Microbiology #2

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Microbiology #2
2012-07-17 01:06:52

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  1. What is sterilization?
    The killing or removal of all viable organisms (including endospore)
  2. What is inhibition?
    Effectively limiting microbial growth
  3. What is decontamination?
    The treatment of an object to make it safe to handle
  4. What is disinfection?
    Directly targets the removal of all pathogens, not necessarily all microorganisms
  5. What are the physical methods of antimicrobial control?
    Heat, Radiation, Filter
  6. What are the chemical methods of antimicrobial control?
    • Used on external surfaces: Sterilants, disinfectants, sanitizers, antiseptics
    • Used internally: antibiotics, antivirals, antifungals
  7. What is the most widely used method of controlling microbial growth?
  8. What do high temperatures do to macromolecules?
    High temperatures denature macromolecules
  9. What is the autoclave?
    • The autoclave is a sealed device that uses stame under pressure:
    • Allows temperature of water to get above 100°C
    • At 15psi, steam reach 121°C, sterilization is achieved in 10-15 min
    • The object being sterilized wil reach this temperature. Not suitable for heat-sensitive object/liquid
    • Not the pressure that kills things, but the high temperatures
  10. What is pasteurization?
    • Pasteurization is the process of using precisely controlled heat to reduce the microbial load in heat-sensitive liquids
    • Does not kill all organisms, it is not a method of sterilization
  11. What pathogens does pasteurization reduce significantly?
    • Listeriamonocytogenes
    • Salmonella enterica
    • Campylobacter
    • E. Coli O157:H7
  12. What is flash pasteurization?
    72°C for 15 seconds
  13. What is bulk pasteurization?
    65°C for 30 mins
  14. How is UV used for antimicrobial control?
    • UV has sufficient energy to cause modifications and breaks in DNA, which inhibit replication, transcription and cause microorganism death
    • UV is useful for decontamination of surfaces
    • Cannot penetrate solid, opaque, or light-absorbing surfaces
  15. What is ionizing radiation?
    • Electromagnetic radiation that produce ions and other reactive molecules
    • Generates electrons, hydroxyl radicals, and hydride radicals
    • Amount of energy required to reduce viability tenfold is analogous to D value
  16. What are some sources of radiation?
    • Cathode ray tubes (electrons)
    • X-Rays
    • radioactive nuclides
  17. What is radiation used for?
    Radiation is used for sterilization in the medical field and food industry
  18. Why is filter sterilization used?
    • Filtration avoids the use of heat on sensitive liquids and gases
    • Pores of filter are too small for organisms to pass through
    • Pores allow liquid or gas to pass through
  19. What are depth filters?
    • Fibrous sheet or mat made from an array of fiber (paper or glass)
    • Use to sterilize liquid, air
    • HEPA filters
  20. What are membrane filters?
    • Function more like a sieve
    • A type of membrane filter is the nucleation track (nucleopore) filter
    • Filtration speed can be increased by syringe, pump, or vacuum
  21. What are the 3 classifications of antimicrobial agents?
    • Bacteriostatic
    • Bacteriocidal
    • Bacteriolytic
  22. What is bacteriostatic?
    What is bacteriodical?
    What is bacteriolytic?
    • Bacteriostatic-Inhibit growth of microorganism
    • Bacteriocidal-Kill microorganism
    • Bacteriolytic-Kill microorganism by inducing lysis
  23. What is disc diffusion assay?
    • Antimicrobial agent added to filter paper disc
    • MIC is reached at some distance
    • Zone of inhibition- area of no growth around disc
  24. What is minimum inhibitory concentration (MIC)?
    • The smallest amount of an agent needed to inhibit growth of a microorganism
    • Varies with the organism used, inoculum size, temp, pH, etc
  25. What is the zone of inhibition?
    Area of no growth around disc
  26. What are the two classifications of chemical antimicrobial agents?
    • Products used to control microorganisms in commercial and industrial applications
    • Products designed to prevent growth of human pathogens in inanimate environments and on external body surfaces
  27. What are some examples of products used to control microorganisms in commercial and industrial applications?
    • Chemicals in foods
    • Air-conditioning cooling towers
    • Textile and paper products
    • Fuel tanks
  28. What are some examples of products designed to prevent growth of human pathogens in inanimate environments and on external body surface?
