Microbiology Midterm 1

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Microbiology Midterm 1
2012-03-21 21:45:15
Microbiology Midterm

Micro Midterm 1
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  1. colony morphology
    • whole
    • edge
    • elevation
    • surface
  2. Flagellum:
    • Properties: long, thin, thread-like appendage attached to the cell membrane
    • Function: responsible for motility and chemotaxis
    • Structure: Filament (main tail-like part), Hook (curved sheath), Basal body (stack of rings firmly anchored to cell wall and membrane)
    • Arrangements:
    • Monotrichous (single flagellum)
    • Lophotrichous (clusters at one end)
    • Amphitrichous (one at each end)
    • Peritrichous (distributed all over surface of cell, slowest moving)
    • Movement: “run” (counterclockwise), “tumble” (clockwise)
  3. Fimbria:
    • Properties: thinner and shorter than flagellum, extend from entire surface of cell
    • Function: attachment to host and other bacteria, enables colonization, enhances the bacteria’s ability to cause disease
  4. Pilus:
    • Properties: longer than fimbriae
    • Function: facilitates conjugation between two bacterial cells
    • Sometimes called sex pilus or F pilus
  5. Basic Cell Structures: Found in all bacteria
    • o Cell wall
    • o Plasma membrane
    • o Chromosome (nucleoid)
    • o Ribosomes
    • o Inclusion (granule)
  6. Special Cell Structures: found only in certain groups of bacteria
    • o Flagellum
    • o Fimbria
    • o Pilus
    • o Glycocalyx
    • o Plasmid
    • o Spore
  7. Spore
    • Gram positive
    • (endospore): a dehydrated, multishelled structure that enables bacteria to survive under harsh environmental conditions, they are NOT dead and can be revived as soon as conditions are good enough
    • o Highly resistant to heat, radiation, attack by most enzymes as well as chemicals
    • o Bacteria that form spores are NEVER Gram Negative
    • o Three layers: Exosporium. Spore coat, cortex
    • o Stain blue
    • o dipicolinic acid, high calcum content
  8. malachite
    • green staining
    • Spore staining: microscopic image of the bacterial spore formation of Bacillus subtilis, magnification:1,000. (green) spores, (red) vegetatives.
  9. Glycocalyx (Specialized):
    • o Properties: jelly-like layer over the cell wall usually made from polysaccharides
    • o Types:
    •  Slime Layer: thin layer loosely organized, irregular
    • • Function: protects cell from drying, allows bacteria to adhere to surfaces (colonize)
    •  Capsule: thick layer, highly organized, regular
    • • Function: protects bacteria from phagocytes, enhances bacteria’s ability to produce disease
    • • Can be visualized by Indian Ink Stain under light microscope
    • o Not required for bacterial growth
  10. Cell Wall:
    • o Provides structural support and osmotic barrier
    • o Major target for antibiotics
    • o Two Types: Gram Positive and Negative
  11. Gram staining process
  12. Staining Comparison
  13. Gram + - Comparison
  14. Gram Positive:
    • • Thicker cell walls due to thick peptidoglycan layer (20-80 nm)
    • o Allows for rigidity and porosity of bacterial wall
    • • Three layers of cell wall: Peptidoglycan, Periplasmic Space, Cell Membrane
    • o Peptidoglycan: unique to both G+ and G- bacteria only
    •  Principle compound of cell wall
    •  Subunit composed of NAG and NAM, tetrapeptide attached to NAM
    •  Subunits bound together by pentaglycine peptide bridge
    • o Periplasmic Space:
    •  Contains enzymes like beta-lactamse that are responsible for degrading antibiotics and other toxins
    • • Tightly bond acidic polysaccharides that play a role in attachment and are essential to cell viability (unique to G+ bacteria)
    • o Teichoic Acid: attaches to peptidoglycan
    • o Lipoteichoic acid: attaches to cell membrane
  15. Lipopolysaccaride (LPS)
    • embedded in the outside layer of the outer membrane, only found in Gram-
    • composed of
    • Lipid A: anchor molecule in outer component of outer membrane; toxic
    • Core polysaccharide: series of sugars constant for a given species
    • O antigen: long repeating 3-5 sugar units, responsible for antigenic diversity
    • LPS, also called endotoxin, is toxic to animals (Lipid A).
    • a powerful inducer of immune response and coagulation factor, leading to fever, sepsis and shock.
