Bacterial Growth and Fermentation

Bacterial Growth

• Bacterial growth occurs primarily by binary fission, where one bacterium splits into two, requiring energy.

Conditions Affecting Bacterial Growth

• Temperature: Most bacteria grow optimally at 37°C, but can vary from 0 to 121°C.
• pH: Most bacteria grow optimally between pH 7-8, but can vary from pH 0 to 11.
• Oxygen:
  • Strict aerobes require oxygen for growth.
  • Facultative aerobes prefer oxygen for growth.
  • Facultative anaerobes prefer no oxygen for growth.
  • Strict anaerobes cannot tolerate oxygen.

Growing Bacteria in the Lab

• Culture media is a substance that supports bacterial growth in the laboratory.
• Media can be:
  • Defined (exact composition known)
  • Complex (extracts from plants or animals; exact composition unknown)
• Additives can be incorporated to enrich or inhibit certain bacteria.

Bacterial Metabolism

• Metabolism is the total of all biochemical reactions occurring inside the cell for survival and reproduction.
• Defined as "from food to energy".
• Bacteria uptake nutrients through:
  • Passive transport (involves nutrient gradient)
  • Active transport (utilizes energy to bring in nutrients)
• Iron uptake is facilitated by siderophores.

Quantifying Bacterial Growth

• Methods for quantifying bacterial growth:
  • Cell counting under a microscope
  • Serial dilution and plating on agar (colony forming units)
  • qPCR (relative or absolute quantification)
• Optical density measurement at 600 nm wavelength is used to interrogate growth dynamics in the lab.

Gut Microbiota

• The stomach has few viable bacteria due to its highly acidic environment
• The small intestine has few viable bacteria due to the presence of acid and bile juices
• The large intestine has a huge diversity of microbiota, with 10^12 cells per gram of stool
• Symbiotic relationships in the gut microbiota:
  • Produce biotin and vitamin K
  • Prevent pathogens from colonizing
  • Aid in the absorption of nutrients

Oral Microbiota

• The mouth is colonized soon after birth
• After tooth eruption, there is an increase in anaerobes such as Porphymonas, Prevotella, and Fusobacterium
• Symbiotic relationship between oral microbiota and the host:
  • Alters taste perception
  • Differs between "super-tasters" and non-tasters
  • Varied tongue microbiota

Changes to the Oral Microbiota

• Nitrate-responsive oral microbiome modulates nitric oxide homeostasis and blood pressure in humans

Homeostasis and Dysbiosis

• Example of dysbiosis: Obesity
• Germ-free mice vs. conventional mice:
  • Same diet, but germ-free mice could eat more and gain less weight
  • Microbiota affected caloric absorption
• Dental conditions caused by microbiome dysbiosis:
  • Dental caries
  • Periodontal disease
  • Oral cancer

Microbe-Host Interaction

• Humans are holobionts, comprising the host and microbiome
• Microbes can either be beneficial or cause harm
• The microbiome across the human body is diverse
• The microbiome can be in flux

Host-Microbe Interactions

• Definitions:
  • Infection/colonization: acquisition of micro-organisms by the host
  • Commensalism: a state of infection that results in no damage to the host
  • Symbiosis/mutualism: a state of infection where both the host and microbe benefit

Humans as Holobionts

• A holobiont consists of the host and microbiome
• The human microbiome includes all microbes that share our body space
• Humans carry 10^14 microbial cells and 10^13 human cells
• Humans need their microbiome for:
  • Beneficial tasks
  • Protection from foreign substances and pathogens

Microbiome - Commensals

• 'Normal' microbes present on the skin and in the oral, respiratory, and gastrointestinal tracts, but not in internal tissues
• Protect the host against pathogens
• Provide nutrients for the host
• Acquired in the first year of life

Microbiome Composition

• Huge diversity in composition, including:
  • Bacteria
  • Fungi
  • Viruses
• Some members are culturable, but most cannot be cultured
• Culture-independent methods are used to study the microbiome

16S Ribosomal RNA Sequencing

• 16S rRNA is a subunit of the ribosome
• Slow rate of evolution
• Highly conserved between bacteria and archaea
• Hypervariable regions for studying diversity

Shotgun Metagenomics

• Sequencing of every nucleic acid within a given sample, including:
  • DNA and/or RNA
  • Bacteria
  • Viruses
  • Fungi
  • Even host
• Allows for extended characterization of the "community", including:
  • Subtyping
  • AMR genes
  • Virulence genes

