Microbiology - Basic Bacteriology

Question 1

Bacterial structures

• For each
  
  • Function
  • Chemical Composition
• Peptidoglycan
• Cell wall/cell membrane (gram positives)
• Outer membrane (gram negatives)
• Plasma membrane
• Ribosome
• Periplasm
• Capsule
• Pilus/fimbria
• Flagellum
• Spore
• Plasmid
• Glycocalyx

Answer 1

• Peptidoglycan
  
  • Gives rigid support, protects against osmotic pressure.
  • Sugar backbone with peptide side chains cross-linked by transpeptidase.
• Cell wall/cell membrane (gram positives)
  
  • Major surface antigen.
  • Peptidoglycan for support. Lipoteichoic acid induces TNF and IL-1.
• Outer membrane (gram negatives)
  
  • Site of endotoxin (lipopolysaccharide [LPS]); major surface antigen.
  • Lipid A induces TNF and IL-1; O polysaccharide is the antigen.
• Plasma membrane
  
  • Site of oxidative and transport enzymes.
  • Phospholipid bilayer.
• Ribosome
  
  • Protein synthesis.
  • 50S and 30S subunits.
• Periplasm
  
  • Space between the cytoplasmic membrane and outer membrane in gram-negative bacteria.
  • Contains many hydrolytic enzymes, including β-lactamases.
• Capsule
  
  • Protects against phagocytosis.
  • Polysaccharide (except Bacillus anthracis, which contains D-glutamate).
• Pilus/fimbria
  
  • Mediate adherence of bacteria to cell surface; sex pilus forms attachment between 2 bacteria during conjugation.
  • Glycoprotein.
• Flagellum
  
  • Motility.
  • Protein.
• Spore
  
  • Resistant to dehydration, heat, and chemicals.
  • Keratin-like coat; dipicolinic acid; peptidoglycan.
• Plasmid
  
  • Contains a variety of genes for antibiotic resistance, enzymes, and toxins.
  • DNA.
• Glycocalyx
  
  • Mediates adherence to surfaces, especially foreign surfaces (e.g., indwelling catheters).
  • Polysaccharide.

Question 2

Cell walls

• Common to both
• Unique to gram-positive organisms
• Unique to gram-negative organisms

Answer 2

• Common to both
  
  • Flagellum
  • Pilus
  • Capsule
  • Peptidoglycan
  • Cytoplasmic membrane
• Unique to gram-positive organisms
  
  • Lipoteichoic acid
    
    • Combination of lipids and teichoic acids
  • Cell wall
• Unique to gram-negative organisms
  
  • Endotoxin / LPS (outer membrane)
  • Periplasm

Question 3

Bacterial taxonomy

• Circular (coccus)
  
  • Gram-positive examples (2)
  • Gram-negative examples (1)
• Rod (bacillus)
  
  • Gram-positive examples (6)
  • Gram-negative examples
    
    • Enterics (13)
    • Respiratory (3)
    • Zoonotic (4)
• Branching filamentous
  
  • Gram-positive examples (2)
• Pleomorphic
  
  • Gram-negative examples (2)
• Spiral (spirochetes)
  
  • Gram-negative examples (3)
• No cell wall
  
  • Gram-positive examples (1)

Answer 3

• Circular (coccus)
  
  • Gram-positive examples
    
    • Staphylococcus
    • Streptococcus
  • Gram-negative examples
    
    • Neisseria
• Rod (bacillus)
  
  • Gram-positive examples
    
    • Clostridium
    • Corynebacterium
    • Bacillus
    • Listeria
    • Mycobacterium (acid fast)
    • Gardnerella (gram variable)
  • Gram-negative examples
    
    • Enterics:
      
      • E. coli
      • Shigella
      • Salmonella
      • Yersinia
      • Klebsiella
      • Proteus
      • Enterobacter
      • Serratia
      • Vibrio
      • Campylobacter
      • elicobacter
      • Pseudomonas
      • Bacteroides
    • Respiratory:
      