  16. β-lactamase
    enzyme responsible for degrading the penicillin group of antibiotic drugs, leading to penicillin resistance
  17. Gram Negative:
    • • Five layers of cell wall: outer membrane, Periplasmic space, peptidoglycan, periplasmc space, cell membrane
    • o Outer Membrane:
    •  Provides protection from adverse environmental conditions
    •  Decreases permeability of membrane
    •  Contains LPS: Lipid A anchor=Toxic, Core Polysaccharide, O antigen (useful in bacterial identification). Also called an endotoxin. LPS is a powerful inducer of immune response and coagulation factor leading to fever, sepsis and shock
    •  Contains Porins: act as protein channels for hydrophilic molecules such as metabolites and small hydrophilic antibiotics
  18. Plasma Membrane: most dynamic bacterial structure, semipermeable, thin and flexible
    • o Lipid bilayer with various proteins dispersed and embedded in the phospholipids
    • o Functions:
    •  synthesis of membrane lipids such as LPS and peptidoglycan
    •  main selective permeability barrier
    •  location of various transport systems and specialized enzyme systems
    •  energy generating functions such as photosynthesis ETC and ATPase
    •  coordination of DNA replication and segregation with septum formation and cell division
    • o usually the target of antimicrobial peptides including defensins produced by our immune cells
    •  increase in permeability usually causes cell death
  19. *** Bacterial Exceptions:
    • 1. Bacteria without cell walls (Mycoplasma)
    •  Contain steroid from host cell
    • 2. Bacteria with chemically unique cell walls (Mycobacterium)
    •  Cell wall surrounded by a waxy substance=Mycolic Acid
    • i. Prevents Gram staining, must use Acid Staining
  20. Acid fast stain
    • (Ziehl-Neelsen Stain)
    • for WAXY bacteria with a high concentration of lipids in their cell wall as they don't stain well with Gram stain
    • procedure:
    • 1. carbol fuchsin (staining) mixed with phenol (fixing)
    • 2. acid/alcohol (decolorization)
    • 3. methylene blue (counterstain)
    • those that resist decolorization are acid fast and they appear pink
  21. Cytoplasm->All basic structures
    • o Chromosome (nucleoid): highly folded DNA suspended in cytoplasm and aggregated into a nucleoid. Lacks histones, instead has DNA gyrase and topoisomerase type IV
    • o Plasmids: small circular double-stranded DNA
    •  Encode proteins responsible for antibiotic resistance and tolerance to toxic metals and toxins
    • o Ribosomes: 2 subunits: 30S and 50S
    • o Inclusion (granule): intracellular storage bodies that vary in size, number and content
  22. Binary Fission:
    • form of asexual reproduction where one cell divides into two cells of the same size
    • Process:
    • 1. Slight enlargement in cell size due to increase in metabolic activities and production of energy and cell parts
    • 2. DNA is duplicated
    • 3. Cell wall and membrane grow inward separating DNA molecules
    • 4. Transverse septum is formed
    • • Due to continued growth of cell wall and membrane
    • • Divides contents of cell and DNA molecules
    • 5. Two daughter cells are formed
  23. Bacterial growth Curve’s Four phases
    • 1. Lag phase: no change in number of cells
    • 2. Log phase: growth in steady state, no cell death, however most susceptible to antibiotics, late log phase, cells are most competent to receive DNA through transformation
    • 3. Stationary phase: rate of reproduction=rate of death
    • 4. Death phase: cells dying in steady state
  24. Requirements for bacterial growth
    • Temperature
    •  Optimum: temp of most rapid reproduction
    •  Mesophiles: 20-45°C (most common)
    •  Thermophiles: 45-110°C
    •  Psychrophiles: 0-20°C
    • pH
    •  Optimum: pH at which maximum growth occurs
    •  Neutrophiles: pH=5-8 (most common)
    •  Acidophiles: pH=0-5
    •  Alkalinophiles: pH=8-12
    • Oxygen gas
    •  Obligate Aerobe: must have aerobic environment for growth
    •  Microaerophile: needs oxygen for growth, but can’t have too much (2-10%)
    •  Obligate anaerobe: no growth in aerobic environment, oxygen is toxic
    •  Facultative: oxygen not required for growth, but utilized when available
    •  Aerotolerant anaerobe: oxygen not required nor utilized, can grow in either environment
    •  Enzymes are capable of detoxifying oxygen radicals in bacteria
    • • Obligate aerobes and most facultative anaerobes and aerotolerant anaerobes contain these enzymes
    • • Obligate anaerobes do not have these enzymes and therefore oxygen is toxic to them
    • Water
    •  All metabolically active bacteria require presence of water
    •  Cells largely comprised of water
    •  Most nutrients, wastes soluble in water to cross cell membrane
    •  Site of metabolic reactions (cytoplasm)
    • Light
    •  Very small group of photosynthetic bacteria (cyanobacteria)UV light
    •  For some bacteria, UV light is lethal (eubacteria)
    • Nutrients
    •  Carbon: chemoheterotrophs get their energy from organic compounds
    •  Nitrogen
    •  Hydrogen
    •  Oxygen
