Introduction to Bacteriology (Bacteria, Shapes & Stains, Bacterial Culture, Special Growth Requirements)

Prokaryotes vs Eukaryotes

- Prokaryotes have no nucleus or membrane bound organelles

- Eukaryotes have a nucleus and membrane bound organelles

- Both can reproduce and respond to the environment

Which type of cells are eukaryotic?

- Plant and animal cells, fungi (modern forms of life)

Which type of cells are prokaryotic?

- Bacteria and Archaea (very old forms of life)

Bacteria

- Single-celled organisms that lack a nucleus; prokaryotes

What is a key component of bacteria? What is its function?

Its cell wall:

- Offers general support

- Maintains osmotic pressure

What component of bacteria is targeted by the immune system and antibiotics?

- Bacterial cell wall

Gram Stain

- A staining method that distinguishes between two different kinds of bacterial cell walls

- Classifies bacteria as either gram +/-

Gram Positive Bacteria

- Bacteria that have a thick peptido glycan cell wall, and no outer membrane. They stain very darkly (purple) in Gram stain.

Take up gram stain → purple

Gram Negative Bacteria

- Bacteria that have complex cell walls with less peptidoglycan but with lipopolysaccharides. Very toxic and hard to treat.

- Take up less of gram stain → pink

How do the cell walls of gram + and gram - bacteria differ?

- Gram +: Much thicker cell wall (takes up more gram stain); have molecules called Lipoteichoic acid

- Gram -: Much thinner cell wall (+ periplasm) as well as an outer membrane

Lipoteichoic Acid

What is unique to gram - bacteria?

- Outer membrane

What is the major structural component of bacterial cell walls?

- Peptidoglycan: Polymer sheets of sugars and peptides; sheets cross linked with other sheets

What are the sugars found in Peptidoglycan?

- N-acetylglucosamine (NAG)

- N-acetylmuramic acid (NAM)

NAM : NAG : NAM : NAG etc...

Are the peptides found in peptidoglycan attached to NAM or NAG?

- Attached to NAM → allows for cross linking

Layer upon layer of NAG : NAM : NAG (linked by peptides) → builds cell wall

Penicillin and Cephalosporin MOA

- Target peptidoglycan in bacterial cell walls → means they will only kill bacteria and not human cells

How many sheets of peptidoglycan are found in Gram +/- bacteria?

- (+): Up to 40 sheets (50%+ of cell wall)

- (-): Very few sheets (5-10% of cell wall)

Which bacteria do not gram stain well? Why?

- Mycoplasma: No cell wall → can't pick up gram stain

- Mycobacteria: Contains mycolic acid → does not gram stain well

- Chlamydia: Lacks muramic acid → can't gram stain

How is the cell wall of mycoplasma unique?

- Cell membrane has sterols for extra stability, makes up for lack of cell wall

What type of stain is used to visualize mycobacteria?

- Ziehl-Neelsen

Cell membrane is found where in bacteria?

- Below cell wall; is a phospholipid bilayer

- Present in Gram +/- bacteria

The cell membrane in bacteria is used for what?

- ETC and oxidative phosphorylation (occurs in cell membrane bc/ they lack mitochondria)

- Has enzymes and carrier molecules that are important for bringing stuff into cell

What is a unique feature of Gram + Bacteria?

- Molecules called lipoteichoic acid (LTA) are found on the surface

What is a major surface antigen that stimulates immune reaction?

- Lipoteichoic acid (LTA) in Gram + → induces cytokine release → binds antibodies and activates the complement cascade

What does LTA induce in animals?

- Arthritis

- Uveitis

- Meningeal inflammation

- Cascades for septic shock & multi-organ failure

What features are unique to gram - bacteria?

- Outer membrane

- Periplasmic space

Periplasm

- The gel-like material that fills the region between the cytoplasmic membrane and the outer membrane of Gram-negative bacteria.

What is located inside the periplasm of gram - bacteria?

- Many enzymes

- MOST IMPORTANT: β-Lactamase → used to inactivate antibiotics

Outer Membrane (Gram -)

- Unique to gram (-); contains lipopolysaccharide (LPS)

Lipopolysaccharide (LPS)

- Molecule found on surface of Gram (-) → MAJOR immune trigger → patients who have Gram (-) bacteremia get VERY sick

Do patients get more sick in the setting of Gram (+) or Gram (-) Bacteremia?

- Gram (-) d/t LPS

What are the components of Lipopolysaccharides?

- Polysaccharide

- Lipid A

- O Antigen

What is the role of lipid A in LPS?

- Highly toxic → component that makes ppl very sick in gram (-) bacteremia

- Triggers cytokine release

What is the role of O antigen in LPS?

