MMI 133 Session 4: Bacterial Toxins, Metabolism, Growth, and Culture

Bacterial Metabolism and Growth: MMI 133 Session 4

Toxin Review

• Endotoxin:
  • Example: Lipid A in Lipopolysaccharide (LPS).
  • Part of the cell structure, anchored in the outer membrane of only Gram-negative bacteria.
  • Is a lipid molecule (fat).
  • Released from the cell when the cell dies and disintegrates.
• Exotoxin:
  • Examples: Tetanus toxin, botulinum toxin.
  • Produced by the bacterial cell (ribosomes synthesize it); not a part of the cell structure.
  • Released from the cell in many ways and damages other cells.
  • Is a protein.
  • Three functional types:
    • Enterotoxin: Affects the gastrointestinal tract.
    • Neurotoxin: Affects the nervous system.
    • Cytotoxin: Damages host cells in general.

Session Objectives

• Describe how bacterial metabolism affects the rate of growth.
• Explain the effect of discussed growth requirements of bacteria (i.e., nutrient, gaseous, pH, temperature).
• Identify Listeria monocytogenes as a problem for foodborne infections and describe its characteristics and pathogenesis.
• Define the kinetics and terminology used for bacterial growth.
• Define and describe differential versus selective media for bacterial growth.

1. Metabolism

Definitions

• Metabolism: The sum total of all chemical reactions within an organism.
  • Catabolic reactions: Release energy, break down organic compounds.
  • Anabolic reactions: Require energy, store energy, build complex organic molecules.
• Enzymes: Biological catalysts.
• Energy Production: Processes include aerobic respiration, anaerobic respiration, and fermentation.

Catabolism

• Chemical reactions that release energy.
• Involve the breakdown of organic compounds.
• Example: Glucose breakdown to carbon dioxide and water.
  • $C<em>6H</em>{12}O<em>6 \rightarrow CO</em>2 + H_2O$
  • This process releases Adenosine Triphosphate (ATP), which is energy.

Anabolism

• Chemical reactions that require energy.
• Involve the building of complex organic molecules from simpler compounds.
• Stores ATP.
• Example: Formation of polysaccharides from simple sugars, like glycogen from glucose.

Reaction Rates & Enzymes

• Temperature Dependence: Reaction rates are generally temperature-dependent; a higher temperature usually leads to a higher reaction rate.
  • Drawback: High temperatures can kill cells by causing denaturation of proteins.
  • Solution: Enzymes.
• Enzymes:
  • Large protein molecules that act as biological catalysts.
  • Increase chemical reaction rates by $100,000,000 \times$ (100 million times) or more.
  • Are substrate-specific, meaning they work on particular molecules.
  • Typically have the suffix "-ase" (e.g., polymerase, amylase).
  • May require cofactors, such as metal ions (e.g., zinc, magnesium).
  • Are recyclable and remain unchanged during the reaction.
• Factors Affecting Enzyme Function (Cellular Controls):
  • Temperature
  • pH
  • Saturation (of substrate)
  • Salt concentration
  • Inhibitors (e.g., mercury, silver)

Energy Production

• Most microbes use carbohydrates as the primary source for energy production.
• Glucose is the most common nutrient source.
• Two main processes for glucose utilization:
  1. Cellular Respiration:
     • Aerobic Respiration: Requires oxygen.
     • Anaerobic Respiration: Does not require oxygen.
  2. Fermentation:
• Both cellular respiration and fermentation use glycolysis (e.g., Embden-Meyerhof pathway), where the final product is pyruvate.
• Bottom Line: Cells need to produce ATP (energy) to run all cellular processes.

Respiration: Aerobic and Anaerobic

• Aerobic Respiration:
  • Produces significantly more ATP (approx. $38$ ATP molecules from one glucose molecule) than anaerobic processes.
  • Bacteria performing aerobic respiration generally grow faster.
  • Bacteria that need oxygen to grow are called obligate aerobes.
• Anaerobic Respiration:
  • Produces less ATP (typically $2-4$ ATP molecules) compared to aerobic respiration.
  • Bacteria performing anaerobic respiration usually grow slower.
  • Bacteria that can only grow without oxygen are called obligate anaerobes.

Other Energy Sources (Besides Glucose)

• Lipids (fats):
  • Broken down by lipases into fatty acids and glycerol.
  • These products then enter the Krebs cycle for energy production.
• Proteins:
  • Broken down by proteases into amino acids.
  • Amino acids can be modified and enter the Krebs cycle for energy production.

2. Factors Affecting Bacterial Growth

A. Physical Factors

1. Temperature: Affects enzyme activity and membrane fluidity.
2. pH: Impacts protein structure and enzyme function.
3. Osmotic Pressure: Influences water movement across cell membranes.
4. Gas (Oxygen): Determines metabolic pathways and growth capabilities.

B. Chemical Factors

• Nutrients: Sources of carbon ($C$), nitrogen ($N$), oxygen ($O$), sulfur ($S$), phosphorus ($P$), and trace elements.
• Organic Growth Factors: Specific organic compounds required for growth, such as yeast extract in artificial media or specific amino acids (e.g., cysteine).