    •  Minerals
  25. Oxygen Radical Enzymes
    • superoxide (02-) & hydrogen peroxide are 2 lethal byproducts of oxygen metabolism; some cells have detoxification enzymes to get rid of these
    • -superoxide dismutase (SD) gets rid of superoxide, and peroxidase & catalase get rid of hydrogen peroxide
    • -obligate anaerobes do not have any of these detoxification enzymes
  26. Metabolism
    All the biochemical reactions within a cell
  27. Radical Oxygen Species
  28. Oxygen and Bacteria
  29. Catabolism:
    • Creates energy by breaking molecules down
    • • Primary energy: ATP, GTP
    • • Secondary energy: NADH (3 ATP), FADH2 (2 ATP)
    • • Aerobic Respiration
    • Glycolysis
    • 1 glucose->2ATP and 2 pyruvates
    • 2. TCA cycle: produces the most various energy storage molecules
    • 3. ETC: converts secondary energy molecules into primary energy molecules and also generates water
    • 4. OVERALL: aerobic respiration= 38 ATP/glucose
  30. Anabolism
    • Catabolic pathways of bacterial cell result in various chemical intermediates, which are used for synthesizing major cellular constituents, i.e., nucleic acid, proteins, lipopolysaccharides, peptidoglycan etc
    • synthesize cellular components from simple precursor molecules
    • use energy-storage molecules
    • examples
    • most biosynthesis, e.g., nucleic acid, proteins, lipopolysaccharides, peptidoglycan etc
  31. Pyruvate
    most common intermediate of catabolism
  32. Anaerobic Respiration
    • 1. Less efficient at energy production than aerobic
    • 2. Only uses glycolysis
    • 3. OVERALL: 2 ATP/glucose molecule
    • • Other catabolic pathways for glucose:
    • 1. Pentose-phosphate pathway
    • 2. Entner-Duodoroff pathway (only in prokaryotic cells)
    •  Anabolism: uses energy by building molecules up from simple precursor molecules
    • • TCA cycle produces key intermediates for the ultimate synthesis of aa, lipids, purines and pyrimidines
  33. Entner-Duodoroff pathway
    • (only in prokaryotic)
    • glucose to pyruvate and glyceraldehyde -3 phosphate
    • mostly in obligate aerobic bacteria
  34. Gene exchange in bacteria-Bacterial Sex
    • • Significance to Bacteria
    •  Beneficial for their own adaptive evolution
    •  Antibiotic resistance
  35. Transformation:
    • DNA enters recipient cell and integrates into DNA
    • • (single strand)ssDNA enters the cell, process requires Calcium ion
    • • cells capable of taking up exogenous DNA = competent cells
    • • RecA protein promotes genetic exchange between a fragment of the donor’s DNA and the recipient’s DNA (RECOMBINATION)
    • • Natural Transformation: in nutritional shortage or adverse condition, develop a capacity to pick up and internalize DNA from the environment (both G+ and G-)
    • 1. Occurs during the end of log phase
    • • Artificial transformation: (in lab), use chemicals and heat to create competent cells
  36. Specialized transduction:
    • only specific host genes next to the phage DNA are transferred and use both the lysogenic and lytic life cycles
    •  Induction of lytic life cycle
    •  Prophage is incorrectly excised from chromosome
    •  Phage DNA incorporating some bacterial genes
    •  Phage replicates and bacterial cell is lysed releasing the phages
    •  Phage infects new host cell
    •  Phage DNA incorporated into chromosome
    •  Host cell chromosome acquires both phage DNA and genes from previous host
    •  New host cell replicates all genes thus producing copies of the phage DNA and old host’s DNA
  37. Generalized transduction
    • any host gene can be transferred randomly via lytic life cycle
    •  Phage injects DNA
    •  Phage enzymes degrade host DNA
    •  Cell synthesizes new phages that incorporate phage DNA and sometimes host DNA
    •  Cell ruptures releasing phages
    •  Transducing phage injects donor DNA
    •  Donor DNA is incorporated into recipients chromosome by recombination
  38. Transduction:
    • phage infects recipient cell, donor DNA integrates into recipient DNA
    • • DNA exchange mediated by bacteriophages (bacterial viruses
    • 1. Generalized transduction: any host gene can be transferred randomly via lytic life cycle
    •  Phage injects DNA
    •  Phage enzymes degrade host DNA
    •  Cell synthesizes new phages that incorporate phage DNA and sometimes host DNA
    •  Cell ruptures releasing phages
    •  Transducing phage injects donor DNA
    •  Donor DNA is incorporated into recipients chromosome by recombination
    • 2. Specialized transduction: only specific host genes next to the phage DNA are transferred and use both the lysogenic and lytic life cycles
    •  Induction of lytic life cycle
    •  Prophage is incorrectly excised from chromosome
    •  Phage DNA incorporating some bacterial genes
    •  Phage replicates and bacterial cell is lysed releasing the phages
    •  Phage infects new host cell
    •  Phage DNA incorporated into chromosome
    •  Host cell chromosome acquires both phage DNA and genes from previous host
    •  New host cell replicates all genes thus producing copies of the phage DNA and old host’s DNA