- Target for antibodies

Overview of the differences between Gram +/- Bacteria

- (+): Cell wall and membrane; Lipoteichoic Acid (LTA)

- (-): Cell Wall/Periplasm + Outer membrane; Lipopolysaccharide (LPS)

Bacterial Capsules

- Sticky, gelatinous layer secreted by bacteria → helps it attach to host cells and protects against phagocytosis

What is the composition of bacterial capsules?

- Mostly water with some polysaccharides

How is Bacillus Anthracis (Anthrax) an exception to the normal composition of bacterial capsules?

- Its capsule is protein (in contrast to polysaccharides) made of d-glutamate → acts as a major virulence factor that allows unimpeded growth

What is the most important encapsulated bug?

- Strep Pneumonia

What are other examples of encapsulated bugs?

- H. influenza

- N. Meningitidis

- E. coli

- Salmonella

- Klebsiella

- Group B strep

Hungry nurses eat spicy Korean grub

How does the body protect itself from encapsulated bacteria?

- B cells secrete capsular antibodies (IgG) → binds capsules; phagocytes recognize Fc receptors on antibodies and swallow em up

Antibodies secreted from B cells that bind capsules are also useful for what?

- Can activate the complement cascade; formation of MAC → death

- Part of cascade (activated) is C3b, can tag pathogens for phagocytosis via opsonization

What happens if you lose antibodies/B-cells/complement?

- Causes recurrent encapsulated bacterial infections

Patients with Asplenia are at risk of what?

- ↑ Risk of sepsis from encapsulated bacteria d/t loss of splenic phagocytes

I.e removed spleen or sickle cell anemia

Which component of bacteria is a basis for many vaccines?

- The capsule; polysaccharides within capsule are often weakly immunogenic

Polysaccharides within capsule, which are targeted by vaccines, are often weakly immunogenic. What is done to the vaccine in order to elicit a larger immune response?

- The polysaccharides within vaccines are "conjugated" to a protein that DOES stimulate an immune response:

1. Diphtheria toxoid, tetanus toxoid, meningococcal outer membrane

Ex: Polysaccharide is weak. Add a toxoid to the polysaccharide within the vaccine → stronger immune response

What are some bacteria that are treated with conjugated vaccines?

- Neisseria meningitides

- Streptococcus pneumonia

- H. Influenza type b

Glycocalyx

- Sugar coat made of polysaccharides (similar to capsule) that is used to adhere to surfaces (i.e. S epidermis → biofilms on catheters)

Some bacteria have capsules, some have glycocalyx

Glycocalyx vs Capsule

- Glycocalyx: Irregular, slimy fuzz layer used to adhere to surfaces

- Capsule: Distinct, firmly attached gelatinous layer

What is the role of the glycocalyx for S. Epidermis?

- Glycocalyx allows it to adhere to surfaces → able to form biofilms on catheters

If you have a central IV line in a patient made of a polymer, S. Epidermis can form biofilm → infection

Biofilm

- A microbial community that usually forms as a slimy layer on a surface

Pili and Fimbria

- Structurally similar to flagella, made of proteins

- Basically an arm/appendage for bacteria → allows them to adhere to surfaces (ordinary pili) // or to other bacteria for conjugation (sex pili)

What is the role of pili in E. coli?

- Allow them to attach to urinary treat → causes UTIs

(Ordinary pili)

What is the role of pili in Neiserria Gonorrhea?

- Pili exhibit a feature known as antigenic variation → bacteria can change the makeup of the proteins in their pili

What is the consequence of Neiserria Gonorrhea's pili exhibiting antigenic variation?

- If a pt has gonorrhea infection, they are at risk for subsequent gonorrhea infections bc/ the antibodies and immune system memory to the first infection does NOT apply to the second

Plasmids

- Small circular DNA molecules that replicate separately from the bacterial chromosome

- Can contain genes for antibiotic resistance, can synthesize toxins, and can be transferred from one bacteria to another

Flagellum

- Long protein arms used for bacteria mobility, pushes them from place → place

Why is it beneficial that bacteria ribosomes have a different structure compared to humans?

- Allows selective toxicity of antibiotics to bacteria, won't kill animal cells

Tetracyclines target what?

- Antibiotics that target the 30S subunit of bacterial ribosomes

Bacteria ribosomes: 50S + 30S

Aminoglycosides target what?

- Antibiotics that interfere with 30S protein synthesis

Bacteria ribosomes: 50S + 30S

What is the dormant state of bacteria?

- Some can enter a dormant state called a spore

Bacterial spore

- A dormant state for some bacteria with no metabolic activity

- Can survive a long period of starvation

- Resistant to dehydration, heat, chemicals

Basically like a seed, in the right condition, can "germinate" and become active

What are the components of a bacterial spore?

