecific efflux pump quinolones, tetracyclines, chloramphenicol
    • Resistance mechanisms: efflux pumpThe efflux pump is a membrane bound protein that"pumps" the antibiotic out of the bacterial cell
    • Microbe LibraryAmerican Society for Microbiology www.microbelibrary.org
    •  Altered permeability › Altered efflux  tetracycline Microbe Library American Society for Microbiology www.microbelibrary.org
    •  Inactivation › ß-lactamase › Chloramphenicol acetyl transferase Microbe Library American Society for Microbiology www.microbelibrary.org
    • Mechanisms of resistanceModification of target sitesaltered PBP (PRSP)new PBP (MRSE, MRSA)Modification in ribosomes (macrolideresistantS.pneumoniae)
    •  Altered target site › Penicillin binding proteins (penicillins) › RNA polymerase (rifampin) Microbe Library American Society for › 30S ribosome Microbiology (streptomycin) www.microbelibrary.org
    • Modification of AB target sites:disruption in protein synthesis
    • Important terms among drug resistant microorganisms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 ß-lactamaseproducing K. pneumoniaePRSP penicillin-resistant S. pneumoniae
    • ß-lactam antibiotics: penicillinscephalosporines carbapenems
    • Alexander Fleming P. chrysogenum (original strain of Fleming) destroy Staphylococcus aureus 1928
    • ß-lactam structure is presented in red and blueSide chain is presented in black
    • PenicillinsCarbapenemsCephalosporins
    • Mechanism of action of ß-lactam antibiotics1ß-lactam abbinds to PBP2. Inhibition ofpeptidoglycansynthesis3. Cell death
    • Structure of peptidoglycanß-lactams inhibit synthesis of crosslinks
    • Penicillins
    • Cephalosporins Initially isolated form the mould Cephalosporium Compared with penicillins: More resistant to ß- lactamase hydrolysis Wider antibacterial spectrum Improved PK-properties
    • Resistance to ß-lactam antibiotics
    • Resistance to ß-lactam antibioticsProduction of ß-lactamases Penicillin-resistant S.aureus (>95%) - Synthetic Penicillins ESBL K.pneumoniae - IV generation cephalosporins, carbapenems Ampicillin-resistant E.coli – cephalosporinsChanges in the structure of PBP (altered PBP) Penicillin-resistant S.pneumoniae - larger doses of penicillin New PBP - MRSA, MRSE . vancomycin
    • Disruption of bacterial cell wallGlycopeptides vancomycin teicoplanin
    • Vancomycin: mechanism of actionMechanism - vancomycin inhibits cross linkage betweenpeptidoglycan layersVancomycin can bind only to D-Ala-D-Ala and not to D-Ala-D-lac
    •  Originally obtained form Streptomyces orientalis Active only against G+ bacteria (large molecule unable to penetrate outer membrane of G+ bacteria) Used for treatment of oxacillin resistant G+ infections
    •  Intrinsic resistance (pentapetide end with D-Ala-D-Lac)  Leuconostoc, Lactobacillus, Pediococcus Or with D-Ala-D-Ser  Enetrococcus gallinarum, Enetercoccus caselliflavus Acquired resistance A thickening of the PG layer, and Modification of the PG termini from D-Ala--D-Ala to D-Ala--D-lactate Gene (vanA, B, C, D, G, E) is carried on plasmids & may be transferred from organism to organism Importance  VRE - vancomycin resistant E. faecium, E.faecalis  VISA - vancomycin intermediately resistant S.aureus  GISA - glycopeptide intermediately resistant S.aureus  VRSA - vancomycin resistant S.aureus (MIC> 32 µg/ml; 1st clinical case reported 2002 in US)
    • Mechanism of Resistance to Vancomycin
    •  Bacitracin (cyclic peptides) is isolated form Bacillus licheniformis  Topically applied agent against G+ bacteria  Interferes with the dephoshorylation and recycling of the lipid carrier responsible for moving peptidoglycan precursors Polymyxin (cyclic polypeptides) derived from Bacillus polymyxa  Interact with the lipopolysaccharides and phospholipids in the outer membrane and thus increase cell permeability  Mostly active against G- bacilli (G+ bacilli do not have outer membrane)
    • Activity of antibiotics to bacterial cell wallpolypeptides ß-lactams glycopeptides G-negative G-positive
    • Inhibition of protein synthesis Aminoglycosides Tetracyclines Oxazolidones Chloramphenicol Macrolides Clindamycin Streptogramins
