Classificationof Bacteria


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Classificationof Bacteria

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  • Chlamydia: No peptidoglycan or murimanic acid, does not stain well with Gram stan (use Giemsa stain)
  • Clinical samples are often mixtures!
  • (A) V.cholera.; (B) H.pylori.; (C) Borellia, (D) Treponema palladum
  • Aerobic: SOD and catalase positive (important for survival) Mouth full of both aerobics and anaerobics (ask why?) Microaerophilics: H. pylori discovery story (2005 Nobel Prize in Medicine) Endospores are formed by certain Gram positives--to overcome adverse growth condition (B. anthracis) Exospores formed by fungi are for the purpose of replication TCBS medium for Vibrio (enrich and differentiate)
  • Yellow color by S. aureus de[ends on culture media Seritia marcesens: red pigment MacConkey: bile salt kills Gram positives EMB: eosin-methylene blue agar, select for enterics (mechanism?), differentiate lac+/- Mannitol salt: high salt (7.5% NaCl) select for certain bacteria, especially Staphylococcus sp., differentiate mannitol+/- Thyayer-Martin medium: contains several antibiotics, select for Nieseria.
  • Extreme cases, single cell bacteria can form fruiting body (any pathogenic bacteria form this???)
  • Different growth media may also affect the colony morphology
  • Ask “what is the function fo the capsules?”--antiphagocytic Shown is B. anthracis (most pathogen that cause systemic/blood stream infections have capsule to protect itself) Klebsialla, P. a., S. pneumonia etc.
  • Original plate is purple color (color of pH indicator) Lac+ turns pink (low pH) Lac- show no color (neutral pH) Contains bile salt, only allow Gram negatives to grow
  • Bacteria secrete protein or metabolites that lyse red blood cells.
  • Mannitol salt agar inoculated with (clockwise from upper left) Staphylococcus aureus showing fermentation of mannitol (yellow medium), Staphylococcus epidermidis showing growth but no fermentation of mannitol (pink medium), and Escherichia coli showing no growth. Mannitol Salt Agar (MSA) is a selective and differential medium. The high concentration of salt (7.5%) selects for members of the genus Staphylococcus , since they can tolerate high saline levels. Organisms from other genera may grow, but they typically grow very weakly.MSA also contains the sugar mannitol and the pH indicator phenol red. ハ If an organism can ferment mannitol, an acidic byproduct is formed that will cause the phenol red in the agar to turn yellow. Most pathogenic staphylococci, such as Staphylococcus aureus , will ferment mannitol. ハ Most non-pathogenic staphylococci will not ferment mannitol.
  • Pathogenic enteric are Lac- (important distinction from non-pathogenic enteric) Citrate slant agar: distinguish two commonly seem Gram negative rods (E.coli vs. Klebsiela) H2S: important in distinguishing Salmonella(+) from Shigella(-)
  • Early: glucose utilization (contains low amount of glucose) Later: if continue to utilize lactose or sucrose, pH keep low. If not (utilize proteins in the medium), then pH returns to neutral (color changes) Also, FeS form dark brown color Gas formation cal also be seen Aerobic and anaerobic growth can be seen
  • Catalase: H2O2---H2O + O2 Oxidase: important distinction of N.g from other Gram negatives cocci Urease: breath test for H. pylori Antibiogram: Bacitracin, optochin Fatty acid profile: seems not affected by growth conditions
  • Color is from indole molecule
  • For enteric identification
  • Phages use different receptors for infection, thus important indicator of presence or absence of particular receptor on the cell surfaces.
  • Ag-Ab complex formation (precipitation)
  • Embedded Ab conjugated with gold nanoparticle(visible red color) Diffusion of the complex
  • Ribotyping: chromosomal DNA digest with restriction enzymes, then probe with rRNA sequences
  • Distinguish different isolates (source of infections)
  • UAMS neonatal intensive care unit: Five children infected with S. aureus, searching for the source of the infections. Used collagen adhesin as probe (Southern blot)
  • Often used in detection of viral infections (intracellular)
  • Primer binding specificity determines if there will be PCR products The length of the PCR product is an important indicator Requires low amount of sample, sensitive and fast.
