03-18 Bacterial Growth and Metabolism

Question 1

OBJECTIVE: What is the source and role of carbon?

Answer 1

role: in all molecules! and used for energy
source: glucose, other sugars, aa’s/peptides/prots, lipids, organic acids and alcohols (can be from other bact), nucleic acids

Question 2

OBJECTIVE: What is the source and role of nitrogen?

Answer 2

role: needed for DNA and protein synth
source: aa’s, peptides/prots, nucleic acids, NH4+ and nitrate

Question 3

OBJECTIVE: What is the source and role of sulfur?

Answer 3

role: proteins (think disulfide bonds)
source: Nucleic acids

Question 4

OBJECTIVE: What is the role of nutrients vs. growth factors?

Answer 4

NUTRIENTS: are metabolized for energy or broken down and used as building blocks.

GROWTH FACTORS: are organic compounds a bacterium uses to make metabolites it cannot make on its own.

• -EXAMPLES: purines, pyrimidines, vitamines, a.a.’s, NAD+, heme, etc.
• -often substances readily available in host environment; bact “stopped making them” in effort to pare down the size of their genomes
• -fastidious bacteria require lots of growth factors
• -prototrophic bacteria synthesize everything they need to grow

Question 5

OBJECTIVE: What physical requirements are needed for bacterial growth?

Answer 5

Correct temp - for pathogens usually = 37°C
Correct pH - most pathogens prefer our 7.4, but can withstand 6.0-8.0
Osmotic conditions - can withstand hypoosmotic conditions w/o bursting better than euk. cells b/c of cell wall

Question 6

OBJECTIVE: Which physical requirements are needed for inhibition of bacterial growth?

Answer 6

Extremes in pH (directly inhibits growth AND increases heat susceptibility) and temperatures; increases in osmolarity

Question 7

OBJECTIVE: How can you measure bacterial growth by colony formation? Describe method and calculation.

Answer 7

a.k.a. Viable (Plate) Counts b/c only measures viable cells

Use serial dilutions plated on agar:

• take 0.1mL of sample and inoculate 9.9mL or buffer (100 fold dilution or 10^x-2); then repeat process w/ inoculated buffer
• *want to have 30-300 colonies/plate

-calulation: (# colonies/# mL’s plated)/[dilution factor]

• given in units of CFUs/mL
• *CFU = colony-forming units

Question 8

OBJECTIVE: What are other methods to measure bacterial growth?

Answer 8

1. optically-measure turbidity by analyzing of light scattered using a spectrophotometer (limit of detection is ~1million bact/mL)
2. measure amt of metab products (e.g. CO2, ATP)
3. direct count under a microscope (though this gives a count of both alive and dead cells)

Question 9

OBJECTIVE: What are the phases of the bacterial growth curve? How does the growth rate relate to dz?

Answer 9

1. Lag - incr metab activity but no growth/division yet
2. Log - a.k.a. exponential growth
3. stationary - point at which cell death rate (due to decr nutrients, oxygen and/or growth factors; build-up of toxic metabolic substances; or, pH ∆) equals cell growth rate
4. death - rate of cell death > growth; bacteria often ∆ shape (more difficult to ID)

Question 10

generation time

Answer 10

time required for the microbial population to double; ranges from 20 mins (e.g. E. coli) to 20 hours (e.g. M. tb)

Question 11

OBJECTIVE: What is the source and role of phosphorous?

Answer 11

role: membrane phospholipids, DNA (and ATP? not in notes)

Question 12

OBJECTIVE: What is the source and role of iron?

Answer 12

role: co-factor for many enzymes, esp those involve w/ metabolism
source: acquired from host by secreting siderophores that bind scantly available host iron. this complex then undergoes receptor-mediated endocytosis

Question 13

How do microbes metabolize nucleic acids?

Answer 13

secrete nucleases to break down RNA and DNA; can use them for their own NA synth or as sources of C, N and P

Question 14

How do microbes acquire aa’s?

Answer 14

Can either uptake aa’s or secrete proteases to breakdown prots (too big by themselves) into aa’s.

–Proteases are a major virulence factor! (per slides)

Question 15

What are phospholipases? What induces their production?

Answer 15

degrade host cell membranes and lung surfactant; up-regulated in low iron and low phosphorous conditions b/c when cells are lysed they release iron

–major virulence factor! (per slides)

Question 16

Give an example of a human pathogen that grows temp lower than 37°C.

Answer 16

Mycobacterium leprae, thus the skin but no internal lesions

Question 17

What is an example of a human pathogen that still grow well with refrigeration? What population is especially susceptible?

Answer 17

Listeria monocytogenes—>causes food poisoning and is a concern in pregnant women b/c can cross the placenta; from meat and animal products

Question 18

How can the osmotic conditions of foods be altered to make them less susceptible to bacterial colonization?

Answer 18

increase sugar and/or salt concentration (i.e. salted fish, jelly)

Question 19

What growth medium allows culture of ~90% of pathogens?

