First Aid USMLE STEP 1: Microbiology

Cell Wall Overall Picture - Compare Gram (+) to Gram (-)

Flagellum on bacteria is composed of what? What is the function?

Composition: Proteins

Function: motility

Pilus/Fimbria on bacteria is composed of what? What is the function?

Composition: Glycoprotein

Function: Mediate adherence of bacteria to cell surface, sex plus during conjugation.

Spore formation is ONLY possible is what type of bacteria? Why is the formation of spores important/beneficial?

What is the composition of a spore?

Gram Positive Only

Function: Resistant to dehydration, heat, and chemicals.

Composition: Keratin-like coat; dipicolinic acid; peptidoglycan.

Capsule will be composed of what on bacteria? What is the benefit of a capsule?

Function: Protects against phagocytosis

Composition: Organized, discrete polysaccharide layer (except Bacillus anthracis, which contains d-glutamate)

Glycocalyx

Function: Mediates adherance to surfaces, especially foreign surfaces (e.g., indwelling catheters)

Composition:Loose network of polysaccharides

Periplasm

Space between the cytoplasmic membrane and outer membrane in gram-negative bacteria

(peptidoglycan in the middle)

Gram Neg only

Accumulates components exiting gram neg cells, including hydrolytic enzymes (β-lactamases)

Outer membrane (gram negatives)

Gram Neg Only

Function:

1. Endotoxin: Lipid A induces TNF and IL-1; O polysaccharide is antigenic

2. Most OMPs are antigenic

Porins: transport across outer membrane

Composition:

1. Outer Leaflet: endotoxin lipopolysaccharide [LPS/LOS]

2. Embedded proteins: porins and other outer membrane proteins (OMPs)

3. Inner Leaflet: phospholipids

Cell wall and Gram Stain

Composition: Peptidoglycan is a sugar backbone with peptide chains cross-linked by transpeptidase

Function: Net-like structure give rigid support, protects against osmotic pressure damage.

Gram Stain:

Thick peptidoglycan layer retains crystal violet = gram +

Thin peptidoglycan layer turns red or pink with counterstain = gram -

Cytoplasmic Membrane

Composition: Phospholipid bilayer sac with embedded proteins (PBP's) and other enzymes.

Lipoteichoic acids (gram Pos+ only) extend from membrane exterior.

Function: Site of oxidative and transport enzymes

PBPs involved in cell wall synthesis

Lipoteichoic acids induce TNF and IL-1

Plasmid

-Contains a variety of genes for antibiotic resistance, enzymes, and toxins

-Composed of DNA

Gram-positive spherical (coccus) bacteria

-Staphylococcus (clusters)

-Streptococcus (chains, pairs)

Gram-positive rod (bacillus) bacteria

-Bacillus

-Clostridium

-Corynebacterium

-Gardnerella (gram variable)

-Lactobacillus

-Listeria

-Mycobacterium (acid fast)

-Propionibacterium

Gram-positive branching filamentous bacteria

-Actinomyces

-Nocardia (weakly acid-fast)

Gram-negative spherical (coccus) bacteria

-Moraxella catarrhalis

-Neisseria

Gram-negative rod (bacillus) bacteria Enterics

Enterics:

1. Bacteroides

2. Campylobacter

3. E. coli

4. Enterobacter

5. Helicobacter

6. Klebsiella

7. Proteus

8. Pseudomonas

9. Salmonella

10. Serrate

11. Shigella

12. Vibrio

13. Yersinia

Gram-negative rod (bacillus) bacteria Respiratory and Zoonotic

Respiratory:

1. Bordetella

2. Haemophilus (pleomorphic)

3. Legionella (silver stain)

Zoonotic:

1. Bartonella

2. Brucella

3. Francisella

4. Pasteurella

Pleomorphic gram-negative bacteria

-Chlamydiae (Giemsa)

-Rickettsiae (Giemsa)

Spiral gram-negative bacteria

Spirochetes:

-Borrelia (Giemsa)

-Leptospira

-Treponema

No cell wall bacteria

Mycoplasma, Ureaplasma (contains sterols, which do not gram stain)

These bugs do not Gram stain well:

Too thin to be visualized.

1. Treponema

2. Leptosipra

Cell wall has high lipid content; mycolic acids in cell wall

detected by carbolfuchsin in acid- fast stain).

1. Mycobacteria

No Cell Wall

1. Mycoplasma

2. Ureaplasma

Primarily Intracellular

1. Legionella pneumophila (silver stain).

2. Rickettsia

3. Chlamydia ( lacks classic peptidoglycan because of low muramic acid)

4. Bartonella

5. Ehrlichia

6. Anaplasma

What is seen with dark-field microscopy and fluorescent antibody staining?