    • Sterilants
    • Disinfectants
    • Sanitizers
    • Antiseptics
  29. What are Sterilants?
    • Destroy all forms of microorganism, including endospore
  30. What are antiseptics?
    • Applied to the surface of living tissues or skin (must not be toxic for animals/humans)
    • Does not kill endospores
  31. What are antimicrobial drugs?
    Antibiotics, antifungals, antivirals: applied outside or inside the body of animals/humans (must not be toxic for animals/humans)
  32. How do phenol/phenolics act as antimicrobial agents?
    Disinfectant/antiseptic- disrupts cytoplasmic membrane, protein denaturant (high concentration)
  33. How do alcohols act as an antimicrobial agent?
    Disinfectant/Antiseptic- Lipid solvent and protein denaturants
  34. How do halogens act as antimicrobial agents?
    Disinfectant/Antiseptic/Sterilant- Chlorine (S/D): oxidizing agent- Iodine (A): iodinate tyrosine residues in protein, oxidizing agent
  35. How do heavy metals act as antimicrobial agents?
    Disinfectant- modify proteins, interact with RNA, DNA...several of different mechanism
  36. How do quaternary ammoniums act as antimicrobial agent?
    Interact with phospholipids of cytoplasmic membrane
  37. How do alkylating agents act as antimicrobial agents?
    Disinfectant/Sterilant- Ethylene gas, formaldehyde, glutaraldehyde. Very toxic
  38. How are antimicrobial drugs classified?
    • Molecular structure
    • Mechanism of action
    • Spectrum of antimicrobial activity
  39. What are the properties of a good antimicrobial drug?
    • NO side effects, must be far more toxic for bacteria than mammalian cells
    • Broad spectrum of activity to facilitate rapid medical intervention
    • Appropriate bioavailability and pharmacokinetic (must reach the site of infection)
    • Low cost to develop and manufacture
  40. What is selective toxicity?
    The ability to inhibit or kill a pathogen without affecting the host
  41. What is salvarsan?
    One of the first antimicrobial drugs, use to treat syphilis (treponema pallidum)
  42. What are growth factor analogs?
    • Structurally similar to growth factors but do not function in the cell
    • Analogs similar to vitamins, amino acids, and other compounds
  43. What are Sulfa drugs?
    • Discovered by Gerhard Domagk in the 1930s
    • Sulfanilamide is an analogue of p-aminobenzoid acid
    • Bacteriostatic
  44. What is isoniazid?
    • A growth analog effective only against Mycobacterium
    • Interferes wwith synthesis of mycolic acid
  45. What are Nucleic acid base analogs?
    • Have been formed by the addition of bromine or fluorine
    • Stop DNA replication, translation
  46. What are quinolones?
    Antibacterial compounds that interfere with DNA gyrase
  47. What are antibiotics?
    Antibiotics are antimicrobial agents naturally produced by a variety of bacteria and fungi to inhibit or kill other microorganisms
  48. Why do gram-positive and gram-negative bacteria very in their sensitivity to antibiotics?
    The cell wall is a major factor
  49. What are beta-Lactam antibiotics?
    • One of the most important groups of antibiotics of all time
    • Include penicillins, cephalosporins, and cephamycins
    • Bactericidal, Bacteriolytic
  50. What are Penicillins?
    • Discovered by Alexander Flemin, isolated from Penicillium chrysogenum (mold)
    • Primarily effective against gram-positive bacteria
    • Some synthetic forms are effective against some gram-negative bacteria
    • Inhibit cell wall synthesis
  51. What are Cephalosporins?
    • Produced by fungus Cephalosporium
    • Same mode of action as the penicillins
    • Commonly used to treat gonorrhea (Neisseria gonorrhea)
  52. What are Aminoglycosides?
    • Kanamycin, neomycin, amikacin, streptomycin
    • Target 30S subunit of ribosome, cause misreading of mRNA
    • Bactericidal
  53. What is Chloramphenicol?
    • Bind to 23S rRNA and block peptide elongation
    • Bacteriostatic
  54. What are Macrolides?
    • Erythromycin
    • Broad-spectrum antibiotic that targets the 50S subunit of ribosome, block protein synthesis