      • ƒƒHaemophilus (pleomorphic)
      • Legionella (silver)
      • Bordetella
    • Zoonotic:
      
      • Francisella
      • Brucella
      • Pasteurella
      • Bartonella
• Branching filamentous
  
  • Gram-positive examples
    
    • Actinomyces
    • Nocardia (weakly acid fast)
• Pleomorphic
  
  • Gram-negative examples
    
    • Rickettsiae (Giemsa)
    • Chlamydiae (Giemsa)
• Spiral (spirochetes)
  
  • Gram-negative examples
    
    • Borrelia (Giemsa)
    • Leptospira
    • Treponema
• No cell wall
  
  • Gram-positive examples
    
    • Mycoplasma (does not Gram stain)

Question 4

Bacteria with unusual cell membranes/walls

Answer 4

• Mycoplasma
  
  • Contain sterols.
  • Have no cell wall.
• Mycobacteria
  
  • Contain mycolic acid.
  • High lipid content.

Question 5

Gram stain limitations:
Bugs that don’t Gram stain well

Answer 5

• These Microbes May Lack Real Color.
• Treponema (too thin to be visualized).
  
  • Treponemes—dark-field microscopy and fluorescent antibody staining.
• Mycobacteria (high lipid content in cell wall detected by carbolfuchsin in acid-fast stain).
• Mycoplasma (no cell wall).
• Legionella pneumophila (primarily intracellular).
  
  • Legionella—silver stain.
• Rickettsia (intracellular parasite).
• Chlamydia (intracellular parasite; lacks muramic acid in cell wall).

Question 6

Stains

• Giemsa
• PAS (periodic acid–Schiff)
• Ziehl-Neelsen (carbol fuchsin)
• India ink
• Silver stain

Answer 6

• Giemsa
  
  • Chlamydia, Borrelia, Rickettsiae, _Try_panosomes, _P_lasmodium.
  • Certain Bugs Really Try my Patience.
• PAS (periodic acid–Schiff)
  
  • Stains glycogen, mucopolysaccharides; used to diagnose Whipple disease (Tropheryma whipplei).
  • PASs the sugar.
• Ziehl-Neelsen (carbol fuchsin)
  
  • Acid-fast organisms (Nocardia, Mycobacterium).
• India ink
  
  • Cryptococcus neoformans (mucicarmine can also be used to stain thick polysaccharide capsule red).
• Silver stain
  
  • Fungi (e.g., Pneumocystis), Legionella, Helicobacter pylori.

Question 7

Special culture requirements

• H. influenzae
• N. gonorrhoeae, N. meningitidis
• B. pertussis
• C. diphtheriae
• M. tuberculosis
• M. pneumoniae
• Lactose-fermenting enterics
• Legionella
• Fungi

Answer 7

• H. influenzae
  
  • Chocolate agar with factors V (NAD+) and X (hematin)
• N. gonorrhoeae, N. meningitidis
  
  • Thayer-Martin (or VPN) media
    
    • Vancomycin (inhibits gram-positive organisms)
    • Polymyxin (inhibits gram-negative organisms except Neisseria)
    • Nystatin (inhibits fungi)
  • “to connect to Neisseria, please use your VPN client”
• B. pertussis
  
  • Bordet-Gengou (potato) agar
  • Bordet for Bordetella
• C. diphtheriae
  
  • Tellurite agar, Löffler medium
• M. tuberculosis
  
  • Löwenstein-Jensen agar
• M. pneumoniae
  
  • Eaton agar, requires cholesterol
• Lactose-fermenting enterics
  
  • Pink colonies on MacConkey agar (fermentation produces acid, turning colony pink)
  • E. coli is also grown on eosin–methylene blue (EMB) agar as colonies with green metallic sheen
• Legionella
  
  • Charcoal yeast extract agar buffered with cysteine and iron
• Fungi
  
  • Sabouraud agar.
  • “Sab’s a fun guy!”

