Lectures%2011%20 12%20 pathogen%20identification


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Lectures%2011%20 12%20 pathogen%20identification

  1. 1. Chapter 10 Prokaryote Identification <ul><li>Microscopy </li></ul><ul><ul><li>Chapter 3 </li></ul></ul><ul><li>Other Phenotypic Characteristics </li></ul><ul><ul><li>Chapters 4, 17 </li></ul></ul><ul><li>Genomic Identification </li></ul><ul><li>Strain Differences </li></ul>
  2. 2. Prokaryote Identification
  3. 3. Microscopy 1670s Antony van Leeuwenhoek
  4. 4. Microscopy
  5. 5. Microscopy: Stains
  6. 6. Gram Stain
  7. 7. Gram Stain: Clinical Specimen
  8. 9. Acid-Fast Stain Mycobacterium species Cell wall composition prevents dye uptake Red dye added heated over boiling water or concentrated dye added Rinse Decolorizing acid-alcohol wash Blue counterstain
  9. 10. Capsule Stain / Negative Stain Cryptococcus neoformans Capsule (glycocalyx) Gel-like layer for protection or attachment Distinct and gelatinous Stains poorly :
  10. 11. Endospore Stain Green dye added heated gently Rinse Red counterstain Bacillus and Clostridium species Endospores resistant to Gram stain
  11. 12. Fluorescent Dyes and Tags Living: green Dead: red Auramine Mycobacterium cell wall <ul><li>Metabolic activity alters fluorescent properties of a dye </li></ul><ul><li>Fluorescent dyes bind to pathogen-specific compound </li></ul><ul><li>Dye-conjugated antibodies: recognize pathogen-specific antigens </li></ul>S. pyogenes protein
  12. 13. Identification by Microscopy
  13. 14. Prokaryote Identification
  14. 15. Enrichment Culture
  15. 16. Streak Plate
  16. 17. Types of Growth Media
  17. 18. Complex vs. Defined Media
  18. 19. Selective & Differential Media Selective Media Inhibits growth of all but target organism Differential Media Target bacteria changes in a recognizable way MacConkey Agar Isolate/identify Gram negative rods from the intestine Selective: bile salts/crystal violet dye inhibit growth of all but Gram negative rods Differential: lactose fermenting bacteria produce pink colonies (pH indicator)
  19. 20. Differential Media: Blood Agar Differential media detects bacteria that lyse red blood cells (hemolysin production) Red blood cell lysis generates a zone of clearing (a) clear zone = beta hemolysis = S. pyogenes (b) greenish zone = alpha hemolysis = non-pathogenic Streptococcus
  20. 21. Prokaryote Identification
  21. 22. Biochemical Tests
  22. 23. Biochemical Tests Catalase test bubbles = catalase activity Sugar fermentation test Gas and acid production = fermentation activity Urease test Gas and ammonia (base) production = urease activity
  23. 24. Dichotomous Key
  24. 25. Biochemical Tests: Commercial Arrays API strip test add liquid culture to dehydrated media Enterotube Innoculation from rod drawn through liquid compartments Other formats: 96 well plates, Miniaturized systems
  25. 26. Prokaryote Identification <ul><li>Serology: </li></ul><ul><li>Recognition of host antibody produced in response to bacterial antigen </li></ul><ul><li>Direct recognition of bacterial antigen </li></ul>
  26. 27. Serology Seronegative No specific antibodies to a pathogen because no prior exposure Seropositive Production of specific antibodies to a pathogen Seroconversion Switch from seronegative to positive Serology In vitro study of antibody-antigen interactions Serum Fluid portion of the blood that remains after blood clots
  27. 28. Serology: Indirect ELISA ELISA Enzyme-Linked Immunosorbent Assay Bacterial antigen affixed to substrate Patient serum added if antibody vs. antigen present, it will stay in the well Peroxidase-conjugated anti-human IgG antibody added color reaction = antibody in serum recognizes antigen Testing for antibody production in response to antigen Often used as HIV test (anti-gp120, anti-p24 antibodies) “ Indirect” because second antibody required for signal
  28. 29. Western Blot Analysis To confirm positive HIV ELISA result HIV proteins run on SDS-PAGE gel protein separation based on size Separated proteins transferred to membrane Test serum added Enzyme-labeled secondary antibody added
  29. 30. Direct ELISA Known antibodies vs. antigen attached to substrate Patient sample added if bacterial antigen is present, it will stay in the well Peroxidase-conjugated antibody added Direct testing for presence of antigen “ Direct” because antibody directly conjugated to peroxidase
  30. 31. ELISA plates 96 well format for ELISA, other high-throughput protocols Antibody titer (concentration): serial dilutions of serum tested reciprocal of last dilution with positive reaction = titer (1:256 dilution = 256 titer)
  31. 32. Fatty Acid Analysis Bacteria differ in the type and quantity of membrane fatty acids Fatty acids removed and converted to methyl ester form (Fatty Acid Methyl Ester) Gas chromatography analysis
  32. 33. Prokaryote Identification
  33. 34. Nucleic Acid Hybridization
  34. 35. Polymerase Chain Reaction Nucleotide sequence must be known for primer design
  35. 36. Polymerase Chain Reaction
  36. 37. Polymerase Chain Reaction 30 cycles = billion-fold amplification
  37. 38. Prokaryote Identification: PCR Could also use DNA probe instead of gel Requires knowledge of a specific nucleotide sequence from the target pathogen
  38. 39. rRNA Sequencing Ribosomes protein + rRNA components S = Svedberg unit measure of sedimentation mRNA translation & protein synthesis important / conserved process Sequence rRNA directly Sequence DNA that codes for rRNA Usually 16S rRNA sequenced
  39. 40. Strain Differences E. coli strains K-12 EPEC EHEC STEC ETEC EIEC EAggEC Strains: related, but not identical, isolates of a species
  40. 41. Detecting Strain Differences
  41. 42. Biochemical Typing <ul><li>Some bacterial strains can be differentiated with biochemical tests </li></ul><ul><li>Biovar/Biotype: </li></ul><ul><li>strain with a characteristic biochemical pattern </li></ul><ul><li>Vibrio cholerae : </li></ul><ul><ul><li>Pandemics 1-6 = Classical Biotype </li></ul></ul><ul><ul><li>Pandemic 7 = El Tor Biotype </li></ul></ul>
  42. 43. Serological Typing <ul><li>Strain differentiation based upon antigenic (protein/carbohydrate) differences </li></ul><ul><li>Serovar/serotype: </li></ul><ul><li>strain with characteristic antigen pattern </li></ul><ul><li>V. cholerae O1 and O139 (LPS O region) </li></ul><ul><li>E. coli O157:H7 </li></ul><ul><ul><li>1 st antigen = carbohydrate (LPS O region) </li></ul></ul><ul><ul><li>2 nd antigen = protein (flagella) </li></ul></ul>
  43. 44. Genomic Typing RFLP: Restriction Fragment Length Polymorphism Strain differences based on subtle differences in DNA sequence
  44. 45. Genomic Typing PulseNet: national database of RFLPs from foodborne pathogens
  45. 46. Phage Typing Strain differences based on bacteriophage susceptibility patterns
  46. 47. Antibiograms Strain differences based on antibiotic susceptibility patterns
  47. 48. Summary Prokaryote Identification / Classification <ul><li>Microscopy </li></ul><ul><ul><li>Chapter 3 </li></ul></ul><ul><li>Other Phenotypic Characteristics </li></ul><ul><ul><li>Chapters 4, 17 </li></ul></ul><ul><li>Genomic Identification </li></ul><ul><li>Strain Differences </li></ul>