Metagenomics Insight into Population Dynamics

• Metagenomics can be used to study population dynamics

Host Microbiome Varies with Body Site

• Skin microbiota:
  • Environment: slightly acidic, high salt, low water content, and inhibitory substances
  • Bacteria on superficial skin (dead cells) and sweat glands
  • Bacteria partially degrade skin oil to volatile fatty acids, producing body odor
• Respiratory microbiota

Infection Process

• Adherence and invasion are the first steps in the infective process
• Entry sites for pathogens include skin, oral, gut, respiratory tract, and eyes
• Adherence can be non-specific or specific, with specific adhesins being pili, fimbriae, and capsules

Invasion Strategies

• Pathogens can actively or passively penetrate mucous membranes or epithelium
• Active invasion and spread involve enzyme production and microbial toxins
• Inhibition of phagocytosis is a key strategy for pathogens
• Bacteremia occurs when pathogens enter the blood, while septicemia is an infection disease in the blood

Surviving Host Defenses

• Pathogens must overcome innate and adaptive immune systems
• Protection strategies include hiding (within capsules, biofilms, or phagocytes), camouflage (coating with host macromolecules), and immune evasion (antigenic variation)
• Examples of hiding include Streptococcus pneumoniae capsules and intracellular pathogens like Mycobacterium tuberculosis
• Camouflage strategies include Streptococci producing streptokinase to coat bacteria with plasmin

Acute and Chronic Infections

• Acute infections are characterized by sudden onset, rapid progression, and severe systemic symptoms
• Chronic infections involve the continued presence of infectious agents following primary infection and may include chronic or recurrent disease

Virulence Factors

• Virulence is the magnitude of harm caused by a pathogen
• Virulence factors increase the pathogen's virulence, including rapid replication, integration of pathogen genome into host, and use of toxins
• Toxins include exotoxins (Gram +ve, heat sensitive) and endotoxins (LPS on Gram -ve, heat stable, causing fever and shock)

Pathogenicity

• Pathogens can cause disease through simple 'hit-and-run' infections or long-term 'co-existence and subversion'
• Strict pathogens always cause disease when present, while opportunistic pathogens only cause disease under certain conditions
• Examples of strict pathogens include Mycobacterium tuberculosis, while opportunistic pathogens include Candida albicans

Opportunistic Infections

• Opportunistic infections occur when the immune system is weakened, such as through frequent antibiotic usage
• Examples of opportunistic infections include oral thrush (Candida albicans) and bacterial endocarditis (Streptococcus viridans)
• Opportunistic infections can occur when a pathogen enters a new niche, such as when Streptococcus viridans infects damaged heart valves

Rate of Infection

• The rate of infection is influenced by virulence and host defenses
• ID50 is the number of micro-organisms required to cause disease in 50% of inoculated hosts

Dental Plaque/Biofilm Formation

• Mature plaque is calcified dental plaque with calcium phosphate mineral salts forming between and within viable microbes
• People with elevated salivary calcium levels form calculus quicker
• A biofilm layer covers the calculus
• Inhibition of calculus formation can be achieved using pyrophosphates, zinc salts, and polyphosphonates

Dental Calculus

• Calcified dental plaque
• Calcium phosphate mineral salts form between and within viable microbes
• People with elevated salivary calcium levels form calculus quicker
• Biofilm layer covers the calculus

Mucosal Microbiota

• Epithelium includes gingival, buccal, and palatal regions
• Desquamation occurs in mucosal microbiota
• Lower volume of microbiota present on mucosa compared to teeth
• Buccal region has P. gingivalis
• Tongue contains papillae, providing protected sites for bacterial colonization
• Tongue has S. mitis, S. salivarius, and medically compromised Candida albicans

Oral Microbiome Development

• At 6 months, tooth emergence and establishment of solid foods provide a non-shedding tooth surface for biofilm maturation and increased diversity of nutrients for the microbial community
• This leads to an increase in the diversity of the oral microbiome, causing it to become more adult-like
• Some of the increasingly abundant bacterial taxa emerging during this period include Fusobacterium, Lactobacillus, Neissera, Gemella, and Haemophillus

Puberty and the Oral Microbiome

• The later childhood oral microbiome is characterized by the further enrichment and increased abundance of bacterial taxa, including periodontal disease pathogens like Porphyromonas gingivalis
• Hormones during puberty provide a nutrient source for gram-negative anaerobes like P. gingivalis

Phagocytosis

• Phagocytosis is the capture and digestion of foreign particles
• Chemokines attract macrophages and neutrophils to infected tissues
• Opsonins attach to microbes to increase phagocyte adhesion (opsonization)