  39. In what growth phase do bacteria develop competence?
    during late log phase, bc of nutritional shortage or adverse conditions
  40. Conjugation:
    • Free plasmid moves from donor to recipient cell via sex (F) pilus, integrated plasmid promotes transfer of genomic DNA, which integrates into recipient DNA
    • • Plasmid (specialized structure): extrachromosomal DNA
    • • Genetic transfer during direct cell-cell contact through sex pilus
    • 1. As ssDNA is transferred to F- cell from the R plasmid, the rolling circle mechanism regenerates the ssDNA of the plasmid so it remains dsDNA
    • • Plays an important role in drug resistance with the transfer of the R plasmid
    • • Occurs only between one F+ and one F- bacterial cell
    • 1. F+: contains the F plasmid, which encodes the genes responsible for making the sex pilus
    •  If the F plasmid is integrated into the chromosome, cell becomes high frequency recombination cell or super F+ cell
    • • These cells will pass the entire chromosome with the Hfr str gene through the pilus
  41. lytic:
    • bacteriophage
    • virulent, resulting in bacterial lysis
  42. lysogenic:
    non-virulent bacteriophage, viral DNA integrates into bacterial chromosome
  43. Transposon:
    • mobile DNA elements that can jump from one place to another on the DNA
    • • Some carry antibiotic resistance genes
    • • Genetic transfer within a cell
    • • If the genes are plasmid located, they can easily transfer from one bacterium to another
    • • Insertion sequence contains transposase gene and/or antibiotic resistance genes
    • • palindromic sequence at ends
  44. Frederick Griffith Experiment
    •  R and S strain streptococcus experiment
    •  Transformation between Rough cell and smooth cell
  45. Consequences of gene transfer in bacteria
    •  Colony morphology changes
    •  Biochemical activity changes
    •  Virulence changes
    •  Drug resistance capabilities acquired
  46. Eukaryotic vs. Prokaryotic
    • differences allow antibiotics to kill bacteria and not human cells
    • • Bacterial presence of a cell wall
    • • Histones vs. DNA gyrase and topoisomerase type IV
    • • RNA pol II vs general RNA pol
    • • Ribosome structure
  47. Peptidoglycan synthesis
    • often a target of antibiotics because it is unique to bacteria
    • • In the cytoplasm: four aa are sequentially added to NAM forming the tetrapeptide
    • • NAM-tetrapeptide is attached to the bactoprenol carrier molecule in cell membrane
    • • NAG is attached to NAM-tetrapeptide on the bactoprenol to complete the monomer
    • • Peptidoglycan monomers are transported into periplasmic space by bactoprenol
    • • In periplasm, autolysins break glycosidic bonds between peptidoglycan monomers
    • 1. Also break the peptide cross-bridges that link the rows of sugars together
    • 2. Now new monomers can be inserted
    • • Transglycosidase (TG) enzymes catalyze the formation of glycosidic bonds between the NAM and NAG of the monomers to the existing NAG and NAM of the peptidoglycan polymer
    • • Transpeptidase (TP) enzymes reform the peptide cross-links (pentaglycine peptides) between the rows and layers of peptidoglycan to make the wall strong
  48. Transglycosidase (TG)
    enzymes catalyze the formation of glycosidic bonds between the NAM and NAG of the monomers to the existing NAG and NAM of the peptidoglycan polymer
  49. Transpeptidase (TP)
    enzymes reform the peptide cross-links (pentaglycine peptides) between the rows and layers of peptidoglycan to make the wall strong
  50. Peptidoglycan Synthesis
    • -think abt: 1) the location of synthesis, and 2) the enzymes involved, in each step
    • -cytoplasm is at top of pic, outside cell is on bottom
    • 1. in cytoplasm, four amino acids are sequentially added to NAM, forming a tetrapeptide (top left)
    • 2. the NAM-tetrapeptide is attached to the bacterial enzyme bactoprenol (like a conveyor belt, which carries the complex) located in the plasma membrane; then, NAG is attached to the NAM-tetrapeptide on the bactoprenol to complete the peptidoglycan monomer
    • 3.Peptidoglycan monomers, bound to bactoprenol, are transported into periplasmic space (located in btw cell membrane & cell wall)
    • 4. in periplasmic space, enzymes called autolysins break down the glycosidic bonds btw the monomers at the point of growth along the existing peptidoglycan (by doing so, there is a space); in this space, new monomers can incorporated
    • 5. transglycolase enzyme- makes bridge btw pre-existing monomers & new monomers, btw NAM and NAG
