Antimicrobial agents and mechanisms of action 2


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Antimicrobial agents and mechanisms of action 2

  1. 1.  Relative or complete lack of effect of antimicrobial against a previously susceptible microbe Increase in MIC
  2. 2. Figure 20.20
  3. 3. Horizontal Gene TransferA = Transformation; B = Conjugation; C = Transduction
  4. 4. • Enzymatic destruction of drug• Prevention of penetration of drug• Alteration of drugs target site• Rapid ejection of the drug
  5. 5.  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
  6. 6. 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
  7. 7. 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
  8. 8. 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
  9. 9. Genetics and spread of drug resistance Viridans Streptococci S.pneumoniae S.Epidermidis S.aureus E.faecium S.aureus
  10. 10. 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
  11. 11. (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
  12. 12. 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
  13. 13. 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
  14. 14. 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
  15. 15.  Altered permeability › Altered influx  Gram negative bacteria
  16. 16. Efflux Mechanisms of resistanceAntibiotics are removed via active efflux pumpUniversal efflux pumpspecific efflux pump quinolones, tetracyclines, chloramphenicol
  17. 17. Resistance mechanisms: efflux pumpThe efflux pump is a membrane bound protein that"pumps" the antibiotic out of the bacterial cell
  18. 18. Microbe LibraryAmerican Society for Microbiology
  19. 19.  Altered permeability › Altered efflux  tetracycline Microbe Library American Society for Microbiology
  20. 20.  Inactivation › ß-lactamase › Chloramphenicol acetyl transferase Microbe Library American Society for Microbiology
  21. 21. Mechanisms of resistanceModification of target sitesaltered PBP (PRSP)new PBP (MRSE, MRSA)Modification in ribosomes (macrolideresistantS.pneumoniae)
  22. 22.  Altered target site › Penicillin binding proteins (penicillins) › RNA polymerase (rifampin) Microbe Library American Society for › 30S ribosome Microbiology (streptomycin)
  23. 23. Modification of AB target sites:disruption in protein synthesis
  24. 24. 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
  25. 25. ß-lactam antibiotics: penicillinscephalosporines carbapenems
  26. 26. Alexander Fleming P. chrysogenum (original strain of Fleming) destroy Staphylococcus aureus 1928
  27. 27. ß-lactam structure is presented in red and blueSide chain is presented in black
  28. 28. PenicillinsCarbapenemsCephalosporins
  29. 29. Mechanism of action of ß-lactam antibiotics1ß-lactam abbinds to PBP2. Inhibition ofpeptidoglycansynthesis3. Cell death
  30. 30. Structure of peptidoglycanß-lactams inhibit synthesis of crosslinks
  31. 31. Penicillins
  32. 32. Cephalosporins Initially isolated form the mould Cephalosporium Compared with penicillins: More resistant to ß- lactamase hydrolysis Wider antibacterial spectrum Improved PK-properties
  33. 33. Resistance to ß-lactam antibiotics
  34. 34. 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
  35. 35. Disruption of bacterial cell wallGlycopeptides vancomycin teicoplanin
  36. 36. 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
  37. 37.  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
  38. 38.  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)
  39. 39. Mechanism of Resistance to Vancomycin
  40. 40.  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)
  41. 41. Activity of antibiotics to bacterial cell wallpolypeptides ß-lactams glycopeptides G-negative G-positive
  42. 42. Inhibition of protein synthesis Aminoglycosides Tetracyclines Oxazolidones Chloramphenicol Macrolides Clindamycin Streptogramins
  43. 43. Protein synthesis
  44. 44. Substance binding to 30S subunit
  45. 45. Antibiotics that act at the level of protein synthesis initiation
  46. 46. Antibiotics that act at the level of theelongation phase of protein synthesis
  47. 47.  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
  48. 48. 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
  49. 49. 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
  50. 50. 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
  51. 51. 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
  52. 52. 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
  53. 53. 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
  54. 54. 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
  55. 55. 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
  56. 56. 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
  57. 57. 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.
  58. 58. 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
  59. 59. Macrolide resistanceResistanceIntrinsic resistance- hydrophobic macrolideshave low permeability through outer membrane(G- bacilli)Acquired resistance Ribosomal modification Efflux pump Enzyme inactivation
  60. 60. 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
  61. 61.  Replacement of a sensitive pathway › Acquisition of a resistant enzyme (sulfonamides, trimethoprim)
  62. 62. 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.
  63. 63. Molecular methods of drug susceptibility testing 1. SequencingUniversal andreliable methodExpensive, time-consuming and notsuitable foreveryday routinetestingApplied asreference method toverify results of othertests.
  64. 64. 2. PCR-based methodsPCR-Single StrandConformationPolymorphism (PCR-SSCP)Mutations causealterations inconformation of single-strand DNA fragmentsand it is registered innon-denaturizing PAGE
  65. 65. Other molecular methods of drug susceptibility testing: Real-Time fluorescent PCRMolecular combines amplification andbeacons detection: minimises amplicon contamination
  66. 66. 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
  67. 67. Molecular tests for the detection of resistance to RIF and INH GenoType® MTBDRplus test procedure
  68. 68. Reaction zones of GenoType® MTBDRplus (examples)
  69. 69.  Exposure to sub-optimal levels of antimicrobial Exposure to microbes carrying resistance genes
  70. 70.  Prescription not taken correctly Antibiotics for viral infections Antibiotics sold without medical supervision Spread of resistant microbes in hospitals due to lack of hygiene
  71. 71.  Lack of quality control in manufacture or outdated antimicrobial Inadequate surveillance or defective susceptibility assays Poverty or war Use of antibiotics in foods
  72. 72.  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
  73. 73.  Infections resistant to available antibiotics Increased cost of treatment
  74. 74.  Methicillin-Resistant Staphylococcus aureus Most frequent nosocomial (hospital-acquired) pathogen Usually resistant to several other antibiotics
  75. 75.  Speed development of new antibiotics Track resistance data nationwide Restrict antimicrobial use Direct observed dosing (TB) Use more narrow spectrum antibiotics Use antimicrobial cocktails
  76. 76. Ecology of Antimicrobial Resistance
  77. 77.  Antimicrobial peptides › Broad spectrum antibiotics from plants and animals  Squalamine (sharks)  Protegrin (pigs)  Magainin (frogs)
  78. 78.  Antisense agents › Complementary DNA or peptide nucleic acids that binds to a pathogens virulence gene(s) and prevents transcription
  79. 79. DID U KNOW T HAT>>>>>>>>>May Allah Bless U in Your Exams!!