Review for Lab Exam #2 Notes

Week 7: Virulence Factors

• Bacteria use virulence factors to defeat and trick our immune system so they can cause disease.

Activity 11: Survey of Virulence Factors

• Adhesion:
  • Proteins help bacteria stick to the host.
  • Often a very specific interaction between host and bacteria.
• Colonization factors:
  • Proteins that allow bacteria to colonize certain parts of the body.
  • Biofilms:
    • Communities of bacteria that take in and share nutrients.
  • Immune response blockers:
    • Requires both adhesion and colonization proteins.
  • Dental plaque on teeth:
    • Cause Chronic infections; antibiotics have a hard time getting in.
• Invasion factors:
  • Allow for host membrane breakdown and for the bacteria to pass into host cells.
• Toxins:
  • Bacteria release poisons such as hemolysins and toxins produced by C. difficile (C. diff).
• Immune response blockers:
  • Inhibit the host immune system defenses.
  • M proteins:
    • Similar to proteins found on the surface of host cells, help bacteria evade phagocytosis.
    • Example: Streptococcus pyogenes.
  • Coagulases:
    • Enzymes that produce fibrin from fibrinogen at the site of infection.
    • The clot offers protection from phagocytosis.
  • Capsule:
    • Glycocalyx that surrounds each individual bacterial cell, preventing phagocytosis.
  • DNAse:
    • Lyse DNA; neutrophils spit out nets of DNA to capture invading bacteria.
    • DNAse is a mechanism used by bacteria to escape these neutrophil nets.
    • Positive result = clear zone around streak of bacteria.

Activity 12: Hemolysis

• Streak bacteria on Sheep blood agar to see if it has the ability to lyse sheep red blood cells.
  • Identify hemolysis from isolated colony.
  • Indicative of the production of hemolysins or, more often, some other kind of cytolysin.
  • They are a virulence factor!
  • Function in the body to lyse white blood cells (no sheep red blood cells in your body!!).
• Types of Hemolysis:
  • Gamma hemolysis:
    • No change around or under isolated colonies.
  • Alpha hemolysis:
    • Change around isolated colonies; brownish or greenish circular zones around colonies (not transparent).
  • Beta hemolysis:
    • Change around isolated colonies; clear zones around colonies (e.g., S. pyogenes and Staph).

Week 9: Immunology

Activity 13: Peripheral Blood Smear

• Components of Blood
• Normal Ranges:
  • Normal count: 5,000-10,000 WBCs/µL
  • WBC counts >10,000/µL indicates INFECTION
  • Normal ranges will be given, or you’ll be told the result is high.
• Neutrophils:
  • 40-75%
  • High numbers indicate localized bacterial infection.
  • Phagocytic cells; eat bacteria and die.
• Lymphocytes:
  • 20-50%
  • High numbers indicate intracellular infections, which are primarily viral infections, or whooping cough, some cancers.
  • Low numbers may indicate HIV.
  • Two Types: T and B cells
• Monocytes:
  • 1-5%
• Eosinophils:
  • ~5%
  • High numbers indicates allergic condition or parasitic helminth (worm) infection.
• Basophils:
  • 0.5%

Activity 14: ABO-Blood Typing

• What do they stand for?
  • Different sugar molecules (glycosyltransferases) on RBC surfaces
• Why do they matter?
  • Blood transfusions
  • If introduce a foreign antigen, the body will create antibodies specific to the newly transferred RBCs
  • Antibody binds to antigen on cell surface: Agglutination and lysis of transferred RBCs, artery blockage, kidney failure à fatal!!!
  • DON’T EVER GIVE A PATIENT AN ANTIGEN THEY DON’T ALREADY HAVE!
• How is blood type determined?
  • Add drop of patient’s blood to known commercial anti-sera
  • If specific surface antigens are present: anti-sera will cause antibody mediated crosslinking of cells and agglutination