Answer 19

Blood agar

Question 20

In what type of clinical sample should you find NO bacteria under normal conditions?

Answer 20

CSF

Question 21

OBJECTIVE: Compare aerobic respiration with fermentation

Answer 21

fermentation: creates a net 2 ATP and recycles oxidizes NADH to NAD+ by turning pyruvate into ethanol or lactic acid.

–Only SUBSTRATE-LEVEL PHOSPHORYLATION

aerobic respiration: uses respiratory chain located in inner membrane to accept electrons from NADH creating proton gradient that drives ATP synthase; O2** is usually the terminal electron acceptor

–This is OXIDATIVE PHOSPHORYLATION

**NOTE: some bacteria can respire anaerobically but still do oxid. phos. by using other e- acceptors, e.g. NO3- reduced to N2

Question 22

OBJECTIVE: How do fermentation products impact host-microbe interactions?

Answer 22

Lactic acid lowers pH and allows competition: e.g. beneficial Lactobacillus spp. in vagina prevents other microbes from growing, esp. Candia albicans which causes yeast infections

Butyric acid and butanol-acetone fermentation: clostridia; form butyric acid, acetic acid, H2 and CO2; wicked smelly but commensal intestinal species may use this pathway to protect us from E. Coli O157:H7

Propionic acid: ferments lactate to acetic acid, CO2 and propionic acid; Swiss cheese, acne

Mixed Acid Fermentation by eneteric microbes:
enterobacteriaceae (E. coli and Salmonella) use pyruvic acid to make acety-CoA and HCOOH (formic acid); formic acid—>CO2 and H2 if enzyme present—>gas gangrene
–end up with acetyl-CoA, formic acid, but also some ethanol, acetic acid and succinic acid thus “mixed”

2,3 butanediol and acetoin are made in addition to the mixed acids above by some non-fecal enterics

Ethanol fermentation: yeasts (Saccharomyces cerevisiae) and fungi (Candida albicans); CO2 appears as gas at low pH and non-gaseous carbonate at higher pH found in some tissue infections

Strickland Reaction: ferment using one amino acid to accept electrons from another a.a.; results in putrefactive derivatives of a.a.’s

Question 23

OBJECTIVE: Explain the biochemical basis for obligates aerobes.

Answer 23

Absolutely need O2 b/c lack ferment enzymes; e.g. Mycobacterium tuberculosis which resides in lungs 1°ly

Question 24

OBJECTIVE: Explain the biochemical basis for obligate anaerobes.

Answer 24

Most gain energy only from ferment b/c they do not have ECT.
–e.g. Bacteroides fragilis

Many contains flavins that create H2O2 which can create ROS and they lack the catalase and superoxide dismutase needed to protect themselves from these ROSs.

Question 25

OBJECTIVE: Explain the biochemical basis for facultative anaerobes.

Answer 25

Can live w/ or w/o O2 b/c have fermentation and respiratory chain; grow faster w/ O2

e.g. staphylococcus and E. coli

Question 26

OBJECTIVE: Explain the biochemical basis for aerotolerant anaerobes.

Answer 26

Anaerobes b/c lack ECT but that can grow in O2.
e.g. the lactic acid fermenters: Lactobacillus and Streptococcus; also Clostridium tetani and botulinum = soil bugs (other Clost. spp. live in intestines) which grow best in anaerobic conditions

Question 27

OBJECTIVE: Explain the biochemical basis for microaerophiles.

Answer 27

Tolerate O2 only at conc lower than air (~20%)

Question 28

OBJECTIVE: Where do anaerobes reside in human hosts?

Answer 28

mouth and urinary tract; can also grow in areas bathed in air if other bacteria nearby are using up all O2

(Also: intestines, deep puncture wounds? as w/ clostridia?)

Question 29

OBJECTIVE: Why is bacterial metabolism important for dx?

Answer 29

We can use known differences in metabolism to ID bacteria. For example, most bacteria use glucose, but some can also use lactose.

-We measure this indirectly by checking for ∆s in pH or gas generation.

Question 30

substrate-level phosphorylation

Answer 30

electrons from glucose are used directly to make ATP

Question 31

To what host proteins is iron bound?

Answer 31

EXTRACELLULAR: dietary iron then bound to transferrin and lactoferrin

INTRACELLULAR: ferritin, hemoproteins

Question 32

What are hemolysins? When are they secreted?

Answer 32

bacterial substances that lyse RBCs; secreted in response to low Fe

Question 33

How does H. pylori survive the acidic stomach environment?

Answer 33

secretes urease to make locally high concentrations of NH3 increasing the pH in their microenvironment.

Question 34

Why use agar?

Answer 34

1. you can count colonies after dilutions
2. you can use colony morphology to aid ID
3. you can ID multiple different species from one sample using points 1 & 2 above

Question 35

How many bacteria per/mL is acceptable in urine?

Answer 35

100,000/mL

Question 36

TNTC means?

Answer 36

Too Numerous To Count