  • Two levels of specificity (1) Binding of the TaqMan probe (2) binding of the PCR primers Therefore, provide highly specific and real time result
  • Rapid Amplification of polymorphic DNA: Use single short primer (9bp) to do PCR Arkansas Children’s Hospital (1997): 3children died of P.a. infections within 2 wk. Ask if they are infected by the same strains? Source? The three kids (#3,4,5) were infected with the same strains, but the source was not found from the hospital environment. Predicted from nurse, but did not look for further (Boston Children’s hospital in 1990, 8 children died within 1month, found nurse had swimmer’s ear infection. After identification, the nurse tried to committee suicide) Learned the lesson, educate nurses for hand washing practice.
  • Depends on the specific nucleotide-nucleotide hybridization (sequence specificity) Specific Ab-Ag binding
  • Chip for pathogen identification (bacterial, viral and fungal) Also, disease markers such as cancer markers
  • For DNA, there are single target sequence per pathogen, while singel pathogen may have multiple copies of Ag (gene product)--more sensitive??
  • Bacitracin: acyclic dodecapeptide antibiotic produced by Bacillus sp. Optochin: Ethylhydrocupreine hydrochloride (EHC) (source?)
  • Rikettissa: typhus, Rocky mountain spotted fever Coxiella: Q fever Erlichia: ehrlichiosm
  • Classificationof Bacteria

    1. 1. Bacterial classification <ul><li>Wall structure </li></ul><ul><ul><li>Gram + </li></ul></ul><ul><ul><ul><li>Staphylococcus , Streptococcus , Clostridium, Bacillus </li></ul></ul></ul><ul><ul><li>Gram - </li></ul></ul><ul><ul><ul><li>Enteric, respiratory and others </li></ul></ul></ul><ul><ul><li>Acid-fast </li></ul></ul><ul><ul><ul><li>Mycobacterium </li></ul></ul></ul><ul><ul><li>Wall-less </li></ul></ul><ul><ul><ul><li>Mycoplasma </li></ul></ul></ul><ul><li>Unusual </li></ul><ul><ul><li>Obligate intracellular </li></ul></ul><ul><ul><ul><li>Rickettsia , Chlamydia, Treponema </li></ul></ul></ul>G+ G- AF WL IC Bacteria
    2. 2. Bacterial classification <ul><li>Cell morphology </li></ul><ul><ul><li>Shapes </li></ul></ul><ul><ul><ul><li>Rod </li></ul></ul></ul><ul><ul><ul><li>Cocci </li></ul></ul></ul><ul><ul><ul><li>Spiral </li></ul></ul></ul><ul><ul><li>Associations </li></ul></ul><ul><ul><ul><li>Individual </li></ul></ul></ul><ul><ul><ul><li>Diplo- </li></ul></ul></ul><ul><ul><ul><li>Staphylo- </li></ul></ul></ul><ul><ul><ul><li>Strepto- </li></ul></ul></ul><ul><ul><ul><li>Filaments </li></ul></ul></ul>G+ G- AF WL IC Bacteria Rod Cocci Rod Cocci Spiral
    3. 3. Gram stains
    4. 4. Gram Stains of Mixtures
    5. 5. Cell shapes
    6. 6. Bacterial classification <ul><li>Growth characteristics </li></ul><ul><ul><li>Oxygen requirement </li></ul></ul><ul><ul><ul><li>Aerobic </li></ul></ul></ul><ul><ul><ul><li>Anaerobic </li></ul></ul></ul><ul><ul><ul><ul><li>Microaerophilic, aerotolerant </li></ul></ul></ul></ul><ul><ul><ul><li>Facultative </li></ul></ul></ul><ul><ul><li>Spore formation </li></ul></ul><ul><ul><li>Intracelular/extracellular </li></ul></ul><ul><ul><li>Fastidious/non-fastidious </li></ul></ul>
    7. 7. Endospore
    8. 8. Classification & Diagnosis <ul><li>Type of colonies </li></ul><ul><ul><li>Appearance </li></ul></ul><ul><ul><ul><li>Color, shape, size and smoothness </li></ul></ul></ul><ul><ul><li>On differential media </li></ul></ul><ul><ul><ul><li>Blood, MacConkey, EMB, Manitol salt agar </li></ul></ul></ul><ul><ul><li>On selective media </li></ul></ul><ul><ul><ul><li>MacConkey, EMB, Manitol salt agar </li></ul></ul></ul>