Rh-Factor

• D-antigen:
  • RBC transmembrane antigen
• Genetically determined:
  • DD, Dd = Rh+, dd = Rh-
• Factor in determining compatible blood transfusions similar to ABO antigens. DO NOT HAVE NATURAL ANTIBODIES!
• Not expressed by pathogens, but can lead to fetal-maternal incompatibility.
  • Mother Rh status ≠ fetus Rh status.
  • Can lead to maternal antibody response against fetal RBCs, complications during pregnancy.
  • Treat mother with blocking antibodies
  • if you’re pregnant and are (-) for Rh factor, you will be given Rhogam
• Uniquely need Rhogam for Rh because maternal anti-Rh ab is IgG and can cross placental barrier

Example Questions:

1. What are the blood types of samples 1 and 2?
   1) A- 2) A+
2. What antigens are on the RBC surface for a given blood type?
   1) A 2) A and Rh
3. What anti-sera will cause agglutination for this type?
   1) A 2) A and Rh
4. What Natural (IgM) Antibodies will be present in the patient’s blood?
   1) anti-B 2) anti-B
5. What blood types can the patient receive?
   1) A- & O- 2) A+, A-, O+, O-

Week 10

Activity 15: Mystery Case #1 & PCR

What are the differences between PCR and PFGE?

PCR

• Purpose:
  • To test for the presence/absence of a gene.
• Start with:
  • Genomic DNA
• Digest all of the DNA using a restriction enzyme or endonucleases
• Amplify a specific region of the DNA (requires polymerase)
• Primers
  • each product (or band on a gel) requires two individual primers
• Run amplified DNA on gel
• Multiplex PCR simply amplifies more than one region at a time

DNA fingerprinting (PFGE)

• Purpose:
  • To compare the relatedness of different isolates.
• Start with:
  • Genomic DNA
• Digest all of the DNA using a restriction enzyme or endonucleases
• The bands represent entire chromosome
• Because there are more bands to separate, DNA must be separated using pulse-field gel electrophoresis (PFGE)

Multiplex PCR and PFGE Gels

• Multiplex PCR:
  • Testing 3 bacterial strains for the presence of two genes
    • Question 1: Which band tells you the organism?
      • 16S rDNA
    • Question 2: The isolates from which patients are methicillin resistant?
      • P2, P3
• DNA fingerprinting (PFGE):
  • Testing if 3 bacterial isolates are from the community or hospital
    • Question 3: Does the isolate from patient 2 match the community-acquired strain or the hospital-acquired strain?
      • Hospital

Example Questions:

1. Are any vial contaminants from the same source as our Vial #1? NO
2. Any vials the same? 2 & 7; 3 & 8
3. If all these vials were produced in the same facility, is there a problem there? They are not from a single source. But YES! Who wants contaminated medication vials?!

Case study #1: logical path

• Gram-stain: first step to identify a BACTERIAL pathogen (found it was a gram + cocci – likely either staph or strep!) – strep cat (-) staph cat (+)
  • Quick review: Gram positive: Purple, Gram Negative: Red/Pink
  • Cocci: Spherical, Bacilli: Rod
• Blood agar plate – beta hemolysis
• Catalase test – positive – Important first test because it differentiates Staphylococcus from Streptococcus
• Coagulase test – positive ID definitive for Staphylococcus aureus
• Kirby-Bauer Assay (antibiotic susceptibilities) – PCR for mecA (methicillin resistance)
• DNA fingerprinting (PFGE) used to identify the common source

Week 11

Activity 16: Handwashing & Hand Sanitizer

• Selective vs Differential Media
  • selective Distinguishes bacteria
    • by preventing some types of bacteria from growing
  • differential Distinguishes bacteria
    • by using some visual marker (e.g. color) but does not inhibit growth in any way.
• These two types of media are not mutually exclusive.
• Mannitol salt agar is both
  • selects for salt tolerant skin flora (GRAM POSITIVE RODS or COCCI) and differentiates between organisms that ferment mannitol and those that don’t.
• MacConkey agar is both
  • selects for gram-negative enteric RODS (gut flora) and differentiates between lactose fermenters and non-lactose fermenters!
• Colonies from skin were grown on TSA or MSA plates before and after washing with alcohol disinfectant or hand soap.
• For MSA:
  • BOTH selective and differential.
  • Looking for skin flora.
  • quadrant contains transient bacteria and normal flora: after wash quadrants