    • Bacteriostatic
  55. What are Tetracyclines?
    • Broad-spectrum inhibition of protein synthesis, bacteriostatic
    • Inhibits functioning of 30S ribosomal subunit, block protein synthesis
  56. What is Daptomycin?
    • Also produced by Streptomyces
    • Used to treat gram-positive bacterial infections
    • Forms pores in cytoplasmic membrane
  57. What is Platensimycin?
    • New structural class of antibiotic
    • Broad-spectrum, effective against MRSA and vancomycin-resistant enterococci
  58. What is antimicrobial drug resistance?
    The acquired ability of a microorganism to resist the effects of a chemotherapeutic agent to which it is normally sensitive
  59. What is producer tolerance?
    • Lack target sites (no peptidoglycan)
    • Modify target sites
    • Lack of uptake mechanism
  60. What is resistance mechanism?
    • Destroy or modify the antibiotic (beta-lactamase)
    • Modify the target site
    • Modify uptake mechanism
    • concentration
    • Efflux pumps: reduce intracellular
  61. How does one acquire resistance?
    • Mutation of target sites
    • Plasmic acquisition
  62. What is an example of a pathogen that has developed resistance to all known antimicrobial agents?
    Methicillin-resistant S. Aureus (MRSA)
  63. How can resistance be minimized?
    Resistance can be minimized by using antibiotics correctly and only when needed (reduce selection)
  64. How do antiviral drugs work?
    • Most antiviral drugs also target host structures, resulting in toxicity (viruses use host cell machinery)
    • Risk to the host may not justify the use of antiviral
  65. What are the most successful and commonly used antivirals?
    Most successful and commonly used antivirals are the nucleoside analogs (eg AZT): block reverse transcriptase and production of viral DNA (RNA viruses)
  66. What are protease inhibitors?
    Inhibit the processing of large viral proteins into individual components
  67. What are fusion inhibitors?
    Prevent viruses from successfully fusing with the host cell
  68. Why do fungi pose special problems for chemotherapy?
    Fungi pose special problems for chemotherapy because they are eukaryotic: much of the cellular machinery is the same as that of animals and humans
  69. What are a few drugs that target unique metabolic processes not found in mammals?
    • Ergosterol synthesis (nystatin, fluconazole)
    • Cell wall synthesis (inhibitor of chitin synthesis)
  70. What is the decimal reduction time?
    The time required to reduce the population by a factor of 10.
  71. What is the thermal death time?
    Thermal death time is the time needed to kill all cells at a given temperature
  72. What is thermal death time dependent on?
    Thermal death time is dependent on the population size of the microorganism tested. Need to standardize the starting number of ells to be able to compare the sensitivity of different microorganisms
  73. What is Minimum Lethal Concentration (MLC)?
    The lowest concentration of an agent that kills a test organism
  74. The four W’s
    • Hospital-acquired infections spread by:
    • • Wind-placement of contaminated ventilators-pneumonia
    • • Water-urethral catheters-UTI
    • • Wounds
    • • Wires-IV lines
  75. Heat Methods of Antimicrobial Control:
    • • Heat
    • • Radiation
    • • Filter
  76. Sterilization:
    The killing or removal of all viable organisms within a growth medium
  77. Inhibition
    Effectively limiting microbial growth
  78. Decontamination
    The treatment of an object to make it safe to handle
  79. Disinfection
    Directly targets the removal of all pathogens, not necessarily all microorganisms.
  80. What is the most widely used method of controlling microbial growth?
    Heat sterilization. High temperatures denature macromolecules.
  81. Denature
    Structural change in macromolecules.
  82. What can survive heat
  83. Autoclave
    • • Sealed device thqt uses steam under pressure
    • • Allows temps to exceed 100C.
    • • It is the heat that kills things, not the pressure
  84. Pasteurization
    • • Process of using precisely controlled to reduce the icrobial load in heat-sensitive liquids
    • • Does not kill all organisms, so it is different than sterilization.
  85. Radiation sterilization Methods:
    • • Microwaves
    • • UV
    • • X-rays
    • • Gamma rays
    • • Electrons
  86. Mechanisms of UV Radiation:
    • • Sufficient energy to cause modifications and breaks in DNA
    • • Useful for decon of surface
    • • Generates thymine dimers
  87. Thymine dimer
    Abnormally chemically bonded adjacent thymine bases in DNA, caused by UV light.