Question 8

Obligate aerobes

• Definition
• Examples

Answer 8

• Definition
  
  • Use an O2-dependent system to generate ATP.
• Examples
  
  • Include Nocardia, Pseudomonas aeruginosa, and MycoBacterium tuberculosis.
    
    • Nagging Pests Must Breathe.
  • Reactivation of M. tuberculosis (e.g., after immune compromise or TNF-α inhibitor use) has a predilection for the apices of the lung, which have the highest Po2.
  • P. aeruginosa is an aerobe seen in burn wounds, complications of diabetes, nosocomial pneumonia, and pneumonias in cystic fibrosis patients.

Question 9

Obligate anaerobes

• Definition
• Examples

Answer 9

• Definition
  
  • They lack catalase and/or superoxide dismutase and are thus susceptible to oxidative damage.
  • 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, pathogenic elsewhere.
  • AminO2glycosides are ineffective against anaerobes because these antibiotics require O2 to enter into bacterial cell.
• Examples
  
  • Include Clostridium, Bacteroides, and Actinomyces.
  • Anaerobes Can’t Breathe Air.

Question 10

Intracellular bugs

• Obligate intracellular
• Facultative intracellular

Answer 10

• Obligate intracellular
  
  • Rickettsia, Chlamydia.
    
    • Stay inside (cells) when it is Really Cold.
  • Can’t make own ATP.
• Facultative intracellular
  
  • Salmonella, Neisseria, Brucella, Mycobacterium, Listeria, Francisella, Legionella, Yersinia pestis.
  • Some Nasty Bugs May Live FacultativeLY.

Question 11

Encapsulated bacteria

• Definition
• Examples

Answer 11

• Definition
  
  • Their capsules serve as an antiphagocytic virulence factor.
    
    • Capsule + protein conjugate serves as an antigen in vaccines.
  • Are opsonized, and then cleared by spleen.
    
    • Asplenics have decreased opsonizing ability and are at risk for severe infections.
    • Give S. pneumoniae, H. influenzae, N. meningitidis vaccines.
• Examples
  
  • Streptococcus pneumoniae, Haemophilus influenzae type B, Neisseria meningitidis, Escherichia coli, Salmonella, Klebsiella pneumoniae, and group B Strep.
  • SHiNE SKi_S_.

Question 12

Catalase-positive organisms

• Definition
• Examples

Answer 12

• Definition
  
  • Catalase degrades H2O2 before it can be converted to microbicidal products by the enzyme myeloperoxidase.
  • People with chronic granulomatous disease (NADPH oxidase deficiency) have recurrent infections with catalase (+) organisms.
• Examples
  
  • Pseudomonas, Listeria, Aspergillus, Candida, E. coli, S. aureus, Serratia.
  • You need PLACESS for your cats.

Question 13

Encapsulated bacteria vaccines

• Definition
• Examples

Answer 13

• Definition
  
  • 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.
• Examples
  
  • Pneumococcal vaccine
    
    • PCV (pneumococcal conjugate vaccine, i.e., Prevnar)
    • PPSV (pneumococcal polysaccharide vaccine with no conjugated protein, i.e., Pneumovax)
  • H. influenzae type B (conjugate vaccine)
  • Meningococcal vaccine (conjugate vaccine)

Question 14

Urease-positive bugs (examples)

Answer 14

• Cryptococcus, H. pylori, Proteus, Ureaplasma, Nocardia, Klebsiella, S. epidermidis, S. saprophyticus.
• CHuck norris hates PUNKSS.

Question 15

Pigment-producing bacteria (examples)

Answer 15

• Actinomyces israelii—yellow “sulfur” granules, which are composed of filaments of bacteria.
  
  • Israel has yellow sand.
• S. aureus—yellow pigment.
  
  • aureus (Latin) = gold.
• Pseudomonas aeruginosa—blue-green pigment.
  
  • Aerugula is green.
• Serratia marcescens—red pigment.
  
