Biology Module 2: Understanding Bacterial Spores and Immune Evasion Mechanisms

virulence

degree to which it evades the immune system, damages host cells and or causing diseases

What are ways that bacteria can evade the immune system?

1. they can coat themselves so that our immune system cannot attack them - capsule, slime layer, or biofilm

2. they can become quiescent (form pores)

T/F: capsules, slime layers, and biofilms are not very antigenic

true

this does not stimulate the complement activation

this does not allow for opsonization of the bacteria and phagocytes won't recognize and kill them

capsule

secreted extracellular materials that increases virulences that is well defined

slime layer

secreted extracellular materials that increase virulence and is not well defined

biofilm

slime layer that envelopes several cells

What does the function of a biofilm?

protects bacterial colony but also helps the colony adhere to the host

Which gram-positive bacteria forms spores?

Clostridium tetani & Clostridium botulinum

spores

dormant cells able to survive the immune attack

produced when cells are starved

can lie dormant for years

survive most environs including heat and toxins

dipicolinic acid

Unique to spores

Calcium dependent (protects cytoplasm)

What can spores not survive?

they cannot survive high pressure

How do autoclaves kill spores?

high pressure from an autoclave

Clostridium tetani

tetanus

spore forming

affects nervous system causing the body to become rigid

Clostridium botulinum

botulism

spore forming

affects nervous system causing the body to become flaccid/droop

Bacillus anthracis

anthrax

sporulation

process of forming a spore in which gram-positive cells develop two plasma membranes

What is the mechanism of sporulation?

1. the cell is divided so that it has one small daughter cell and a large mother cell

2. the mother engulfs the daughter cell to make an endospore

3. the spore synthesizes a cortex, and an exosporium

4. the spore is released and remains dormant until exposed to favorable conditions when it can become "reactivated"

T/F: the spore of a gram-positive cell has two membrane bilayers - similar to gram negative cells

true

What are the 5 layers of a spore envelope?

inner

1. cytoplasmic membrane

2. cortex: peptidoglycan layers that lack water and lives in arid conditions, has dehydrated muramic acid

3. outer membrane

4. keratin protein coat

5. exosporium outer layer: outer layer will stain gram-positive even though it is not a typical gram-positive cell

pili

hair-like structures on the surface of the bacteria that are smaller than flagella

required to produce a biofilm, but not always present with a biofilm

streptococcus have pili

naked pili

not encased in biofilm

highly antigenic because they are made of mannose and activate the complement cascade via the lectin-mannose pathway

toxins

proteins that can damage host cells

endotoxin

toxins that are part of the bacterial membrane

cause damage

exotoxin

proteins secreted by bacteria

lipid A

endotoxin which is a part of LPS on gram-negative pathogens

shed continually from live cells

released in large amounts from lysed cells

Why can antibiotics make the patient's symptoms worse temporally?

when cells lyse, they release large amounts of lipid A

can cause septic shock

shock

blood pressure dangerously low

What is septic shock caused by?

bacteremia (bacteria in the blood)

often leads to organ failure and death (40-60% mortality rate)

How does septic shock cause vasodilation?

1. endotoxins released from dead bacteria directly stimulate vasodilation causing hypotension/shock

2. bacteria in the blood can stimulate complement activation causing vasodilation and hypotension through an indirect mechanism

T/F: exotoxins are released from all Gram-positive pathogens except Listeria

true

neurotoxin

exotoxin that causes paralysis

What are some examples of neurotoxins?

tetanus toxin: causes spastic paralysis where all muscles are contracted

botulinum toxin: causes flaccid paralysis where the muscles don't have any tone

pyrogenic exotoxins cause

fever

tissue invasive exotoxins

allow bacteria to burrow into tissue

ex: group A strep secretes SLO and SpeA

What are the minimum growth requirements for all bacteria?

1. carbon source like glucose so that the bacteria can produce ATP, NADH, & NADPH

2. nitrogen so that the bacteria can produce amino acids and nucleic acids

3. optimal temperature: about 37 deg C for most

4. various ions, especially iron

siderophores

proteins that are secreted and capture iron from the environment to aid in oxygen manipulation

T/F: lysed blood cells can provide minimum growth requirements for bacteria

true

What are enzymes that degrade ROS?

superoxide dismutase

peroxidase + NADH2

catalase

Which enzymes are present in obligate aerobes?

SOD

catalase

peroxidase

* all in high concentrations

Which enzymes are present in facultative anaerobes?

SOD

catalase

peroxidase

*all in low concentrations

obligate anaerobes

will die in O2

lack SOD, catalase, and peroxidase

must use fermentation/anaerobic respiration to produce ATP

What is the final electron acceptor in obligate aerobes?