  88. Sources of Radiation Sterlization
    • • Cathode ray
    • • X-ray
    • • Radioactive Nucleotides
  89. Ionizing Radiation Sterilization
    • • Electromagnetic radiation that produces ions and other reactive molecules
    • • Generates electrons, hydroxyl radiacals, and hydride radicals
    • • Some microorganisms are more resistant to radiation than others.
  90. Uses of radiation Sterilization
    • Medical field and food industry.
    • Approved by the WHO for decon of foods susceptible to contamination:
    • • Hamburger
    • • Chicken
    • • Spices
  91. Filter Types
    • • Depth filters (HEPA)
    • • Membrane filter-like a sieve
    • • Membrane filter- can be accomplished by syringe, pump or vacuum
    • • A type of membrane filter is the nucleation track (nucleopore) filter
  92. Nucleopore Filter
    Nuclepore® polycarbonate track-etched membranes are made from high quality polycarbonate film and have sharply defined pore sizes, high flow rates and excellent chemical and thermal resistance.
  93. Chemical Antimicrobial Agent Classifications
    • • Bacteriostatic-inhibits growth of bacteria
    • • Bactericidal-kills bacteria
    • • Bacteriolytic-kills and destroys bacteria
  94. Minimum Inhibitory Concentration (MIC)
    • Smallest amount of an agentneeded to inhibit growth of a microorganism.
    • Varies with the organism used, , inoculum size, temp, pH, etc.
  95. Disc diffusion assay
    • Antimicrobial agent added to filter paper discMIC is reached at some distance
  96. Zone of inhibition
    • Area of no growth around disc
    • Small paper discs are added to culture tray
  97. Antiseptic
    • Chemical agents that kill or inhibit growth and are safe enough to be applied to living tissue
    • • Alcohol-lipid solvent and protein denaturant
    • • Quat disrupts phospholipid bilayer
    • • H202 Oxidizing agents remove electrons
    • • Betadine replaces hydrogen to inactivate molecules
  98. Sterilants
    Destroy most forms of microbial life where heat or radiation cannot be used.
  99. Folic Acid metabolism Antibacterial drugs:
    Trimethoprim, Sulfonamides
  100. Cell Wall Synthesis Inhibitors
    • • Beta-lactam drugs are the most widely used
    • • Primarily effective against gram-positive bacteria
    • • Vancomycin
    • • Bacitracin
    • • Penicillin
    • • Cephalosporins
  101. Protein Synthesis inhibitors
    • • Macrolide Antibiotics
    • • Erythromycin binds to 50s RNA subunit
    • • Tetracycline prevents tRNA binding
    • • Clindamycin obstructs peptide bond formation
  102. Antimicrobial Drug Resistance Mechanisms
    • • Mutation
    • • Inactivation
    • • Efflux
  103. Transfer Methods
    • • Transformation
    • • Conjugation
    • • Transduction
  104. Role of pharmaceutical sales reps
    Results in symptomology treatment instead of lab tests to determine actual bug.
  105. Folic Acid Metabolism Inhibitors
    • • Folic acid (THF) is a vitamin supplement
    • • Humans require folic acid for synthesis of nucleic acids
    • • Bacteria synthesize their own folic acid
    • • Trimethoprim. Sulfa
    • • Stephens Johnson symdrome: separation of epidermis and dermis
  106. What are Cell Wall Synthesis Inhibitors?
    • • Target Cell Wall Synthesis
    • • Lactam Ring binds to transpeptidase, which make peptide cross-links
  107. Carbapenems
    Beta lactam from Streptomyces BACTERIA
  108. Floroquinolones
    • • DNA gyrase/topoisomerase inhibitors-binds to the enzyme
    • • Cipro
    • • Nalidixic Acid
  109. Helicase…DNA gryase
    Splits the DNA strand….Un-super coils the build up below the helicase
  110. Koch’s Molecular Postulates
    • • Used to establish the link between a particular virulence factor and the pathogenicity of that microbe.
    • • The phenotype or property under investigation should be associated with pathogenic members of a genus or pathogenic strains of a species
    • • Specific inactivation of the gene(s) associated with the suspected virulence trait should lead to a measurable loss in pathogenicity or virulence, or the genes should be isolated by molecular methods. Specific inactivation or deletion of the genes should lead to loss of function in the clone.