    • 6. transpeptidase- forms peptide cross-links (ie pentaglycine peptides) btw the layers/rows of peptidoglycan
    • -remember, know the enzyme name & location for each step
  51. Bactericidal:
    • killing of all bacteria
    • 1. ALL cell wall synthesis inhibitors
    • 2. ALL DNA synthesis inhibitors
    • 3. ALL RNA synthesis inhibitors
    • 4. ALL membrane integrity inhibitor
    • 5. Aminoglycosides are the ONLY bactericidal protein synthesis inhibtors
  52. Bacteriostatic:
    • suppressing the growth of bacteria
    • 1. All the rest of protein synthesis inhibitors and ALL folic acid synthesis inhibitors
  53. Antimicrobial agents
    • • Three groups:
    • 1. Natural agent: “antibiotic” produced by certain groups of microorganisms
    •  Molds: Penicillian
    • • Beta lactam antibiotics
    •  Streptomyces species (ART)
    • • Aminoglycosides
    • • Tetracyclines
    • • Rifamycins
    •  Bacillus species
    • • Polymyxin and bacitracin
    • 2. Semi-synthetic agent: hybrid substance of natural and synthetic
    • 3. Synthetic agent: produced entirely by chemical means
  54. Cell wall synthesis (antibiotics):
    • a. Beta-lactam antibiotics (have beta-lactam ring): block peptidoglycan synthesis by binding and inhibiting TP and TG (penicillin binding proteins, PBPs). This weakens the cell wall and causes lysis, generally bactericidal
    • i. Penicllins
    • ii. Cephalosporins
    • iii. Monobactems
    • iv. Carbapenems
    • b. Vancomycin: inhibits peptidoglycan synthesis by binding to the Ala at the terminus of the tetrapeptide and preventing TG enzymes from forming the glycosidic bonds between sugars and TD from forming peptide cross-links. Weakens cell wall and is generally bactericidal. Inactive against gram-negative cells due to presence of outer-membrane and decreased permeability.
    • i. Does not inhibit the enzymes, but prevents them from performing their duties sterically
    • c. Bacitracin (topical use only): inhibits the bactoprenol from transporting the peptidoglycan monomers across the cell membrane.Therefore no building blocks are available for peptidoglycan synthesis
    • d. Acid Fast: isoniazid, ethionamind, ethambutol, cycloserine
  55. Beta-lactam antibiotics
    • (have beta-lactam ring): block peptidoglycan synthesis by binding and inhibiting TP and TG (penicillin binding proteins, PBPs). This weakens the cell wall and causes lysis, generally bactericidal
    • i. Penicllins
    • ii. Cephalosporins
    • iii. Monobactems
    • iv. Carbapenems
  56. Cell membrane integrity (antibiotics)
    a. Polymyxins: effective mainly against G- bacteria. Is a neurotoxin, therefore cannot be ingested. Generally bactericidal.
  57. Polymyxins
    • produced by Bacillus polymyxis
    • binds to membrane phospholipids and thereby causes cleavages (openings) in cell membrane, resulting in loss of cell contents
    • effective mainly against gram-negative bacteria; cannot effectively diffuse through the thick peptidoglycan layer in gram-positives
    • usually limited to topical usage.
    • bactericidal
  58. Nucleic acid synthesis (antibiotics)
    • a. DNA Replication inhibitor: Quniolones
    • i. Bind to bacterial DNA gyrase and topoisomerase type IV
    • b. RNA transcription inhibitor: Rifamycins
    • i. Bind to bacterial DNA-dependent RNA polymerase thus inhibiting txn initiation
  59. Protein synthesis (antibiotics)
    • a. 30S subunit-binding antibiotics
    • i. Aminoglycosides
    • 1. Irreversibly bind to 30S ribosome leading to
    • a. Inhibition of protein synthesis
    • b. Production of aberrant protein synthesis
    • 2. Bactericidal
    • ii. Tetracyclines
    • 1. Bind reversibly to 30S subunit blocking the binding of aminoacyl-tRNA
    • 2. Bacteriostatic
    • b. 50S subunit-binding antibiotics
    • i. Chloramephenical
    • ii. Clindamycin
    • iii. Macrolides
    • iv. Oxazolidinones
  60. Biosynthesis pathways (antibiotics)
    • a. Folic Acid synthesis inhibitors (truly synthetic antibiotic)
    • i. Sulfonamide
    • ii. Trimethoprim
  61. Antibiotc mechanisms:
    • 1. Cell wall synthesis
    • 2. Cell membrane integrity
    • 3. Nucleaic acid synthesis
    • 4. Protein synthesis
    • 5. Biosynthesis pathways
  62. Vancomycin
    • Originally obtained from Streptomyces orientalis
    • inhibits bacterial cell wall synthesis by binding to the peptidoglycan precursor at the Ala terminus, thereby preventing both the formation of glycosidic bonds and the formation of pentaglycine peptide bonds.