Oxygen Independent and Dependent Pathways

• Oxygen-independent pathway involves lysosomal enzymes (e.g. collagenase, gelatinase, phospholipases or serine proteases)
• Oxygen-dependent pathway involves oxidative burst, producing reactive oxygen and nitrogen species (RONS)

White Blood Cells (WBCs)

• Neutrophilic PMN (most common WBC): kills bacteria, makes pus
• Monocytic PMN (second most common WBC): kills bacteria, eats debris, attacks intracellular pathogens, becomes macrophages
• Lymphocytes (many types: T, B, NK): specific immunity, immunity against viruses, tumor protection
• Eosinophils (rarest variety of WBC): becomes mast cells, contributes to allergy

Chemotaxis and Opsonisation

• Chemotaxis: directional movement of neutrophils towards bacterial products (e.g. fMLP, peptidoglycan) detected by surface receptors
• Opsonisation: microorganisms must be coated before being bound and engulfed by phagocytic cells, using complement system or other coating agents

Innate Responses

• Cells involved: antigen-presenting cells (APCs), phagocytic cells (e.g. neutrophils, macrophages)
• Pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs)
• Effects of activating TLRs: cytokine production, chemokine production, activation of bacterial killing mechanisms, and activation of dendritic cells

MHC and Antigen Recognition

• MHC class I displays self-antigens, while MHC class II displays foreign antigens
• All nucleated cells display MHC class I, while immune cells display both MHC class I and II
• Each MHC displays one antigen at a time
• High expression levels of peptide-MHC complexes increase the propensity to activate T-cells

Characteristics of MHC

• MHC class I: 6-16 amino acids
• MHC class II: up to 30 amino acid residues

T-Cell Activation

• MHC-TCR engagement requires co-stimulation: T-cell coreceptors CD28 and CD80/B7.1 and CD86/B7.2 on the APC
• IL-2 is immediately released by T cells and promotes proliferation
• 3 signals for T-cell activation: TCR engages antigen on MHC, CD4 or CD8 binds MHC, and CD28 binds CD80 or CD86
• Further engagement with ICOS, 4-1BB, and OX40 strengthens activation
• 'Brakes' are applied through CD152, which limits expansion

Cytokines and T-Cell Identity

• Exposure to IL-12 from APC → Th1
• Exposure to IL-4 from APC → Th2
• Exposure to IL-6, IL-23 from APC → Th17

Adaptive (Specific) Immunity

• Specificity: every clone of T and B lymphocytes recognizes a different antigen
• Acquired immunity: has a memory, provides improved protection on next exposure, and is the basis for immunity against re-infection and immunization

Characteristics of Antigen-Presenting Cells

• Dendritic cells, macrophages, B cells, and Langerhans cells are professional APCs
• APCs express MHC class II and are highly effective at presenting antigens

Immune Responses

• Innate immune response: reduces pathogen load, immediate, and part of homeostasis
• Adaptive immune response: has a memory, provides improved protection on next exposure, and is the basis for immunity against re-infection and immunization

Antigen Presentation to T-Cells

• Antigen presentation on MHC is a fundamental link between innate and adaptive immunity
• Memory: accelerated and larger response, with fewer 'checkpoints' required for reactivation

Antigen Recognition and B-Cell Activation

• T-dependent antigens: require T-cell 'help', use costimulatory molecule CD40, and need cytokines
• T-independent antigens: do not require T-cell help
• BCRs recognize antigens in their native unprocessed forms (does not require peptide bound to MHC molecules)
• Each B-cell is decorated with IgM with a unique specificity, with 10 billion combinations
• Affinity maturation: decrease in the dissociation constant (Kd) following repeated stimulation, involving class switching from IgM to IgG

Fluid Balance and Osmosis

• Body fluid balance and distribution are crucial, and the movement of water in the body needs to be controlled to prevent swelling or dehydration.
• Biological membranes act as a barrier for water movement, controlling the movement of substances into and out of cells, and regulating the composition within individual cells.