    • • Reversion or alleic replacement of the mutated gened should lead to restoration of pathogenicity, or the replacement of the modified gene(s) for its alleic counterpart in the strain of origin should lead to loss of function and loss of pathogenicity or virulence. Restoration of pathogenicity should accompany the reintroduction of the wild-type gene/s.
    • • To assess molecular Koch’s postulates, a system for site-specific genetic manipulation of that microbe is required.
  111. Scott Samuels
    • • Genetic Transformation of Borrelia burgdorferi (Lyme disease) with coumarin-resistant gyrB
    • • First to genetically manipulate the microbe
    • • Site-directed mutations were successfully introduced using a PCR product
    • • A PCR product was introduced
  112. Techniques of Genetic Manipulation
    • • Plasmid Transformation
    • • Mutagenesis (insertional, replacement)
  113. Definition: Disease
    any deviation from or interruption of the normal structure or function of any body part, organ, or system
  114. Definition: Infection
    Invasion by and multiplication of pathogenic microorganisms in a bodily part or tissue, which may produce subsequent tissue injury and progress to overt disease through a variety of cellular or toxic mechanisms.
  115. Definition: Normal Flora
    Bacteria that live in the body, many performing necessary functions
  116. Definition: Pathogen
    A disease causing organism
  117. Definition: Pathogenicity
    Ability of an organism to cause disease
  118. Definition: Opportunistic
    Normal flora that causes an infection when the body’s immunity is lowered
  119. Definition: Frank Pathogen
    pathogen-organism that causes disease in the host by direct interactions with the host
  120. Definition: virulence
    the ability of the organism to invade the tissues of the host.
  121. Definition: Lethal dose and LD50
    Dose to kill; dosage that kills 50% of a population. There are variables.
  122. Definition: Virulence Factors
    the properties (i.e., gene products) that enable a microorganism to establish itself on or within a host of a particular species and enhance its potential to cause disease
  123. Mechanisms of virulence Factors (9):
    • 1. Adherence
    • 2. Antiphagocytic
    • 3. Invasion
    • 4. Toxins
    • 5. Spreading/invasion factors
    • 6. Lytic toxins
    • 7. Protein synthesis blocking toxins
    • 8. Toxins blocking nerve function
    • 9. Iron Acquisition
  124. Adherence
    • • Pili (N. gonorrhea, E. coli)
    • • Flagella (V. cholerae)
    • • Cell wall component (S. Pyogenes M-Protein)
  125. Antiphagocytic factors:
    • Capsule (s. pyogenes)
    • Cell surface proteins
  126. Invasion factors
    • • Cell surface proteins (invasion of Y. pestis)
    • • Motility (flagella)
  127. Toxins
    • • Endotoxin is a part of cell released on lysis (LPS and septic shock)
    • • Exotoxin is manufactured and secreted from cell (hemolysins)
  128. Invasion toxins (spreading factors) (5)
    • • Hyaluronidase (breaks hyaluronic acid, a basement membrane component)
    • • Streptokinase (clots)
    • • Collagenase
    • • Elastasse
    • • Proteases (a variety)
  129. Lytic toxins
    • • Lecithinase (phospholipase)
    • • Protein pores (Staph alpha toxin, streptolysin O)
  130. Protein synthesis blocking toxins
    • • Diptheria toxin, A1-B1, target is elongation factor 2
    • • Cholera toxin, [cAMP] increase
  131. Toxins blocking nerve function
    Clostridium tetani inhibition of glycine release
  132. Iron acquisition ‘toxins’
    Siderophores are used to steal Fe from our Lactoferrin and Transferrin
  133. 1. Which of the following extracellular enzymes produced by Group A streptococci is called "spreading factor," an enzyme important in skin and soft tissue infection?
    A. Streptokinase
    B. Hyaluronidase
    C. M Protein
    D.  Deoxyribonuclease C
    E. None of the above
    . D
  134. 3. All of the following statements about the M-protein of Group A Streptococci are correct EXCEPT :
    A. The amino terminal portion (distal portion) is variable, accounting for over 80 distinct serotypes.
    B. M proteins allow streptococci to resist phagocytosis. C. Antibodies to M protein confer type-specific immunity.
    D. M protein is the major virulence factor of Group A streptococci.
    E. M protein is the major constituent of the capsule of Group A streptococci.
  135. 12. Movement of DNA from one bacteria to another through a tubular bridge or pilus:
    A. Conjugation
    B. Transposition
    C. Transfection
    D. Transduction
  136. 38. Specific organisms that are considered strict anaerobes include:
    A. E. coli and Streptococcus pneumoniae
    B. Bacteroides and Klebsiella
    C. Fusobacterium and Clostridium
    D. Peptostreptococcus and Nocardia
  137. What bacteria causes 90% of ulcers?
    Helicobacter pylori
  138. Adhesins
    • The cell wall component of a bacterium which allows it to adhere to a cell and cause disease.
