Module 3 Prerecorded Lecture Notes

Shapes and morphology of bacteria

Cocci - round/spherical

Bacilli - rod shaped

Spirochaetes - spiral shped

Other shapes:

• filamentous

• curved

• square

• pleomorphic

Different arrangements of bacteria

Clusters (staphylo-)

Chains (strepto-)

Pairs (diplo-)

Tetrads (micro-)

Nomenclature of bacteria

Genus name + Species name

Genus = group with similar overall characteristics

Species = subgroup with same biochemical characteristics

Ex,, Staphylococcus Aureus

• Staphylococcus = GENUS

• Aureus = SPECIES

Process of gram staining

1. Crystal Violet is used once bacteria is fixated and stains all bacteria purple

2. Crystal violet is removed as much as possible using decolourizing agent (usually alcohol iodine)

3. Bacteria then stained with Safranin (counterstain) which turns gram (-) bacteria pink and keep gram (+) bacteria purple

Why do Gram (+) stay purple and gram (-) go pink

Gram-positive bacteria retain the crystal violet stain due to their thick peptidoglycan layer in the cell wall

Gram-negative bacteria lose the stain (bc of no thick cell wall) and take up the safranin counterstain, appearing pink.

What are the three things needed to grow bacteria in a lab

1. Equipment

2. Media

3. Colony isolation

What are the “equipment” that are used to grow specific bacteria

• Agar plate

• Deep agar tube - for anaerobic bacteria

• Broth - for initial bacterial growth

• Agar slant

Different Media used when growing bacteria

1. Undefined media

2. Chemically defined media

3. Functional media

4. Physical nature media

Undefined media

used to grow ALL species of bacteria in a host sample (not selective)

Chemically defined media

media with know chemical components that will select for a specific bacteria

Functional media

once targeted bacteria is identified, nutrients and other functional measures will be added to ensure selected bacteria growth and death to other bacteria

Physical nature media

media that can vary in its physical properties to selectively grow a bacteria

Isolated Colonies

extraction of specific colony of bacteria which enables future research and test

Aerobes

bacteria that use oxygen

Anearobes

bacteria that die in the presence of oxygen and thrive in CO2 environment

*Anaerobes are very prevalent in the oral cavity

Aerotolerant

thrive in CO2 environment, but are not affected by O2 environment

Microaerophiles

only can grow in low concentration O2 environments

Ways of quantifying bacterial growth

1. Cell counting

2. Serial dilution and plating

3. Optical density of culture

4. qPCR

Cell counting

counting cells using a gridded slide to estimate the size of the entire colony

Serial dilution and plating

continue diluting solution until there are few enough bacteria to count - then extrapolate to find the colonies in units/ml

Optical density of culture

use spectrophotometer at 600nm to determine number of cells in a liquid culture by measuring light absorption.

qPCR

indirect measurement that provides the quantity of DNA or RNA in a sample by amplifying it through polymerase chain reaction and fluorescent light

Traditional methods of identifying bacterial species

Identify bacteria based on:

• morphology

• gram stain

• standard biochemical testing

Novel ways of identifying bacteria

PCR: makes primers and detect bacteria by scanning the 16S subunit of ribosomal RNA

MALDI-TOF: “Matrix Assisted Laser Desorption/Ionization Time of Flight” - uses spectrophotometry to measure time of flight of a particular protein

• still need to have an idea for what bacteria you are looking for to do this technique

Features of the Bacterial Growth Curve

1. Lag Phase

2. Exponential/Logarithmic phase

3. Stationary phase

4. Death/Lysis phase

Lag phase

Few cells present, bacteria is looking for nutrients and is adapting to their environment

Exponential/Logarithmic phase

Nutrients are found and bacteria begin to rapidly grow and divide at exponential rate

Stationary phase

Number of cells growing = number of cells dying

• due to nutrient competition and toxic byproducts (metabolic waste toxins) that restrict further growth

Death/Lysis phase

More dying bacterial cells due to a loss of nutrients and increase toxins in environment

Disease-causing Gram (+) bacteria

• Streptococcus Mutans

• S. Sanguinis

• S. Oralis

• S. Mitis

• S. Gordonii

• S. Parasanguinis

• S. Salivarius

• S. Anginosus

Disease-causing Gram (-) bacteria

• fusobacterium

• nucleatum

• porphyromonas gingivalis

• tannerella forsynthia

• aggregatibacter actinomycetemcomitans

• treponema denticola (Spirochaetes)

Streptococci bacteria traits

• Gram (+)

• cocci shape

• major genus of bacteria found in mouth

• some can lyse red blood cells

Streptococci related to poor oral health

• S. gordonii

• S. salivarius

• S. sanguis

Streptococci related to dental caries

• S. pyogens

• S. mutans

Spirochaetes that cause disease

Bacteria that causes Gingivitis and Periodontis

• Treponema denticola and Porphyromonas ginigivalis

  • mainly responsible for chronic gingivitis and periodontitis

• Fusobacterium nucleatum

• Prevotella intermedia

• Selenomonas sputigena

Bacteria that cause juvenile periodontitis

Microaerophile Bacteria:

• actinobacillus actinomycentemcomitans

Biofilm

• matrix-encased community of microbes, held together by polymers and fibrils, that accumulates at tooth enamel surface

  • plaque = example of biofilm

• biofilm allows ease of nutrient transfer between bacteria and protection from host immune response

Stages of microbial colonization of the oral cavity

1. Adhesion

2. Early colonizers

3. Late colonizers

4. mature plaque

Adhesion

Pellicles are salivary glycoproteins that are found on teeth after brushing

Bacterial surfaces contain glucose binding proteins which facilitate the bacteria binding to the tooth surface

What forces must the bacteria overcome to adhere to the tooth

1. Salivary forces (movement of saliva)

2. Shear forces (mouth movements)

Types of bacterial bonding to teeth

Initially,

• Non-specific/low affinity bonding

  • quite weak and easily broken (ionic bonds, hydrophobic bonds, H-bonds, Van der Waals forces)

Later,

• Specific/high affinity bonding

  • bacteria use their own proteins to bind to each other and to the teeth

Early and Late Colonizers

Early colonizer bacteria attach to pellicle, usually strepto bacteria

• Examples

  • S. mitis

  • S. gordonni

  • S. sanguinis

  • S. oralis

Bacteria then aggregate using coadhesion to join biofilm

• Examples

  • propionibacterium

  • haemophilus

  • actinomyces species

Late colonizers will continue to attach to the biofilm

• Examples

  • fusobacterium

  • nucleatu

  • prevotella intermedia

  • haemophilus prarainfluenzae

Mature Plaque

Plaque is now surrounded by matrix and combines with host DNA and polymers to make plaque ‘sticky’

• morphology appears as ‘corn cob’, ‘test tube brushes’, or ‘hedgehogs’

Mature plaque is self sustainable and can break off into separate colonies

Growth of plaque is influenced by prevalence of salivary glycoproteins and diet

Aerobic bacteria (outer layer) → microaerophiles (middle) → anaerobes (inner layer of plaque)

*O2, nutrients, and pH decrease as you move from exterior to interior of the plaque

Beneficial bacteria-bacteria interactions (Veillonella with Streptococci)

Streptcocci produce lactate, which the Veillonella will consume

• will reduce acidity of the mouth

Beneficial bacteria-bacteria interactions (S. gordonii with P. gingivalis and F. nucleatum)

S. gordonii will facilitate redox reactions that make the oral environment more anaerobic → increased growth of anaerobic bacteria like P. gingivalis and F. nucleatum.

Beneficial bacteria-bacteria interactions (F. nucleatum with P. gingivalis and T. forsythia)

Interaction reduces oxygen levels and increases pH → makes it easier for harmful anaerobic bacteria to thrive

Beneficial bacteria-bacteria interactions (P. gingivalis with T. forsythia, T. denticola, and P. intermedia)

Metabolize succinate and incorporate heme groups → increase in survival of pathogenic bacteria

Calculus

Calcium phosphate deposits within the biofilm that hardens and must be removed by a professional

• accentuated by individuals with increased levels of Ca in saliva

Bacteria found on the tongue

• prevotella

• Veillonella

• actinomyces

Bacteria found on the hard palate

• prevotella

• veillonella

• actinomyces

• gemella

Bacteria found in the bacterial mucosa

• prevotella

• veillonella

• actinomyces

• gemella

• streptococcus

Bacteria found in the throat, tonsils, and saliva

• prevotella

• veillonella

• actinomyces

• gemella

• streptococcus

Advantages for bacteria living in a biofilm

1. Antimicrobial resistance - biofilm will block antibiotics from entering the matrix

2. Food sharing - bacteria can exchange nutrients and metabolites with one another

3. Communication - cells can coordinate gene expression and virulence

4. Competence - transfer of DNA between bacteria to tolerate new environments

Disadvantages for bacteria living in a biofilm

1. Slow diffusion - matrix slows metabolism, nutrients are restricted to the deepest parts of the biofilm

2. Concentrating chemicals - chemicals are hard to remove from biofilm therefore bacteriotoxins can build up

3. Bacteriocins - release of proteins that kill other bacteria in the biofilm

Oral ecology

microorganisms and their interactions between each other and with their environment

Specific Plaque Hypothesis (SPH)

Walter J. Loesche (1976) - only specific cariogenic bacteria like S. mutans and lactobacilli are responsible for dental caries

• relies on culture based techniques and microscopy to identify and target specific pathogens

Non-specific Plaque Hypothesis (NSPH)

Walter Loesche (1976)

• initially stated that caries formed from the quantity of plaque accumulation rather than specific bacteria (as argued in SPH)

• abandoned this and revised the NSPH in 1986 to suggest quantity and bacterial virulence cause caries and advocates for mechanical removal to prevent disease

Ecological Plaque Hypothesis (EPH)

Proposed by Marsh (1994)

• integrates elements of SPH and NSPH

• suggests that dysbiosis (imbalance of oral microflora) = disease

• changes in pH, oxygen, and nutrient availability contribute to the disease

• suggests using sugar alternatives to remove risk factors for disease

Keystone Pathogen Hypothesis (KPH)