Treponemes are seen with FTA-ABS

Giemsa staining

Chlamydia, Borrelia, Rickettsia, Trypanosomes, Plasmodium

PAS (periodic acid-Schiff)

Stains glycogen, mucopolysaccharides

Used to diagnose Whipple disease (Tropheryma whipplei).

Ziehl-Neelsen (carbol fuchsin)

Acid-fast bacteria

1. Nocardia

2. Mycobacteria - stains mycelia acid in cell wall

Protozoa

1. Cryptosporidium oocysts

-Alternative is auramine-rhodamine stain for screening (inexpensive, more sensitive but less specific).

India ink

Cryptococcus neoformans (mucicarmine can also be used to stain thick polysaccharide capsule red).

Silver stain

Fungi (e.g., Coccidioides, Pneumocystis jirovecii)

Legionella

Helicobacter pylori

H. influenzae special culture requirements

Chocolate agar: Factors V (NAD+) and X (hematin)

N. gonorrhoeae, N. meningitidis special culture requirements

Thayer-Martin agar:

-Vancomycin inhibits gram+ organisms

-Trimethoprim and Colistin inhibits gram(-) organisms except Neisseria

- Nystatin inhibits fungi

"Very Typically Cultures Neisseria"

B. pertussis special culture requirements

Bordet-Gengou agar (Bordet for Bordetella)

-potato

Regan-Lowe medium

-charcoal, blood, and antibiotic

C. diphtheriae special culture requirements

Tellurite agar

Löffler medium

M. tuberculosis special culture requirements

Lowenstein-Jensen agar

M. pneumoniae special culture requirements

Eaton agar - requires cholesterol

Lactose fermenting enterics special culture requirements

MacConkey agar - fermentation produces acid, causing colonies to turn pink

E. coli special culture requirements

Eosin-methylene blue (EMB) agar

-Colonies with green metallic sheen

Legionella special culture requirements

Charcoal yeast extract agar buffered with cysteine and iron

Fungi special culture requirements

Sabouraud agar

"Sam's a fun guy!"

What are aerobes? Examples?

Use an O2-dependent system to generate ATP

Examples include:

1. Nocardia

2. Pseudomonas aeruginosa

3. MycoBacterium tuberculosis

Reactivation of M. tuberculosis (e.g., after immunocompromise or TNF-α inhibitor use) has a predilection for the apices of the lung, which have the highest Po2.

What are anaerobes? Examples?

Can't grow in the presence of O2

Examples include:

1. Fusobacterium

2. Clostridium

3. Bacteroides

4. Actinomyces.

They lack catalase and/or superoxide dismutase and are thus susceptible to oxidative damage. ("Anaerobes Frankly Can't Breathe Air")

Generally foul smelling (short-chain fatty acids), are difficult to culture, and produce gas in tissue (CO2 and H2)

Anaerobes are normal flora in GI tract, typically

pathogenic elsewhere

AminO2glycosides are ineffective against anaerobes because these antibiotics require O2 to enter into bacterial cell.

Obligate intracellular bugs

Rickettsia, chlamydia, coxiella

Rely on host ATP.

Facultative intracellular bugs

Salmonella, Neisseria, Brucella, Mycobacterium, Listeria, Francisella, Legionella, Yersinia pestis.

Encapsulated bacteria

1. Streptococcus pneumoniae

2. Haemophilus influenzae type B

3. Neisseria meningitides

4. Escherichia coli

5. Salmonella

6. Klebsiella pneumoniae

7. Group B Strep.

Their capsules serve as an antiphagocytic virulence factor.

Capsular polysaccharide + protein conjugate serves as an antigen in vaccines.

How are encapsulated bacteria killed?

They are opsonized, and then cleared by spleen.

Asplenics have decreased opsonizing ability and thus increased risk for severe infections.

Give S. pneumoniae, H. influenzae, N. meningitidis vaccines.

Encapsulated bacteria vaccines

Some vaccines containing polysaccharide capsule antigens are conjugated to a carrier protein, enhancing immunogenicity by promoting T-cell activation and subsequent class switching.

A polysaccharide antigen alone cannot be presented to T cells

Pneumococcal vaccine

PCV (pneumococcal conjugate vaccine, i.e., Prevnar)

PPSV (pneumococcal polysaccharide vaccine with no conjugated protein, i.e., Pneumovax)

H. influenzae type B vaccine

Conjugate vaccine

Meningococcal vaccine

Conjugate vaccine

Urease-positive organisms

Cryptococcus

H. pylori

Proteus

Ureaplasma

Nocardia

Klebsiella

S. epidermis

S. saprophytic.

Potentiate struvite (ammonium magnesium phosphate) stones.

