Antimicrobial agents and mechanisms of action 2

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Antimicrobial agents and mechanisms of action 2

  1. 1. Antimicrobial agents andantimicrobial resistance<br />BLS 206 Lecture<br />Hoza, A . S<br />
  2. 2. Antimicrobial Resistance<br />Relative or complete lack of effect of antimicrobial against a previously susceptible microbe<br />Increase in MIC<br />
  3. 3. Antibiotic Resistance<br />Figure 20.20<br />
  4. 4. Horizontal Gene Transfer<br />A = Transformation; B = Conjugation; C = Transduction<br />
  5. 5. Mechanisms of Antibiotic Resistance<br /><ul><li>Enzymatic destruction of drug
  6. 6. Prevention of penetration of drug
  7. 7. Alteration of drug's target site
  8. 8. Rapid ejection of the drug</li></li></ul><li>Antimicrobial Drug ResistancePrinciples and Definitions<br /><ul><li>Clinical resistance vs actual resistance
  9. 9. Resistance can arise by mutation or by gene transfer (e.g. acquisition of a plasmid)
  10. 10. Resistance provides a selective advantage
  11. 11. Resistance can result from single or multiple steps
  12. 12. Cross resistance vs multiple resistance</li></ul>Cross resistance -- Single mechanism-- closely related antibiotics<br />Multiple resistance -- Multiple mechanisms -- unrelated antibiotics<br />
  13. 13. Antibiotic Selection for Resistant Bacteria<br />
  14. 14. Terminologies<br /><ul><li>Resistant organism
  15. 15. MICs of organism are higher than achieved drug concentrations in tissues
  16. 16. Intermediately resistant
  17. 17. the antibiotic may still be effective but higher doses should be used
  18. 18. Highly resistant
  19. 19. the antibiotic tissue concentrations are likely not to exceed MICs of the microorganisms</li></li></ul><li>Types of resistance<br /><ul><li>Intrinsic or natural resistance
  20. 20. G-neg bacteria are resistant to vancomycin (large molecule)
  21. 21. Tetracyclines are hydrophobic, G-neg bacilli are resistant
  22. 22. Acquired resistance
  23. 23. Mutations (PBP)
  24. 24. Disseminated by plasmids and transposons
  25. 25. Spontaneous mutations</li></li></ul><li>Mechanisms of antibiotic resistance<br />1. Production of enzymes<br />destroying and modifying AB ß-lactamasesAG modifying enzymes<br />2. Decrease of cell membrane permeability<br />3. Active efflux of AB from cell<br />4. Modification of AB target sites<br />
  26. 26. Genetics and spread of drug resistance<br />ViridansStreptococci <br />S.pneumoniae<br />S.EpidermidisS.aureus<br />E.faeciumS.aureus<br />
  27. 27. <ul><li>Transposon.
  28. 28. genes moving from one point to another (jumping genes)
  29. 29. Bacteriophage
  30. 30. virus, infecting bacteria (virus of bacteria)
  31. 31. Integron
  32. 32. slice(s) of DNA, cassette of gene that may be entered into other cell
  33. 33. Plasmid
  34. 34. circular double stranded DNA molecule, located separately of the chromosomal RNA</li></li></ul><li>(1) Mechanisms of resistance<br /><ul><li>Production of enzymes inactivating (destroying) antibiotics
  35. 35. ß-lactamases
  36. 36. Main mechanism of resistance in ß-lactamantibiotics
  37. 37. Penicillin-resistant S.aureus
  38. 38. Ampicillin-resistant E.coli
  39. 39. Production of enzymes modifying antibiotics
  40. 40. Aminoglycosides, chloramphenicol</li></li></ul><li>Resistance mechanisms: inactivating enzymes (2)<br />Degrading enzymes will bind to the antibiotic and essentially degrade it<br />or make the antibiotic inactive<br />Blocking enzymes attach side chains to the antibiotic that inhibit its function.<br />E.g. ß-lactamases<br />
  41. 41. PBP & ß-lactamase<br /><ul><li>Serine proteases (PBP) a metalloenzymes (Zn-binding thiolegroup as coenzyme)