- Coat

- Cortex/core wall

- Diplicolinic acid

What is the role of the coat in the spore?

- Outermost layer made of keratin-line protein

- Impermeable to many chemicals + bacterial agents

What is the role of dipicolonic acid in the spore?

- Large amounts are found inside spores

- May help with heat resistance

What are the key spore forming bacteria?

- Bacillus Anthracis/Cereus

- Clostridium Perfringens/Tetani/Botulinum

How are bacteria typically identified?

- Their shape

- What color they stain

- Special tests

Common Bacterial Shapes

- Coccus: Spherical

- Bacilli: Rod shaped

- Coccobacillus: Oval

- Spirilli: Spiral-shaped

Cocci vs Diplococci vs Streptococci vs Staphylococci

- Cocci: 1 ball

- Diplococci: 2 balls

- Strep: Many balls in chain

- Staph: Bunches/clusters of balls

Streptococci vs Staphylococci

- Strep: Cocci in chain

- Staph: Bunches of cocci (grapes, yum!)

Are most cocci gram + or -?

- Most are gram (+)

- i.e strep and staph

Which gram - bugs are cocci?

- Neisseria (meningitides/gonorrhea)

- Moraxella Catarrhalis

Most cocci are gram (+)

Are most rods/bacilli gram + or -?

- Most are gram (-)

Which rods are gram +?

- Corynebacterium

- Clostridium

- Listeria

- Bacillus (anthrax)

Most rods are gram (-)

Branching/Filamentous bacteria: appearance and common ones

- Long/branching (resemble fungi)

- Actinomyces, Nocardia

Sphirochetes

- Spiral shaped bacteria

Which bacteria are spirochetes?

- Treponema (Syphilis)

- Borrelia (Lyme disease)

- Leptospira (leptospirosis)

Vibrio: appearance and common ones

- Comma shaped bacteria

- Vibrio cholerae

Pleomorphic Bacteria

- Can assume several shapes

What are key pleomorphic bacteria?

- Rickettsia

- Chlamydia

Why are Rickettsia and Chlamydia often grouped together?

- Two intracellular pathogens (obligate intracellular)

Simple Stains

- Utilized just to see bacteria under microscope (can see # of bacteria and their shapes)

- I.e. Methylene blue, safranin, crystal violet

Stains all bugs the same, nonspecific

What is the gram stain?

- Helps differentiate between gram +/- bacteria

- Purple → Gram +

- Pink → Gram -

Stain based on characteristics of their cell wallq

What are the steps of gram staining?

1. Fixation

2. Crystal violet

3. Iodine

4. Decolorization: Washes away purple from everything except Gram +

5. Safranin (Counter stain)

Gram (+) pick up crystal violet → purple

Gram (-) washes away crystal violet, stained by safranin (counter stain) → pink

Why do gram + bacteria pick up crystal violet?

- Because of their thick cell walls (peptidoglycan)

Why does Treponema (Syphilis) not gram stain well?

- Cell wall is too thin

Why do mycobacteria (tuberculosis) not gram stain well?

- Contains mycologic acid in cell wall (waxy, makes them impermeable to gram stain)

Acid fast stains used instead

Why do Mycoplasma not gram stain well?

- No cell wall (have cell membrane instead)

Why do Rickettsia/Chlamydia/Legionella not gram stain well?

- They are intracellular bacteria; grow inside other cells

Giemsa Stain

- Chlamydia, Borrelia, Rickettsia, Trypanosomes, Plasmodium

USED TO STAIN INTRACELLULAR BACTERIA

Ziehl-Neelsen Stain

- Use to stain Acid-fast bacteria (Nocardia, Mycobacterium)

Silver Stain

Special stain for 3 organisms:

- Pneumocystis Pneumonia (fungal infections seen in HIV/AIDS)

- Legionella (pnemonia, contaminates water → outbreaks in nursing homes)

- H Pylori (causes gastric ulcers)

India Ink

- Negative stain (makes the background dark, not the organism)

- Used in fungi/yeast (Cryptococcus neoformans)

Some bacteria can produce what as they grow?

- Pigments, can give colonies unique colors

Staph Aureus color

- Golden/yellow color

Aura = glowing = yellow

Pseudomonas Aeruginosa Color

- Blue-green

Serratia Color

- Red

Serratia = Sriracha = RED

Actinomyces

- Long filamentous bacteria that "cements" together

- Colonies have yellow/orange appearance

Where is Actinomyces commonly found?

- Normal oral flora but can form abscess infections in jaw

Agar Plate

- A petri dish filled with a jello-like nutrient material used to grow bacteria upon

Bacteria don't usually consume agar (from seaweed), can grow on top of it