    • Inactive against gram-negative cells
    • Last line of defense against MRSA (methicillin resistant S. aureus)
  63. Bacitracin
    • cell wall synthesis inhibitor
    • produced by Bacillus species
    • prevents the peptidoglycan monomers from being transported across the cell membrane by inhibiting bactoprenol (the conveyer of peptidoglycan monomers). As a result, no building blocks are available for peptidoglycan synthesis.
  64. Inhibitors of acid-fast cell wall synthesis
    • Isoniazid, ethionamide, ethambutol, cycloserine (for mycobacteria (waxy)
    • isoniazid blocks the incorporation of mycolic acid into acid-fast cell walls
    • ethambutol interferes with the incorporation of arabinoglactan.
    • mycolic acid is a major component
  65. Inhibitors of protein synthesis
    • in your 30s, it’s All Terrific”; “in your 50s, your Career is Climbing and your Marriage is ‘Ohmygod
    • 30s subunit-binding antibiotics
    • Aminoglycosides
    • Tetracyclines
    • 50s subunit-binding antibiotics
    • Chloramphenical
    • Clindamycin
    • Macrolides
    • Oxazolidinones
  66. Aminoglycosides
    • Products of Streptomyces species and are represented by streptomycin, kanamycin, neomycin, tobramycin, gentamicin etc.
    • Irreversibly bind to 30s ribosome, leading to 1) inhibition of protein synthesis and 2) production of aberrant protein synthesis
    • Penetration through the outer membrane requires O2 (anaerobes are resistant)
    • Bactericidal
  67. Tetracyclines
    • Products of Streptomyces and are represented by tetracycline, doxycycline, minocycline etc.
    • Bind reversibly to 30s subunit, blocking the binding of aminoacyl-tRNA to the 30S ribosome-mRNA complex
    • Bacteriostatic
    • Effective against Chlamydia, Mycoplasma, Richettsia
  68. quinolones
    • DNA replication inhibitor
    • bind to bacterial DNA gyrase (topoisomerase II) and topoisomerase type IV- these are the targets of quinolones
  69. rifamycins
    • binds to beta subunit of DNA-dependent RNA polymerase
    • RNA polymerase in bacteria is 4 subunits, but in humans is 12 subunits
  70. Sulfonamide
    biosynth-dihydropteroate synthetase
  71. Trimethoprim
    biosynth-dihydrofolate reductase
  72. Sensitivity Testing:
    • in vitro testing of antibiotics against isolated strains
    • Disk Diffusion method (Kirby-Bauer Method):Grow bacterial lawn in the presence of many different antibiotics to determine most effective
    • Broth or Agar dilution method to measure MIC and MBC
    • • MIC: Minimum Inhibitory Concentration: lowest concentration of an antibiotic that inhibits the visible growth of a microorganism after overnight incubation
    • • MBC: Minimum Bactericidal Concentration: is the lowest concentration of an antibiotic that completely kills microorganism
    • • NOTE: MIC may be different that MBC
    • o E-Test to measure MIC
    • • MIC of antibiotic is determined by where the growth of the bacteria starts on an agar plate with a gradient of antibiotic on a stick
    • • Antibiotic Resistance
  73. Mechanisms of Antibiotic Resistance:
    • • Reduced drug accumulation
    • • Decreasing permeability
    • • Increasing drug efflux
    • • Drug inactivation or modification
    • • B-lactamses, aminoglycosidases-modifying enzymes
    • • Alteration of drug target site
    • • Alteration of PBPs (penicillian binding proteins), gyrase, ribosome, RNA pol
    • • Alteration of biosynthesis
    • • Some sulfonamide-resistant bacteria
  74. Origin of Antibiotic Resistance
    • • Inherent (natural, intrinsic) resistance
    • • Ex/G- bacteria is naturally resistant to Vancomycin due to presence of outer membrane
    • • Acquired resistance driven by four genetic processes
    • • Transformation
    • • Conjugation
    • • Transduction
    • • Spontaneous mutation
    • • NOTE: Transposition CANNOT result in resistance
  75. Mechanisms of B-Lactam Resistance
    • • Alterations in PBPs leading to decreased binding affinity
    • • Production of B-lacatamase enzymes that destroy ring
  76. Mechanisms of Vancomycin Resistance
    • • Naturally inactive against G- bacteria
    • • Production of non-compatible tetrapeptide terminus which does not bind to vancomycin
    • o Ex/ lactate terminus replaces Ala
    • o Some bacterias have VanA and VanB that modify the Ala and prevent Vancomycin from binding
    • • Bacterial Relationships with Host
  77. Symbiotic Relationships:
    • • Commensalism: neither party benefits or is harmed from the association