  42. 42. 200 different enzymes e.g. penicillinases, cephalosporinases, ESBL, AmpC
  43. 43. ESBL - extended spectrum ß-lactamases(broad spectrum of activity);</li></ul>encoded in plasmids, can be transferred from organism to organism<br />
  44. 44. Production of ß-lactamases: mechanism of action<br />Examples<br /><ul><li>TEM-1 is a widespread ß- lactamase of Enterobacteriaciae that attacks Penicillin G and narrow spectrum cephalosporins
  45. 45. >50% AmpRE.coliisolates are caused by TEM-1</li></li></ul><li>Antimicrobial Drug ResistanceMechanisms<br />Altered permeability<br />Altered influx<br />Gram negative bacteria<br />
  46. 46. Efflux Mechanisms of resistance<br /><ul><li>Antibiotics are removed via active efflux pump
  47. 47. Universal efflux pump
  48. 48. specific efflux pump
  49. 49. quinolones, tetracyclines, chloramphenicol</li></li></ul><li>Resistance mechanisms: efflux pump <br /><ul><li>The efflux pump is a membrane bound protein that "pumps" the antibiotic out of the bacterial cell</li></li></ul><li>Microbe Library<br />American Society for Microbiology<br />www.microbelibrary.org<br />
  50. 50. Microbe Library<br />American Society for Microbiology<br />www.microbelibrary.org<br />Antimicrobial Drug ResistanceMechanisms<br />Altered permeability<br />Altered efflux<br />tetracycline<br />
  51. 51. Microbe Library<br />American Society for Microbiology<br />www.microbelibrary.org<br />Antimicrobial Drug ResistanceMechanisms<br />Inactivation<br />ß-lactamase<br />Chloramphenicol acetyl transferase<br />
  52. 52. Mechanisms of resistance<br /><ul><li>Modification of target sites
  53. 53. altered PBP (PRSP)
  54. 54. new PBP (MRSE, MRSA)
  55. 55. Modification in ribosomes (macrolideresistant</li></ul>S.pneumoniae)<br />
  56. 56. Microbe Library<br />American Society for Microbiology<br />www.microbelibrary.org<br />Antimicrobial Drug ResistanceMechanisms<br />Altered target site<br />Penicillin binding proteins (penicillins)<br />RNA polymerase (rifampin)<br />30S ribosome (streptomycin)<br />
  57. 57. Modification of AB target sites:<br />disruption in protein synthesis<br />
  58. 58. Important terms among drug<br />resistant microorganisms<br /><ul><li>VRE . vancomycin-resistant enterococci
  59. 59. 70% of E. faecium strains in USA
  60. 60. GISA . glycopeptide intermediately susceptible S.aureus
  61. 61. VISA . vancomycin intermediately susceptible S.aureus
  62. 62. VRSA & VRSE . vancomycin-resistant S.aureus and S.epidermidis
  63. 63. (MIC> 32 mcg/ml; 1st clinical case described in 2002 in USA)
  64. 64. ESBL producing K.pneumoniae . Extended spectrum ß-lactamase producing K. pneumoniae
  65. 65. PRSP penicillin-resistant S. pneumoniae</li></li></ul><li>
  66. 66. Interference with cell wallsynthesis<br />ß-lactam antibiotics: <br />penicillins<br />cephalosporines<br />carbapenems<br />
  67. 67. Alexander Fleming<br />P. chrysogenum<br />(original strain of Fleming)<br />destroy Staphylococcus aureus 1928<br />
  68. 68. <ul><li>ß-lactam structure is presented in red and blue
  69. 69. Side chain is presented in black</li></li></ul><li>Penicillins<br />Carbapenems<br />Cephalosporins<br />
  70. 70. Mechanism of action of ß-lactam antibiotics<br />1ß-lactam ab<br />binds to PBP<br />2. Inhibition of<br />peptidoglycan<br />synthesis<br />3. Cell death<br />
  71. 71. Structure of peptidoglycan<br />ß-lactams inhibit synthesis of crosslinks<br />
  72. 72. Penicillins<br />
  73. 73. Cephalosporins<br />Initially isolated form<br />the mould Cephalosporium<br />Compared with penicillins:<br />More resistant to ß- lactamase hydrolysis<br />Wider antibacterial spectrum Improved PK-properties<br />
  74. 74. Resistance to ß-lactam<br />antibiotics<br />
  75. 75.