    9. 9. Colony morphology/color
    10. 10. Bacterial colony morphology
    11. 11. Capsule
    12. 12. MacConkey Agar Plate
    13. 13. Blood Agar Plates and Hemolysis
    14. 14. Mannitol salt agar
    15. 15. Classification & Diagnosis <ul><li>Metabolism </li></ul><ul><ul><li>Utilization of specific substrates </li></ul></ul><ul><ul><ul><li>Lactose ( Sal / Shi / Yer /) - </li></ul></ul></ul><ul><ul><ul><li>Citrate ( E.coli - / Klebsiela + ) </li></ul></ul></ul><ul><ul><li>Production of certain end products </li></ul></ul><ul><ul><ul><li>Fermentation end products </li></ul></ul></ul><ul><ul><ul><ul><li>Acid (acetate, propionic acid, butyric acid etc .) </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Aceton </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Alcohol </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Amine </li></ul></ul></ul></ul><ul><ul><ul><ul><li>H 2 S </li></ul></ul></ul></ul>
    16. 16. Triple Sugar Iron Agar Slants <ul><li>TSI </li></ul><ul><li>Fermentation of glucose, lactose, and/or sucrose </li></ul><ul><li>Reduction of sulfur to hydrogen sulfide </li></ul><ul><li>Gas formation </li></ul><ul><li>Used for Enterobacteriaceae </li></ul>
    17. 17. Classification & Diagnosis <ul><li>Specialized tests </li></ul><ul><ul><li>Immunological </li></ul></ul><ul><ul><ul><li>O-, H- & K-Ag (serotype) </li></ul></ul></ul><ul><ul><ul><li>Precipitation, agglutination </li></ul></ul></ul><ul><ul><li>Specialized enzymes </li></ul></ul><ul><ul><ul><li>Catalase--- Staph + . vs. Strep - . </li></ul></ul></ul><ul><ul><ul><li>Coagulase--- S. aureus + vs. S. epidermidis - </li></ul></ul></ul><ul><ul><ul><li>Oxidase--- Neisseria gonorrhoea + </li></ul></ul></ul><ul><ul><ul><li>Urease--- Proteus + , Helicobacter + </li></ul></ul></ul><ul><ul><li>Antibiogram pattern </li></ul></ul><ul><ul><li>Phage typing </li></ul></ul><ul><ul><li>Fatty acid profile </li></ul></ul>
    18. 18. Urease Test Urea CO 2 + NH 3 NH 4 + + OH - H 2 O
    19. 19. Indole Test
    20. 20. API test strip
    21. 21. Bacteriophage Plaques
    22. 22. Conventional method <ul><li>Depend on ability to culture </li></ul><ul><ul><li>Treponema pallidum </li></ul></ul><ul><li>Slow, esp. for fastidious species </li></ul><ul><ul><li>Mycobacterium spp . </li></ul></ul><ul><li>Not always definitive </li></ul>
    23. 23. Immunological detection ELISA
    24. 24. Instant View tests
    25. 25. Molecular diagnosis <ul><li>Ribotyping </li></ul><ul><li>Restriction fragment length polymorphism (RFLP) </li></ul><ul><li>DNA hybridization </li></ul><ul><li>PCR, RT-PCR and RAPD </li></ul><ul><li>Nucleic acid sequence analysis </li></ul><ul><li>Phage-GFP (TB) </li></ul>
    27. 27. DNA hybridization
    28. 28. In situ Hybridization
    29. 29. Rate of increase 2 n PCR
    30. 30. Real time PCR Q R 5' 3' 5' 3' 5' 3' 3' 5' Q R 5' 3' 3' 5' Q R Q 5' 3' 3' 5' R Primer Primer Probe Taq DNA Pol
    31. 31. RAPD of P. aeruginosa
    32. 32. DNA and protein chips A B C D
    33. 33. Microarray array mediated pathogen detection
    34. 34. Ab chip mediated pathogen detection  -O157:H7  -Salmonella  -O157:H7  -Salmonella E.coli O157:H7 Salmonella typhimurium
