1. Module 1 covers immune-microbe interactions, antigen presentation pathways, and early life immune responses. It also covers defenses against extracellular and intracellular bacteria. 2. The adaptive immune system defends against intracellular bacteria through dendritic cell antigen presentation to CD4 T cells, which activate macrophages to kill bacteria via ROS/RNS. Intracellular bacteria enter host cells through mechanisms like T3SS injection and phagocytosis induction. 3. Intracellular bacteria evade immunity by preventing phagolysosome formation, inhibiting neutrophil free radical production, and escaping the phagosome into the cytosol. The adaptive immune system and inflammation contribute to defenses against extracellular bacteria through antibodies, cytokine production, and acute inflammation.

1. Module 1 Objectives
Module 1.1 – Immune:Microbe Interactions
1. Recall the key concepts of the immune system: innate
vs. adaptive immunity
Feature Innate Adaptive
Time of Response Rapid (minutes/hours) Slower (days/weeks)
Diversity Limited to classes or groups
of microbes
Very large; specific for each
unique antigen
Microbe Recognition General patterns; nonspecific Specific to microbes and
antigens
Nonself recognition Yes Yes
Response to repeated
infection
Similar with each exposure Immunologic memory; more
rapid and efficient response
with subsequent exposure
Defense Barriers (skin, mucous
membranes), phagocytes,
inflammation, fever
Cell killing; tagging of antigen
by antibody for removal
Cells Phagocytes (macrophages,
neutrophils), natural killer
cells, dendritic cells
T and B lymphocytes
Molecules Cytokines, complement
proteins, acute-phase
proteins, soluble mediators
Antibodies, cytokines
2. Know the key steps and consequences of the two
antigen presentationpathways
The Cytosolic (Endogenous) Pathway

2.  This pathway utilizes MHC I and is often used for viruses. You can remember that the
Cytosolic (endogenous) pathway is used for viruses by remembering that a virus must
first be inside the cell (e.g. cytosol), or inject its contents into the cell, to produce an
effect. MHC I is on almost all cells.
 The first step occurs when the cell notices foreign proteins and tags them with ubiquitin.
This tagging causes the proteins to be degraded by a proteasome, forming small
peptides.
 Transporter Associated with Antigen Processing (TAP) will then take these peptides and
transport them to the endoplasmic reticulum, where it will encounter MHC I.
 MHC I takes the foreign peptides from TAP, passes through the Golgi, and presents
them at the cell’s plasma membrane.
 Presentation of the antigen activates CD8 cytotoxic T cells and release perforin and
granzyme. Perforin forms a pore, granzyme enters. With this, the cell is killed.
The Endocytic (Exogenous) Pathway

3.  In this pathway, the main contributor is MHC II and usually starts with bacteria outside
the cell. MHC II is only used in professional antigen-presenting cells (dendritic cells, B
cells, etc)
 First, the microbe is phagocytosed into an endosome (phagosome) and this phagosome
fuses with a lysosome, forming a phagolysosome. This is where the bacteria are
degraded into peptide fragments.
 Separate from the phagolysosome in the ER and the Golgi, MHC II is being
formed/processed.
o The two chains are formed (MHC-II alpha and beta) and connect with CD74 – a
protein that rests in the antigen binding portion of MHC II, effectively preventing
peptide binding.
o When the MHC II-CD74 complex fuses with the phagolysosome, Cathepsin S
cleaves CD74 into CLIP (Class II associated invariant chain peptide).
o Here, the peptide fragment can take the place of CLIP with the help of HLA-DM.
 HLA-DM leaves and the MHC II and antigen complex are presented at the plasma
membrane.
 The antigen is eventually recognized by CD4 T cells that become activated and begin to
stimulate B cells to produce plasma cells (antibodies), and memory B cells.
3. Know the key features of early life immune responses
and relevance to vaccination

4.  In fetal life, monomer IgG is the only immunoglobulin capable of crossing the placenta.
 As a neonate, the maternal IgG levels decline 3-6 months after birth as the baby begins
to activate its own immune system.
 While breastfeeding, maternal IgA, immune cells, cytokines, chemokines and growth
factors are passed in breast milk.
o Levels are much greater in colostrum than mature milk.
o IgA is involved in mucosal immunity, protecting against GI and respiratory
infections.
 Babies have a greater response to peptide antigens than polysaccharide antigens.
o Thus, the Hepatitis B vaccine is given at birth and the polio vaccine given at 2
months (both are peptides)
o Polysaccharide-based pneumococcal vaccine is given at age 2.
Module 1.2 – Extracellular Bacteria
1. Know how the innate immune systemdefends
against bacteria at body’s surface
Skin
Our keratinized skin offers a relatively impermeable barrier for bacteria. We have glands that
secrete lysozyme, toxic lipids (sebum), and hydrogen ions that alter pH.
Mucosa (mechanical and bactericidal mechanisms)
 These cells are not keratinized and areas include the GI, nasopharynx, upper airway,
urinary and reproductive tracts.
 Pathogenic bacteria can reside in mucosa without causing issue, however, bacteria are
also able to passively enter tissue to cause infection.
 Local bacterial proliferation is controlled by mechanical cleaning and lack of available
free iron, which a lot of microbes depend on.
 In the GI tract, we are protected from bacteria via:
o Peristaltic motility
o Mucus secretion
o Stomach acid
o Bile (used as a detergent)
 In the lower respiratory tract, a sterile environment is maintained via constant mucosal
movement and removal via cilia. Additionally, mucosal epithelial cells do not mount an
inflammatory response against bacteria that colonize there.
o Epithelial damage from smoking or viral/bacterial infections reduces mucociliary
movement and promotes opportunistic infections
 Bactericidal agents in mucosal fluids include:

5. o Lactoferrin – binds free iron, upon which microbes rely
o Defensin – forms pores in bacterial membranes to allow efflux of iron/nutrients
o Lysozyme – breaks bacterial peptidoglycan
o IgA – interferes with bacterial adhesion, inactivates toxins, promotes bacterial
aggregation for removal by mucus
2. Know how the innate immune systemdefends
against extracellular bacteria inside the body
The innate immune systemprovides a rapid response for removal of bacteria.
 In the blood stream, the complement system and neutrophils are used
 In the tissue, macrophages engulf bacteria and then neutrophils infiltrate.
The complement system
 A collection of about 20 inactive proteins made by the liver and are abundantly present
in the blood
 There are three complement pathways
o 1) Lectin pathway: has Mannose Binding Lectin that recognizes bacterial poly-
mannose
o 2) Classical pathway: has Complement C1 that recognizes IgG-tagged bacteria
o 3) Alternative pathway: has complement C3 that interacts with bacteria
 Pathways generates the membrane attack complex (MAC) in the bacteria membrane,
which allows water to enter and lyse the bacteria.
 Pathways promote formation of C3a that tags bacteria for phagocytosis
 Pathways promote formation of C3a and C5a to induce neutrophil infiltration (C5a is a
chemoattractant)
Neutrophils
 The most abundant immune cell
 They become activated by using their toll like receptors (TLRs)
o TLR2 recognizes the peptidoglycan of gram-positive bacteria
o TLR4 recognizes the LPS of gram-negative bacteria
 They have Complement receptors to recognize complement-tagged bacteria
 They have Fc receptors to recognize Ig-tagged bacteria
 Neutrophils phagocytose and kill bacteria, and infiltrate tissue in response to
chemoattractant signals from tissue macrophages.
 Neutrophil weaponry – Granules
o Primarily hydrolytic enzymes
o Secondary: enzymes, receptors, oxidases
o Tertiary: enzymes, receptors, oxidases

6. o Secretary: membrane markers, enzymes, receptors, oxidases
 Examples of neutrophil weaponry:
o Enzymes: lysozyme, NADPH oxidase complex
o Receptors: TLRs, complement receptors, Fc receptors, Formyl peptide receptor
(recognizes bacterial fMLP)
o Defensins: small proteins forming pores to lyse gram negative and gram-positive
bacteria, as well as fungi, yeast, and certain viruses.
o Lactoferrin: an iron chelator; makes iron unavailable for microbial growth
o Proteinases: MMP-8 and MMP-9 are matrix metalloproteinases that break down
collagen. Elastases break down elastin.
o Neutrophil free radicals: NADPH oxidase (comprised of p22, gp91 [membrane
bound] and p40, p47, and p67 [cytosolic] subunits) generates superoxide anions.
 Superoxide anions can be converted to hydroxy radicals via SOD and Fe2+
via a Fenton reaction.
 Cellular bleach (hypochlorous acid) is created from hydrogen peroxide
using free chloride ions and the myeloperoxidase enzyme.
 These radicals attack nucleic acid, protein and lipid molecules to destroy
the ingested microbe.
 There is a disease associated with the inability of neutrophils to produce superoxide
radicals called Chronic Granulomatous Disease (CGD)
o The disease is an inherited mutation effecting the NADPH oxidase subunits and is
X-linked, so mostly effects boys.
o These patients cannot fight off bacterial or fungal infections that are usually mild
or nothing to worry about in normal individuals. They are susceptible to
repeated infections.
3. Know how extracellular bacteria evade the immune
system
Bacterial evasion of skin and mucosal mechanisms and barriers:
 Physical injury to skin
 Mucosal bacteria can passively enter tissue
 Mucosal bacteria can also actively invade deeper tissues through multiple mechanisms
including using enzymes to destroy tight junctions.
 H. pylori produces urease to generate ammonia to neutralize stomach acid
 Inflammation caused by initial bacterial infection could lead to tissue damage, allowing
bacterial entry.
Bacterial evasion while inside the body:

7.  Bacteria use their surface molecules as ligands to establish strong adhesion to the
surfaces of host epithelial cells.
 Bacteria can compete with host cells for iron and blood (for nutrients)
o N. meningitidis can steal iron from the host’s transferrin
o E. coli can secrete high affinity iron chelators (siderophores) to steal soluble iron
in the local environment before taking it up and releasing it into their cytoplasm.
 Bacteria can utilize capsules to reduce immune cell recognition
o Capsules can allow for mimicry of other cells, making them look like eukaryotic
cells
o Capsules can mask their surface antigenic epitopes so that our receptors cannot
detect them
o Capsules can render antibodies and complements ineffective by making the
respective structures they bind to inaccessible, making them safe from
phagocytosis
o Capsules can be responsible for phase variation, where the capsule is found in
high amounts when needing to evade host immune responses, low amounts
when needing to adhere to or enter host cells, and high amounts again after
gaining access to deeper tissues to protect against leukocytes within those
tissues.
 Bacteria can reduce tagging and phagocytosis
o Along with masking and making their receptors inaccessible, proteins in the
bacteria’s outer layer can inhibit complement deposition
 E.g. Proteins PspA and PspC in pneumococci
 E.g. C3 binding protein in S. pneumoniae
o Proteins in the outer layer can also prevent antibody binding
 E.g. protein A in staph aureus
o Bacteria can also activate Immunoglobulin proteases.
 E.g. in Neisseria, the proteases remove the Fc fragment. This effectively
prevents opsonization AND covers up its bacterial antigen!
 Bacteria also have various ways to reduce their chance of getting killed
o S. pneumoniae can use pneumolysin to deplete complement proteins (which
would reduce MACs)
o S. pyogenes and B. streptococci can use C5a peptidase to reduce neutrophil
infiltration
o S. aureus uses antioxidants to inactivate free radicals
o E. Coli uses catalase to convert hydrogen peroxide to water – preventing
formation of hypochlorous acid and hydroxy free radicals

8. 4. Know the contribution of adaptive immunity and
inflammationto the defense against extracellular
bacteria.
Adaptive Immunity
 Antibodies generated can inactivate virulence factors, inactivate toxins, increase
phagocytosis and provide antigen-specific IgA for mucosal defense
Inflammation
 Acute (local) inflammation
o Local vasculature response to limit injury/infection
o Neutrophil infiltration kills bacteria
 Systemic inflammation
o Acute phase protein production to tag or kill invading bacteria (e.g. C-reactive
protein production)
o Release of cytokines to further activate immune cells
o Stimulation of WBC production
o Fever generation to reduce bacterial activity
o Increased serum transferrin to bind free iron
o Increased tissue iron storage
Module 1.3 – Intracellular Bacteria
1. Know how the adaptive immune system defends
against intracellular bacteria
The defenses against intracellular bacteria are not well understood and the responses are not
as effective when compared to extracellular bacteria.
Briefly, the innate response can utilize an inflammasome that can detect intracellular bacterial
fragments in macrophages that can induce cytokine production and apoptosis of the infected
cell.
The Adaptive systemis the major contributor to intracellular defense.
 Dendritic cells
 Dendritic cells are not often targets for intracellular bacterial infection, although
dendritic cells in the spleen can be directly infected by L. monocytogenes. They
would then present MHC I and be killed by CD8 T cells.

9.  More often, the dendritic cells will acquire fragments of infected cells and bacteria.
When Salmonella enterica uses its T3SS to enter epithelial cells, dendritic cells will
phagocytose fragments of the infected epithelial cell and then present as MHC II to
CD4 T cells.
o Dendritic cells induce activation of CD4+ T cells, causing themto produce
IFN-gamma
o IFN-gamma activated bacteria-infected macrophages to enhance ROS and
NOS production to kill the bacteria.
 Macrophage ROS produced by NADPH oxidase. Their RNS are
generated by iNOS (inducible nitric oxide synthase).
 ROS (O2-) and RNS (NO) can form even more toxic
peroxynitrite (ONOO-)
 Patients with low CD4+ T cells (HIV/AIDS patients) are particularly
susceptible to infection with M. tuberculosis, M. avium complex and
certain species of Salmonella.
2. Know how intracellular bacteria enter host cells
First, it’s important to know which cells bacteria target most frequently, and those include:
 “First encounter” barrier cells – epithelial cells in the mucosa, GI and respiratory tracts
 “First encounter” immune cells – macrophages (monocytes), neutrophils, etc.
 “Barrier/lining” cells – endothelial cells
 Other cells including hepatocytes, erythrocytes and Schwann cells
When the immune cells phagocytose the bacterium due to recognition of its PAMPs, some
bacteria have mechanisms that allow them to survive inside the cell. Oftentimes the bacteria
stay inside vacuoles in the cytoplasm, leaving them free to replicate and feed off the host cell’s
nutrients.
When non-immune cells are targets for bacteria it gets a little different. Non-immune cells
cannot actively recognize and phagocytose bacteria. Instead, bacteria induce their own
phagocytosis.
As an example, consider the Salmonella enterica bacteria. When this bacterium encounters the
GI epithelial cell surface, it produces a “needle” complex (a type 3 secretion system, T3SS). The
needle goes entirely through the epithelial cell membrane and into its cytosol, where it then
injects effector proteins into the epithelial cell that induce cytoskeleton rearrangement. This
results in engulfment of the bacterium. While in the host cell, it remains in its original vacuole
and replicates.

10. 3. Know how they evade the host immune system
 Prevention of the phagolysosome formation that is normally responsible for degrading
the bacteria into peptides. Specifically, lysosomal acidification can be impaired, resulting
in inhibition of fusion between the lysosome and phagosome (mycobacterium)
 Prevention of free radical-mediated killing in neutrophils via inhibiting proper NADPH
oxidase assembly, preventing free radical generation (anaplasma phagocytophylum)
 Escaping from phagosome and entering the cytosol – some bacteria can rupture the
phagosome (listeria monocytogenes via listeriolysin)
o Advantage of this mechanism:
 The bacterium can replicate rapidly because of the abundant nutrients in
the cytosol
 The bacterium can spread easily to neighboring host cells
o Disadvantage of this mechanism:
 Bacterial proteins may be processed and presented via MHC I, resulting in
killing of both the host cell and the bacterium.
o The success of controlling infection depends both on the rate of bacterial
proliferation and abundance of CD8 T cells (e.g. Herpes virus and mycobacterium
tuberculosis replicates very slowly and so less chance of being detected and
producing an immune response). Not ideal for a rapidly replicating bacterium,
such as Salmonella enterica – which would produce a robust immune response.
Module 1.4 – Viruses
1. Know the types of viral infections
“Hit and run” (e.g. influenza)
 Virus infects the host, replicates rapidly, spreads to next host easily
 Requires robust immune response to control infection
“Hit and hide” (e.g. HIV)
 Virus infects host and then viral load drops due to an immune response but does not kill
all of the virus
 Virus remains in host and the host either clears the virus or becomes a victim of it

11. 2. Describe innate immunity against viral infections
Cytokines
 Immune cells use TLR-3,7,8,9, and 13 to recognize viruses. TLR recognition activates
cytokine production in the immune cell
 Viral infection causes almost all cell types to produce the type I cytokine IFN-1, mainly
coming from dendritic cells, and the type II cytokine IFN-gamma is produced from T and
NK cells.
o IFN-1 is highly potent against viruses. It can:
 Reduce host cell viral receptor expression to reduce viral entry
 Inhibit viral protein synthesis in the host cell
 Activate macrophages and NK cells
 Promote overall immune responses
 The viral dsRNA can also be recognized by enzymes (PKR [protein kinase R], RNAse L-2-
5A-dependent ribonuclease) that can inhibit viral protein synthesis and induce cytokine
production.
NK Cells
 Have KIR (Killing Inhibiting Receptors) CD159a, CD158,
CD94
 Have KAR (Killing Activating Receptors) CD314
 Have SHP-1: an SH2 domain-containing the phosphatase
1 protein (inactive when free, becomes an active
phosphatase enzyme when bound to KIRs)
 Have DAP10: activates PI3 kinase when bound to the
CD314 KAR
 Have perforin and granzyme that kill infected cells.
Notice the KIR, KARs, and
DAP10 are membrane bound