  76. 76. Resistance to ß-lactamantibiotics<br /><ul><li>Production of ß-lactamases
  77. 77. Penicillin-resistant S.aureus (>95%) - Synthetic Penicillins
  78. 78. ESBL K.pneumoniae- IV generation cephalosporins, carbapenems
  79. 79. Ampicillin-resistant E.coli – cephalosporins
  80. 80. Changes in the structure of PBP
  81. 81. (altered PBP) Penicillin-resistant S.pneumoniae - larger doses of penicillin
  82. 82. New PBP - MRSA, MRSE . vancomycin</li></li></ul><li>Disruption of bacterial cell wall<br />Glycopeptides<br />vancomycin<br />teicoplanin<br />
  83. 83. Vancomycin: mechanism of action<br /><ul><li>Mechanism - vancomycin inhibits cross linkage between peptidoglycan layers
  84. 84. Vancomycin can bind only to D-Ala-D-Ala and not to D-Ala-D-lac</li></li></ul><li><ul><li>Originally obtained form Streptomycesorientalis
  85. 85. Active only against G+ bacteria (large molecule unable to penetrate outer membrane of G+ bacteria)
  86. 86. Used for treatment of oxacillin resistant G+ infections</li></li></ul><li>Glycopeptide resistance<br /><ul><li>Intrinsic resistance (pentapetide end with D-Ala-D-Lac)
  87. 87. Leuconostoc, Lactobacillus, Pediococcus</li></ul> Or with D-Ala-D-Ser<br /><ul><li>Enetrococcusgallinarum, Enetercoccuscaselliflavus
  88. 88. Acquired resistance
  89. 89. A thickening of the PG layer, and
  90. 90. Modification of the PG termini from D-Ala--D-Ala to D-Ala--D-lactate
  91. 91. Gene (vanA, B, C, D, G, E) is carried on plasmids & may be transferred from organism to organism
  92. 92. Importance
  93. 93. VRE - vancomycin resistant E. faecium, E.faecalis
  94. 94. VISA - vancomycin intermediately resistant S.aureus
  95. 95. GISA - glycopeptide intermediately resistant S.aureus
  96. 96. VRSA - vancomycin resistant S.aureus (MIC> 32 µg/ml; 1st clinical case reported 2002 in US)</li></li></ul><li>Mechanism of Resistance to Vancomycin<br />
  97. 97. Polypeptides<br /><ul><li>Bacitracin (cyclic peptides) is isolated formBacillus licheniformis
  98. 98. Topically applied agent against G+ bacteria
  99. 99. Interferes with the dephoshorylation and recycling of the lipid carrier responsible for moving peptidoglycan precursors
  100. 100. Polymyxin (cyclic polypeptides) derived from Bacillus polymyxa
  101. 101. Interact with the lipopolysaccharides and phospholipids in the outer membrane and thus increase cell permeability
  102. 102. Mostly active against G- bacilli (G+ bacilli do not have outer membrane)</li></li></ul><li>Activity of antibiotics to bacterial cell wall<br />polypeptides<br />ß-lactams<br />glycopeptides<br />G-negative<br />G-positive<br />
  103. 103. Inhibition of protein synthesis<br /><ul><li>Aminoglycosides
  104. 104. Tetracyclines
  105. 105. Oxazolidones
  106. 106. Chloramphenicol
  107. 107. Macrolides
  108. 108. Clindamycin
  109. 109. Streptogramins</li></li></ul><li>Protein synthesis<br />
  110. 110. Substance binding to 30S subunit<br />
  111. 111. Antibiotics that act at the level of protein synthesis initiation<br />
  112. 112. Antibiotics that act at the level of the elongation phase of protein synthesis<br />
  113. 113. Aminoglycosides<br /><ul><li>Consists of aminosugars that are linked through glycosidic rings
  114. 114. Origin
  115. 115. Streptomyces - streptomycin,
  116. 116. neomycin, kanamycin, tobramycin
  117. 117. Micromonospora - gentamicin, Sisomicin
  118. 118. Synthetic derivates
  119. 119. Amikacin = kanamycin
  120. 120. Netilmycin = sisomycin</li></ul>Mainly active against G-negative bacteria<br />Gentamycin<br />
  121. 121. Aminoglycoside: mode of action<br /><ul><li>AG pass through cell wall, cytoplasmic membrane to cytoplasma (mainly of Gbacteria, no penetration through cytoplasmic membrane of strepto- and entrococci)
  122. 122. Bind irreversible to the 30S subunit of bacterial ribosomes and block the attachment of the 50S subunit to the initiation complex