    • Protein synthesis
    • Substance binding to 30S subunit
    • Antibiotics that act at the level of protein synthesis initiation
    • Antibiotics that act at the level of theelongation phase of protein synthesis
    •  Consists of aminosugars that are linked through glycosidic rings  Origin  Streptomyces - streptomycin,  neomycin, kanamycin, tobramycin  Micromonospora - gentamicin, Sisomicin  Synthetic derivates  Amikacin = kanamycin  Netilmycin = sisomycin Mainly active against G-negative bacteriaGentamycin
    • Aminoglycoside: mode of action AG pass through cell wall, cytoplasmic membrane to cytoplasma (mainly of Gbacteria, no penetration through cytoplasmic membrane of strepto- and entrococci) Bind irreversible to the 30S subunit of bacterial ribosomes and block the attachment of the 50S subunit to the initiation complex As a result production of aberrant proteins and misreading of RNA occurs
    • Aminoglycoside: mode of action1. Passage through cytoplasmic membrane of G- bacteria (no penetration through cytoplasmic membrane of strepto- and enterococci)2. Binding to 30S subunit3. Misreading the codon along mRNA4. Inhibition of protein synthesis
    • Aminoglycoside resistanceEnzymatic modification (common) of the drug  High level resistance >50 enzymes identified Genes encoding resistance located in plasmids Gene transfer occurs across species Reduced uptake or decreased permeability of bacterialcell wall  Resistance in anaerobes (transport through cytoplasmic membrane depends on anaerobic respiration)Altered ribosome binding sites (rare) Microbes bind to multiple sites Low level resistance
    • TetracyclinesOrigin Tetracyclin, oxytetracyclin isolated from Streptomyces  Minocyclin, doxycyclin are synthetic Broad spectrum bacteriostatic antibioticsAntibacterial spectrum similar to macrolides (incl. Clamydia,Mycoplasma, Rickettsia)Resistance (widespread)Energy dependent efflux pump (most common)Alteration of ribosomal target (ribosome protection)Enzymatic change
    • Tetracyclines The tetracyclines block bacterial translation by binding reversibly to the 30S subunit and distorting it in such a way that the anticodons of the charged tRNAs cannot align properly with the codons of the mRNA
    • Oxazolidones: linezolidNewest class of antibiotics; completely syntheticNarrow spectrum of activity (G+ bacteria, includingVRE,MRSA) G-neg bacteria resistant due to efflux pumpMode of action: unique mechanism among antibiotics;interferes with the initiation complex at the 50S ribosomesubunit (V domain of 23S rRNA)Resistance confers to mutation at 23S rRNAResistance is rare; cross-resistance unlikely because 23SrRNA is encoded by several genes
    • Oxazolidones: mode of actionInhibit the formation of an initiation complex by binding to the 50Sribosomal subunit (domain V of the 23S rRNA), disrupting the preliminaryphases of protein synthesis
    • ChloramphenicolBinds irreversible to peptidyl transferase component of 50Sribosome and blocks peptide elongation, thus interferes withprotein synthesisBacteriostatic antibiotic with broad spectrum of antibacterialactivityInterferes with the protein synthesis of bone marrow cellscausing aplastic anaemiaLimited clinical use in Western world due to side EffectResistance is associated with producingacetyltransferase which catalyses acetylation of 3-hydroxygroup of chloramphenicol
    • Macrolides (1)Erythromycin was derived from Streptomyces erythreusThe basic structure is a lactone ring14-membered lactone ring . erthromycin, clarithromycin, roxithromycin,telithromyin (ketolide)15-membered lactone ring . Azithromycin16-membered lactone ring . spiramycin, josamycinAcitivity . broad spectrum G+ bacteria and some G- bacteria including Chlamydia, Mycoplasma, Legionella, Rickettsia, Neisseria Azithromycin, Clarithromycin active against some mycobacteria
    • Macrolides: mode of action Blocking Translation during Bacterial Protein Synthesis erythromycinThe macrolides bind reversibly to the 50S subunit.They can inhibit elongation of the protein by the peptidyltransferase, theenzyme that forms peptide bonds between the amino acids.