12. When NK cells recognize a normal healthy cell:
 The KIRs (CD159a, CD158, CD94) bind to HLA (the
“self” version of MHC I)
 SHP-1 bound to KIRs dephosphorylates, confirming
“selfness” & inhibits NK cell activation.
When NK cells recognize a virus-infected cell:
 If a host is infected, the HLA self-molecules go
away, resulting in inactivation of the inhibitory
pathway (because KIR’s no longer have
anything to bind to)
 Now, however, MHC-I is present in the form of
MIC A/B (which is not present on the surface of
healthy cells).
 This MIC A/B is bound by the KAR CD314
located on the NK cell.
 The KAR is bound by DAP10, activating PI3
Kinase, which tells the NK cell to activate and
kill the infected cell with perforin/granzyme

13. 3. Describe adaptive immunity against viral infections
The adaptive systemis more useful in secondary or continuing infections since it takes about 2
weeks to make antibodies following an infection.
Dendritic cells present to CD4 T cells, which activate B cells to make antibodies.
Antibody effects on viruses:
 Neutralizes viral pathogens
 Inhibits viral entry
 IgG able to cross placenta to protect fetus
 IgG and IgA in breast milk to protect newborn
4. Know key features of viral immune evasive
mechanisms
Escape by hiding
 Select mutants that change molecular identity
 Residing in a site that lacks many immune cells.
Escape by destroying immune cells
 Killing off T cells via pyroptosis – an inflammasome induced and caspase-1 mediated cell
death
Interfering with innate immunity response signaling pathways
 Viruses can interfere with TLR-mediated activation and inflammatory response
 IFN signaling pathway
Escape by altering host cell MHC expression to avoid NK cell killing of the infected host cell
Interfering with MHC I expression to avoid presentation and antibody generation

14. Module 2 Objectives
Module 2.1 – Introduction to Microbiology
1. Describe the morphologyof bacteria
Characteristics:
 Prokaryotic
 Unicellular
 1-5um in diameter (smaller than
WBCs at 12-15um)
 No membrane-bound organelles
 The nucleoid contains both DNA and
RNA
 They have a rigid cell wall with
peptidoglycan
 Some have motility
 Bacteria replicate via binary fission
Morphology:
 Cocci (circular)
 Bacilli (rods)
 Spirochete (spiral)
 Pleomorphic (lack a distinct shape)
“KNOW”:
Morphology Gram-positive Gram-negative
Cocci (circular)  Staphylococcus
(clusters)
 Streptococcus
(chains)
 Enterococcus
(pairs and chains)
 Neisseria
(diplococci)
Bacillus (rod)  Listeria
 Clostridium
 E. Coli
 Shigella
 Klebsiella

15.  Serratia
 Pseudomonas
 Bacteroides
Spiral   Treponema
Branching filamentous
growth (like fungi)
 Actinomyces
nocardia

2. Recall major infections caused by key bacteria
Organism Major infection
Staphylococcus aureus  Boils
 Impetigo
 Wound infection
 Osteomyelitis
 Bacteremia
 Pneumonia
 Endocarditis
Staphylococcus saprophyticus  Urinary tract infections
Streptococcus pyogenes  Tonsillitis
 Impetigo
 Cellulitis
 Necrotizing fasciitis
 Rheumatic fever
 Acute pharyngitis (strep throat, most
common)
Streptococcus algalactiae  Most common cause of Neonatal sepsis
Streptococcus pneumoniae  Pneumonia, meningitis, otitis media
Listeria monocytogenes  Neonatal sepsis
 Meningitis
E. coli  UTIs
 Bacteremia

16.  Neonatal meningitis
 Intra-abdominal infections
 Wounds
Klebsiella oxytoca  UTIs
 Intra-abdominal infections
Klebsiella pneumoniae  UTIs
 Intra-abdominal infections
Proteus mirabilis  UTIs
Serratia marcescens  Wounds
Shigella dysenteriae  Infectious diarrhea
Neisseria meningitidis  Meningitis
 Bacteremia
Neisseria gonorrhea  Gonorrhea
 Pelvic inflammatory disease
Pseudomonas aeruginosa  Pneumonia
 Bacteremia
Clostridium tetani (+)  Tetanus
Clostridium perfringens (+)  Gas gangrene
 Food poisoning
 Wound infections
Clostridium difficile (+)  Diarrhea
Bacteroides fragilis (-)  Intra-abdominal infections
Staphylococci (gram-positive cocci in clusters)
Streptococci (gram-positive cocci in chains)
Non-spore forming Bacillus (rods)
Enterobacteriaceae (gram-negative rods)
Gram-negative cocci and cocci-bacilli
Pseudomonads (gram-negative rod)
Anaerobes
3. Recognize the components of a bacterial cell and
their function

17. Structure Function Chemical
Composition
Peptidoglycan Gives rigid support Sugar backbone with
peptide side chains
that are cross-linked
**Found only in
bacterial cell wall
**D and L amino acids
(D AA’s not found in
eukaryotic cell walls)
**Major target for
antimicrobials
Surface fibers of gram-
positive bacteria
A major surface
antigen, although
rarely used in
laboratory diagnosis
Teichoic acid
Outer membrane of
gram-negative bacteria
 Lipid A
 Polysaccharide
An endotoxin
Major surface antigen
frequently used in
laboratory diagnosis
Cytoplasmic membrane Site of oxidative and
transport enzymes
Lipoprotein bilayer
without sterols
Ribosome Protein synthesis, site
of action for antibiotics
RNA and protein 50S
and 30S subunits
Nucleoid Genetic material DNA
Mesosome Cell division Invagination of plasma
membrane

18. Periplasm Contain hydrolytic
enzymes that can
destroy antibiotics
Space between plasma
membrane and outer
membrane
4. Compare and contrast gram-positive and gram-
negative bacteria cell wall
Component Gram-positive Cells Gram-negative cells
Peptidoglycan Thicker, multilayered Thinner, single layer
Teichoic acids Yes No
Lipopolysaccharide
(endotoxin)
No Yes
Lipid content Low lipid content High lipid content
Periplasmic space No Yes
Porin channel No Yes
Response to
lysozyme and
penicillin attack
Vulnerable to lysozyme
and penicillin attack
*because there is no outer
membrane layer*
Resistant to lysozyme and
penicillin attack
*protected by its outer
membrane layer*
Note they are exact opposites. Just remember one column and you’ll know the other.

19. 5. List bacterial virulence factors
Brief definitions:
 An endotoxin is released when the bacteria undergoes lysis
 An exotoxin is a protein released by both gram negative and positive bacteria. Can be a
neurotoxin or enterotoxin.
LPS
 Located on the outer membrane of the cell wall of gram-negative bacteria.
 Is an endotoxin
 Causes fever and shock secondary to infection
 Lipid-A is responsible for its toxic effects
Capsule
 Limits phagocytosis
 Allows adherence to human tissue
 Can be used to identify bacteria as composition is species specific
 Can be used as an antigen in some vaccines
Flagella

20.  Can help propel bacteria up the urethra into bladder
 Found primarily in gram-negative rods
 Can be used to identify bacteria
Pili (Fimbriae)
 Hair-like filaments that are shorter than flagella
 Found mainly on gram-negative organisms
 Mediate attachment to specific receptors of human cells
 Facilitates attachment between male (donor) and recipient (female) bacteria during
conjugation
Glycoclayx
 Adhere to structures
Spores
 Highly resistant
 Found in Bacillus and Clostridium species
 Spore forms when nutrients are depleted to protect against unfavorable conditions
 Spores have no metabolic activity and can remain dormant for years
6. Describe the growth and metabolic characteristics of
bacteria
Stages of Growth (4 phases)
 Phase I – Lag phase
o Metabolic activity but cells do not divide
 Phase II – Log phase
o Rapid cell division – binary fission
o Beta-lactamdrugs work in this phase as the bacteria make peptidoglycan
 Phase III – Stationary phase
o Nutrient depletion or toxic products cause growth to slow
o Steady state
 Phase IV – Death phase
o Decline in number of organisms

21. Types of Growth
 Obligate aerobes (A) – require oxygen, oxygen is the hydrogen acceptor
(mycobacterium tuberculosis)
 Obligate anaerobes (B) – cannot grow in the presence of oxygen. Response varies, some
die quickly, and others are able to survive but not grow. (clostridium tetani)
 Facultative anaerobes (C) – utilize oxygen to generate energy by respiration if it is
present, but they can use the fermentation pathway to synthesize ATP in the absence of
oxygen (Escherichia coli)
 Aerotolerant anaerobes (D) – can survive in the presence of oxygen, but utilizes the
fermentation pathway to generate ATP (streptococcus mutans)
 Microaerophiles (E) – requires environments with low levels of oxygen to survive
(helicobacter pylori)
Metabolism
 ATP generation via cellular respiration
o Fermentation (anaerobic glycolysis)
o Aerobic (glycolysis)
o Lactic acid fermentation

22. Alternative Methods of Growth/DNA transfer
Transformation
 Naked DNA fragments from one bacterium bind to cell wall and are taken up by
another bacterium
Transduction
 Virus that infects bacteria (bacteriophage), carries a piece of bacterial DNA from
one bacterium to another
Conjugation
 DNA is transferred directly by cell-to-cell contact
7. Distinguishpathogenic bacteria fromthose which are
considerednormal flora
Normal Flora Location
Bacteroides spp Colon
Clostridium Colon
Enterococcus faecalis Colon
Escherichia coli and other
coliforms
Colon, vagina, outer urethra
Propionibacterium acnes Skin
Staphylococcus aureus Skin, nose
Staphylococcus epidermidis Skin, nose

23. Module 2.2 – Blood Culture Review
1. Describe the role of blood cultures in diagnosing
Infections
Blood culture testing allows identification of pathogens to direct proper choice of antimicrobial
therapy. Blood cultures test for the possibility of bacteremia (bacteria in blood).
Cases where a culture would be warranted:
 Hyper (>38C) or Hypothermic (<36C)
 Neutropenia
 Sepsis or septic shock
 Known site of infection
 Leukocytosis
 Clinical suspicion of infection
 Fever of unknown origin
2. Describe the bloodculture collectionand organism
identificationprocess
Details:
 The best time to collect is around the time of a fever
 The best site to draw from is the peripheral veins
o With limited access to this site, central venous catheters or arterial lines can also
be used
o Catheters can be tunneled, non-tunneled, implanted, peripherally inserted, or
inserted at the midline.
o A catheter can be colonized at the site of entry or within the lumen, assess
carefully.
 There are two sets taken, each set has two bottles – 4 total
o One bottle supports aerobic growth, the other anaerobic (increased CO2/N)
o While two sets should be used to avoid false positives/contamination.
Sometimes the size/condition of the patient prohibits drawing four 10mL bottles.
 Children are adjusted at 1mL per year of age up to 10 years or based on
weight at roughly 1% of total blood volume.
 Blood cultures drawn while on antibiotics will decrease yield
Incubation of Organisms

24. 1. After blood is collected, if organisms are present then the bottle will increase levels of
CO2 from the bacteria’s metabolic activity.
2. The CO2 will react with a dye in the vial that acts as a sensor
3. There is fluorescent material in the dye which is modulated/activated by LED light.
4. Once activated, the data is detected by a computer and analyzed by a row board.
Positive results are displayed audibly, on screen, and with the Positive Vial system
indicator.
 TTP (time to bottle positivity) varies amongst organisms and this helps differentiating
pathogens from contaminants. TTP can be influenced by:
o Intrinsic growth rates of the organism
o Inoculum size
o Presence of antibiotics
o Liver failure or neutropenia
 Most aerobic bacteria grow within 48 hours. Anaerobes, yeast and contaminated
specimens grow slower
 For rapid growing organisms, knowing TTP can aid in antibiotic de-escalation (narrowing
antibiotic therapy instead of broad use of antibiotics)
Organism Identification
 Automated organism identification and susceptibility
o Identifies and determines susceptibility within 24hr
o Four primary systems:
 MicroScan WalkAway
 BD Phoenix Automated Microbiology System
 Vitek 2
 Sensititre ARIS 2X
 Manual Systems (does not give susceptibility info)
o RapID uses chemical reactions to determine organism
 Takes about 4 hours
 Uses color reactions to detect more than 400 organisms
 PCR platforms (does not give susceptibility info)
o Film array: Organisms lysed, their nucleic acids released. Reverse transcriptase is
used to create DNA, then primers are added to amplify target DNA.
o The target DNA is analyzed to identify the organism.
o This systemcan also tell you the presence of resistance in some organisms
 MALDI-TOF (mass spectrometry; Matrix Assisted Laser Desorption/Ionization – Time of
Flight) (does not give susceptibility info)
o UV light evaporates solid compounds into ions
o Ions drift toward a detector
o The ion’s TOF is proportional to its molecular weight. A spectrum is generated.
o This spectrum is compared to a library of organisms to identify the bacterium.

25. 3. Interpret a gram stain
If a blood culture result is positive, a gram stain is warranted.
 Gram positive organisms will show purple hue as the crystal violet is retained in their
thick peptidoglycan layer
 Gram negative organisms will show red when the crystal violet is washed away and red
carbol fuschin counterstains the bacteria.
 You’ll also want to look at the shape and arrangement of the bacteria to help identify
the species.
4. Recognize commonsolidmedia preparations used for
bacterial growth
The organism from the positive result must be grown to help identify it.
 Blood Agar – almost all bacteria grow on this agar. Differentiates hemolytic organisms
(alpha, beta and gamma hemolytic)
 Chocolate Agar (lysed RBC’s) – identifies fastidious organisms (organisms with specific
nutrient requirements) such as Neisseria or Haemophilus
 MacConkey Agar (contains bile salts) – Gram-negative bacteria
o The bile salts inhibit growth of gram-positive bacteria
 Mueller-Hinton – used for determining antimicrobial susceptibility
5. Identify variables associatedwith contaminated
specimens
 Contamination can result in a false positive
 Rate of false positives results should not exceed 3%
 Variables affecting contamination rates:
o Improper use of skin antisepsis
o Improper culture bottle preparation
o Collection from a vascular catheter
 Organisms likely to be contaminates:
o Coagulase-negative staphylococci
o Corynebacterium species
o Bacillus species (but not anthrax)
o Micrococcus

26. Module 2.3 – Urine Culture and Urinalysis
1. Interpret urinalysis results
pH
 Normal urine pH is 5.5-6.5 (slightly acidic)
 Urea splitting organisms may raise urine pH (proteus species)
Specific gravity
 Normal is 1.003 – 1.03
 Correlates with urine osmolality and gives insight into hydration status
 Not helpful for UTI
If pyuria present
 Defined as WBC count >10 cells per high power field
 Indicates inflammation
 Leukocyte esterase will be present
o False positives seen with vaginal fluid, presence of trichomonas, or eosinophils in
urine
 Absence of pyuria eliminates presence of an infection
 Presence of pyuria does not confirm infection
Nitrites
 Organisms that convert nitrates to nitrites can be detected in urine
o Enterobacteriaceae converts
o Gram positive organisms (enterococcus spp, staph spp) and pseudomonas do not
convert.
 A false positive may result if a patient uses phenazopyridine (a urinary analgesic)
 Test is not 100% reliable for identifying or predicting organisms. E. coli
(Enterobacteriaceae) may show up as nitrite negative
Hematuria
 Normal: <5 RBCs/hpf
 Can be seen with cystitis or pyelonephritis
Proteinuria
 May be present in UTI
Microscopic analysis

27.  Squamous epithelial cells
 Casts may be seen in severe infection
 May see presence of bacteria
2. Describe the role of urine cultures to diagnose
infections
Urine cultures help investigate the possibility of a UTI.
A urine culture is indicated when a patient presents with:
 A complicated UTI
 Relapse of recurrent UTI
 Diabetes
 Kidney transplant
 Pregnancy
 Pyelonephritis
 A recent catheterization
 Men can also get UTIs
Culture Media: Blood and MacConkey agar are preferred for gram negative bacteria.
Colony Counts
 Considered significant bacteriuria if voided collection counts are:
o Women:  105 cfu/mL
o Men:  104 cfu/mL
o Lower colony counts ( 103 cfu/mL) may be considered if symptomatic or a
common uropathogen is present.
o If a catheter is used to collect specimen, significant bacteriuria is considered to
be defined as anything 103 cfu/mL
3. Identify variables associatedwith contaminated
specimens
 When collecting urine using a catheter, the catheter may be colonized and lead to a
false positive. Only the port should be used to collect a sample. Never collect from the
collection bag.
 Urine is an excellent medium for growing bacteria
o Process the sample rapidly

28. o Refrigerate if possible
o Preservatives like boric acid maintain quality for 24-48 hours
 Contamination can arise from improper storage, catheter insertion, cleansing,
processing, etc.
 Clues to contamination:
o Low colony counts of an organism
o 2 or more isolates of an organism (polymicrobial infections uncommon, reassess)
 Presence of skin flora
 Large quantity of epithelial cells ( >20 cells/hpf)
o An abnormal value is > 5 – 10 cells/hpf
Module 2.4 – Respiratory Cultures
1. Describe the role of respiratory cultures for
diagnosing pneumonia
A diagnosis of pneumonia is oftentimes made with clinical presentation and a positive culture.
In up to 50% of patients, the causative agent is not identified.
2. Describe the respiratoryculture collection process
Oral secretions often result in false positives due to contamination, resulting in overtreatment
and thus increased resistance in respiratory organisms.
Methods of collection of specimen (chosen by severity of the illness):
 Non-invasive
o Expectorated sputum (from a deep cough)
 Most controversial collection method, large variation in quality, storage
and laboratory delivery times. Also, about 1/3 of patients can’t produce
sputum.
 Ways to improve collection quality:
 Obtaining specimen prior to antibiotic therapy
 Rinsing mouth prior to expectoration
 No food up to 2 hours prior to specimen collection
 Prompt inoculation of culture media
o Endotracheal aspirate (if the patient is intubated)
 Invasive
o Bronchoscopy techniques
 Using a bronchiolar lavage
 Using a protected specimen brush