  123. 123. As a result production of aberrant proteins and misreading of RNA occurs</li></li></ul><li>Aminoglycoside: mode of action<br />Passage through cytoplasmic membrane of G- bacteria (no penetration through cytoplasmic membrane of strepto- and enterococci)<br />2. Binding to 30S subunit<br />3. Misreading the codon along mRNA<br />4. Inhibition of protein synthesis<br />
  124. 124. Aminoglycoside resistance<br /><ul><li>Enzymatic modification (common) of the drug
  125. 125. High level resistance
  126. 126. >50 enzymes identified
  127. 127. Genes encoding resistance located in plasmids
  128. 128. Gene transfer occurs across species
  129. 129. Reduced uptake or decreased permeability of bacterial cell wall
  130. 130. Resistance in anaerobes (transport through cytoplasmic membrane depends on anaerobic respiration)
  131. 131. Altered ribosome binding sites (rare)
  132. 132. Microbes bind to multiple sites
  133. 133. Low level resistance</li></li></ul><li>Tetracyclines<br /><ul><li>Origin
  134. 134. Tetracyclin, oxytetracyclin isolated from Streptomyces
  135. 135. Minocyclin, doxycyclin are synthetic
  136. 136. Broad spectrum bacteriostatic antibiotics
  137. 137. Antibacterial spectrum similar to macrolides(incl. Clamydia, Mycoplasma, Rickettsia)
  138. 138. Resistance (widespread)
  139. 139. Energy dependent efflux pump (most common)
  140. 140. Alteration of ribosomal target (ribosome protection)
  141. 141. Enzymatic change</li></li></ul><li>Tetracyclines<br /><ul><li>The tetracyclinesblock 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</li></li></ul><li>Oxazolidones: linezolid<br /><ul><li>Newest class of antibiotics; completely synthetic
  142. 142. Narrow spectrum of activity (G+ bacteria, includingVRE, MRSA)
  143. 143. G-neg bacteria resistant due to efflux pump
  144. 144. Mode of action: unique mechanism among antibiotics; interferes with the initiation complex at the 50S ribosome subunit (V domain of 23S rRNA)
  145. 145. Resistance confers to mutation at 23S rRNA
  146. 146. Resistance is rare; cross-resistance unlikely because 23S rRNA is encoded by several genes</li></li></ul><li>Oxazolidones: mode of action<br /><ul><li>Inhibit the formation of an initiation complex by binding to the 50S ribosomal subunit (domain V of the 23S rRNA), disrupting the preliminary phases of protein synthesis</li></li></ul><li>Chloramphenicol<br /><ul><li>Binds irreversible to peptidyltransferase component of 50S ribosome and blocks peptide elongation, thus interferes with protein synthesis
  147. 147. Bacteriostatic antibiotic with broad spectrum of antibacterial activity
  148. 148. Interferes with the protein synthesis of bone marrow cells causing aplastic anaemia
  149. 149. Limited clinical use in Western world due to side Effect
  150. 150. Resistance is associated with producing</li></ul>acetyltransferase which catalyses acetylation of 3-hydroxy group of chloramphenicol<br />
  151. 151. Macrolides (1)<br /><ul><li>Erythromycin was derived from Streptomyceserythreus
  152. 152. The basic structure is a lactone ring
  153. 153. 14-membered lactone ring . erthromycin, clarithromycin, roxithromycin, telithromyin (ketolide)
  154. 154. 15-membered lactone ring . Azithromycin
  155. 155. 16-membered lactone ring . spiramycin, josamycin
  156. 156. Acitivity .
  157. 157. broad spectrum G+ bacteria and some G- bacteria including Chlamydia, Mycoplasma, Legionella, Rickettsia, Neisseria
  158. 158. Azithromycin, Clarithromycin active against some mycobacteria</li></li></ul><li>Macrolides: mode of action<br />Blocking Translation during Bacterial Protein<br />Synthesis<br />erythromycin<br /><ul><li>The macrolides bind reversibly to the 50S subunit.
  159. 159. They can inhibit elongation of the protein by the peptidyltransferase, the enzyme that forms peptide bonds between the amino acids.</li></li></ul><li>Mode of Action of Macrolides in Blocking<br />Translation during Bacterial Protein<br />Synthesis<br /><ul><li>The macrolides bind reversibly to the 50S subunit.