    • • Mutualism: both parties benefit from the association
    • • Opportunism: Potential pathogens arise from normal flora when inoculated into the wrong places or when the host becomes weakened immunologically, causes disease
  78. Tissue Specificity
    • • Most members of the normal bacterial flora prefer to colonize certain tissues and not others due to properties of both the host and the bacterium
    • • Tissue Tropism: different factors for bacterial growth
    • • Specific Adherence
    • • Bacterial Pathogenesis
    • o Definitions:
    • • Pathogen: a microorganism that is able to cause disease in a plant, animal or insect
    • • Pathogenicity (Virulence): terms that refer to an organism’s ability to cause disease
    • • Infection: the colonization and/or invasion and multiplication of pathogenic microoransisms in the host with or without the manifestation of disease
    • • Disease: an abnormal condition of body function(s) or structure that is considered to be harmful to the affected host
  79. Acquisition of Infectious Agents
    • • Ingestion
    • • Inhalation
    • • Direct Penetration
    • • Trauma or Surgical procedure
    • • Needlestick
    • • Arthropod bite
    • • Sexual Transmission
    • • Transplacental
  80. General Stages of Bacterial Infection
    • • Bacterial Colonization/Adhesion
    • • Successful occupation of a new habitat not normally found in this niche
    • • Factors: fimbria, glycocalyx, Teichoic acids and lipoteichoic acids, adhesins
  81. Adhesins:
    bacterial surface proteins that bind to specific receptor molecules on the surface of host cells
  82. Bacterial Invasion
    • • Entry and spread throughout cells and/or tissues of the host
    • • Factors: Invasins (bacterial extracellular proteins)
    • o Act locally to damage host cells
    • o Act against the host by breaking down primary or secondary immune defenses of the body
    • • Bacterial Toxigenesis: ability to produces toxins
    • • Exotoxins: released from bacterial cells
    • o Toxins act on cell surface: affect receptors or form pores in membrane
    •  Superantigens (bind MHC Class II and TCR receptors thus stimulating T cells independently of antigen)
    •  Hemolysins and Leukotoxins (form pores)
    • o Toxins that need to enter cell: A/B toxins (A=toxin, B=binds receptor)
    •  Diphtheria toxin, Shinga toxin, cholera toxin, heat-labile toxin, botulinum toxin, tetanus toxin, pertussin toxin
    • o Toxins directly delivered into host cell: Type III and IV secretion
    •  Apoptotic toxins: Yop J/P
    •  Protein kinases and phosphorylases: YopH and E
    • • Endotoxins: cell-associated substances that are structural components of the cell walls of G- bacteria
    • o Lipid A on LPS
  83. Bacterial Multiplication
    • • Influenced by immunologic status, nutrient availability
    • • Factors: bacterial ability to compete for iron and other nutrients
    • • Bacterial ability to avoid being killed or removed by host immune mechanisms
    • • Host defense mechanisms against bacterial infection
    • • First Line: Physical and chemical barriers
    • • Second Line: Innate Immunity
    • • Third Line: Adaptive Immunty
    • • NOTE: Disease develops ONLY if pathogens overcome multiple host defense mechanisms
  84. Bacterial ability to bypass or overcome host defense mechanisms
    • • Inhibition of phagocytosis
    • o Inhibiting formation of phagolysosome and resistant to killing by lysosomes
    • o Glycocalyx inferferes with opsonization
    • o Escape from the phagosome by lysing phagosome membrane
    • o Producing proteins that kill or damage phagocytes
  85. Destruction of immune function
    • o Antigenic Variation: periodically changing antigens
    • o Produce proteases that degrade complement proteins and antibodies
  86. Intracellular growth (parasite)
    • o Have the ability to multiplicate/replicate inside of host cells
    • o Protects bacteria from immune clearance
    • • Disease develops only in the right host and under the right conditions
  87. Invasiveness (bacterial ability to invade cells/tissues)
    • colonization factors
    • Invasion factors
    • ability to bypass or overcome host defense mechanisms
  88. 2. Toxigenesis (bacterial ability to produce toxins)
    • Exotoxins: released from bacterial cells
    • Endotoxins: cell-associated substances that are structural components of the cell walls of Gram-negative bacteria; lipopolysaccharide (LPS)
  89. Exotoxins
    • Toxins produced by bacteria and secreted to the outside of the bacteria cell.