29. Direct examination of the collection/specimen
 Confirms material submitted is representative or acceptable
 Identifies cellular components and debris
 Identifies suspected organisms
 Assists with empirical antibiotic therapy
 Gram stains
o Assess the suitability of the specimen for continued processing.
o Not helpful in patients with mycoplasma, chlamydia, or legionella (can’t be seen
on gram stain)
o May be useful in determining the causative agent (determining
morphology/arrangement)
 Cellular components determine specimen quality
o >10 Squamous epithelial cells per low power field suggests contamination
o Look for >25 WBCs per low power field
o Basically, you want high amounts of WBCs and very few squamous cells to help
determine whether the sample is quality or not.
Quantification of the collection/specimen
 Quantification can be semiquantitative or quantitative
 Semiquantitative
o Reported as 1+, 2+, 3+, or 4+ where the agar is divided into 4 quadrants. E.g. if
reported as 3+, colonies can be found in 3 quadrants.
 Quantitative – usually for those who have received a bronchoscopic approach to
specimen collection
o Reported as # of colony forming units per mL
o BAL (bronchoalveolar lavage) > 104 cfu/mL is consistent with pneumonia
o PSB (protected specimen brush) > 103 cfu/mL is consistent with pneumonia
 Note: sputum can be directly tested using PCR. This method is used for organisms that
are difficult to culture, including:
o Bacteria: Chlamydia, Bordetella, Legionella, Mycoplasma
o Viruses: influenza, parainfluenza, respiratory syncytial virus
3. Recognize commonsolidmedia preparations used for
bacterial growth
 Blood agar
o Supports growth of gram-positive cocci and gram-negative bacilli
o Detects hemolytic patterns (alpha, beta, gamma)
 Chocolate agar

30. o Permits recovery of H. influenzae and other fastidious organisms
 MacConkey agar
o Permits recovery of gram-negative organisms
o Classifies organisms as lactose positive or negative via color change
4. Identify variables associatedwith contaminated
specimens
 Proper optimization of antimicrobial therapy for pneumonia/influenza depends greatly
on respiratory cultures
 Contamination can occur during collection, transport, and storage of the sample
 Gram stains help determine quality of a sample and help drive proper antimicrobial
selection by assessing the suitability of the specimen for continued processing and
helping identify the causative organisms.
 Overtreatment is associated with contaminated or poorly collected specimens.
Module 2.5 – Wound Cultures
1. Recall commonskin microbiota
By location:
Dry areas Moist areas Sebaceous areas
Proteobacteria Staphylococcus spp Propionibacterium spp
Corynebacterium spp Pseudomonas spp Staphylococcus spp
Bacteroides Corynebacterium Streptococcus spp
Propionibacterium Proteobacteria
Staphylococcus spp
Streptococcus spp
2. Describe the wound culture collectionprocess

31. Although the most common and least expensive technique is the swab, collection technique
depends on the type of wound
Collection w/ Cellulitis
 Needle aspiration
o Performed at the center or leading edge of the wound
o The area is injected with a small volume of saline and aspirated
o Poor yield, particularly because there is such little fluid/drainage in the first
place.
Collection with an Abscess
 Involves incision and drainage. Pus is aspirated from the wound and sent to lab in an
anaerobic transport tube.
 Swabbing of skin is not recommended with an abscess due to high contamination risk
Collection from bone
 A bone biopsy can help direct antimicrobial therapy
 A common method with chronic wounds, post-surgical assessment of infection, or if
antimicrobial therapy has failed
Collection from Joints
 Synovial fluid is withdrawn directly from the infected joint and inserted into an aerobic
(unlike an abscess) tube
 Use of a blood culture is not recommended (inability to perform a cell count)
 Joint fluid is gram stained and cultured, and also sent off for fluid analysis (e.g. cell count
and crystal analysis)
 Fluid analysis is used to differentiate between infection.
o There will be a predominance of white blood cells and neutrophils
Culturing a Chronic Wound
 The best option (gold standard) is a tissue biopsy as it removes tissue deep within the
wound
o The reason this is the best method is because if you just swab the surface and
culture the surface bacteria, that specific bacteria may not actually be causing
the infection (a chronic wound always has some bacteria on it). This may lead us
to target therapy to an organism that isn’t actually causing the problem.
 Other methods include using a swab or a needle aspiration (although not useful if it’s a
dry wound).

32. 3. Recall joint fluid analysis values used to assess
infectionand inflammation
Joint Fluid Analysis:
4. Identify variables associatedwith contaminated
specimens
 With chronic wounds, any open wound is at least somewhat contaminated on the
surface. This does not mean the wound is infected.
o This means it would be unnecessary to culture all chronic wounds, because all
would have at least some degree of “infection.”
 Once the organisms multiply enough for an immune response to be elicited
(swelling/redness), the wound can be considered infected.
 The biggest driver of infection is the volume of bacteria, virulence of the bacteria, and
the host response.
 When assessing colonization of bacteria in a wound, it is important to assess whether or
not the host is having a response when determining antibiotic needs. Most wounds are
contaminated, but if there is no host response then therapy may not be needed.
Module 2.6 – Interpreting Bacterial Susceptibilities

33. 1. Define qualitative and quantitative antimicrobial
testing methodologies
Qualitative
 Disk diffusion (Kirby-Bauer method)
o Here, antibiotics are placed on a disk and the diameter of
the empty space around the disk is measured. The
diameter of the zone depends on:
 The quality of the antibiotic disk
 How susceptible the bacteria is
 The concentration of the bacteria
 The molecular weight of the antibiotic (high MW
means the antibiotic can’t diffuse well into the agar)
 Doesn’t work well for vancomycin, colistin, or macrolide
antibiotics.
o Only gives information about susceptibility (meaning you only know if an
organism is susceptible, intermediate, or resistant to a drug)
o Does not give exact MIC (minimum inhibitory concentration)
o Although simple and cost effective with a wide variety of antibiotics to choose
from, the test is open to interpreter variability
Quantitative
All these tests give susceptibility info and the exact MIC
 Broth dilution
o Macrodilution and microdilution
 Macro: the antibiotic is diluted 50% (1 tube dilution) and lowest the
concentration at which growth does not occur on agar is considered the
MIC.
 Micro: Same as macro, but the agar is in gradient dilutions as well. The
gold standard in MIC determination. Used primarily in research settings.
 Agar dilution

34.  E-test
o Provides precise scale of MICs
o Can be used for resistance detection
o The test strip is in gradient dilutions of antibiotics
o Expensive, but variety of antibiotics to choose from
o In the example to the right, the MIC would be around 1.5
because that is where you first see some empty space
where bacterial growth is inhibited.
 Vitek-2 Automated system
o Provides identification AND susceptibility testing, as well as MICs.
o Uses colorimetric technology
o Results in 2-18 hours (variable)
2. Provide examples of qualitative and quantitative
testing
See above
3. Describe the process for determining MICs

35. See above
4. Interpret MICs and apply it to antibiotic selection
Three interpretations of a MIC:
1) Resistant
 Proper therapy not achievable with concentration of drug at recommended dose at the
site of infection.
2) Intermediate
 A buffer. Accommodates for technical variability. Higher doses can treat at some sites.
Prolonged perfusion/duration of the antibiotic may be effective, etc.
3) Susceptible
 Proper therapy is achievable with concentration of drug at recommended dose at the
site of infection.
 MICs are unique for a given pathogen to an antibiotic
 MICs are not directly comparable to one another – the same pathogen can have a
different MIC when compared to two different antibiotics.
 MICs need to be interpreted against standardized concentrations
 ***MICs should only be used as tools that help with antibiotic selection. One MIC may
be lower, but it may be a broad-spectrum antibiotic. As a healthcare provider we need
to be aware of broad spectrum use and resistance development, and, when possible,
choose the best targeted therapy option that concentrates at the site***
List of common broad-spectrum agents:
 Aztreonam
 Ceftazidime
 Cefepime
 Piperacillin/tazobactam
 Meropenem
 Doripenem
 Ceftolozane/tazobactam
 Ceftazidime/avibactam
 Meropenem/vaborbactam
 Levofloxacin
 Ciprofloxacin

36. 5. Describe the factors consideredin the setting of
breakpoints
Breakpoints are concentrations of antibiotic that best predict clinical success.
Factors:
 Clinical outcomes – at what MIC do patients have clinical success vs. what MIC do
patients have clinical failure?
 Pharmacokinetic (drug effect vs. time) and pharmacodynamic (concentration vs time)
data
 Epidemiological cut-offs – where several bacterial MICs are looked at, we should see a
distribution/bell curve. Based on these MIC distributions, breakpoints are set.
The FDA requires initial breakpoint data from manufactures but does not and cannot require
them to reassess the data thereafter.
Thus, the CLSI (Clinical and Laboratory Standards Institute, a non-federal standard-setting
organization) has FDA representatives as advisors and publishes the M100 document annually
in January. This document contains updated breakpoint data.
Module 2.7 – Rapid Diagnostic Tests Used in the Microbiology Lab
1. Discuss the roles of rapid diagnostic tests for
management of infections
They enhance the function of clinical microbiology labs by providing accurate organism
identification and timely antimicrobial susceptibility data. They also help personalize patient
treatment and provide early detection of resistance.
2. Describe differentrapid diagnostic tests available for
diagnosing infection
Rapid diagnostic tests utilize antigen/antibody tests, where a specific antibody is used for each
antigen. The tests are always performed on the serum.
Types of antibody/antigen tests and what they are used to detect:

37. o Agglutination reactions (Latex, coagulase tests)
 The coagulase test differentiates between staph aureus (agglutination
will occur, coagulase positive) and staph epidemidis (coagulase negative)
o Immunochromatographic tests
 Legionella (BinaxNow)
 Malaria (BinaxNow)
 Cryptococcal Antigen (IMMY CrA LFA)
 Qualitative or semi-quantitative results via serial dilutions
 Rotavirus (ImmunoCard Stat)
 Ebstein-Barr (Acceava Mono II)
 Influenza (Various products)
o Immunofluorescent assays
 Herpes viruses
 Herpes simplex virus
 Varicella zoster
 Cytomegalovirus
 Respiratory viruses
 Influenza
 Adenovirus
 Parainfluenza
 Respiratory Syncytial virus
 Bacteria
 Bordetella
 Legionella
o Enzyme-linked immunosorbent assay (ELISA)
 Clostridium difficile via toxin detection or GDH
Agglutination reactions (Latex Agglutination)
 Antibodies are bound to latex beads.
When mixed with serum, the antibody-
latex complex will bind with the antigen
and agglutinate. Example 
 Classifies:
o Coagulase (?)
o Streptococcus Lancefield
classification
 Identifies:
o Streptococcus pneumoniae
o Haemophilis influenzae

38. o Ebstein-barr Virus (mononucleosis)
Immunochromatographic tests (lateral flow assays)
 Results can be qualitative or semi-quantitative and require minimal training. Great test
for point-of-care or field-use applications.
How the strip is set up:
 A dye-labelled antibody, specific for target antigen, is present on the lower end of a
nitrocellulose strip or in a plastic well provided with the strip.
 There is also a bound antibody that is specific for the target antigen that forms a test
line on the strip.
 There is a control strip that will contain the antibody or the antigen. This control strip
should always turn positive.
How the test is performed:
1) The sample (which may or may not contain the antigen) is placed on the sample pad, where
it mixes with antibody
2) Assuming the proper antigen was present from the sample, capillary action causes the
sample-antibody complex to move across the membrane
3) The sample eventually reaches the test band, which contains detector molecules and bound
antibody. IF the sample-antibody complex was formed (i.e. if the sample contained the proper
antigen), the detector molecules in the test band will turn the strip red/purple.

39. Influenza Immunoassay Tests
 Quick (30min), simple, can be used at bedside/office
 False-negatives are common, false positives also occur.
 Cannot distinguish influenza A vs B viruses and cannot provide strain subtype
 Highly dependent upon proper sample collection, transport, storage and timing of
sample from onset of illness
 Works best with 104 – 106 infectious particles
Immunofluorescent Assays
 Used to help detect organisms that are more difficult to grow
 Antigen is placed on a slide. Antigen-specific antibodies are fixed with a fluorophore and
added to the slide, where they bind to antigen.
 After about 15-30min of incubation, the prep is washed and viewed with a fluorescence
microscope.

40. ELISA
1) Enzyme conjugates (antibody plus enzyme) are added to a well with the sample (antigen) in
it.
 The enzyme linked to the antibody is eventually used to visualize that the antibody has
bound to the antigen in the well.
2) Enzyme conjugate and sample are incubated for 45-60min
3) The preparation is washed to remove excess proteins or antibodies that didn’t bind anything
4) Chromogen is added. Chromogen contains a coloring agent and is a substrate for the enzyme
from step 1 – allowing visualization of antigen-antibody binding.
5) A stopping agent is added that halts the reaction. Results are interpreted.
Module 2.8 – Nucleic Acid Testing
1. Discuss the role of rapid diagnostic tests for the
management of infections

41. Nucleic acid tests are highly sensitive and specific and are slowly replacing many rapid
diagnostic tests.
2. Describe differentrapid diagnostic tests available for
diagnosing infection
The nucleic acid tests can be broken down into 3 categories: target amplification, probe
amplification and signal amplification
Target amplification techniques
PCR (polymerase chain reaction)
 Amplifies and detects small amounts of DNA
 Identifies an organism
 Assists with antimicrobial susceptibilities by detecting specific genetic markers (but does
not give the whole susceptibility profile, so oftentimes combined with other tests)
 Filmarray system:
o Organisms lysed, releasing their nucleic acids
o Reverse transcriptase binds to create target DNA from target RNA
o Primers and fluorescent dyes are added. The primers amplify the target DNA, the
fluorescent dye is added to identify the organism
o The sample is then heated and the DNA melts. Each organism’s DNA has a
different melting point, so this acts as a confirmation/secondary identification
method of the organism.
o Identifies:
 17 viruses and 3 bacteria of the respiratory tract
 24 organisms and 3 resistance mechanisms from blood cultures
 22 bacteria, parasites and viruses from the GI
Probe Amplification techniques
Peptide Nucleic Acid-fluorescence in situ hybridization (PNA FISH)
 Fishes for chromosomes – detects and localizes the presence or absence of specific DNA
or RNA sequences
 Offers specific target binding, short hybridization time (probe binding with rRNA), and is
reproducible
 How it works:
o The systemuses hydrophobic fluorescent PNA probes that bind highly conserved
rRNA sequences in the nucleus of the cell.

42. o When the bound rRNA is amplified and fluorescence detected, a unique color
will appear for the specific species or organism.
 Available tests (as with PCR, helps identify but does not provide much info on
susceptibility):
o Candida
o Differentiates staphylococcus aureus vs. coagulase negative staphylococci by
detecting the mecA gene, of which is commonly found in S. aureus (MRSA)
o Enterococcus
o Gram-negative panel
 E. Coli
 Klebsiella pneumoniae
 Pseudomonas aeruginosa
 This test can also be used to identify STDs
o Chlamydia trachomatis, Neisseria gonorrheae, Trichomonas vaginalis
 Detecting Clostridium difficile toxin
o Nucleic acid testing is used to identify the presence of toxin b gene (tcdB)
o One potential issue is that the test doesn't say the patient has the infection, it
only says that the toxin genetic material is present. It could be detecting live cells
or dead cells. So it's very good at determining the organism, but you still have to
bring it back to whether or not the patient has an active disease/infection
o Tests that can detect the tcdB gene:
 Cepheid Gene Xpert
 BD GeneOhm
 Prodesse ProGastro
Module 2.9 – Candida Diagnostics
1. Reviewcommondiagnostic tools used for identifying
Candida species
Germ tube test (triptacase soy broth) – differentiates albicans vs non-albicans yeast within 4
hours when viewed under the microscope
Trehalose -- differentiates glabrata vs. non-glabrata yeast (if glabrata is present, the solution
will turn from blue to yellow within 3 hours)
API 20 – identifies species of Candida by analyzing their use of specific carbohydrates. By
analyzing their physiological/biochemical profiles, the systemcan interpret what species is
present within 48-72 hours.

43. Sensititre YeastOne – A colorimetric microdilution test
 Each plate/well is dosed with antifungal agents in specific dilution concentrations and a
colorimetric indicator.
 Results are read manually. Identifying a change from blue to red suggests fungal growth.
 Thus allows a person to determine the lowest concentration of a drug required to inhibit
growth of the Candida spp.
2. List direct examination tools for Candida diagnosis
For cutaneous infections, direct examination of the sample by wet prep with potassium
hydroxide is used.
 The KOH digests proteins and makes visibility of fungal elements clearer
 Another chemical called Calcafluor white is added. This chemical binds to chitin and
cellulose in fungal cell walls and fluoresces in response to UV light.
 The test does not tell species, just identifies that there is yeast present.
3. Identify commonculture media used for Candida
diagnosis
Identification using culture methods usually takes 5-7 days.
CHROMagar – each different candida species will use different nutrients/enzymes on the media
and turn a different color on this agar.
Sabouraud agar (SAB orSDA) – an agar specifically used to maximize yeast growth while
minimizing bacterial growth via low pH
Cornmeal – really specific for yeast species.