    35. 35. Molecular diagnosis <ul><li>Reduce reliance on culture </li></ul><ul><li>Faster </li></ul><ul><li>High sensitive </li></ul><ul><li>More definitive </li></ul><ul><li>More discriminating </li></ul><ul><li>Techniques adaptable to all pathogens </li></ul><ul><li>Technically demanding </li></ul><ul><li>Relatively expensive </li></ul><ul><li>Can be too sensitive </li></ul><ul><li>Provides no information if results are negative </li></ul>
    36. 36. Bioterrorism <ul><li>Pathogen detection </li></ul><ul><ul><li>Fast and accurate </li></ul></ul><ul><ul><li>Mobile </li></ul></ul><ul><ul><li>Inexpensive </li></ul></ul><ul><li>Source investigation </li></ul>
    37. 37. Biosensor Mediated Pathogen Detection Step 1: Attach Capture Antibody to Fiber Optic Waveguide Step 2: Apply Sample. Target Organism Binds to Waveguide Step 3: Wash Away Other Cells and Debris Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y
    38. 38. Step 1: Attach Capture Antibody to Fiber Optic Waveguide Step 2: Apply Sample. Target Organism Binds to Waveguide Step 3: Wash Away Other Cells and Debris Step 4: Add Specific Detection Antibody Step 5: Turn on Laser. Detection Antibody Fluoresces, Indicating a Positive Sample. Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Biosensor Mediated Pathogen Detection Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y
    39. 39. Biosensors Analyte 2000 TM for laboratory research and development RAPTOR TM automated version for field analysis
    40. 40. Differentiating Staphylococci from Streptococci <ul><li>Gram stain and morphology </li></ul><ul><ul><li>Both Gram + </li></ul></ul><ul><ul><li>Staphylococci : bunched cocci </li></ul></ul><ul><ul><li>Streptococci : chained cocci </li></ul></ul><ul><ul><ul><li>S. pneumoniae : diplococcus </li></ul></ul></ul><ul><li>Enzyme tests </li></ul><ul><ul><li>Staphylococci : catalase + </li></ul></ul><ul><ul><li>Streptococci : catalase - </li></ul></ul><ul><li>Growth </li></ul><ul><ul><li>Staph .: large colonies (non-fastidious), some hemolytic </li></ul></ul><ul><ul><li>Strep .: small colonies (fastidious), many hemolytic (  or  ) </li></ul></ul>
    41. 41. Staphylococci <ul><li>S. aureus : coagulase + </li></ul><ul><li>S. epidermidis : coagulase - </li></ul>
    42. 42. Streptococci
    43. 43. Differentiating the Gram - bacteria <ul><li>Cocci </li></ul><ul><ul><li>Neisseria </li></ul></ul><ul><li>Rods </li></ul><ul><ul><li>Type of disease they cause </li></ul></ul><ul><ul><li>Enteric Gram - rods </li></ul></ul><ul><ul><ul><li>API test </li></ul></ul></ul><ul><li>Curved </li></ul><ul><ul><li>Vibrio , Campylobacter , Helicobacter </li></ul></ul><ul><li>Spiral Gram - organisms </li></ul><ul><ul><li>Spirochetes </li></ul></ul>
    44. 44. Gram negative Straight rods Curved rods Lactose+ Lactose- Citrate+ Citrate- H 2 S+ H 2 S- Klebsiella E.coli Salmonella Shigella Campy blood agar 42 o C+ 25 o C- Campylobacter TCBS agar Yellow Oxidase+ Vibrio
    45. 45. Bacteria Gram+ Cocci Rod Staph. Strep. Non-spore Spore S. a. S. e. S. s. A B Pn Vir Fil Rod A.i. C.d. L. m. +O 2 -O 2 B.a. B.c. C.b. C.t. C.p. C.d. Acid Fast M.t. M.l. N.c. Intra Cellular Rickettsia Coxiella Erlichia Chlamydia Wall Less Mycoplasma Gram- Cocci Rod Spiral Straight Curve +O 2 -O 2 +/-O 2 Other Treponema Borrelia Leptospira Neisseria Moraxella P.a. Enteric Bact. Resp. Zoo GU Bordetella. H. influenzae Legionella Yersinia Pasteurella Brucella Francislla Streptobacillus H. ducreyi Gardnerella Calymmatobacterium Vibrio Campylobacter Helicobacter