    • Three types of exotoxins
    • 1. Toxins act on cell surface
    • by binding to certain receptors
    • Superantigens (toxic shock)
    • by forming pores in the cytoplasmic membrane
    • Hemolysins
    • Leukotoxins
    • 2. Toxins that need to enter cell : A/B toxins
    • Diphtheria toxin, Shiga toxin etc.
    • 3. Toxins directly delivered into host cell – Type III and IV secretion systems
    • Apoptotic toxins
    • Protein kinases and phosphorylases
  90. Superantigens
    • Bacterial exotoxins that stimulate T-cells independently of antigen
    • Mediate aberrant cross-linking of MHC II and T-cell receptor, resulting in activation of T-cells in absence of specific peptide.
    • Leads to massive release of cytokines, IL-1 and TNF, causing systemic reaction of fever, blood clots, diarrhea, decreased blood pressure and shock.
    • The best characterized superantigens are the microbial toxins from Staphylococcus aureus and Streptococcus pyogenes, causing Toxic shock syndrome.
  91. A/B toxins
    • Two subunits, A and B
    • B subunit binds to receptor
    • A subunit is transferred into the interior of the cell
    • BOTOX
    • Diphtheria toxin (diphtheria)
    • Cholera toxin (cholera)
    • Heat-labile enterotoxin (diarrhea)
    • Shiga toxin (dysentery)
    • Botulinum toxin (botulism)
    • Tetanus toxin (tetanus)
    • Pertussis toxin (whooping cough)
  92. Type III and IV secretion systems
    • Needle-like structure enables bacteria to inject exotoxins into host cells
    • Secreted proteins (Yersinia bacterium)
    • YopH: dephosphorylate proteins required for phagocytosis
    • YopE; disrupt actin filaments
    • YopJ/P: initiate apoptosis of macrophage
  93. Germ Theory of Disease (Koch’s Postulates):
    • o The microorganism must be found in abundance in all organisms suffering from the disease, but not in healthy organisms
    • o The microorganism must be isolated from a diseased organism and grown in pure culture
    • o The cultured microorganism should cause disease when introduced into a healthy organisms
    • o The microorganism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent
  94. Laboratory Diagnosis of Bacteria
    • • Purpose: to provide patients with first rate care (therapy/treatment)
    • • Procedure:
    • o Specimen collection: two types, body fluids and discharges
    • o Identification (classification) of bacteria
    •  Can classify the bacteria using many different methods, however the most accurate is genomic
    •  If the bacteria grows very slowly, can use direct identification (without culture)
    •  If direct detection is not an option, must use pure culture methods
    • • Morphologic Characteristics: colony and cellular morphology
    • o Staining reactions such as simple stains, differential stains and special stains
    • • Growth Characteristics: effects of temp, oxygen, pH, antibiotics on growth
    • o Generally human pathogens are mesophiles and neutrophiles
    • o GasPak jar provides a no oxygen environment (anaeorbes) and the Candle jar provides ~10% oxygen for microarophiles
    • o Can use in vitro testing of antibiotics against isolated strains (disk diffusion)
    • • Biochemical characteristics
  95. Hemolysis test (hemolysin activity
    •  B-hemolytic: complete lysis of RBCs
    •  G-hemolytic: non-hemolytic, no effect on RBCs
    •  A-hemolytic: incomplete lysis of RBCs
  96. Fermentation Assay
    •  Phenol Red (PR) carbohydrate broth
    • • Detects if acid is produced during fermentation
    • • If PR is red then pH>7
    • • If PR is yellow then pH<7 (carbs broken down by fermentation)
  97. Coagulase Test
    some bacteria produce coagulase: if there is a fibrin clot, the sample contains coagulase
  98. Catalase Test
    nullifies toxic oxygen radicals; if there are bubbles, it means the bacteria has catalase enzyme
  99. Serological Characteristics: Antigen/antibody reaction
    •  ELISA
    •  Western Blot
    •  Radioimmunoassay
  100. Genomic Characteristics:
    •  By comparing DNA restriction patterns (RFLP)
    •  By detecting specific bacterial gene(s) using PCR or hybridization
    •  By determining genomic DNA sequences (16S ribosomal RNA gene sequencing)
    • • fastestr Most accurate genetic test