44. 4. List non-culture methods used to improve Candida
diagnosis
(1,3)-Beta-D-Glucan Assay - used for early detection
 A serum assay used in combination with fungal blood cultures in patients at risk for
invasive candidiasis
 The main structural constituents of the fungal cell wall are polysaccharides in the
Candida spp, mainly Glucan.
o The Glucan is composed of glucose polymers linked in a linear arrangement by
carbons 1 and 3
 When soluble (1-3)-Beta-D-Glucan is released into circulation, it is detected and a value
of > 80pg/mL helps confirm that yeast is present
 Does not tell specific fungi species.
Amplification techniques
 PNA FISH (probes that target the specific rRNA)
 Filmarray (PCR)
o Requires growth within the culture bottle
o Available fast, results in 1 hour after the blood culture is deemed positive
o Can identify the specific yeast species (albicans, glabrata, krusei, parapsilosis,
tropicalis) based on their specific melting points
Direct from Blood Testing – T2 Candida System
 Combines NMR and PCR assays to directly detect and identify Candida spp. from whole
blood samples
 The yeast are lysed and species specific super magnetic probes amplify DNA for
detection
o Each species has a specific signal for detection

45. Module 3 - Gram Positive Bacteria
Streptococcus Species Summary

46. All spp: Gram positive cocci in chains, catalase negative, coagulase negative, facultative
anaerobes.
Streptococcus pyogenes
Hemolysis: beta
Lancefield: Group A antigen (GAS)
Optochin: N/A – only used if alpha hemolytic
Normal microbiota?: yes, on skin
Virulence factors:
 Capsule and cell wall
o A hyaluronic acid capsule prevents phagocytosis from leukocytes/macrophages
due to its similarity to human connective tissue.
o Allows binding to epithelial cells, disrupting intercellular junctions
o M protein
 A filamentous macromolecule that binds to host proteins and promotes
adherence to human cells and inhibition of the complement pathway.
 Enzymes
o Streptokinase – activates plasmin (which then digests fibrin), allowing escape
from blood clots
o Deoxyribonuclease – degrades DNA, liquifies pus, facilitates spread
o Hyaluronidase – splits hyaluronic acid in human connective tissue, facilitates
spread
 Toxins
o Pyrogenic exotoxins (A, B, and C) stimulate T cell cytokine release -> shock/tissue
injury
o Hemolysins (streptolysin O and S)
 Streptolysin O: lyses erythrocytes, leukocytes, and platelets
 Streptolysin S: lyses erythromycin, leukocytes, and platelets and
stimulates the release of lysosomal contents after it is engulfed, killing
the phagocyte.
 Ability to diffuse though body rapidly
Infections:
 Skin and soft tissue infections
o Erysipelas
o Cellulitis
o Necrotizing fasciitis (gangrene)
o Impetigo (pyoderma)
 Pharyngitis (strep throat)
 Puerperal fever
 Scarlet fever
 Toxic shock syndrome

47.  If not treated quickly/delayed:
o Glomerulonephritis – occurs 1 week after skin infection or pharyngitis
o Rheumatic fever - most serious, occurs 1-5 weeks after pharyngitis. Caused
primarily by the M protein.
Treatment (DOC only) *
 Penicillin, Amoxicillin
*he did not say “only know these,” these will be all I list on the study guide, however.
Streptococcus pneumoniae
Hemolysis: alpha
Lancefield: none
Optochin: sensitive (susceptible)
Normal microbiota?: yes, upper respiratory tract
Virulence factors:
 Capsule polysaccharide
o The capsule is covalently bound to the peptidoglycan and teichoic acid
 Cell wall
o Helps evade phagocytosis
o Impairs opsonization -> evades complement
o Prevents mechanical clearance by mucous secretion
o Assists the bacteria’s transit to epithelial surface
o Restricts its own autolysis
o Proteins
 Surface protein A
 Inhibits complement pathway (C3B to factor B)
 Allows binding to epithelial membranes
 Surface protein C (choline binding protein A)
 Inhibits complement pathway (factor H)
 Inhibits transportation of antibodies across the epithelial cells by
inhibiting polymeric immunoglobulin receptor (which is
responsible for transporting IgA across the epithelial cell)
o Teichoic acid
o Peptidoglycan
 Enzymes
o Pneumolysin (an exotoxin)
 This exotoxin is produced by invasive species and is released during
autolysis, causing host cells to lyse as well.
 Inhibits ciliary action of epithelial cells
 Impairs phagocyte respiratory burst
 Promotes inflammation via stimulating chemokine/cytokine release
Pathogenesis:

48.  An extracellular bacterial pathogen
 Adheres to cells and replicates
 Disease caused by its ability to produce an intense inflammatory response
Infections:
Strep. pneumoniae is the number one cause of MOPS:
 Meningitis
 Otitis media
 Pneumonia
 Sinusitis
Can also cause:
 Bronchitis
 Bacteremia
 From the invasive species (with pneumolysin):
o Causes classical respiratory, blood, and CNS infections
Treatment (DOC only)
 Best option is prevention with vaccines (PPSV23 and PCV13)
 Penicillin/amoxicillin/azithromycin/clarithromycin, but starting to see resistance
Streptococcus algalactiae
Hemolysis: beta
Lancefield: Group B strep (GBS)
Optochin: N/A
Normal microbiota?: Lower GI tract (5-30%), vagina
Virulence factors:
 Antigenic structure
o A capsule polysaccharide that allows evasion of phagocytosis and complement
inhibition
o Also has surface proteins that allow adhesion and invasion
 Cytokine release induction (TNF-alpha, IL-8, IL-1beta and IL-6) that cause inflammation
and damaged tissue.
Pathogenesis:
 It’s an extracellular bacterial pathogen
 The bacteria colonizes mucosal surfaces and then breaches them to enter sites that are
usually sterile, such as the blood
 The bacteria also adheres to vaginal epithelium, placental membranes, and respiratory
tract epithelium
 It can also cross epithelial barriers paracellularly and has pili surface proteins
 Infections:
o Pregnancy related:
 Bacteremia
 Sepsis in neonates

49.  #1 cause of meningitis in neonates
o Non-pregnancy related
 Bacteremia
 Female genital tract infection
 Skin and soft tissue infection
 Osteomyelitis
 Arthritis – Knee, shoulder, hip joints
Treatment (DOC only) `
 Group A and group B strep are both highly susceptible to penicillin and amoxicillin
Viridans strep (mitus, salivarius,mutans)
Hemolysis: alpha
Lancefield: none
Optochin: resistant
Normal microbiota?: upper respiratory tract, female genital tract, GI tract, oral cavity
Infections:
 Endocarditis
 Intra-abdominal infections
 Dental caries (supragingival plaques)
Treatment
 Penicillin - DOC, but starting to see some resistance. Ceftriaxone and vancomycin can
also be used.
Streptococcus gallolyticus (aka bovis)
Hemolysis: gamma (none)
Lancefield: Group D antigen
Optochin: N/A
Normal microbiota?: GI tract – colorectal mucosa
Infections:
 Endocarditis
 Intra-abdominal infections (most common)
Treatment
 Penicillin - DOC, but starting to see some resistance. Ceftriaxone and vancomycin can
also be used

50. Staphylococci Species Summary
Staphylococcusaureus
Morphology & Characteristics
 Gram positive
 Cocci
 Clusters
 Beta-hemolytic
 Golden pigment
Diagnostic tests
 Catalase positive
 Coagulase positive
Virulence factors
 Protein A
o Proteins in the outer layer can also prevent antibody binding
 A component of the Staph. aureus cell wall that can bind the Fc portion of
immunoglobulins. This prevents complement molecules from binding
that immunoglobulin and thus prevents opsonization and phagocytosis.
Pathogenesis
Exotoxin release
 Gastroenteritis (can be caused by rapid onset food poisoning from ingesting the bacteria
with its pre-formed toxins)
 Scalded skin syndrome
 Toxic Shock Syndrome Toxin (TSST) – a superantigen that causes overactivation of the
immune system/cytokines.
Direct organ invasion
 Pneumonia
 Most common cause of osteomyelitis in adults
 Most common cause of septic arthritis in adults
 Meningitis
 Endocarditis (usually acute/rapid onset)
 Skin infection
 Blood/catheter infection
Resistance mechanisms
 Penicillinase – renders penicillin resistant. Led companies to produce methicillin,
oxacillin, dicloxacillin, nafcillin. Bulked up the molecule and altered beta-lactamring.

51.  MecA gene acquisition – PBP2 (penicillin binding protein) mutated to PBP2A.
o Granted resistance to methicillin, oxacillin, dicloxacillin, nafcillin, cephalosporins
and carbapenems
 Cell wall thickening – led to VISA (vancomycin intermediate staph aureus), meaning the
staph was susceptible to vancomycin but not yet resistant
 vanA gene acquisition – staph became resistant to vancomycin
 Most resistant strains are hospital based
Healthcare-associated vs. community associated MRSA
Treatment

52. Once a patient has MRSA
colonization, decolonization
occurs via intranasal mupirocin
and chlorhexidine baths
Staphylococcus epidermidis
Considered normal skin flora and a contaminant in blood cultures (can be a pathogen as well)
Commonly associated with prosthetic devises and intravascular catheters
Diagnostic tests
 Catalase positive
 Coagulase negative
Virulence factors
 Polysaccharide capsule
 Biofilms – help adhere to the devices and resist antibiotics
DOC: vancomycin
Staphylococcus saprophyticus
Leading cause of UTIs in young women
Diagnostic tests
 Catalase positive

53.  Coagulase negative

54. Bacillus Species Summary
All spp: saprophytic (needs only carbon and nitrogen for energy/growth), usually not
associated with human infections. All are gram positive rods, facultative anaerobes, and
acid-fast test negative.
The bacillus species virulence:
 Spore forming, allowing withstanding of tough environments
 Anti-phagocytic capsule
Bacillus anthracis
 Gray to white, “ground glass” appearance
 Hemolysis: gamma
 Motility: non-motile
 Anthracis specific virulence
o Toxins
 Protective antigen (PA)
 Allows binding to a cell and forms a membrane channel
 Edema factor (EF)
 Lethal factor (LF)
 PA and EF combine to form edema toxin – causes cell and tissue edema
 PA and LF combine to form lethal toxin to cause host cell death
 PA and LF also impair innate and adaptive immunity and help with
bacteria proliferation
 Infections/diseases/health risks:
o Cutaneous anthrax (95%)
o Inhalation [pulmonary] anthrax (5%)
o GI anthrax (rare)
o Biological warfare/bioterrorism
 Anthrax most commonly seen in Asia/Africa
 Treatment:
o Ciprofloxacin (fluoroquinolones) OR Doxycycline plus 1-2 of the following:
 Rifampin
 Vancomycin
 Penicillin
 Imipenem
 Clindamycin
 Clarithromycin
o Anthrax prophylaxis with short-lived vaccines

55. Bacillus cereus
 Gray to white, “ground glass” appearance
 Hemolysis: beta
 Motility: swarming – group movement
 Cereus specific virulence:
o Toxins (depends on the food)
 Moreso intoxication rather than food borne illness
 Emits an emetic toxin (a cyclic peptide) that is quick acting
 Spores germinate and vegetative cells produce the toxin during
the log phase.
 Associated with rice, milk, and pasta
 Emits a diarrheal toxin (an enterotoxin)
 The spores germinate and secrete the toxin. It induces fluid
accumulation in the small intestines.
 Associated with sauces and meat dishes
 Infections:
o Food poisoning (emetic type in 1-5hrs, diarrheal type in 1-24hrs)
o Eye infections
o Considered a contaminant in blood cultures
 Treatment:
o There is no treatment for food poisoning.
o Ciprofloxacin OR Doxycycline plus 1-2 of the following:
 Rifampin
 Vancomycin
 Penicillin
 Imipenem
 Clindamycin
 Clarithromycin
Corynebacterium and ListeriaSummary
Corynebacterium spp
 Gram positive bacilli (rods), although sometimes club shaped or irregular.
o When you think about a gram-positive rod, an acid fast test should come to mind

56.  Acid fast test: negative
 Facultative anaerobe
 Non-sporulating
 Non-motile
 Commonly found on skin and in mucous membranes of the respiratory tract.
Corynebacteriumdiphtheriae
 Most common species
 Does not have a capsule
 Virulence:
o Produces an exotoxin that inhibits protein synthesis by inhibiting elongation
factor, leading to cell death
o Interaction with beta-phage promotes epithelium destruction and a superficial
inflammatory response (on surface).
o The necrotic epithelium, along with RBCs/WBCs and fibrin, produce a
pseudomembrane film with a grayish appearance. Commonly found on the
tonsils, pharynx or larynx.
 Infections:
o Respiratory diphtheria – most common and can spread leading to:
 Myocarditis
 HF, arrhythmias, death
 Neurotoxicity
 Neuropathy
o Cutaneous diphtheriae related infections
 Treatment:
o Diptheria antitoxin – neutralizes the toxin before it can bind tissue
o Antimicrobials – penicillin and erythromycin
o Best method is prevention with vaccination with a nontoxic immunogenic toxoid
 Children given DTaP
 Booster with Td every 10 years

57. Listeria
 Commonly found in soil, water, poultry, cattle, and raw milk – but not often in humans.
 Gram positive rod
 Acid fast test: negative
 Catalase test: positive
 Facultative anaerobe
 Non-sporulating
Listeria monocytogenes
 Can overcomefood preservation and safety barriers with its ability to grow
at:
o Low pH environments
o High salt concentrations
o Wide ranges of temperatures
 Virulence:
o The bacterium enters the GI after ingestion of contaminated food
o Adhesion proteins on the bacterium allow binding to hostcells.
o Italso has surfaceproteins (internalins A and B) that:
 Promote phagocytosis
 Once it’s phagocytosed, it will be placed in the familiar
phagolysosome. Thephagolysosomeis normally a low pH.
Unfortunately for the hostcell, this low pH activates the
bacteria’s listeriolysin O.
 LLO lyses the phagolysosomeand now the bacterium is free in
the cytosolwhereit can grow intracellularly and evade
immune systemdetection.
 Infections:
o Food borne illness - usually self-limiting gastroenteritis for 1-3 days
o The real problemis in the immunocompromised (neonates, elderly,
etc)
 Can go fromGI to the blood or CNS, leading to meningitis or
bacteremia.
 Treatment:
o Ampicillin
o Bactrim (sulfamethoxazole-trimethoprim)

58. Actinomycetes Summary
 A bacterium that appears like a fungi and form hyphae-likestructures
 Make long, beaded, branching filaments
 Grampositive
Actinomyces israelii
 Description: “Anaerobic, gram positive branching rods with sulfur granules”
 Normal flora?: oral cavity and GI tract
 The disease is named according to the area the resultant abscess erodes:
o Cervicofacial actinomycosis
o Abdominal actinomycosis
o Thoracic actinomycosis
 Form hard yellow granules called sulfur granules (not actually sulfur), mostly in face and
neck
 Pelvic actinomycosis is possible in women with an intrauterine device
 The infection is non-communicable.
 Treatment:
o Penicillin, no resistance
o There is no vaccine or prophylactic drug available
Nocardia asteroides
 Aerobic, gram positive branching rods or filaments that are weakly acid-fast
 Responsible for lung infections and disseminates in immunocompromised hosts
 Pathogenesis: inhaled, causes pulmonary infections, lung abscesses and cavitations.
o Can erode into pleural space and the infection may become blood born.
 Highest risk patients are immunocompromised and long term steroid users.
 Treatment:
o Bactrim with surgical drainage, drug resistance may occur.
o No vaccine or prophylactic drug available.
Propionibacterium (NOT in actinomycetes family)
 Description: Anaerobic, gram-positive rods with a pleomorphic (irregular) shape
 Normal flora? Yes, skin and GI tract.
 Virulence:
o Propionibacterium acnes contains lipase that contributes to acne generation.
o Biofilmcan protect it against antibiotics
 Infections:

59. o Causes device-related infections, endocarditis, septic arthritis, and corneal
ulcers.
 Treatment: macrolides, cephalosporins, tetracycline, penicillin – but increasing
resistance worldwide with macrolides.
Clostridium Species Summary
 Species description: Anaerobic, gram-positive, spore-forming rods
Clostridium botulinum
 Emits a neurotoxin that blocks ACh release from the presynaptic terminal, leading to
flaccid muscle paralysis.
 SNARE proteins are on the plasma membrane of the muscle fiber that bind incoming
vesicles full of ACh from the presynaptic neuron. Botulinum toxin cleaves these proteins
and prevents vesicular fusion with the plasma membrane, preventing ACh diffusion.
 Adult botulism
o Spores float in air and can land on food. When this food is canned, it grows and
matures in this anaerobic environment. The person then ingest the pre-formed
toxins. Cooking kills spores.
o Clinical manifestations:
 N/V/D
 Abdominal cramps
 Diplopia
 Dysphagia
 Muscle weakness
 Respiratory paralysis
 Death
o Treatment of adult botulism:
 Heptavalent antitoxin
 Contains antibodies of 7 botulinum types (A-G)
 Only available via request to CDC
 The antitoxin is made from horse serum, so it is recommended to
do a skin test prior to administration to make sure the patient is
not allergic to the antitoxin
 Infant botulism
o Acquired when an infant ingests contaminated food, i.e. fresh honey
contaminated with spores. The spores germinate and colonize the infant’s GI
tract, leading to:
 Constipation (2-3 days)
 Muscle weakness with a “floppy baby” appearance
o Treatment of infant botulism:
 Human botulism Immunoglobulin IV (BIG-IV)

60.  Obtainable only from the California Department of Public Health
o Note that infant botulism is caused by ingestion of spores. Adult botulism is due
to ingestion of already matured spores that germinated in the anaerobic
environment – the spores cannot survive/compete against the gut flora in the
adult GI
 Wound botulism:
o Least common. Acquired from puncture wounds of deep space infections with
exposure to spores from the soil or environment.
o Similar presentation to adult botulism but without GI symptoms and the
incubation period is longer
o Treatment of wound botulism:
 Combination of surgical management (e.g. making sure the wound is
clean), anti-toxin therapy, and antibiotics
 Botulinum neurotoxins are available and FDA approved for use in cosmetics, axillary
hyperhidrosis (severe sweating), chronic migraines, and neurogenic detrusor
overactivity
o Brands include Botox, Dysport, and Xeomin. Each of these has different
durations of action, dosage effectiveness, and immunogenicity and are therefore
not interchangeable.
Clostridium tetani
 Tetanus is a neurotoxin that causes sustained contraction of muscles via inhibition of
the inhibitory GABA and glycine terminals.
o Causes trismus (lockjaw), and risus sardonicus (causes a grin-like appearance)
 Tetanus is acquired from puncture wounds that are contaminated with spores. The
spores germinate in anaerobic conditions.
 Spores are commonly found in soil and animal feces
 Vaccines/Treatment:
o DTaP and Tdap contain reduced diptheria toxoids, tetanus toxoids, and acellular
pertussis toxoids. The two products contain different amounts of each antigen.
o Although milk products are used to make the vaccines, milk allergies are not a
contraindication for their administration
o DTaP
 For infants and children (<6y/o)
 Should receive 5 doses of DTaP
 DTaP can be combines with IPV and HepB vaccines
o Tdap
 For children and adults 11 years or older
 If lack of vaccination history or Tdap not received, give 1 dose of
Tdap followed by a Td booster every 10 years.
 Pregnant women should receive 1 dose of Tdap each pregnancy at 27-36
weeks.