    • Mode of Action of Macrolides in Blocking Translation during Bacterial Protein Synthesis The macrolides bind reversibly to the 50S subunit. They can inhibit elongation of the protein by blocking the translocation of the ribosome to the next codon on mRNA
    • Macrolide resistanceResistanceIntrinsic resistance- hydrophobic macrolideshave low permeability through outer membrane(G- bacilli)Acquired resistance Ribosomal modification Efflux pump Enzyme inactivation
    • Clindamycin, lincomycinFamily of lincosamide antibiotics originally isolated fromStreptomyces lincolnensisMode of action: bind 50S ribosome subunit and blocksprotein elongationResistance is related to 23S ribosomal RNA MethylationActive against staphylococci and G-ve anaerobic bacilli.No activity against aerobic
    •  Replacement of a sensitive pathway › Acquisition of a resistant enzyme (sulfonamides, trimethoprim)
    • Molecular Drug Susceptibility Testing• Genotypic methods: the drug target and nature of the gene mutation are known• Usually molecular amplification of target DNA or RNA followed by some means of detecting mutation in the product.
    • Molecular methods of drug susceptibility testing 1. SequencingUniversal andreliable methodExpensive, time-consuming and notsuitable foreveryday routinetestingApplied asreference method toverify results of othertests.
    • 2. PCR-based methodsPCR-Single StrandConformationPolymorphism (PCR-SSCP)Mutations causealterations inconformation of single-strand DNA fragmentsand it is registered innon-denaturizing PAGE
    • Other molecular methods of drug susceptibility testing: Real-Time fluorescent PCRMolecular combines amplification andbeacons detection: minimises amplicon contamination
    • PCR+hybridizationBased on amplification of fragments of genesresponsible for drug resistance developmentfollwed by hybridization with oligonucleotideprobes immobilized on membranes;Both commercial kits and in-house macro-arrays have been reported to demonstratehigh sensitivity and specificity
    • Molecular tests for the detection of resistance to RIF and INH GenoType® MTBDRplus test procedure
    • Reaction zones of GenoType® MTBDRplus (examples)
    •  Exposure to sub-optimal levels of antimicrobial Exposure to microbes carrying resistance genes
    •  Prescription not taken correctly Antibiotics for viral infections Antibiotics sold without medical supervision Spread of resistant microbes in hospitals due to lack of hygiene
    •  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
    •  Infections resistant to available antibiotics Increased cost of treatment
    •  Methicillin-Resistant Staphylococcus aureus Most frequent nosocomial (hospital-acquired) pathogen Usually resistant to several other antibiotics
    •  Speed development of new antibiotics Track resistance data nationwide Restrict antimicrobial use Direct observed dosing (TB) Use more narrow spectrum antibiotics Use antimicrobial cocktails
    • Ecology of Antimicrobial Resistance
    •  Antimicrobial peptides › Broad spectrum antibiotics from plants and animals  Squalamine (sharks)  Protegrin (pigs)  Magainin (frogs)
    •  Antisense agents › Complementary DNA or peptide nucleic acids that binds to a pathogens virulence gene(s) and prevents transcription
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