C. Environmental Factors

• Intracellular Growth: Some bacteria grow inside host cells.

1. Temperature

• Bacteria are classified based on their optimal growth temperature ranges:
  • Psychrophiles: Grow best in cold temperatures (typically $-5\text{°C}$ to $+15\text{°C}$).
  • Psychrotrophs: Can grow in refrigeration temperatures but have optimal growth at moderate temperatures (e.g., $20-30\text{°C}$). Listeria monocytogenes is an example.
  • Mesophiles: Grow best at moderate temperatures (typically $25-45\text{°C}$). Most human pathogens fall into this group.
  • Thermophiles: Grow best at high temperatures (typically $45-70\text{°C}$).
  • Hyperthermophiles: Grow best at extremely high temperatures (typically $70-110\text{°C}$).

Listeria monocytogenes

• Characteristics:
  • Gram-positive bacillus (rod-shaped bacterium).
  • Non-spore forming.
  • Psychrotroph (can grow at refrigerator temperatures).
  • Facultatively anaerobic (can grow with or without oxygen).
  • Motile (can move).
  • Grows well on complex media, such as Blood Agar Plates (BAP).
  • Can grow over a wide temperature range ($3-42\text{°C}$).
  • Can grow over a wide pH range ($\le 5.5 - 9.5$).
  • Can grow in high concentrations of NaCl (up to $10\%$).
• Normal Habitat:
  • Widely distributed in nature; soil is likely its natural reservoir.
  • Found in animal intestines through contaminated feed.
  • Is hardy, surviving in low temperatures, high acidity, and high salt concentrations.
• Clinical Presentation of Listeriosis:
  • Causes disease in both humans and animals.
  • In Animals:
    • Central Nervous System (CNS) infections (e.g., in goats, cattle, causing "circling disease").
    • Septic abortion.
  • In Humans:
    • Foodborne disease.
    • CNS infections/Death (e.g., meningitis, encephalitis).
    • Abortion in pregnant women.
• Virulence and Pathogenesis (How it causes disease):
  • Ingestion of contaminated food.
  • Invasion of gastric epithelial cells: The bacteria rearrange host cell actin to propel themselves from one cell to another like a rocket. The gene ActA codes for this invasin protein.
  • Intracellular growth in macrophages: Destroys the phagolysosome (membrane-bound compartment where bacteria are taken into the cell) using listeriolysin.
  • Produces two other hemolysins (phospholipases) that destroy cell membranes.
  • Exhibits both intracellular and extracellular growth.
• Clinical Infection:
  • Can cause meningitis, encephalitis, and septicemia.
  • Incubation period can be as long as $2$ months.
  • The elderly and immunocompromised individuals are at greater risk.
  • Mortality rate for CNS infections is high ($20-50\%$).
  • Difficult to diagnose this type of meningitis.
  • Pregnant women: A special case; can experience an influenza-like bacteremic illness that may result in abortion or stillbirth.
• Transmission:
  • Fecal-oral route.
  • Foods implicated as vehicles of infection: Coleslaw, soft unpasteurized cheeses, turkey wieners, cold cuts, mushrooms, prepackaged salads, milk, etc.
• Epidemiology:
  • Hard to track due to the long incubation period and underreporting.
  • Estimated to be responsible for approximately $1700$ cases of invasive disease in the US each year (a significant number).
  • Fatal in about $1/3$ of cases, even with appropriate antibiotic treatment.
  • Notable incident: Maple Leaf in $2008$ (Canada), with $57$ severe infections and $23$ deaths.
  • Food recalls due to Listeria contamination are not uncommon.
  • Example: $2024$ recall of plant-based milks (almond or oat milk) due to equipment contamination, resulting in over $20$ deaths.
• Prevention/Treatment:
  • No vaccine is available.
  • Antibiotics are used for treatment, but timely diagnosis is crucial for effectiveness.

2. pH

• The usual activity zone for human pathogens is between pH $5-8$ (neutrophiles).
• Extreme high or low pH can denature proteins, affecting their structure and function, which negatively impacts bacterial growth and survival.

3. Osmotic Pressure

• Isotonic Solution: The concentration of particles per milliliter in the solution is equal to that inside the cell (e.g., $0.85\%$ NaCl is isotonic to many cells).
• Hypotonic Solution: Fewer particles per milliliter in the solution than inside the cell.
  • Water enters the cell to balance the osmolality (concentration of particles).
  • Causes the cell to expand and potentially explode (lysis).
• Hypertonic Solution: More particles per milliliter in the solution than inside the cell.
  • Water leaves the cell, causing the cell to dehydrate and the cell membrane to collapse.
  • This process is called plasmolysis and inhibits growth.
• Practical Use of Osmotic Pressure Knowledge: This understanding is vital for the food industry (e.g., using salt or sugar to preserve food by inhibiting microbial growth) and medicine (e.g., intravenous fluid formulations).
• Bacterial Adaptations to Hypertonic Environments:
  • Obligate Halophiles: Require high salt concentrations for growth (e.g., Dead Sea halophiles need $30\%$ salt). These are not typically human pathogens.
  • Facultative Halophiles: Can tolerate high salt concentrations (e.g., $2\% - 15\%$), but do not require them. Examples include some Enterococcus species, Vibrio species (enteric bacteria), and some Staphylococci (often found on skin).