61. o DT (diptheriae and tetanus)
 For use in children 7 years or younger
 This vaccine is used when patients have some kind of contraindication to
the pertussis vaccine
 The concentration of diptheria toxoid is higher in DT compared to Td (↓)
o Td
 This is the booster vaccine against diptheria and tetanus that is for ages
7+
o In summary, children 7 years and younger should receive DTaP or DT, while older
children and adults should receive Tdap and Td.
 Wound management:
o Step 1: establish the patient’s vaccination history
o Step 2: Decide whether or not to administer tetanus immune globulin (TIG).
 TIG only administered when the patient has an unknown vaccination
history, or <3 primary vaccine series, or if it has been >5 years from last
tetanus vaccine and the wound is not clean or minor.
o Step 3: Decide whether or not to administer DTaP, Tdap or Td
 In all cases (clean, minor wound or all other wounds), if the patient has
had 3 or more primary vaccine series or if it has been less than 5 years
from their last tetanus vaccine, they should not receive TIG nor DTaP,
Tdap, or Td.
 DTaP, Tdap or Td should be given if the patient has an unknown
vaccination history, or <3 primary vaccine series, or if it has been >5 years
from last tetanus vaccine with any type of wound.
Clostridium perfringens
 Causes gas gangrene
 Spores are found in soil and mature in anaerobic conditions, producing gas.
 The spores contaminate wounds, where exotoxins destroy tissue and promote
anaerobic environment production to further the bacteria’s growth.
 There are three types of infection
1) Cellulitis/wound infection
 The bacteria grows and causes local tissue damage
 Upon palpation will be a moist, spongy, crackling consistency due
to gas buildup (crepitus)
2) Clostridium myonecrosis
 When the bacteria is inoculated into muscle during trauma and its
exotoxin destroys the muscle
 The anaerobic bacteria ferments carbohydrates, resulting in gas
formation
 A thin, black fluid forms
 Often fatal

62. 3) Diarrheal illness
 Occurs when the spores germinate in food
 Can lead to toxin production in the gut and watery diarrhea
Clostridium difficile
 Spores are resistant to acidic environment of the GI and are able to germinate in the
small intestine
 Form 2 exotoxins: A and B
 Pathogenesis:
1) After disruption of normal gut flora (e.g. from antibiotics), C. difficile colonizes
and produces toxin A and B
2) Toxin A and B bind to apical side (closest to GI lumen) of the cell and stimulate
an inflammatory response that attracts neutrophils
3) This neutrophil infiltration disrupts tight junctions and leads to a
pseudomembrane (pseudomembranous colitis)
 Nosocomial (hospital acquired) C. difficile infection:
o Upon receiving antibiotics, a patient may be exposed to spores. There are three
typical results of this exposure:
 The patient is asymptomatically colonized with non-toxigenic C. difficile
 The patient is asymptomatically colonized with toxigenic C. difficile as IgG
successfully responds to Toxin A.
 The patient experiences a C. difficile infection (CDI) because IgG does not
respond properly to the bacteria’s Toxin A.
o There has been a large increase in CDIs in recent years
o Previously C. difficile was generally a nosocomial pathogen, but now even more
cases are seen in the community setting.
 CDI Risk Factors:
o Age > 65 y/o
o Prior antibiotic use
 Highest CDI risk: Clindamycin, fluoroquinolones, cephalosporins (2nd and
3rd gen), aztreonam, carbapenems
 Moderate CDI risk: macrolides, Bactrim, penicillins
 Lowest risk: tetracyclines
o Prior hospitalization
o Immunocompromised patients
o GI surgery
o Tube feeding
o Use of PPIs
 The decreased acidity results in inadequate sterilization of ingested
organisms and allows colonization of the normally sterile upper GI tract
 PPIs may have a negative effect on leukocyte activity
 Clostridium difficile-associated diarrhea (CDAD) should be considered as
a possible diagnosis for PPI users with diarrhea that does not improve.

63.  Advise patient to seek care if he or she experiences watery stool that
does not go away, abdominal pain, and fever while taking PPI
 Advise patient to use lowest dose and shortest duration of PPI
 Definition of CDI:
o Presence of diarrhea – 3 or more unformed stools in 24 hours or less
o Stool test result positive for C. difficile or its toxins
o Colonoscopic or histopathologic findings demonstrating pseudomembrane colitis
 Treatment of CDI:
o Vancomycin OR fidaxomicin
o Metronidazole may be used if the other two are not available
 Infection control and prevention:
o Hand washing with soap and water (not alcohol)
Enterococci Species Summary
Description: Gram positive, aerobic cocci in chains.
Catalase: negative
Hemolysis test: gamma (none)
Lancefield: Group D
Normal microbiota? Yes, GI tract
Virulence factors:
 Due to its cell surface (an antigenic structure)
o Has an aggregative substance that increases adherence and
internalization into eukaryotic cells
o Also adheres to extracellular matrix proteins fibronectin, fibrin and
collagen type I
 Enzymes
o Gelatinase and serine proteases
 Facilitate invasion by altering immunoglobulins and/or
complement molecules
 Help develop biofilms by regulating autolysis and releasing high
MW extracellular DNA
 Degrades host connective tissues and exposes ligands for the
bacteria to attach
 Toxins
 Bacterial toxin that lyses eukaryotic cells
 Resistance to antibiotics

64. o The species is genetically (intrinsically) resistant to cephalosporins
o Most are penicillin resistant
o Vancomycin resistance is also an issue (moreso with E. faecium)
 This resistance is associated with vanA and vanB gene expression
 If a species is vancomycin resistant, then they are also likely to be
ampicillin resistant
o In general, E. faecium is more resistant than E. faecalis.
 Infections:
o Enterococcus is the most common pathogen to cause healthcare-
associated infections
o UTIs
o Wound infections
o Intra-abdominal infections
o Endocarditis
o Sometimes, infections with enterococcus can be polymicrobial
 Associated with intra-abdominal infections and wound infections
 Treatment:
o Gentamicin in combination with a drug like ampicillin or vancomycin
o Telavancin – only useful if the bacteria has the vanB gene. With vanA it
will be resistant.
o Tigecycline
o Daptomycin
o Linezolid
o Vancomycin
Enterococci faecalis
Specific virulence factors:
 E. faecalis surface protein
o Allows adhesion in the formation of biofilms
o The pili also play a role in biofilm formation
 Specific Treatment DOC:
o Ampicillin
Enterococci faecium
Specific treatment DOC:
 Quinupristine-dalfopristin, tigecycline, linezolid

65. Module 4 – Gram-Negative Bacteria
Enterobacter Family Overview
Includes Escherichia, Klebsiella, Enterobacter, Citrobacter, Serratia, etc.
 Gram-negative, facultative anaerobes
 Lactose positive
 Oxidase positive ????
 Antigenic features:

66. o The cellular envelope which consists of an inner membrane, periplasmic space,
and the outer membrane
 Inner membrane:
 Polar molecules are impermeable – protects from antibiotics
 Periplasmic space:
 Between the inner and outer membrane, contains the
peptidoglycan
 Outer membrane
 Has phospholipids in the inner leaflet and the endotoxin LPS in the
outer leaflet.
o LPS:
 The toxic portion of LPS is lipid A. Lipid A is
recognized by immune cells and promotes an
inflammatory response
o The capsule
 Usually bound to LPS
 Extends out via repeating trisaccharide units, allowing for masking of O
antigen from host antibodies
 Virulence factors:
o LPS and Capsule
o Adhesins
 Allow binding to specific host receptors
 Fimbriae can bind to mannose residues of host cell
 Outer membrane proteins and surface carbohydrates also serve as
adhesins
o Secretion systems and toxins
 SPATE: Serine Protease Autotransporters of Enterobacteriaceae
 These can cause lysis of host cell via specific secretion systems:
o Type I secretion: releases hemolysins
o Type II secretion: Releases lytic enzymes (e.g. chitinase)
o Type III secretion: exports important cell nutrients from
host cell or injects toxins into host cell
o Plasmids
 Plasmids can provide and transfer antibiotic resistance genes, even
between different genera.
 As with resistance, plasmid genes may also play major roles in
pathogenesis
 Genes for type II and III secretion systems and pilus genes may be
acquired.
o Iron acquisition

67. Escherichia Species Summary (Enterobacter family )
 Very distinguishable from other Enterobacter spp. because it is motile and indole
positive (produces indole from tryptophan)
 Does not usually cause disease in otherwise healthy individuals
Escherichia Coli
 Characteristics:
o Gram negative rod
o Facultative anaerobe
o Positive for lactose fermentation
o Negative oxidase test
o Hemolytic
o Voges-Proskauer reaction test: negative
o Commonly found in the microbiota of the GI
 Pathogenesis of Diarrhea due to E. Col I (caused by 3 main types of virulence):
o Enteropathogenic E. Coli (EPEC)
 Seen in developing countries and infants
 Related to a watery diarrhea with vomiting and
fever
 Caused by a pilus that acts as an adherence
factor to mucosal cells of the small intestine.
 The pili attach and form actin pedestals. From
there, effector proteins are injected into the
mucosal cells.
 Note that no toxins used here
o Enterotoxigenic (ETEC)
 Related to traveler’s diarrhea and children < 5 y/o in developing countries
 As with EPEC, also adhere to SI cells except with flagellum and not pilus.
 Enterotoxins that are heat labile or heat stable will cause reduction in
sodium absorption and increased chlorine secretion into the lumen,
leading to watery diarrhea (lots of solutes draw water into lumen).
 Plasma mediated – bacteria can receive this ability to be enterotoxigenic
from plasmids
o Shiga toxin-producing E. Coli (STEC)

68.  Varying levels of severity – simple mild diarrhea/food poisoning to
hemorrhagic colitis and death.
 Caused by production of cytotoxins: Shiga-like toxin-1 and toxin-2
 These block protein synthesis and induce host cell apoptosis
 These are released during stressful conditions (e.g. exposure to
antibiotics)
 O157:H7 is most common serotype (strain) of E. Coli responsible
for this type of infection
o This serotype is found as normal microbiota in cattle and
can be acquired from undercooked meat and
unpasteurized food, but can also be found in cooked
burgers that have been handled poorly
 Infections other than Diarrhea:
o UTIs
 The bacteria can go from the GI tract to the urinary tract and cause a
bladder infection, cystitis, pyelonephritis, or bloodstream infection
o Intra-abdominal infections
 Treatment:
o Ampicillin/sulbactam
o Amoxicillin/clavulanate
o Cefazolin (possible resistance, follow up with susceptibility test)
o Cephalexin (possible resistance, follow up with susceptibility test)
o Cefuroxime
o Ceftriaxone
o Cefdinir
o Cefepime
o Piperacillin/tazobactam
o Ciprofloxacin
KlebsiellaSpecies Summary (Enterobacter family)
 Common in community and hospital settings
 Pneumonia from this bacteria differs from streptococci in that an x-ray will reveal
bulging interlobar fissures
Klebsiella pneumoniaeand Klebsiella oxytoca
 Characteristics

69. o Gram negative facultative anaerobe
o Lactose fermentation positive
o Oxidase test negative
o Hemolysis negative
o Voges-Proskauer reaction positive
o Non motile
o Mucoid growth (colonies are sticky/viscous)
o Normally found in GI
 Virulence factors
o LPS
o Plasmids
o Pili that allow cell adherence
o Polysaccharide capsule
 70 different variants
 Responsible for mucoid phenotype
 Inhibits phagocytosis
o Antibiotic resistance to amoxicillin and ampicillin and penicillin within its genome
due to penicillinase
 Nosocomial isolates may also be resistant to other types of antibiotics
due to plasmids. For example:
 A plasmid can be picked up that gives the bacteria extended
spectrum beta-lactamase production (ESBL), an enzyme granting
resistance to all cephalosporins.
 Resistance to fluoroquinolones may be acquired (ciprofloxacin)
 Resistance to aminoglycosides may be acquired (gentamicin)
 Infections
o UTIs (main concern with Klebsiella)
o Respiratory tract infections
o Intra-abdominal infections
 Liver abscesses
 Biliary tract infections
 Peritonitis
o Wound infections
 Treatment
o Penicillins, amoxicillin, and ampicillin shouldn’t be used
o Cephalosporins
 Cefazolin*
 Cephalexin *
 Cefuroxime*
 Ceftriaxone*
o Piperacillin/tazobactam*
o Meropenem

70. o *When the bacteria are producing the ESBL enzyme, such as in some nosocomial
isolates, you can only use meropenem
Enterobacter, Citrobacter,and SerratiaSpecies
Summary (Enterobacter family)
Virulence factors (for all species)
 All contain an inducible ampC gene that codes for beta-lactamases that can degrade
antibiotics, giving resistance to:
o Ampicillin
o Cefazolin
o Cephalexin
o Cefuroxime
 Each can carry plasmids encoding resistance to multiple antibiotics (e.g. some may carry
plasmids for ESBLs, etc)
Treatment (for all species)
 Cefepime
 Meropenem
 Ciprofloxacin
Enterobacter cloacae and Enterobacter aerogenes
Characteristics:
 Gram negative bacilli, facultative anaerobes
 Lactose fermentation positive
 Oxidase test negative
 Hemolysis negative
 Voges-Proskauer reaction positive
 Motile
 Mucoid growth (colonies are sticky/viscous)
 Normally found in the GI
Infections:
 Mostly associated with respiratory tract infections (pneumonia)
 UTIs (with indwelling [inside the body] catheter use)
 Intra-abdominal infections
 Wounds or burns – skin infections

71. Citrobacter koseri and Citrobacter freundii
Characteristics:
 Can use citrate as their only carbon source
 Produce H2S
 Gram negative, facultative anaerobes
 Lactose fermentation positive
 Oxidase test negative
 Voges-Proskauer reaction negative
 Commonly found in GI tract of animals and humans
 Commonly found in soil and water
Infections:
 Mostly associated with UTI’s (with indwelling [inside the body] catheter use)
 Respiratory tract infections
 Intra-abdominal infections
 Wound infections
 Osteomyelitis
Serratia marcesens
Characteristics:
 Widespread in the environment due to its saprophytic characteristics, although not
common in human fecal flora
 Produces the exotoxin DNase
 Produces a red pigment – prodigosin
 Gram negative bacilli, facultative anaerobe
 Lactose fermentation positive
 Oxidase test negative
 Hemolysis negative
 Voges-Proskauer reaction positive
 Normal flora, but mostly found in the environment
Serratia-specific Virulence:
 Contain fimbriae that allow adherence to uroepithelial cells and can be cytotoxic to
some the tissue cells
 Can survive harsh conditions, even disinfectants
 Its ampC gene produces higher levels of beta-lactamases during antibiotic therapy

72. Infections:
 Mostly associated with UTIs (with indwelling [inside the body] catheter use)
 Respiratory tract infections
 Wound infections
 Osteomyelitis
 Intra-abdominal infections
Other Important Enterobacteriaceae Summary
(Enterobacter family)
Salmonella
There are two species
 Salmonella bongori
 Salmonella enterica
o Subspecies:
 Salmonella enterica salamae
 Salmonella enterica arizonae
 Salmonella enterica diarizonae
 Salmonella enterica houtenae
 Salmonella enterica indica
 Salmonella enterica enterica
 Sub-subspecies (serotypes) that cause enteric fever:
o S. paratyphi A (serogroup A)
o S. paratyphi B (serogroup B)
o S. choleraesuis (serogroup C1)
o S. typhi (serogroup D)
 Infections from Salmonella:
o Overview:
 Usually cultures will be from stool or blood. Hospitals will be able to
determine if Salmonella is present, but to identify the serotype, the
sample is sent to a state or regional reference lab.
 Usually the four serotypes enter the body orally with contaminated food
or drinks.
 Our body’s protective factors:
 Gastric acidity
 Normal intestinal microbiota
 Local intestinal immunity