    • Example-Gonoccoccus either has pili or not-pili+means disease
  139. Cell Wall components of adhesins
    • LTA-LipoTeichoic Acid acts as an adhesin
    • M-Protein(membrane protein) are antiphagocytic. Example: S. pyogenes.
  140. General Antiphagocytic Factors
    • A. Capsules-are polysaccharides, the are slippery, antiopsionic and decrease phagocytosis. S. pyiogenes if capsule+ is virulent.
    • B. Invasion-The ability to invade and get to the target site
    • INVASIN is a little protein that helps to invade i.e. Y. enterococoticia
    • Flagella help to invade target tissues
  141. What happens if Ampicillin is administered with gram negative septicemia?
    Leads to shock and death due to cell wall synthesis inhibiition-endotoxins are released into thebloodstream.
  142. Endotoxins
    • usually a cell wall component of gram -, LPS (lipopolysaccharide)a
    • One big sx of bugs breakiong up is fever
    • Low amounts of lps leads to fever, inflammationm
    • hight amounts of lps leads to shock, intravascular clotting and death
  143. Exotoxins
    • are proteins produced by both gram + and gram-
    • Exotoxins are released by the bacteria to help dissolve nearby proteins so they can eat.
    • Example S. aureus has several different toxins associated with different diseases.
  144. Exotoxins target...
    • Tissues
    • Some are localized such as leukocidins (kill WBC)
    • Some are disseminated (diptheria killls cells indiscriminately)
  145. Invasion toxins AKA:
    • Hyaluronidase breaks down hyaluronic acid (part of the basement membrane)
    • Streptokinase breaks up clots--is a spreading factor
  146. Clostridium spreading factors (3):
    • Collagenases-breks up collagen
    • Elastases-breaks up fibrous elastin
    • Proteases-breaks up protein
  147. Lytic toxins:
    • \Leukocidins have lecithinase (a phospholipase) that puts holes indiscrimately into cells.
    • RBC attacked means anemia.
  148. S. aureus has A toxin-describe:
    similar to complement (the complement cascade that makes holes in cells)
  149. S. pyogenes has what lytic toxin?
    Streptolysin O which forms a channel by binding to cholesterol in mammal membranes.
  150. Protein Synthesis blocker toxin
    A-B toxins A=activity-does the work, B is the binding molecule
  151. Diphteria toxin how does it work? 4 steps
    • The A-B toxin is taken into the target cell by endocytosis
    • The phagosome swallows the toixn
    • The lysosome and phagosome work together and the acidic pH breaks up the vacualized toxin...(vacuole)
    • The result is that A part of AB toxin is an Elongation factor #2 which is an mRNA translation blocker
  152. Toxins that block nerve function
    • Clostridium--C. tetani causes inhibition of glycine release and leads to musculr contraction
    • C. botuliuum inhibits ACH and results in inability to contract (rubber neck)
  153. Stapholococcus 7 qualities
    • 2nd most common nosocomial intection (E. coli is first)
    • 80% of pus filled lesions are caused by S.a aureus
    • Opportunistic-always present on skin
    • Morphology-Gram+, thick cell wall
    • Forms in 3D clusters like grapes
    • Looks yellow on plate
    • Normal flora on the skin and nostrils
  154. Staph infections: 5 items
    • Abcess small to large-pimple-boil-carbuncle, contained in a wall of fibrin beneath the skin
    • Wound lesions-sutures, slivers
    • Scalded skin syndrome-common in nurseries-Toxin exfsolitin causes skin to slough-Scaleded skin syndrome
    • Blood-septicemia
    • Food poisoning-Common source is guacamole
  155. Which two pathogens form spores?
    Bacillus and Clostridium
  156. Cholera toxin has
    cAMP which is responsible for massive rice-water diarrhea of cholera