  • Think red maraschino cherries.

Question 16

Bacterial virulence factors

• Definition
• Protein A
• IgA protease
• M protein

Answer 16

• Definition
  
  • 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.

Question 17

Main features of exotoxins and endotoxins

• Source
• Secreted from cell?
• Chemistry
• Location of genes
• Toxicity (high/low)
• Clinical effects
• Mode of action
• Antigenicity
• Vaccines
• Heat stability
• Typical diseases

Answer 17

• Source
  
  • Ex: Certain species of some gram-positive and gram-negative bacteria
  • En: Outer cell membrane of most gram-negative bacteria
• Secreted from cell?
  
  • Ex: Yes
  • En: No
• Chemistry
  
  • Ex: Polypeptide
  • En: Lipopolysaccharide (structural part of bacteria; released when lysed)
• Location of genes
  
  • Ex: Plasmid or bacteriophage
  • En: Bacterial chromosome
• Toxicity
  
  • Ex: High (fatal dose on the order of 1 μg)
  • En: Low (fatal dose on the order of hundreds of micrograms)
• Clinical effects
  
  • Ex: Various effects
  • En: Fever, shock (hypotension), DIC
• Mode of action
  
  • Ex: Various modes
  • En: Induces TNF, IL-1, and IL-6
• Antigenicity
  
  • Ex: Induces high-titer antibodies called antitoxins
  • En: Poorly antigenic
• Vaccines
  
  • Ex: Toxoids used as vaccines
  • En: No toxoids formed and no vaccine available
• Heat stability
  
  • Ex: Destroyed rapidly at 60°C (except staphylococcal enterotoxin)
  • En: Stable at 100°C for 1 hr
• Typical diseases
  
  • Ex: Tetanus, botulism, diphtheria
  • En: Meningococcemia; sepsis by gram-negative rods

Question 18

Bugs with exotoxins

• Inhibit protein synthesis (4)
• Increase fluid secretion (3)
• Inhibit phagocytic ability (1)
• Inhibit release of neurotransmitter (2)
• Lyse cell membranes (2)
• Superantigens causing shock (2)

Answer 18

• Inhibit protein synthesis
  
  • Corynebacterium diphtheriae
  • Pseudomonas aeruginosa
  • Shigella spp.
  • Enterohemorrhagic E. coli (EHEC), including O157:H7 strain
• Increase fluid secretion
  
  • Enterotoxigenic E. coli (ETEC)
  • Bacillus anthracis
  • Vibrio cholerae
• Inhibit phagocytic ability
  
  • Bordetella pertussis
• Inhibit release of neurotransmitter
  
  • Clostridium tetani
  • Clostridium botulinum
• Lyse cell membranes
  
  • Clostridium perfringens
  • Streptococcus pyogenes
• Superantigens causing shock
  
  • Staphylococcus aureus
  • Streptococcus pyogenes

Question 19

Bugs with exotoxins:
Inhibit protein synthesis

• For each
  
  • Toxin
  • Mechanism
  • Manifestation
• Corynebacterium diphtheriae
• Pseudomonas aeruginosa
• Shigella spp.
• Enterohemorrhagic E. coli (EHEC), including O157:H7 strain

Answer 19

• Corynebacterium diphtheriae
  
  • Toxin: Diphtheria toxin*
  • Mechanism: Inactivates elongation factor (EF-2)
  • Manifestation: Pharyngitis with pseudomembranes in throat and severe lymphadenopathy (bull neck)
• Pseudomonas aeruginosa
  
  • Toxin: Exotoxin A*
  • Mechanism: Inactivates elongation factor (EF-2)
  • Manifestation: Host cell death
• Shigella spp.
  