73.  Beware of plasmid-based antibiotic resistance. Follow up treatment with
susceptibility tests.
 Prevention of infection via hand washing, cooking food, and typhoid
vaccine
o Diseases:
 Enteric fever (typhoid fever)
 S. typhi
 Seen frequently in southeast Asia
 The bacteria enters from the small intestine to the lymphatics and
blood
 After about 10-14 days, patients may have lesions/necrosis of
lymphoid tissues (the Peyer’s patches)
 Treatment:
o Ciprofloxacin
o Ceftriaxone
o Bactrim
 Bacteremia with focal lesions (lungs/bones etc)
 S. choleraesuis
 Treatment:
o Ciprofloxacin
o Ceftriaxone
o Bactrim
 Enterocolitis
 S. typhimurium or S. enteritidis
 Most common seen in US
 Faster incubation period at around 8-48 hours after ingestion,
resolving in around 2-3 days thereafter
 Associated with N/V, Ha and profuse dh
 Treatment: rehydration (nonpharmacologic therapy)
S. typhi and S. paratyphi
 Characteristics
o Gram negative rods
o Facultative anaerobe
o Lactose fermentation negative
o Oxidase test negative
o Voges-Proskauer reaction negative
o Motile
o Non-spore forming
o Produces H2S
o NOT generally part of human microbiota

74. Shigella
 Infection with shigella is often limited to the GI tract with invasion of the
mucosal cells in the intestines.
 Infection can cause micro abscess formation
 Infection can cause necrosis, ulcerations and bleeding leading to
pseudomembrane formation.
Shigella dysenteriae, Shigella flexneri, Shigella boydii, Shigella sonnei
Characteristics
 Gram negative small rod
 Facultative anaerobe
 Lactose fermentation negative (ferments, but very slowly – delayed)
 Oxidase test negative
 Voges-Proskauer reaction test negative
 Non-motile
 Non-spore forming
 NOT part of normal microbiota
Shigellosis (caused by the Shigella family, not just one specific species)
 Tested for with a stool culture or rectal swab
 Upon autolysis of the Shigella, LPS endotoxin is released and irritates the
intestinal wall
 S. dysenteriae has a unique exotoxin that is heat labile, produces diarrhea,
inhibits sugar and amino acid absorption in the SI, and can act as a
neurotoxin to result in a coma or pseudomeningitis (meningism)
 Treatment:
o Usually self-limiting
o Nonpharmacologic: rehydration
o In severe cases, may use ciprofloxacin and Bactrim
o Beware of plasmid-mediated resistance, follow up with susceptibility
tests
Proteus
Proteus mirabilus
Characteristics
 Gram negative rod, facultative anaerobe
 Lactose fermentation negative

75.  Oxidase test negative
 Voges-Proskauer reaction positive
 Motile (swarming, cilia)
 Normal GI microbiota
Morganella
Morganella Morganii
Characteristics
 Gram negative rod, facultative anaerobe
 Lactose fermentation negative
 Oxidase test negative
 Voges-Proskauer reaction negative
 Nonmotile
 Normal GI microbiota
Providencia
Providencia stuartii and Providencia rettgeri
Characteristics
 Gram-negative rods, facultative anaerobes
 Lactose fermentation negative
 Oxidase test negative
 Voges-Proskauer reaction negative
 Nonmotile
 Normal GI microbiota
Morganella, Providencia and Proteus are all associated with:
 UTIs (more specific to Proteus mirabilis)
 Bacteremia
 Pneumonia
 Intra-abdominal infections
Morganella, Providencia and Proteus can be treated with:
 Cefazolin
 Cephalexin
 Ceftriaxone
 Bactrim
 Ciprofloxacin
 Ampicillin or Penicillin cannot be used.

76. Pseudomonas aeruginosa Summary
Pseudomonasaeruginosa
 Characteristics
o Gram-negative rod, facultative anaerobe
o Oxidase test positive
o Catalase positive
o Lactose fermentation negative
o Flagella at the pole of the cell
o Grows at 37-42C
Continued:
o Can grow as a single cell or be found in pairs
o Often produces non-fluorescent bluish-green pigment. Pigment color can
help determine virulence
o Colony morphology can vary
o Can develop biofilms
o Can grow on many types of media and smell sweet like grapes or tacos (wut)
o Opportunistic nosocomial pathogen
o Most important pathogen concerning Cystic fibrosis patients
o Many antibiotic resistance mechanisms because it has a large genome
relative to other bacteria
o Found in soil, water, food, skin, hospital environments, and even diesel and
jet fuels!
 Virulence
o Pili – allow attachment to host cells
o Alginate production – produces mucoid colonies in cystic fibrosis patients
o LPS
o Elastases, proteases, and hemolysins (extracellular enzymes)
o Exotoxin A – produces by most strains, results in tissue necrosis
o Type III secreted toxins (four of them) – results in host cell death and
immune systemdysfunction
o Quorum sensing – communication with other cells with Pseudomonas
Quinolone Signal (PQS)
o Resistance mechanisms
 Beta-lactamase enzymes (ampC gene)
 Efflux pumps
 Lower outer membrane permeability
 Mutation in antibiotic target sites

77.  Outer membrane changes resulting in a less net negative charge,
resulting in less attractive force for positively charges antibiotics like
polymyxins and aminoglycosides
 Enzymes able to modify aminoglycosides
 Pathogenesis
o Only pathogenic when host defense is impaired
 Neutropenic patients
 Skin wounds
 Mucosal damage
 IV/Catheters
o Attaches to mucous membranes, invades host locally and then systemically
using pili, enzymes and toxins. LPS causes fever and sepsis
 Infections
o Pneumonia and other respiratory tract infections, usually from contaminated
respirators. May lead to lung damage and death
o In cystic fibrosis and COPD patients, may cause chronic respiratory tract
infections because of biofilm formation
o Bloodstream infections (including febrile neutropenia) can lead to sepsis and
death
o Wound and burn infections
o Meningitis
 Treatment
o Beta-lactamantibiotics that inhibit cell wall synthesis
 Carbapenems (meropenem, imipenem, doripenem, not ertapenem)
 Extended spectrum penicillins (piperacillin)
 Cephalosporins (ceftazidime, cefepime)
 Monobactams (aztreonam)
o Aminoglycosides
o Polymyxins
o Fluoroquinolones
o Fosfomycin

78. Acinetobacter baumannii Summary
Acinetobacterbaumannii
 Characteristics
o Gram negative, aerobic rod
o Oxidase test negative
o Grows on most types of media
Continued:
o Ubiquitous
o Usually commensal (doesn’t cause infection) but can cause nosocomial
infections, especially since it is difficult to kill in hospital environments.
o 63% are MDR
o Associated with respiratory tract infections, bacteremia, and wound
infections
 Virulence
o Does not have flagella, but has pili that are involved in cell adhesion and
biofilm formation
o LPS
o Extracellular enzymes and toxins
o Polysaccharide capsule provides methods of evasion from immune system
o Can survive under dry and iron-deficient conditions
o Resistance mechanisms
 The outer membrane is much less permeable to antibiotics such as
beta-lactams relative to other species
 Beta-lactamase enzymes (OXA and ampC)
 Efflux pumps
 Target site mutations
 Outer membrane changes resulting in a less net negative charge,
resulting in less attractive force for positively charges antibiotics like
polymyxins and aminoglycosides
 Aminoglycoside-modifying enzymes
 Pathogenesis
o Pathogenic when host defense is impaired
o A. baumannii attaches to mucous membranes, invades local tissues, and then
becomes systemic using pili, enzymes and toxins. LPS causes fever and shock
 Infections
o Respiratory tract infections (i.e. pneumonia)
o Bloodstream infections (usually from devices like catheters)

79. o Wound infections from combat
o UTIs
o Meningitis
 Treatment
o There are pretty much no drugs available for multi-drug resistant isolates
o For susceptible wild-types:
 Beta lactams
 Carbapenems
 Aminoglycosides
 Polymyxins
 Fluoroquinolones
Burkholderiaand Stenotrophomonas Summary
Burkholderia cepacia
 Characteristics
o Gram-negative, aerobic rod
o Oxidase test positive
o Lactose fermentation negative
o Motile
o Non-spore forming
o Ubiquitous
 Virulence
o Mutated genes provide antibiotic resistance
o Enzymes
 Metalloproteinases – break down collagen and fibronectin in lung to
assist invasion
 Lipase
 Serine proteases
o Quorum sensing
 Bacterial communication allows them to express resistance to
prepare for immune system or antibiotic attacks.
 Pathogenesis
o Vulnerable patients include:
 Cystic fibrosis patients
 Transmitted from one CF patient to another via close contact
 Asymptomatic infection develops into pneumonia
 Bronchiectasis patients
 Chronic granulomatous disease patients
 Infections

80. o Nosocomial infections
o Respiratory tract infections (pneumonia)
o Catheter-related bloodstream infections
 Treatment
o Often multi-drug resistant – susceptibility testing must be performed
o Bactrim
o Meropenem
o Ciprofloxacin
o Minocycline
Stenotrophomonasmaltophilia
 Characteristics
o Gram-negative, aerobic rod
o Oxidase test negative (differentiate from burkholderia)
o Lactose fermentation negative
o Non-spore forming
o Ubiquitous
 Virulence
o Multidrug resistance via metallo-beta-lactamase
o Enzymes
 Lipases
 Proteases
 DNase
 RNase
o Can adhere to plastic via positive charge at physiologic pH
o Because the bacteria are ubiquitous, there is often difficulty in differentiating
colonization vs. a true infection of S. maltophilia
 Infections
o Nosocomial infections
o Bloodstream infections (catheter and non-catheter related)
o Respiratory tract infections (pneumonia)
o UTIs, ophthalmologic infections, skin soft tissue infections, etc
 Treatment
o Bactrim
o Moxifloxacin (prone to resistance)
o Ciprofloxacin (prone to resistance)
o Minocycline

81. Gram-negative anaerobes Summary
 Pay attention to where the following bacteria are located in the body and what type
of infections they are related to
 Anaerobic bacterial infections are common and often polymicrobial. These bacteria
are often found as normal microbiota in the skin, mouth, or GI.
 Treatment (all spp)
o Control and drain the abscess (since allcan form abscesses)
o DOC: metronidazole
o
Bacteroides spp (Bacteroides fragilis)
 Characteristics
o Gram negative bacilli (may appear as coccobacilli)
o Non-spore forming
o Commonly found in the GI (SI and LI) and mouth
 Virulence factors
o Capsular polysaccharides induce abscess formation
o Enzymes
 Proteases, neuraminidases
 Cause hemolysis of erythrocytes
 Superoxide dismutase
 Remove oxygen in the environment and converts it to
hydrogen peroxide with the help of iron.
o By removing oxygen it also helps maintain its
anaerobic environment
 Infections
o Intra-abdominal infections due to intestinal wall disruption (surgery, trauma,
etc)
o Abscess formation
 CNS
 Pelvic inflammatory infections
 Ovarian abscesses
 Dental caries (since its found normally in the mouth)
o Bloodstream infection
Prevotella spp (Prevotella melaninogenica)
 Characteristics
o Gram negative bacilli (may appear as coccobacilli)

82. o Non-spore forming
o Commonly found in the GI (LI), mouth, and genitourinary tract
 Virulence factors
o Surface appendages allow adhesion (a difference from Bacteroides)
o Enzymes
 Neuraminidase
 Breakdown of mucous
 Proteases
 Cleavage of IgG and IgA
 Hemolysin
 Degradation of erythrocytes
 Infections
o Usually more related to the upper respiratory tract (i.e. periodontal disease)
o Other species can cause abscesses in the brain, lungs, ovaries, etc.
Fusibacterium spp (Fusibacterium necrophrum)
 Characteristics
o Gram negative bacilli, or pleomorphic (a long rod with round ends)
o Non-spore forming
o Commonly found in the mouth
 Virulence
o Surface LPS endotoxin
o Leukotoxin (activates leukocyte apoptosis causing necrosis )
o Enzymes (hemolysin)
 Infections
o More related to head and neck infections
 Lemierre’s disease
 This syndrome presents jugular vein septic thrombophlebitis
o A throat infection with F. necrophrum can progress to
abscess formation. If the abscess ruptures, the bacteria
once within the abscess can infect nearby structures
and even the blood stream. Inflammation and
compression surrounding the jugular vein can lead to
blood clots. Pieces of the clot can break off and
metastasis to the lungs, liver, mediastinum, pleural
space, etc.
 Gingivitis/periodontitis
o Intra-abdominal infections

83. BordetellaPertussis Summary
 Causes severe respiratory illness
 Highly contagious (spreads through droplets)
 Common in young children and adults due to poor immunity
Bordetella pertussis
 Characteristics
o Gram-negative, aerobic coccobacilli
o Small, gray, shiny, looks like a pearl
o Non-motile
o Grows on Bordet-gengou or Regan-lowe medium
 Diagnosis
o Collect a specimen with a nasopharyngeal swab or aspirate
o Gram-stain should be gram-negative coccobacillus
o Culture media - should see growth on Bordet-gengou or Regan-Lowe
o Rapid diagnostic tests – PCR
 Virulence
o BvgAS regulatory system
 Modifies organism response to the environment and promotes adhesion
and toxin production when activated
o Adhesions
 Filamentous hemagglutinin and fimbrae mediate adhesion to ciliated
epithelial cells
o Toxins
 Pertussis toxin – promotes lymphocytosis, disrupts signal transduction
 Adenylate cyclase toxin (ACT) – inhibits phagocyte function
 Tracheal cytotoxin – kills respiratory cells
 LPS
 Pathogenesis
o The bacteria survive only a few hours outside of human hosts
o Spreads via respiratory droplets
o Adheres to respiratory epithelial cells
 Interferes with ciliary clearance
 Releases toxins that promote coughing and cell necrosis

84. o There is a two-week incubation followed by a period characterized in 3 stages
 S1: “catarrhal”
 Mild coughing and sneezing, shedding
large numbers of organisms
 S2: “paroxysmal”
 Explosive cough with “whoop” during
inspiration
o Cough is predominant at night
 S3: “convalescent” – slow recovery phase
 Typically, high white blood cell counts
between 15K-30K will be seen
 Treatment
o Oftentimes susceptibility testing is not performed. Treatment is often empiric.
o DOC’s and B. pertussis resistance mechanisms:
Macrolides
Azithromycin
Clarithromycin
Erythromycin
Altered binding to 23S
ribosomal RNA component of
the 50S subunit (alteration to
the macrolide’s binding site)
Fluoroquinolones
Levofloxacin
Moxifloxacin
Altered binding to DNA gyrase
Tetracyclines
Tetracycline
Doxycycline
Minocycline
Efflux mechanisms – tet(A) and
tet(C)
Bactrim N/A
o Prevention:
 DTaP given at 2, 4, 6, and 15 months. Adults receive a pertussis booster
once during their life.
 Post-exposure prophylaxis
 Administered within 21 days to high-risk contacts (infants <1
y/o or pregnant patients)

85. Helicobacter pylori Summary
 Most frequent and persistent infection worldwide
 Can be hard to grow/detect. Detection of H. pylori may be invasive, but stool serology
and breath tests can be useful.
Helicobacter pylori
 Characteristics
o Gram-negative, microaerophilic, spiral-shaped rod
o Difficult to culture/grow, but can grow in Skirrow’s medium with vancomycin,
polymyxin B, and trimethoprim
o Grows in 3-6 days when incubated in a microaerobic environment
o Catalase positive
o Motile
o Can be identified via PCR, enzyme immunoassays for H. pylori antigens in stool, a
urea breath test, or histology where the bacteria are stained and rabbit
antibodies are used to see if H. pylori is present.
 Virulence
o Flagella – contains 4-6 for motility. Allows organism to penetrate mucous layer of
stomach and enter epithelial lining
o Urease production – produces ammonia to buffer gastric pH. The elevated pH
helps with de-gelling mucin layer.
o LPS – cell adhesion
o Cytotoxins
 CagA helps with epithelial cell attachment
 VacA helps cause tissue damage and cellular dysfunction
o Catalase helps protect from hydrogen peroxide
 Pathogenesis
o Transmission
 Human to human via oral/oral or fecal/oral
o Colonization/disease
 Colonization induces gastritis, but few develop disease
 Acute gastritis – feeling full, N/V, inflammation of stomach
mucosa
 After colonization, persistent infections may result in complications and
diseases such as ulcer disease or gastric cancer
 Treatment
o Treatment is typically a combination of 3-4 agents
 Macrolides such as clarithromycin serve as the foundation, although
resistance may be present in the form of altered binding to the 23S rRNA

86.  Beta-lactams (aminopenicillins) are also important in treatment of
H. pylori, although resistance may be present in the form of
altered penicillin-binding proteins.
 Metronidazole and tetracyclines may also be used.
 Triple therapy includes clarithromycin, amoxicillin, and a PPI. A fourth
medication may be added if high clarithromycin resistance is present.
 Treatment is 14 days

87. NeisseriaeSpecies Summary
 Characteristics and general facts:
 Gram-negative diplococci
 Grow in Thayer-Martin (chocolate [lysed] sheep blood and antibiotics)
o Growth is slow and performs better in CO2 rich environments
 Colonies appear translucent, nonpigmented, and glisten (mucoid
growth) and are hemolysis negative. Colonies are also oxidase positive
(turns purple)
 Quick comparison between species (same information is presented below in list format)