O2

Why are obligate aerobes more likely to evade the immune system?

since they contain a lot of ROS reducing enzymes, they are more likely to survive ROS bombardment by our PMNs and macrophages

facultative anaerobes

prefer to grow aerobically but have the faculty to grow in anaerobic conditions

can use both O2 for metabolism and fermentation to generate ATP

T/F: obligate anaerobes are killed by ROS

true

How do bacteria use the pentose-phosphate pathway?

to produce NADPH (energy)

What is the lifecycle of bacteria?

1. initiation period: bacteria elongates

2. chromosomal replication period

3. division: plasma membrane pinches inward in the middle of the elongated cell producing two daughter cells

What is the generation time for bacteria?

the amount of time that it takes for bacteria to double (divide)

What are two ways to measure growth of bacteria?

1. liquid culture, turbidity or cloudiness is measured

2. colony forming units are measured on solid media

What are the phases of growth for bacteria?

1. lag phase: cells are active but not dividing

2. log phase: exponential cell division and growth

3. stationary phase: division has stopped because an essential nutrient has become depleted. Bacteria are still alive but not dividing

4. death phase: cells begin to die or self-digest in solution

# dead cells > # of living cells

What type of antibiotic is most effective during the log phase?

bacteriostats because the bacteria cannot synthesize peptidoglycan walls

bacteriostats work to stop bacterial division and give the immune system time to kill the bacteria

What are the indications for culturing bacterial keratitis?

if lesion is large >2 mm

if lesion is central: in or near the visual axis

if lesion is not smooth

if lesion has poor response to therapy

When do you preform a culture in treatment of bacterial keratitis?

culture before starting antibiotics

What are the benefits of using blood agar to culture?

has nutrients like carbon, nitrogen and iron

Which type of bacteria loves blood agar?

streptococcus pyogenes

Which bacteria prefer chocolate agar?

Haemophilus influenzae

Neisseria gonorrhea

What are the different types of liquid media?

general nutrient media: grows most bacteria

thioglycolate: grows anaerobic bacteria

M4: grows chlamydia and Herpes

How do you take a bacterial sample from the cornea?

1. anesthetize the cornea

2. tangentially hold spatula or blade to the eye

3. gently scrape surface of ulcerated area

4. try to get some epithelial tissue along with the specimen

How do you culture bacteria from the conjunctiva?

1. use sterile cotton swab: no anesthetic

2. swipe inferior and superior tarsal plates

3. plate

How do you culture bacteria from a contact lens wearer?

plate the contacts themselves and or the storage case

cut CL into pieces to plate out on separate plates

bag the case and send it or solution to the lab

How does a laboratory determine the identity of bacteria?

O2 growth requirements

texture of colonies

hemolysis: lysis of blood

What information can be said about a bacteria from Gram-staining?

1. cell envelope of bacteria: gram neg or gram pos

2. morphology of cell colony: clusters or chains

3. morphology of the individual cell

What is the procedure for Gram staining?

1. bacteria dried and fixed to slide

2. sample is treated with crystal violet, dye is fixed with Gram's iodine * at this point both gram-positive and gram-negative bacteria are purple

3. the stain is washed with alcohol or acetone

* gram-pos cells the stain gets stuck and cells remain purple

* gram-neg cells the stain does adhere and the cells are now clear

4. cells are treated with safranin red dye

* gram-pos cells are purple

*gram-neg cells are pink

What color is Mycobacterium stained using Gram-stain?

pin because the acid stain still sticks to the bacteria

What is the procedure for the Kirby-Bauer Agar Diffusion Assay?

1. the pathogen is spread on a plate and grown under appropriate conditions

2. the bacteria is allowed to grow and cover the plate

3. then antibiotic-soaked paper discs are placed on the plate

4. if the bacteria are susceptible: there will be a clear zone of dead cells around the disc. if cells are resistant: bacteria will grow around disc

What is the best method to identify bacteria?

genetic sequencing

but also most expensive so not used

What gene is most commonly sequenced in genetic sequencing to identify bacteria?

16S rRNA gene

sterilization

kills almost all bacteria and spores

autoclave at 121 deg C with pressure for 15 min

What organisms are not killed by sterilization?

prions

Pseudomonas aeruginosa: can be introduced during cataract surgery using sterilized tools and can cause vision-threatening conditions

bacteriostatic antibiotics

stop cell growth long enough for the immune system to kill bacteria

often used prophylactically before an infection is established or during an infection

may not be best for immunocompromised patients

bactericidal antibiotics

kill bacteria when an infection is already established