  • Toxin: Shiga toxin (ST)*
  • Mechanism: Inactivates 60S ribosome by removing adenine from rRNA
  • Manifestation: GI mucosal damage Ž–> dysentery; ST also enhances cytokine release, causing hemolytic-uremic syndrome (HUS)
• Enterohemorrhagic E. coli (EHEC), including O157:H7 strain
  
  • Toxin: Shiga-like toxin (SLT)*
  • Mechanism: Inactivates 60S ribosome by removing adenine from rRNA
  • Manifestation: SLT enhances cytokine release, causing HUS; unlike Shigella, EHEC does not invade host cells
• *Toxin is an 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.

Question 20

Bugs with exotoxins:
Increase fluid secretion

• For each
  
  • Toxin
  • Mechanism
  • Manifestation
• Enterotoxigenic E. coli (ETEC)
• Bacillus anthracis
• Vibrio cholerae

Answer 20

• Enterotoxigenic E. coli (ETEC)
  
  • Toxin:
    
    • Heat-labile toxin (LT)*
    • Heat-stable toxin (ST)
  • Mechanism:
    
    • Overactivates adenylate cyclase (increases cAMP) Ž–> increased Cl- secretion in gut and H2O efflux
      
      • Labile in the Air (Adenylate cyclase)
    • Overactivates guanylate cyclase (increases cGMP) –> decreased resorption of NaCl and H2O in gut
      
      • Stable on the Ground (Guanylate cyclase)
  • Manifestation: Watery diarrhea
• Bacillus anthracis
  
  • Toxin: Edema factor
  • Mechanism: Mimics the adenylate cyclase enzyme (increases cAMP)
    Manifestation: Likely responsible for characteristic edematous borders of black eschar in cutaneous anthrax
• Vibrio cholerae
  
  • Toxin: Cholera toxin*
  • Mechanism: Overactivates adenylate cyclase (increases cAMP) by permanently activating Gs –> increased Cl- secretion in gut and H2O efflux
  • Manifestation: Voluminous “rice-water” diarrhea
• *Toxin is an 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.

Question 21

Bugs with exotoxins:
Inhibit phagocytic ability

• For each
  
  • Toxin
  • Mechanism
  • Manifestation
• Bordetella pertussis

Answer 21

• Bordetella pertussis
  
  • Toxin: Pertussis toxin*
  • Mechanism: Overactivates adenylate cyclase (increases cAMP) by disabling Gi, impairing phagocytosis to permit survival of microbe
  • Manifestation:
    
    • 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
• *Toxin is an 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.

Question 22

Bugs with exotoxins:
Inhibit release of neurotransmitter

• For each
  
  • Toxin
  • Mechanism
  • Manifestation
• Clostridium tetani
• Clostridium botulinum

Answer 22

• Clostridium tetani
  
  • Toxin: Tetanospasmin
  • Mechanism: Protease cleaves SNARE proteins required for neurotransmitter release
  • Manifestation:
    
    • Spasticity, risus sardonicus, and “lockjaw”
    • Toxin prevents release of inhibitory (GABA and glycine) neurotransmitters from Renshaw cells in spinal cord
• Clostridium botulinum
  
  • Toxin: Botulinum toxin
  • Mechanism: Protease cleaves SNARE proteins required for neurotransmitter release
  • Manifestation:
    
    • Flaccid paralysis, floppy baby
    • Toxin prevents release of stimulatory (ACh) signals at neuromuscular junctions –>Ž flaccid paralysis

Question 23

Bugs with exotoxins:
Lyse cell membranes

• For each
  
  • Toxin
  • Mechanism
  • Manifestation
• Clostridium perfringens
• Streptococcus pyogenes

Answer 23

• Clostridium perfringens
  
  • Toxin: Alpha toxin
  • Mechanism: Phospholipase (lecithinase) that degrades tissue and cell membranes
  • Manifestation: Degradation of phospholipids Ž–> myonecrosis (“gas gangrene”) and hemolysis (“double zone” of hemolysis on blood agar)
• Streptococcus pyogenes
  
  • Toxin: Streptolysin O
  • Mechanism: Protein that degrades cell membrane
  • Manifestation:
    