88. Neisseria gonorrhoeae
 Characteristics
o Oxidase positive
o Nonmotile
o Tends to prefer to colonize the urethra in men and endocervix in women
o NOT normal flora
 Identification/Diagnosis methods
o 1) Specimen collection (swabbing endocervix or urethra in men)
o 2) Testing – nucleic acid amplification testing, culturing, or microscopy
 Virulence
o Contain pili (for attachment)
o Contain opa (enhances adhesion, minimizes recognition)
o Contain lipooligosaccharide (cell toxicity, mimicry – evading immune system)
o Contain IgA proteases
o Does not have a capsule
o Different antibiotics and N. gonorrehoeae’s resistance mechanism:
 Beta-lactams and 3rd generation cephalosporins – altered binding to PBP
 Penicillin – Penicillinase
 Fluoroquinolones – point mutations in DNA gyrase or topoisomerase IV
 Macrolides – altered binding to 23S rRNA of 50S subunit
 Tetracyclines – ribosome protection (tetM)
 Pathogenesis
o Acquired via sexual or mucosal contact where it attaches to the epithelium and
penetrates into the cells. Can penetrate through the genitourinary tract, eye,
rectum, and throat. Symptoms vary by location of infection:
 Genitourinary
 Dysuria
 Urethral discharge
 Testicular or scrotal pain
 Vaginal discharge or bleeding
 Rectal
 Itching
 Rectal discharge
 Painful bowel movements
 Pharyngeal
 Sore throat
 Bone/Joints
 Arthritis

89.  Tenosynovitis
 Eye
 Conjunctivitis
 Keratitis
 Treatment
o 3rd generation cephalosporins
 Ceftriaxone
 Cefixime
AND
o Macrolides – a single dose of azithromycin
OR
o Tetracyclines – doxycycline
Neisseria meningitidis
 Characteristics
o Oxidase positive
o Nonmotile
o Tends to prefer to colonize respiratory tissue and cerebrospinal fluid
o Normally found in the upper respiratory tract
 Identification/Diagnosis methods
o 1) Collect specimen from blood/CSF
o 2) Gram-stain, culture and perform rapid diagnostic tests (PCR, MALDI-TOF)
 Virulence
o Contain pili (for attachment)
o Contain opa (enhances adhesion, minimizes recognition)
o Contain lipooligosaccharide (cell toxicity, mimicry – evading immune system)
o Contain IgA proteases
o Has a capsule
o Different antibiotics and N. meningitidis’ resistance mechanism:
 Beta-lactams and 3rd generation cephalosporins – altered binding to PBP
 Penicillin – Penicillinase
 Fluoroquinolones – point mutations in DNA gyrase or topoisomerase IV
 Macrolides – altered binding to 23S rRNA of 50S subunit
 Tetracyclines – ribosome protection (tetM)
 Sulfonamides – alteration in folic acid synthesis enzymes (DHPS)
 Pathogenesis

90. o Acquired via respiratory droplets where the bacteria attaches to mucosal cells and
enters bloodstream
 Capsule allows evasion of immune cells
o Generally seen with F/N/V, but different symptoms depending on location infected:
 Eye
 Photophobia
 CNS
 Altered mental status
 Neck stiffness
 Hip/knee flexion with neck flexion
 Inability to straighten leg with hip flexion
 Skin
 Petechial or purpuric rash
 Cardiovascular
 Hypotension
 Shock
 Treatment
o 3rd generation cephalosporins (DOC)
 Ceftriaxone
 Cefotaxime
o Penicillins:
 Penicillin G
 Ampicillin
o Carbapenems
o Prevention
 Conjugate meningococcal vaccines
 Quadrivalent product contains serogroups A, C, Y, W (Menactra)
o Given to:
 All 11 and 12 y/o, booster given at age 16
 Complement deficient patients
 Asplenic patients
 Travelling patients
 Crowded areas like schools or bootcamps
 There is also a Serotype B product available (Bexsero)
o Given to:
 Adolescents and young adults (16-23 y/o)
 Complement deficient, asplenic, crowded areas

91. ChlamydiaSpecies Summary
 Gram-negative cocci or rods
 Obligate intracellular bacterium that infect human cells
 Require host energy to function and reproduce
 Growth in lab and detection is difficult
 Developmental cycle
o Chlamydia exist in two forms
 1) Elementary body (EB)
 2) Reticulate body (RB)
o The EB makes contact with host cell and
gets endocytosed into a lysosome
o The lysosome travels to Golgi, where the
EB transforms to the RB
o Cell division occurs. A micro colony forms
o Nutrients and ATP are scavenged from
the host cell
o Host cell dies, EB are released
 Organism characteristics and virulence factors
o Surface proteins – avoid antibody detection to survive outside host cells
o Inclusion body formation – inhibits release of cytochrome C required for apoptosis
o Low potency LPS – reduces activation of host cells, decreases inflammatory response
o TNF secretion – induces T cell apoptosis, decreases immune response
Chlamydia pneumoniae
 Identification/Diagnosis methods
o Respiratory specimen collection
o PCR/serologic testing
 Pathogenesis
o Acquired via aerosols
o Onset: Gradual
o Symptoms:
 Asymptomatic or mild respiratory illness
 Bronchitis/pneumonia, low grade fever, cough
 Pharyngitis, sinusitis (uncommon)
o Complications:
 The bacteria target damaged cells
 May be involved in heart disease

92.  Treatment (including specific resistance mechanisms)
o Macrolides (altered binding to 23s rRNA of 50S subunit)
o Tetracyclines (efflux)
o Fluoroquinolones
Chlamydia trachomatis
 Characteristics
o There are 19 variants (serovars) that cause disease at different locations
 Ocular infections – A, B, C
 STD – D through K
 Lymphogranuloma venereum – L1- L3
 Identification/Diagnosis methods
 First catch urine or endocervix swab (women) or urine/urethral swab (men)
 Nucleic acid amplification or PCR
 Pathogenesis
o Acquired via sexual contact
o Onset: weeks after exposure
o Symptoms:
 >50% of infections are asymptomatic
 Women:
 Purulent vaginal discharge
 Pyuria
 Dysuria
 Abdominal/pelvi pain
 Men:
 Mucoid or watery urethral discharge
 Dysuria
 Testicular pain or swelling
o Complications:
 Pre-term delivery if pregnant; neonatal conjunctivitis
 Treatment (including specific resistance mechanisms)
o Macrolides – azithromycin (altered binding to 23s rRNA of 50S subunit)
o Tetracyclines – doxyclycline (Efflux)

93. Legionella Species Summary
Legionella pneumophila
 Characteristics
 Fastidious, gram-negative, aerobic rod
 Motile
 Of 15 serogroups, serogroup 1 is most prevalent in humans
 Poorly stain and are hard to detect from respiratory samples
 Grow in buffered charcoal yeast agar (contains cysteine and antibiotics)
o Growth is slow and takes > 3 days
o Colonies are round and flat with minimal color (may be light pink
or blue)
 Found in ubiquitous, freshwater habitats
 Can live freely, in biofilms, or in vegetative states inside amoebas
 Thrive in tepid water, but can survive in growth-restrictive temperatures
o Found in potable water networks within hospitals, hotels, cruise ships, etc
 Identifications/Diagnosis methods
o Collect specimen via sputum
o Culture in buffered charcoal yeast extract
o Utilize antigen detection via urine antigen test (BinaxNow)
o Utilize rapid diagnostic tests (PCR, MALDI-TOF)
 Virulence
o Attachment and motility with pili, flagella, and attachment proteins
o LPS
o Outer membrane vesicles – inhibit phagolysosome formation (MHCII)
o Enzymes – break down lung surfactant, signal macrophage recruitment
 Pathogenesis
o Acquired via inhalation of aerosols where the organism invades and grows within
alveolar macrophages
 They reside in phagosomes
 Cellular machinery surrounds phagosome, limiting cellular destruction
 Multiple using host cell’s nutrients until the cell dies
o Symptoms:
 Respiratory illness, most patients are asymptomatic
 Disease typically seen in immunocompromised hosts or those with structural
lung disease
 Fever, chills, malaise, nonproductive cough, diarrhea

94.  May develop multilobar pneumonia, cavitary lesions, or pleural
effusions
 Treatment (resistance mechanism)
o Macrolides (efflux, in contrast to binding issues associated with other bacteria)
o Fluoroquinolones (point mutations in DNA gyrase)
o Tetracyclines
MycoplasmaSpecies Summary
 Pleomorphic
 They are the smallest self-replicating organisms
 Enriched medium (called PPLO) and a stereomicroscope can be used to visualize
o They require sterols for growth (horse serum)
o Yeast extract in the medium serves to provide the mycoplasma with nucleic acid
precursors.
o Difficult to grow
Mycoplasma pneumoniae
 Identification/DiagnosticMethods
o ID’ing is sometimes difficult as it is difficult to grow
o Specimen collection via serum or sputum
o Testing IgG and IgM antibodies
o Can be rapidly identified via nucleic acid amplification
 Virulence
o Cytoadherence properties
 Allow adherence to cilia of a cell and then to the cell’s surface via an
attachment organelle
o Community-acquired respiratory distress syndrome toxin is produced
 Promotes cell death and airway hyperactivity
o Production of H2O2 (damages host cells)
 Pathogenesis
o Human to human via respiratory droplets, where the organism attaches to
ciliated respiratory epithelial cells
 The attachment organelle stabilizes the mycoplasma and minimizes
removal by cilia.
 Cytotoxic events then occur via adhesins and H2O2/superoxide radical
production  leads to respiratory cell death  results in decrease or loss

95. of cilia
 Clinical presentation
o Responsible mainly for mild upper and lower respiratory infections, with a 3-
week incubation period
o Presents as tracheobronchitis with dry or productive cough. May have headache,
sore throat, or coryza.
o Elderly patients may progress to pneumonia
 Treatment (resistance mechanism)
Empirical treatment consists of
o Macrolides (altered target site binding)
o Tetracyclines
o Fluoroquinolones
MycobacteriaSpecies Summary
Non-tuberculosis Mycobacteria
 Characteristics
o Ubiquitous
o Seen in higher frequency in HIV and elderly patients (“Lady Windemere
Syndrome”)
 Treatment (not all listed)
o Macrolides
o Rifamycin
o Ethambutol
o Treatment for active TB requires multiple drugs (4 drugs for 2 months)
 Isoniazid
 Rifampin
 Pyrazinamide
 Ethambutol
o And then 2 drugs for another 4 months
 Isoniazid
 Rifampin
o Latent TB is treated with Isoniazid for 9 months
Mycobacterium tuberculosis
 Characteristics

96. o Lipid rich cell wall resists gramstain dyes, but respond to acid fast stain
o Aerobic
o Non-motile
o Grows on solid growth media in about 4-8 weeks (slow doubling time)
 Lowenstein-Jensen agar
 Contains antibiotics, eggs, and nutrients to optimize growth
o Can also be grown in liquid medium in around 2 weeks (MGIT agar)
 As the mycobacterium use oxygen, the agar turns orange
o Leading cause of death by a bacterial pathogen
 Identification/DiagnosticMethods
o The Mantoux tuberculin skin test (Purified protein derivative) is used
o Blood tests (interferon gamma release assays) can also be used to measure
release of IFN when exposed to TB antigens.
o If positive for the above tests, a chest radiograph should be performed
o Identification can be confirmed with sputum smears, acid fast microscopy,
culturing, etc.
 Virulence
 Pathogenesis
o Transmitted via inhalation (sneezing, coughing) where droplets are inhaled and
eventually deposit into the alveoli. Normally, macrophages and T cells protect
against infection. In elderly/immunocompromised patients, the bacteria can
eventually escape the alveoli and invade other organ systems/the bloodstream.
o Symptoms:
 Fever, fatigue, night sweats, weight loss
 Prolonged cough (>3 weeks)
 Hemoptysis
 Chest pain
 Treatment (not all listed)
o Isoniazid
o Rifamycin
o Ethambutol
o Treatment for active TB requires multiple drugs (4 drugs for 2 months)
 Isoniazid
 Rifampin
 Pyrazinamide
 Ethambutol
o And then 2 drugs for another 4 months
 Isoniazid
 Rifampin
o Latent TB is treated with Isoniazid for 9 months

97. Candida Species Summary
 General characteristics
o Normal microbiota (Oral, GI, vagina)
o Rarely associated with disease
o Oval, buddying yeast that can grow pseudohyphae (chains of budding yeast that
never detach from the spore)
o Candida albicans are dimorphic (can exist in spore and hyphal forms)
o They produce soft and cream-colored colonies and the pseudohyphae grow
often beneath the agar
 Identification/Diagnosticmethods
o Microscopic examination
o Culturing
o 1,3-Beta-D-Glucan Assay – an antigen test detecting the presence of glucan
o T2 Candida systems – lyses yeast, amplifies their DNA
o PCR – detects nucleic acids released by cells
 Virulence
o Agglutinin-like sequences -> form proteins that promote adhesion
o Aspartyl proteinases -> break down cell walls
o Invasins -> proteins that stimulate endocytosis and cellular uptake
o Hydrolases -> facilitate cell penetration
o Biofilmformation -> protect colony from antifungals, facilitate adhesion
o It is a dimorphic pathogen -> both the spore and hyphae are involved with
dissemination and tissue invasion
 Pathogenesis
o Risk factors that can increase chance for infection:
 The use of antifungals (can allow for Candida overgrowth)
 Sex
 High estrogen birth control
 DM
 Damp/moist areas within skin folds
 Immunocompromised patients (chemo or corticosteroids)
 Malignancy
 Foreign devices like IV catheters
o Cutaneous infection are driven by Candida overgrowth causing local tissue
damage
o Systemic infections arise from a variety of sources:
 Translocation across the GI tract

98.  Invasive devices
 Localized sources of infection (e.g. pyelonephritis)
o Symptoms of cutaneous infection:
 Thrush
 Vulvovaginal candidiasis
 Localized skin reactions
o Symptoms of systemic infection:
 Candidemia
 Infection of internal organs like kidneys, heart, brain
 Candida may infect hardware (e.g. prosthetic heart valves)
 Treatment options and theirresistance mechanisms
o Triazoles (fluconazole)
 Efflux out of yeast cells
 Altered drug binding site
 Alternative ergosterol pathways
o Polyenes (amphotericin B or nystatin)
 Alternative ergosterol pathways or low ergosterol levels in the cell wall
o Echinocandins (micafungin)
 Altered binding to target site
Haemophilus Influenzae Summary
HaemophilusInfluenzae
 Characteristics
o Gram-negative, facultative coccobacillus, pleomorphic (morphology
changes with length of incubation)
o Considered normal respiratory flora
o Grows best on chocolate agar (lysed blood) and in the presence of CO2
 Requires presence of X (hemin) and V (nicotinamide adenine
dinucleotide [NAD]) factors for growth which are contained in
the chocolate agar
o Colonies appear large, flag and colorless (or gray)
o Non-hemolytic
o Odor
o Encapsulated forms appear mucoid
o Can be found as a satellite organismfor pathogens that lyse RBC’s (e.g.
staph aureus) This means that if staph aureus is growing on a plate and
breaking down RBC’s within the chocolate agar, haemophilus may be

99. found growing next to the staph as it is using the nutrients from those
broken down RBC’s
 Identification/Diagnosticmethods
o Specimen collection via sputum, CSF, or blood
o Gram stain
o Culture on chocolate agar in CO2-rich environment
o Rapid ID with PCR, MALDI-TOF, or RapID NH panel
o Testing susceptibility:
 Disk diffusion can report susceptibility, but does not correlate
with outcomes
 Beta-lactamase can be detected with Nitrocef disks. When the
enzyme is present, it will turn red.
 Virulence
o Encapsulated – antiphagocytic
o Mucociliary interactions
 Membrane proteins P2 and P5 help bind mucous
 Lipooligosaccharides alter ciliary function
o Respiratory mucosal attachment via pili and adhesins
o Evasion of mucosal antibodies (IgA)
o Intracellular survival/invasion of local tissue – can survive within
macrophages and epithelial cells
 Pathogenesis
o Encapsulation is the primary driver of invasive disease
 Allows avoidance of phagocytosis
 Helps penetrate epithelium and invade capillaries
 Antigenic strain type B is the most common cause of severe
invasive infections
o Transmitted via respiratory droplets, infection can be caused by all H.
influenzae strains
 Treatment and resistance mechanisms
o Beta-lactams
o Fluoroquinolones
 Point mutation in DNA gyrase or topoisomerase IV
o Macrolides (azithromycin)
 Efflux pumps
o Tetracyclines
 Efflux pumps via tetB gene
o Prevention:
 H. influenzae type b conjugate vaccine
 A 3 or 4-dose series depending on vaccine

100.  Typically administered soon after birth, at age 2, 4, and
6 months. Booster at 12-15 months
 Available as monovalent vaccine or combination with
others
Haemophilusducreyi
 Virulence/pathogenesis
o Enters via a break in the epithelium
o An uncommon cause of genital, anal and perianal ulcers
o Presents as a papule that ruptures and results in a painful ulcer filled
with purulent exudate
 Growth:
o Same as H. influenzae but does not require NAD (only X factor –
hemin) to grow
 Identification/diagnostic methods
o Specimen collected from scrapings from the base of the ulcer
 Treatment
o Macrolides (azithromycin)
o 3rd generation cephalosporins (ceftriaxone)
o Fluoroquinolone (ciprofloxacin)
o Susceptibility testing rarely performed
o Should also test for HIV as Chancroid is a common co-infection