Week 12

Activity 17: The Sepsis Unknown

• Blood culture – (+)! first step to identify a BACTERIAL PATHOGEN

  1. Gram Stain
  2. Growth on Primary media
     1. Sheep Blood agar plate -Hemolysis
     2. MacConkey agar plate (can use growth on this plate to verify gram (–) rod)
     3. Chocolate Agar for fastidious organisms
     4. TSA (just in case)

• Sheep blood agar

• MacConkey (LF & NLF)

Week 13

Activity 18: ELISA and Activity 19: Throat Culture

Antibody vs Antigen

• A Pathogen contains many antigens
  • Antigens:
    • specific components of the pathogen which are recognized by the immune system
    • Ex.) the spike protein of SARS CoV-2
• Antibodies recognize and bind to specific antigens. They are bound by the epitope.
  • Epitope:
    • The specific nucleotide sequence expressed on the antigen.

Activity 18: ELISA

• Enzyme-Linked-Immuno-Sorbent Assay
• Enzyme necessary for reaction to produce detectable signal (ie color change)
• This Assay requires the use of antibodies, at least one of which is linked to enzyme.
• Requires process of adsorbing components to a solid WHY?
  • Some pathogens are not easily grown on media.
  • The disease may be asymptomatic.
  • The patient had the disease a while ago.

Types of ELISAs

• Direct ELISA:
  • Detecting pathogen directly by using a capture antibody that binds specifically to an antigen present on pathogen.
    • "do they have this antigen in their body?"
• Indirect ELISA:
  • Detect antibodies produced in response to a pathogen by presenting a viral antigen as bait (looking for evidence of infection)
    • "did they mount an immune response?"

Example Questions:

• Where is the antigen from the patient sample?
• Which antibody is from serum?

ELISA HIV screen

*If positive titer is 25 and above, which patients are negative?
*What further test should be done to confirm the positive results?

Activity 19: The Throat Culture Group A Strep

• Streptococcus pyogenes
  • strep throat, scarlet fever, rheumatic fever
  • Beta-hemolytic
  • Bacitracin-sensitive
• Two main methods diagnosis:
  • Blood agar throat culture
  • QuickVue Rapid strip test

QuickVue Rapid Strep A Test

• Positive test-antibiotic treatment
• Negative test-perform the throat culture no antibiotics yet!

Blood Agar Throat Culture

• Beta (S. pyogenes)
• Gamma
• Normal throat bacteria
• Bacitracin disk

Week 14

Activity 20: Mystery Case #2

Case study #2 STOOL CULTURE

• Hektoen-enteric agar
• MacConkey agar
• Campy agar
• Sorbitol MacConkey agar
• Ova and parasite exam

For stool cultures: NON- fermenting is BAD

• PFGE to identify cause

Selective vs Differential Media

• Selective:

  • Distinguishes bacteria by preventing some types of bacteria from growing

• Differential:

  • Distinguishes bacteria by using some visual marker (e.g. color)
  • Remember that all of the bacteria still grow on the plate

• These two types of media are not mutually exclusive

• Mannitol salt agar (MSA) is both!

  • selects for salt tolerant skin flora (GRAM POSITIVE rods or cocci) and differentiates between organisms that ferment mannitol and those that don’t

• So is Macconkey agar

  • selects for Gram-negative rods and differentiates between lactose fermenters and non-lactose fermenters!

• And Hektoen-Enteric agar (HE)

• selective for Gram- negative rods and differentiates between lactose fermenters and non-lactose fermenters, also for NLF shigella is pale green, Salmonella has black centers

Lab Media