Biological Membranes

• Functions of biological membranes:
  • Control the movement of substances into and out of cells
  • Regulate the composition within individual cells
  • Control the flow of information between cells (recognizing or sending signals)
  • Capture and release energy (e.g., mitochondria)
  • Cell adhesion
  • Synthesize steroids, etc.
• Structure and function of plasma membrane:
  • Bi-layer of phospholipids (phosphate group and 2 chains of fatty acids)
  • Amphipathic (head is hydrophilic, tail is hydrophobic)
  • Selective permeability (some molecules are allowed in, while others are kept out)
  • Permeable to lipid-soluble substances (e.g., O2)
  • Impermeable to charged molecules (e.g., ions)

Body Fluid Compartments

• Intracellular fluid: ~25 L
• Interstitial fluid: ~12 L
• Plasma: ~3 L
• Extracellular fluid (ECF): plasma + interstitial fluid

Osmosis and Water Movement

• Osmosis: water moves through a semi-permeable membrane to equalize the concentration of a solution
• Osmotic pressure (OP): created by a concentration difference of dissolved substances between two sides of a bio-membrane
• Hydrostatic pressure: movement of water stops
• Equilibrium is reached once sufficient water has moved to equalize the solute concentration on both sides

Membrane Transport

• Passive movement: follows the concentration gradient, does not require energy
  • Driving forces: concentration gradient, electrical potential
• Active transport: against the concentration gradient, requires energy
  • Examples: endocytosis, exocytosis
• Diffusion: passive movement across membranes, forming equilibrium
• Facilitated diffusion: diffusion for substances that cannot cross lipid bi-layer due to size/charge/polarity, uses carrier proteins

Membrane Proteins

• Transport proteins: channels, pumps, and carriers
• Enzymes
• Intercellular junctions
• Cell-cell recognition
• Receptors
• Adhesion to extracellular matrix

Membrane Transport Proteins

• Channels: allow passive movement of solutes through the membrane down their concentration gradient
  • Examples: ion channels, aquaporins
  • Gated: open or closed in response to local changes (e.g., ligand-gated, voltage-gated)
• Pumps: active transport, against the concentration gradient, catalyze the hydrolysis of ATP to ADP
  • Examples: Na+/K+-ATPase, important for controlling movement of water and electrolyte balances
• Carriers/Transporters: facilitate diffusion or active transport, bind specific molecules and transfer them across the membrane
  • Examples: uniporter, symporter, antiporter

Cellular Transport

• Exocytosis: vesicles containing material for export, waste removal, secrete hormones
• Endocytosis: take in material, the reverse of exocytosis, white blood cell engulfing bacteria/viruses

Water Imbalance and Treatment

• Oedema: accumulation of excess water in the tissues, water leakage from capillaries
• Intravenous fluid therapy: maintain body fluid and electrolytes balance, various types of fluid for different purposes

Osmosis

• Isotonicity: concentration of dissolved substances is the same in the solution as in the cell, same OP for both sides
  • Isotonic solution of animal cells: 0.9% w/v NaCl solution (saline)
• Hypotonicity: outside solution has lower OP (less solute) than cell interior, water moves into cell
  • Example: 0.45% saline
• Hypertonicity: outside solution has higher OP (more solute) than cell interior, water moves out of cell
  • Example: 3% saline

Function of Proteins

• Enzymes speed up reactions in the body
• Hormones direct specific activities, such as regulating blood glucose levels (insulin)
• Antibodies and complement system are involved in the immune system to defend the body from antigens (foreign invaders)
• Structural and mechanical support proteins provide strength and flexibility to body tissues
• Carrier proteins shuttle substances such as oxygen, nutrients (e.g., iron), and waste products through the blood and into and out of cells
• Proteins help maintain fluid balance and acid-base balance in the body
• Proteins provide energy when necessary

Specialized Roles of Amino Acids

• Glycine, GABA, and dopamine are chemical messengers (neurotransmitters)
• Histamine is a potent local mediator of allergic reactions
• Thyroxine is a thyroid hormone
• Citrulline, ornithine, and homocysteine are intermediates in various metabolic processes

Specialized Roles of Non-Standard Amino Acids

• Azaserine is an antimicrobial, antifungal, antineoplastic, and immunosuppressive agent
• β-Cyanoalanine is involved in the removal of cyanide from nature and is used in treating acute childhood leukemia

Central Dogma - Flow of Genetic Information

• The sequence of mRNA is translated into a sequence of amino acids
• mRNA reads the sequence of codons (AUG, AAG, CCG, etc.)
• tRNA reads the sequence of anti-codons and brings amino acids to the ribosome
• Ribosomes are composed of rRNA and ribosomal proteins and have two subunits (large and small)

Components of Translation

• mRNA carries the genetic information
• Amino acids are the building blocks of proteins
• tRNA is the adapter molecule that brings amino acids to the ribosome
• Ribosomes are the action stations where protein synthesis occurs
• Enzymes and energy are necessary for translation

Synthesis of Proteins from RNA

• Initiation: starts with the start codon AUG (methionine)
• Elongation: addition of amino acids by peptide bonds
• Termination: ends with the stop codon (UAA, UAG, or UGA)