101. Module 5 – Viruses
Module 5.1 – Virology Basics
1. Describe the general properties,classification,and
replicationstrategies of viruses
General:
 Viruses are infectious, obligate intracellular parasites. They are made of a protein
coat with genetic material inside. Viruses are not alive and are dependent on host
cells to replicate
 Viruses are small and most cannot be viewed with a light microscope (although
specific viruses can). They are usually between 20 – 250nm
 Viruses are specifically DNA or RNA based, never both
 A naked virus surrounds its nucleic acid with a capsid that is composed of
capsomeres
 An enveloped virus features an envelope made of glycoproteins
 The capsid and nucleic acid make up the nucleocapsid
Classification:
 Viruses are classified based on size/morphology, nucleic acid type (DNA or RNA), their
mode of replication, their target host organism and the type of disease they cause
 Viruses can be helical, icosahedral, or complex shaped.
 The Family of a virus ends in -viridae
o The Genus of a virus ends in -virus
Replication
 Replication specific to RNA viruses (2 types)
o Positive-sense RNA viruses
 These viruses are identical to mRNA and are instantly ready for translation
into proteins once inside the host cell.
o Negative-sense RNA
 The genomes of these viruses are complementary to mRNA. RNA polymerase
converts this viral genome, allowing it to be translated.
 Replication specific to DNA viruses (6 steps)
o 1) Attachment
o 2) Penetration
o 3) Uncoating

102. o 4) Synthesis
o Consists of transcription/translation of viral mRNA and viral genome replication
o 5) Assembly
o 6) Release
o ***Important: the viral DNA needs to be shuttled into the host cell nucleus. Therefore,
most replication for DNA viruses occurs in the host cell nucleus where its DNA can be
transcribed to RNA**
2. Discuss the pathogenesis of viral infections
Tropism
 Tropism is the specificity or affinity of a virus for a target (e.g. receptor) on a host
cell/tissue. Tropismeffects the attachment phase of replication.
 Tropism directly determines the host range and types of cells that can be infected
(e.g. Tobacco Mosaic Virus cannot infect human cells because it has a strict tropism
to plants)
 KNOW (Virus: Tropism)
o Influenza: Lungs
o Herpes: Neurons
o Rotavirus: Small Intestine
o Epstein Barr: B cells
Transmission
 To successfully infect a host cell, there needs to be enough virus available, the
appropriate cell type must be accessible and local antiviral defenses must be absent
or overcome.
 Methods of transmission
o Respiratory – the easiest mode of transmission (Influenza, cold viruses)
o Fecal-Oral
 Calcivirus (Norwalk)
 Rotavirus
 Adenovirus
 Picornavirus (Hep A, Poliovirus)
o Blood-borne – HIV, Hep C, Hep B
o Sexual – HIV, Hep B, Zika
o Vertical (Mom to fetus) – HIV, Hep C, Hep B
o Arthropods/bugs – West Nile, Dengue, Zika
o Animals – Rabies
Infections
 Most viral infection do not lead to serious complications
 Acute infections are rapid and self-limiting (influenza, norovirus, Zika, Measles)

103.  Persistent infections may be long term or for the life of the host and occur when the
primary infection is not cleared by the immune system (HIV, Hep C, Epstein Barr)
3. List diagnostic tests to identify viral infections
Various Methods
 Direct examination with an electron microscope (not commonly used)
 Antigen detection with enzyme-linked assays
o Low sensitivity and specificity. Tedious & expensive. Relies on good
specimens
 Serology
o Often uses ELISA
o To diagnose a primary infection, there should be:
 Presence of IgM
 Seroconversion
 A 4+ fold increase in IgG between acute and convalescent sera
o To diagnose a reinfection, there should be:
 An absence of slight increase in IgM
 A 4+ fold increase in IgG between acute and convalescent sera
o Problems with serology
 There is a long period of time between acute and convalescent paired
sera (the technique requires paired samples from the two phases)
 Mild local infections may not produce detectable antibodies because
the infection may not be systemic
 Cross-reactivity between viruses may occur because some viruses are
very similar
 Immunocompromised patients may not produce enough antibody to
be detected
 Indirect examination via culturing and isolating the virus and observing for
cytopathic effects
o Problems:
 Takes a long time
 Not all viruses grow in cell cultures
 There is no universal cell line and multiple must be used (meaning
since viruses have specific cell tropisms, you may need to use lung
cells or stomach cells, etc, for growth)
 Specimens must be retrieved promptly and transported to preserve
its infectivity
 Prone to contamination

104.  PCR
o Can be used to detect viruses that are not cultivatable
o Provides rapid ID and can be useful for detecting viral load
o High sensitivity, easy to set up, and fast turnaround
o Contamination is a problem, quantitative assays are more difficult to
conduct, and positive results may be difficult to interpret because the result
may be positive but the disease may not be present
4. Identify mechanisms of action for antiviral agents
Strategy is to block any vital step in the viral life cycle
 Entry inhibitors – block viral entry, similar method of protection to vaccines
(Enfuvirtide)
 Prevention of uncoating (thus preventing viral RNA from being released into the
host cell) (Adamantanes)
 Viral protease inhibitors
 Preventing viral replication (sofosbuvir, cidofovir for DNA viruses)
 Neuraminidase inhibitors (block release of the virus)
 Reverse transcriptase inhibitors and integrase inhibitors
Module 5.2 - Influenza
1. Describe the basic characteristics of influenzavirus
Characteristics
 In the Orthomyxovirux family, comprised of single-stranded negative-sense RNA
 This negative-sense RNA genome is made up of 8 segments
 The virus is enveloped, spherical, pleomorphic and about 80-200nm in diameter
 There are three types of influenza viruses:
o Influenza A – Highly infectious and most serious, causes moderate to severe
disease
o Influenza B – Less severe than type A. Primarily effects children and does not
effect birds or animals
o Influenza C - rare and usually subclinical effects, only infects humans

105. 2. Discuss the pathogenesis and clinical features of
influenza infection
Pathogenesis
 There are two main antigenic determinants (the antigens our immune systemdetects)
of the flu virus: Hemagglutinin and Neuraminidase
o Hemagglutinin proteins (H1-H17) determine the ability of the virus to infect a
host and the specific subtype of influenza.
o Neuraminidase also drives/determines the species of influenza with its subtypes
(N1-N9) e.g. H1N1 is a specific species of influenza.
 The susceptibility of a person to the virus is dependent on the presence and distribution
of sialic acid receptors to which the virus’ hemagglutinin protein binds
o These sialic acid receptors are called SAa2,6GAL and are found in the epithelial
cells of the trachea
o SAa2,3Gal are in the intestinal tract of avian species and are the site of
replication for the avian flu virus.
 Humans DO have a2,3 receptors as well and although not common,
transmission can be direct from avian to human.
o Pigs can acquire both human flu virus and avian flu virus because they have a2,6
and a2,3 receptors (more later)
 The virus is highly contagious and can even live on inanimate objects for 4-9hrs
Replication of the virus
 1) The virus attaches to sialic acid receptors
 2) The virus is endocytosed into a vesicle in the cytoplasm
 3) Fusion with the vesicle occurs and the viral particles are released into the cytoplasm
 4) Viral particles in the cytoplasm transport to the nucleus (uncommon for RNA viruses)
 5) The negative sense RNA gets converted to positive sense RNA by viral-dependent
RNA polymerase
 6) This newly formed positive RNA is shuttled out of the nucleus where proteins are
translated from it and the positive RNA is also used as a template for negative-strand
RNA replication.
 7) The new negative strands are packed into virions (8 segments total) that exocytose
and infect new cells.
Mutations contributing topathogenesis
 Antigenic drift
o Viral-dependent RNA polymerase does not have any proofreading mechanisms
and mutations occur often. This could lead to small variations in the subtype
(this is why we need a flu shot each year) but does not change the subtype (ie
H1N1 will stay H1N1 but will be slightly mutated)

106. o Our immune systemdoesn’t completely recognize the mutant, resulting in only
partial immunity/incomplete protection
 Antigenic shift
o Major change. Results in formation of new viruses
o Occurs when avian flu and human flu infects a pig, and reassortment occurs in
the pig that results in a new virus.
o Assuming the virus can efficiently infect and transmit from human to human, the
result is a pandemic because no one has even partial immunity to this new
strain.
o Antigenic shift occurs most frequently where humans, birds, and pigs are in close
contact.
Clinical features of the flu
 Respiratory illness with fever
 Abrupt onset
 A fever of 101-103 in adults, higher in children
 Headache, muscle aches
 Runny/stuffy nose
 Sore throat
 Extreme fatigue
 Nonproductive coughs
 Don’t confuse with influenza-like illness which is mild in nature with its symptoms
 Uncomplicated flu is usually self-limiting
Complications with flu
 Although usually self-limiting, complications most often occur in:
o Adults >50, children <2
o Pregnant women in 2nd/3rd trimester or 2 weeks postpartum
o People with underlying conditions (immunosuppressed etc)
 These people have a higher likelihood of developing pneumonia directly related to the
flu virus or secondarily from bacteria.
3. List diagnostic tests used to identify influenza
Rapid diagnostic tests
 Results in <15min
 Sensitivity 50-70%, specificity 90-95%
 Predictive value depends on prevalence
Viral cultures
 Gold standard, but not used clinically because results take 7 days

107.  Great for providing information on circulating strains and subtypes and determining
drug susceptibilities
Serology
 Requires paired acute and convalescent sera and results take >2 weeks
 Not generally recommended
 Intranasal vaccinations will affect results of this test
RT-PCR
 This is the most sensitive test
 The turnaround time may not be ideal for clinical environments as most doctors do not
have PCR machines readily available and a sample would need to be sent to a lab.
4. Recall the types of influenzavaccine formulations and
the common side effects associatedwith vaccination
In all cases, it takes about 2 weeks to develop immune protection post-vaccination.
Intramuscular vaccine
 An Inactivated Influenza Vaccine (IIV)
 Adults get 0.5mL
 Elderly >65 get 0.5mL but the dose is 4x stronger than the adult version
 Children <3 years get 0.25mL dose
o For a child’s very first flu shoe, they will get 2 doses 4 weeks apart
 Contraindications/Precautions
o Allergies to vaccine (eggs)
o Moderate or severe acute illness
o Hx of Guillain-Barré syndrome within 6 weeks following a previous influenza
vaccine
Intranasal
 Live attenuated influenza vaccine (LAIV)
 Contraindications/Precautions:
o Pregnant women or immunosuppressed
o Children taking ASA/salicylates
o Children 2-4 yrs with asthma/wheezing
o Those on anti-influenza agents in the last 48hrs
o Caregivers of immunosuppressed patients (They could pass the live virus on to
them)
o Same as IIV options

108. Intradermal (IIV)
Cell-culture trivalent (ccIIV) and recombinant trivalent (RIV) options are available for those with
egg allergies.
5. Identify commonclasses of antivirals used for the
treatment of influenza
Neuraminidase inhibitors (Oseltamivir, Zanamivir, Peramivir)
Module 5.3 - HIV
1. List diagnostic tests used to identify HIV
There are two types of HIV – HIV-1 and HIV-2 testing can be broken down as follows:
1) A general test is done that tests for both HIV-1 and HIV-2 antibodies in an immunoassay
2) A differentiation assay is performed to determine if the patient has HIV-1 or HIV-2
3) A confirmation test if performed:

109.  Testing the HIV viral load (its RNA)
 Using a Western Blot
o A WB detects antibodies specific to HIV proteins
o The result is positive if 2 or more bands are seen
At home tests
 Home Access HIV-1 Test System
o Blood is collected and then sent to a lab. Results can be in a week or even a day.
 OraQuick In-Home HIV test
o Utilizes a swab of the mouth that is then inserted into the kit. Results in 20min.
o Positive results need follow-up testing. There are false negatives in about 1 in 12
tests because these tests check for antibodies to the HIV virus. If the patient's
immune systemis suppressed or if the infection is acute and the body has not
yet had time to mount antibodies to the viral infection, the test may show up as
negative.
Monitoring
 HIV is monitored via CD4 T-cell counts and these are used to asses immune function and
start/stop prophylaxis for opportunistic infections
 HIV RNA (viral load) is tested for to see how a patient is responding to treatment
Resistance Tests
 Genotyping – detects drug resistance in select genes
o Done for patients starting treatment and patients with treatment failure
 Phenotyping – measures ability of virus to grow in certain concentrations of
antiretrovirals and is reserved for treatment-experienced patients with treatment
failure
Other Tests
 HLA-B*5701 Test
o This tests screens for abacavir hypersensitivity (a patient should not take
abacavir if they are positive to this test)
 Co-Receptor Tropism Assay
o There is an HIV drug that will only work if the CCR5 co-receptor is present. This
assay tests for presence of this receptor.
 Hep B and C Serology Tests
o These are necessary because hepatitis infections will affect the drugs selected
for HIV treatment
2. Discuss the pathogenesis and types of infections
caused by HIV
Pathogenesis

110.  Transmitted by blood and bodily fluids
o Most common route is sexual contact
o Injection drug users, needlesticks, newborns of infected mothers
 The virus targets CD4 T cells and infiltrates lymphoid organs
 The virus is an RNA virus that uses reverse transcription to turn into DNA, where it
integrates with the host cell genome.
Symptoms/Infections
 Acute infection symptoms are usually mild to severe, last ~14 days, and include:
o Fever
o Fatigue
o Lymphadenopathy
o Headache
o Rash
 As the disease progresses and the immune system is suppressed, the patient is more
prone to opportunistic infections such as:
o Mycobacterium avium complex
o Pneumocystis jirovecii pneumonia (PCP)
o Toxoplasmosis of the brain
 There are also diseases/illnesses that HIV-infected patients are more prone to
acquiring:
o Invasive cervical cancer
o Kaposi’s sarcoma
o Multiple forms of Lymphoma
o Progressive multifocal leukoencephalopathy
o Wasting syndrome due to HIV
3. Recall commonclasses of antivirals used for the
treatment of HIV
Nucleoside Reverse Transcriptase Inhibitors (NRTIs)
 These bind directly to the active site of the reverse transcriptase
 Abacavir, tenofovir, emtricitabine, lamivudine
Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs)
 These bind to an allosteric site on the enzyme, not the active site
 Efavirenz, etravirine, rilpivirine
Protease Inhibitors (PIs)
 Atazanavir, lopinavir, darunavir

111. Integrase Strand Transfer Inhibitors (INSTIs)
 These block integration of viral DNA into host DNA
 Raltegravir, Elvitegravir, Dolutegravir
CCR5 Antagonist (an Entry Inhibitor)
 Maraviroc
Fusion Inhibitor
 Blocks fusion of the HIV envelope with the CD4 cell
 Enfuvirtide
Module 5.3 – Hepatitis A B C
1. List diagnostic tests used to identify hepatitis
Hep A
 The only way to diagnose is with antibody tests
o Anti-HAV positive = currently infected or immune (have been vaccinated)
o Anti-HAV positive = Susceptible (never been infected nor vaccinated)
Hep B
 Tests are done to differentiate between acute and chronic infections and whether a
patient has been vaccinated or is susceptible. Take note of the differences

113. Hep C
 Testing for antibodies and viral load for proper diagnosis

114. 2. Discuss the pathogenesis and types of infections
caused by hepatitis viruses
Hep A
 Pathogenesis
o Transmits via Fecal-oral route
o ONLY causes acute infections, never chronic (2-6mo)
 Infections/Symptoms
o Fever
o Fatigue
o Loss of appetite
o N/V
o Abdominal pain
o Dark urine/clay colored stool
o Jaundice
Hep B
 Pathogenesis
o Contact with blood or bodily fluids
o Chronic infection can occur (most frequently in infants)
 Infections/Symptoms
o Same as Hep A
o Extrahepatic manifestations including:
 Serum sickness-like conditions
 Necrotizing vasculitis
 Glomerulonephritis
Hep C
 Pathogenesis
o Mostly Blood-blood transmission, not other bodily fluids (most commonly
injection drug use)
o Most develop chronic infection
 Infections/Symptoms
o Acute infection:
 Mostly asymptomatic or nonspecific symptoms (weakness, jaundice, etc)
o Chronic infection
 Mostly asymptomatic until late stage
 Patient can develop cirrhosis and hepatocellular carcinoma (HCC)
 Extrahepatic manifestations:
 Cryoglobulinemia
 Renal disorders
 Skin conditions
 Insulin resistance, T2DM

115.  Lymphomas
3. Recall commonclasses of antivirals used for the
treatment of hepatitis
Hep A
 NO treatment
 Prevention
o Good hygiene/cooking food well etc
o Vaccination
 HAV only vaccine (Havrix, Vaqta)
 2 shots given at 0 and 6mo
 HAV & HBV vaccine (Twinrix)
 3 shot series given at 0, 1, 6mo but can be adapted for travelers
o Immune Globulin
 Used only when vaccination is not an option
 0.02mL/kg IM within 2 weeks of exposure. Can also be given with vaccine
but generally no need to do so
Hep B
 NO treatment for the acute infection
 Chronic infection:
o Monitoring liver function tests
o Treatment with inferferon and/or nucleos(t)ide analogs such as Tenofovir,
entecavir, lamivudine, emtricitabine, adefovir
 There is resistance to some of these analogs due to mutations
 Prevention
o HBV only vaccines (Engerix-B, Recombivax HB) or HAV+HBV vaccine (Twinrix)
 Both are 3 shot series given at 0, 1, and 6mo
o For peds:
 HBV+Hib (Comvax)
 HBV + Hib +DTaP + IPV (Pediarix)
Hep C
 NO vaccine
 Antiviral treatment is available:

116. Summary of pathogenesis/treatment