    • 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)

Question 24

Bugs with exotoxins:
Superantigens causing shock

• For each
  
  • Toxin
  • Mechanism
  • Manifestation
• Staphylococcus aureus
• Streptococcus pyogenes

Answer 24

• Staphylococcus aureus
  
  • Toxin: Toxic shock syndrome toxin (TSST-1)
  • Mechanism: Bring MHC II and TCR in proximity to outside of antigen binding site to cause overwhelming release of IFN-γ and IL-2 –>Ž shock
  • Manifestation:
    
    • Toxic shock syndrome: fever, rash, shock
    • Other toxins cause scalded skin syndrome (exfoliative toxin) and food poisoning (enterotoxin)
• Streptococcus pyogenes
  
  • Toxin: Exotoxin A
  • Mechanism: Bring MHC II and TCR in proximity to outside of antigen binding site to cause overwhelming release of IFN-γ and IL-2 –>Ž shock
  • Manifestation: Toxic shock syndrome: fever, rash, shock

Question 25

Endotoxin

• Definition
• Associations

Answer 25

• An LPS found in outer membrane of gramnegative bacteria (both cocci and rods).
• Associations: ENDOTOXIN
  
  • C3a –> hypotension & Edema
  • Nitric oxide –> hypotension
  • Coagulation cascade –> D**IC/**Death
  • Outer membrane
  • TNF-α –> fever & hypotension
  • O-antigen
  • eXtremely heat stable
  • IL-1 –> fever
  • C5a –> Neutrophil chemotaxis

Question 26

Transformation

Answer 26

• Ability to take up naked DNA (i.e., from cell lysis) from environment
  
  • Also known as “competence”
• A feature of many bacteria, especially S**. pneumoniae, **H. influenzae type B, and N**eisseria (**SHiN).
• Any DNA can be used.
• Adding deoxyribonuclease to environment will degrade naked DNA in medium Ž–> no transformation seen.

Question 27

Conjugation

• F+ × F–
• Hfr × F–

Answer 27

• F+ × F–
  
  • F+ plasmid contains genes required for sex pilus and conjugation.
    
    • Bacteria without this plasmid are termed F–.
  • Plasmid (dsDNA) is replicated and transferred through pilus from F+ cell.
    
    • No transfer of chromosomal genes.
• Hfr × F–
  
  • F+ plasmid can become incorporated into bacterial chromosomal DNA, termed high-frequency recombination (Hfr) cell.
  • Replication of incorporated plasmid DNA may include some flanking chromosomal DNA.
    
    • Transfer of plasmid and chromosomal genes.

Question 28

Transposition

Answer 28

• Segment of DNA (e.g., transposon) that can “jump” (excision and reintegration) from one location to another, can transfer genes from plasmid to chromosome and vice versa.
• When excision occurs, may include some flanking chromosomal DNA, which can be incorporated into a plasmid and transferred to another bacterium.
• Examples include antibiotic resistance genes on R plasmid.

Question 29

Transduction

• Generalized
• Specialized
  
  • Definition
  • Encoded genes

Answer 29

• Generalized
  
  • A “packaging” event.
  • Lytic phage infects bacterium, leading to cleavage of bacterial DNA.
  • Parts of bacterial chromosomal DNA may become packaged in viral capsid.
  • Phage infects another bacterium, transferring these genes.
• Specialized
  
  • An “excision” event.
    
    • Lysogenic phage infects bacterium
    • Viral DNA incorporates into bacterial chromosome.
    • When phage DNA is excised, flanking bacterial genes may be excised with it.
    • DNA is packaged into phage viral capsid and can infect another bacterium.
  • Genes for the following 5 bacterial toxins are encoded in a lysogenic phage (ABCDE):
    
    • ShigA-like toxin
    • Botulinum toxin (certain strains)
    • ƒƒCholera toxin
    • ƒƒDiphtheria toxin
    • Erythrogenic toxin of Streptococcus pyogenes