Urease hydrolyzes urea to release ammonia + CO2 which increases pH to become alkaline

Catalase-positive organisms

Catalase degrades H2O2 into H2O and bubbles of O2 before it can be converted to microbicidal products by the enzyme myeloperoxidase.

People with chronic granulomatous disease (NADPH oxidase deficiency) have recurrent infections with certain catalase ⊕ organisms.

Examples: Nocardia, Pseudomonas, Listeria, Aspergillus, Candida, E. coli, Staphylococci, Serrate, B Cepacia, H. Pylori

Actinomyces israelii pigment

Yellow "sulfur" granules, which are composed of filaments of bacteria

"Israel has yellow sand"

S. aureus pigment

Yellow pigment

Aureus (latin) = gold

Pseudomonas aeruginosa pigment

Blue-green pigment

"Aerugula salad is green"

Serratia marcescens pigment

Red pigment

"Think red maraschino cherries"

Staph epidermidis biofilm location

Catheter and prosthetic devices

Viridian's Streptococci (S. Mutans, S. Sanguinis) biofilm location

dental plaques

infective endocarditis

P. aeruginosa biofilm

Respiratory tree colonization in cystic fibrosis patients, contact lens-associated keratitis

Nontypeable unencapsulated H influenza biofilm

Otitis Media

Bacterial virulence factors

These promote evasion of host immune response.

Protein A

Binds Fc region of IgG. Prevents opsonization and phagocytosis. Expressed by S. aureus.

IgA protease

Enzyme that cleaves IgA

Secreted by S. pneumoniae, H. influenzae type B, and Neisseria (SHiN) in order to colonize respiratory mucosa.

M protein

Helps prevent phagocytosis. Expressed by group A streptococci.

Shares similar epitopes to human cellular proteins (molecular mimicry); possibly underlies the autoimmune response seen in acute rheumatic fever.

Type III secretion system

Also known as "injectisome."

Needle-like protein appendage facilitating direct delivery of toxins from certain gram-negative bacteria (e.g., Pseudomonas, Salmonella, Shigella, E. coli) to eukaryotic host cell.

Exotoxin source

Certain species of gram-positive and gram-negative bacteria

Exotoxin secreted from cell?

Yes

Exotoxin chemistry

Polypeptide

Location of exotoxin genes

Plasmid or bacteriophage

Exotoxin toxicity

High (fatal dose on the order of 1 μg)

Exotoxin antigenicity

Induces high-titer antibodies called antitoxins

Exotoxin vaccines

Toxoids used as vaccines

Heat stability of exotoxins

Destroyed rapidly at 60°C (except staphylococcal enterotoxin)

Typical exotoxin diseases

Tetanus, botulism, diptheria

Endotoxin source

Outer cell membrane of most gram-negative bacteria

Is endotoxin secreted from the cell?

No

Chemistry of endotoxin

Lipid A component of Lipopolysaccharide (structural part of bacteria; released when lysed)

Endotoxin Toxicity

Low

Fatal dose on the order of 100s of μg

Location of endotoxin genes

Bacterial chromosome

Clinical effects of endotoxin

Fever, shock (hypotension), DIC

Mode of action of endotoxin

Induces TNF, IL-1, and IL-6

Antigenicity of endotoxin

Poorly antigenic

Endotoxin vaccines

No toxoids formed and no vaccine available

Endotoxin heat stability

Stable at 100°C for 1 hr

Typical endotoxin diseases

Meningococcemia; sepsis by gram-negative rods

Corynebacterium diphtheriae exotoxin

Diphtheria toxin

Inhibits protein synthesis via inactivation of elongation factor (EF-2).

Presents as pharyngitis with pseudomembranes in throat and severe lymphadenopathy (bull neck)

Pseudomonas aeruginosa exotoxin

Exotoxin A

inhibits protein synthesis via inactivation of elongation factor (EF-2)

Manifests itself in host cell death

Shigella spp. exotoxin

Shiga toxin (ST)

Inactivation of 60S ribosome by removing adenine from rRNA

Manifestations: GI mucosal damage --> dysentery; ST also enhances cytokine release, causing hemolytic- uremic syndrome (HUS)

Enterohemorrhagic E. coli (EHEC) exotoxin

Shiga-like toxin (SLT)

Inactivation of 60S ribosome by removing adenine from rRNA

Manifestations: SLT enhances cytokine release, causing HUS (prototypically in EHEC serotype O157:H7). Unlike Shigella, EHEC does not invade host cells

Enterotoxigenic E. coli (ETEC) exotoxin

Heat-labile toxin (LT): overactivates adenylate cyclase (increasing cAMP) --> increasing Cl− secretion in gut and H2O efflux