  160. 160. They can inhibit elongation of the protein by blocking the translocation of the ribosome to the next codon on mRNA</li></li></ul><li>Macrolide resistance<br /><ul><li>Resistance
  161. 161. Intrinsic resistance- hydrophobic macrolides have low permeability through outer membrane (G- bacilli)
  162. 162. Acquired resistance
  163. 163. Ribosomal modification
  164. 164. Efflux pump
  165. 165. Enzyme inactivation</li></li></ul><li>Clindamycin, lincomycin<br /><ul><li>Family of lincosamide antibiotics originally isolated from Streptomyceslincolnensis
  166. 166. Mode of action: bind 50S ribosome subunit and blocks protein elongation
  167. 167. Resistance is related to 23S ribosomal RNA Methylation
  168. 168. Active against staphylococci and G-ve anaerobic bacilli.
  169. 169. No activity against aerobic</li></li></ul><li>Antimicrobial Drug ResistanceMechanisms<br />Replacement of a sensitive pathway<br />Acquisition of a resistant enzyme (sulfonamides, trimethoprim)<br />
  170. 170. Molecular Drug Susceptibility Testing<br /><ul><li>Genotypic methods: the drug target and nature of the gene mutation are known
  171. 171. Usually molecular amplification of target DNA or RNA followed by some means of detecting mutation in the product.</li></li></ul><li>Molecular methods of drug susceptibility testing <br />1. Sequencing<br />Universal and reliable method<br />Expensive, time-consuming and not suitable for everyday routine testing<br />Applied as reference method to verify results of other tests.<br />
  172. 172.
  173. 173. 2. PCR-based methods<br />PCR-Single Strand Conformation Polymorphism (PCR-SSCP) <br />Mutations cause alterations in conformation of single-strand DNA fragments and it is registered in non-denaturizing PAGE<br />
  174. 174. Other molecular methods of drug susceptibility testing:<br />Real-Time fluorescent PCR combines amplification and detection: minimises amplicon contamination<br />Molecular <br />beacons<br />
  175. 175. PCR+hybridization<br />Based on amplification of fragments of genes responsible for drug resistance development follwed by hybridization with oligonucleotide probes immobilized on membranes;<br />Both commercial kits and in-house macro-arrays have been reported to demonstrate high sensitivity and specificity<br />
  176. 176. Molecular tests for the detection of resistance to RIF and INH<br />GenoType® MTBDRplus test procedure<br />
  177. 177. Reaction zones of GenoType®MTBDRplus (examples)<br />
  178. 178. What Factors Promote Antimicrobial Resistance?<br />Exposure to sub-optimal levels of antimicrobial<br />Exposure to microbes carrying resistance genes<br />
  179. 179. Inappropriate Antimicrobial Use<br />Prescription not taken correctly<br />Antibiotics for viral infections<br />Antibiotics sold without medical supervision<br />Spread of resistant microbes in hospitals due to lack of hygiene<br />
  180. 180. Inappropriate Antimicrobial Use<br />Lack of quality control in manufacture or outdated antimicrobial<br />Inadequate surveillance or defective susceptibility assays<br />Poverty or war<br />Use of antibiotics in foods<br />
  181. 181. Antibiotics in Foods<br />Antibiotics are used in animal feeds and sprayed on plants to prevent infection and promote growth<br />Multi drug-resistant Salmonella typhi has been found in 4 states in 18 people who ate beef fed antibiotics<br />
  182. 182. Consequences of Antimicrobial Resistance<br />Infections resistant to available antibiotics<br />Increased cost of treatment<br />
  183. 183.
  184. 184. MRSA “mer-sah”<br />Methicillin-Resistant Staphylococcus aureus<br />Most frequent nosocomial (hospital-acquired) pathogen<br />Usually resistant to several other antibiotics<br />
  185. 185. Proposals to Combat Antimicrobial Resistance<br />Speed development of new antibiotics<br />Track resistance data nationwide<br />Restrict antimicrobial use<br />Direct observed dosing (TB) <br />Use more narrow spectrum antibiotics<br />Use antimicrobial cocktails<br />
  186. 186. Ecology of Antimicrobial Resistance<br />
  187. 187.
  188. 188. The Future of Chemotherapeutic Agents<br />Antimicrobial peptides<br />Broad spectrum antibiotics from plants and animals<br />Squalamine (sharks)<br />Protegrin (pigs)<br />Magainin (frogs)<br />
  189. 189. The Future of Chemotherapeutic Agents<br />Antisense agents<br />Complementary DNA or peptide nucleic acids that binds to a pathogen's virulence gene(s) and prevents transcription<br />
  190. 190. DID U KNOW THAT>>>>>>>>><br />Doctors are men who prescribe medicines of which they know little: to cure diseases of which they know less: in human being of who they know nothing’’<br />May Allah Bless U in Your Exams!!<br />

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