4. Gaseous Requirements for Growth (Oxygen)

• Oxygen requirement is a key criterion for bacterial classification:
  • Strict (Obligate) Aerobe: Requires oxygen for growth.
  • Facultative Anaerobe: Can grow with or without oxygen, but grows better in the presence of oxygen.
  • Strict (Obligate) Anaerobe: Cannot tolerate oxygen and grows only in its absence.
  • Aerotolerant Anaerobe: Does not use oxygen but can tolerate its presence.
  • Microaerophile: Requires oxygen but only at concentrations lower than atmospheric levels ($\sim 2-10\%$ oxygen).
• Anaerobic Conditions in the Lab: Tools like anaerobic jars and anaerobic boxes are used to create oxygen-free environments for culturing obligate anaerobes.

B. Chemical Factors

• Sources of Essential Elements: Bacteria require sources of carbon ($C$), nitrogen ($N$), oxygen ($O$), sulfur ($S$), and phosphorus ($P$), along with various trace elements.
• Organic Growth Factors: Some bacteria require specific organic compounds that they cannot synthesize themselves.
  • Examples: Yeast extract in artificial media, specific amino acids like cysteine.

C. Environmental Factors (Intracellular vs. Extracellular Microorganisms)

• Intracellular Organisms:
  • Grow and replicate inside host cells.
  • Evasion of Defense Mechanisms: Can evade host immune responses (e.g., white blood cells).
  • Antibiotic Treatment: Require antibiotics that can penetrate host cell membranes.
  • Cultivation: Some cannot be grown on artificial media and must be cultured in living cell cultures (e.g., Chlamydia).
  • Host Dependence: May be dependent on host cells for energy (e.g., Chlamydia lacks enzymes for ATP production).
  • Example: Chlamydia is an obligate intracellular pathogen.

3. Bacterial Growth

I. Bacterial Reproduction

• Binary Fission: The primary method of bacterial reproduction, where one cell divides into two identical daughter cells.

II. Generation Time (Doubling Time)

• Definition: The time it takes for one bacterium to divide into two.
• Factors: Dependent on the culture medium and growth conditions.
• ATP Production & Growth Rate: Aerobic bacteria usually grow much faster than anaerobic bacteria due to higher ATP production.
• Examples (not to memorize):
  • Escherichia coli: $15-20$ minutes
  • Staphylococcus aureus: $27-30$ minutes
  • Mycobacterium tuberculosis: $16-24$ hours
  • Mycobacterium leprae: $2$ weeks
  • Treponema pallidum: $33$ hours
• Logarithmic Expression of Bacterial Number: Bacterial populations are expressed logarithmically.
  • Example: If E. coli has a generation time of $\sim 20$ minutes, one cell would ideally grow to $\sim 1 \times 10^{23}$ bacterial cells in $24$ hours.

III. Bacterial Growth Kinetics (Growth Curve)

• When bacteria are grown in a closed system (e.g., a batch culture), their population growth typically follows four distinct phases:
  • Lag Phase: Little or no cell division; bacteria adapt to the new environment, synthesize necessary enzymes, and prepare for growth.
  • Exponential (Log) Phase: Cells divide at a constant, maximal rate. The population increases logarithmically.
  • Stationary Phase: The growth rate slows down, and the number of new cells produced equals the number of dying cells. This is due to nutrient depletion, accumulation of waste products, and limited space.
  • Death (or Decline) Phase: The number of dying cells exceeds the number of new cells. The population declines exponentially as nutrients are exhausted and waste products become toxic.

IV. Biofilms

• Definition: Bacteria often live in communities called biofilms.
• Prevalence: More than $85\%$ of human infections involve biofilms.
• Dynamic Nature: Bacteria in biofilms are not permanently fixed; they can detach and disseminate, starting new foci of infection.
• Structure: A slimy matrix composed of polysaccharides, proteins, DNA, and bacterial cells.
• Immune Evasion: White Blood Cells (WBCs) often cannot penetrate the biofilm matrix.
• Antibiotic Resistance: Antibiotics are often ineffective against bacteria living within biofilms due to limited penetration and altered metabolic states of the bacteria.

V. Bacterial Culture: How to Grow Bacteria in the Lab

• Nutrient Medium: Requires nutrient material, which can be solid, semi-solid, or liquid.
• Sterility: The nutrient medium must be sterile initially to prevent contamination.
• Agar: A complex polysaccharide from algae used as a solidifying agent for solid culture matrices; it resembles gelatin.
• Nutrient Enrichment: Animal blood (e.g., sheep, horse blood) is often added to media to provide additional nutrients.
• Colonies: Bacteria grow in