Heat-stable toxin (ST): overactivates guanylate cyclase (increases cGMP) --> decreased resorption of NaCl and H2O in gut

Both increase fluid - presents as watery diarrhea

"labile in the Air (Adenylate cyclase), stable on the Ground (Guanylate cyclase)"

Edema toxin

Bacillus anthracis exotoxin

- Increase Fluid Secretion

Mimics the adenylate cyclase enzyme (increase cAMP)

Likely responsible for characteristic edematous borders of black eschar in cutaneous anthrax

Cholera toxin

Vibrio cholerae exotoxin

-Increases fluid secretion

Overactivates adenylate cyclase (increasing cAMP) by permanently activating Gs --> increasing Cl− secretion in gut and H2O efflux

Voluminous "rice-water" diarrhea

Pertussis toxin

Bordetella pertussis exotoxin

Inhibits phagocytic ability

Overactivates adenylate cyclase (increasing cAMP) by disabling Gi, impairing phagocytosis to permit survival of microbe

Whooping cough—child coughs on expiration and "whoops" on inspiration (toxin may not actually be a cause of cough; can cause "100-day cough" in adults)

Tetanospasmin

Clostridium tetani exotoxin

Inhibits release of neurotransmitter

Protease that cleaves SNARE (soluble NSF attachment protein receptor), a set of proteins required for neurotransmitter release via vesicular fusion

Spastic Paralysis, risus sardonicus, and "lockjaw"; toxin prevents release of inhibitory (GABA and glycine) neurotransmitters from Renshaw cells in spinal cord

Botulinum toxin

Clostridium botulinum exotoxin

Inhibits release of neurotransmitters

Protease that cleaves SNARE (soluble NSF attachment protein receptor), a set of proteins required for neurotransmitter release via vesicular fusion

Flaccid paralysis, floppy baby; toxin prevents release of stimulatory (ACh) signals at neuromuscular junctionsflaccid paralysis

ADP ribosylating A-B toxin

B (binding) component binds to host cell surface receptor, enabling endocytosis

A (active) component attaches ADP-ribosyl to disrupt host cell proteins

Examples: Botulinum, Tetanospasmin, Pertissis toxin, Cholera toxin, Edema toxin, Heat-labile toxin (LT), Shiga-like toxin (SLT), Shiga toxin (ST), Endotoxin A, Diphtheria toxin.

Alpha toxin

Clostridium perfringens exotoxin

Lyses cell membrane

Phospholipase (lecthinase) that degrades tissue and cell membranes

Manifests as a degranulation of phospholipids --> myonecrosis ("gas gangrene") and hemolysis ("double zone" of hemolysis on blood agar)

Streptolysin O

Streptococcus progenies exotoxin

-Lyses Cell Membrane

Protein that degrades cell membrane

Lyses RBCs; contributes to β-hemolysis;

host antibodies against toxin (ASO) used to diagnose rheumatic fever (do not confuse with immune complexes of poststreptococcal glomerulonephritis)

Toxic shock syndrome toxin (TSST-1)

Staphylococcus aureus exotoxin

Superantigen causing shock

Binds to MHC II and TCR outside of antigen binding site to cause overwhelming release of IL-1, IL-2, IFN-γ, and TNF-α ---> shock

Toxic shock syndrome: fever, rash, shock; other toxins cause scalded skin syndrome (exfoliative toxin) and food poisoning (enterotoxin)

Exotoxin A

Streptococcus pyogenes exotoxin

Superantigen causing shock

Binds to MHC II and TCR outside of antigen binding site to cause overwhelming release of IL-1, IL-2, IFN-γ, and TNF-α causing shock

Toxic shock syndrome: fever, rash, shock

Endotoxin

LPS found in outer membrane of gram-negative bacteria (both cocci and rod).

Composed of:

O antigen + core polysaccharide + lipid A (the toxic component)

Released upon cell lysis or by living cells by blebs detaching from the outer surface membrane (vs exotoxin which is actively secreted)

Endotoxin action

1. Activate macrophages(TLR4) ---> they secrete:

- IL-, IL-6 (fever)

-TNF-α (fever and hypotension)

- NO (hypotension)

2. Activates complement --> releases

- C3a (Hypotension, edema, histamine release)

- C5a (Hypotension, Edema, Histamine release, neutrophil chemotaxis)

3. Activates tissue factor -> coagulation cascade -> DIC

ENDOTOXINS

pneumonic

Edema

Nitric Oxide

Dic/Death

Outer Membrane

TNF-alpha

O-antigen+core polysacc + lipid A

eXtremely heat stable

IL-1 and IL-6

Neutrophil Chemotaxis

Shock