Hajishengallis et al. (2012)

• proposes that certain pathogens, known as keystone pathogens, can disrupt the host's microbial community balance, leading to dysbiosis and disease, even in low abundance

Polymicrobial Synergy and Dysbiosis (PSD)

• complements KPS

• emphasizes that keystone pathogens interact with other polymicrobes that lead to dysbiosis and contribute to disease progression

Which hypothesis is considered to be the best right now

Ecological Plaque Hypothesis due to consideration of environmental factors

Summary of Plaque Hypotheses

Factors that cause changes in the oral microbiota

• bidirectional dynamic relationship

• Intrinsic host factors (saliva, oral pH, host immune response)

• Extrinsic host factors (lifestyle, diet, oral hygiene, environment)

Caries process (caries ecological hypothesis)

• extensions of EPH to include caries formation and factors; pH, nutrients, oxygen that are included in the caries process

What are the three stages of caries ecological hypothesis

1. Dynamic stability stage

2. Acidogenic stage

3. Aciduric Stage

Dynamic stability stage

• occurs when non-mutans streptococci and actinomyces dominate the biofilm

• acid begins being released and mouth pH decreases

  • generally, reversible cascade can return pH to normal

  • if bacteria left long enough, low pH will not be reversible

Acidogenic stage

increase non-mutans strep. and actinomyces as pH continues to decreases

early demineralization of enamel

Aciduric stage

as pH crashes, mutans bacteria emerge along with non-mutans aciduric bacteria (lactobacilli)

further demineralization of enamel

Proteolytic stage

responsible for caries in dentine and root

• stage exists between acidogenic and aciduric stage

• demineralizes organic matrix (collagen) in dentine and roots

• activates salivary matrix metalloproteinases (MPP) and cathepsins to further demineralize dentine and root

Generalist carious bacteria

Non-mutans streptococci

• can adapt to variety of conditions in the biofilm

• contain their own adhesion proteins for binding to pellicle

• produce polysaccharides (glucans and glycosidases) to reinforce plaque matrix and lower pH

Specialist carious bacteria

• specifically produce water-insoluble glucans to recruit more bacteria

  • contributes to pH falling close to 4

What is the critical pH of the mouth

5.5

Bacterial fermentation of carbohydrates

Bacteria perform glycolysis via the Emben-Meyrhof-Parnas (EMP) pathway

• Glucose → pyruvate (ATP and NADH produced)

• Pyruvate → lactate (S. mutans and lactobacilli produced - decrease pH)

Glucose metabolism produces other acids:

• acetic

• lactic

• formic

• propionic

Types of salivary glands

1. Intrinsic

2. Extrinsic

Intrinsic salivary glands

• numerous and small (500-1000 of them)

• found in mucosa/submucosa of oral cavity, tongue, oropharynx, upper resp. tract

• Function:

  • mucous-secreting

  • saliva

  • lubrication

  • digestion

Extrinsic salivary glands

• three pairs that are larger and located outside the oral cavity

  • Parotid gland - serous secretion

  • Submandibular gland - mixed secretion

  • Sublingual gland - mucous secretion

Main type of intrinsic salivary gland and where it’s found

Von Ebner’s Gland (found on tongue)

Label the extrinsic salivary glands

Sublingual Gland

Submandibular gland

Parotid gland

Describe the parotid gland

• sits lateral to the ramus of the mandible and masseter

• enclosed in parotid capsule

  • parotid capsule receives external carotid, retromandibular vein, facial nerve

• parotid duct leaves gland superficial to the masseter and through the buccinator into the vestibule near the second molar (parotid papilla)

Innervation of the parotid gland

Parasympathetic: glossopharyngeal (CN IX) via the auriculotemporal nerve (a part of CN V3 that runs through the foramen ovale)

Sympathetic: external carotid plexus

Sensory: auriculotemporal nerve and greater auricular nerve (branch of the cervical plexus)

Describe the submandibular gland

• medial to the body of the mandible

• Has superficial and deep parts as it wraps around the posterior border of the mylohyoid muscle

  • extraoral lobe sits below the mylohyoid muscles and the intraoral lobe wraps around the mylohyoid

• duct opens into the sublingual papilla as it travels into the oral cavity

Innervation of the submandibular gland

Parasympathetic: chorda tympani (branch of the facial nerve CN VII)

Sympathetic: external carotid plexus

Sensory: lingual nerve (mandibular branch of trigeminal nerve CN V3)

Describe the sublingual gland

• located between oral mucosa of the floor of mouth and the mylohyoid in the sublingual fossa

• opens directly in the oral cavity proper via 8-10 ducts in the sublingual fold of the alveolar sulcus

Functions of saliva

1. wound healing

2. buffer

3. teeth mineralization

4. food digestion

5. lubrication

6. anti-viral/antibacterial/antifungal

Composition of saliva

• mostly water

• high K+ and HCO3-

• low Na+ and Cl-

• digestive enzymes (salivary amylase, lingual lipase)

• mucin

• lysozyme

• IgA antibodies

What are the components that saliva is categorized into

1. Water and Electrolytes

2. Proteins

3. Small organic molecules

4. Hormones

Saliva components (1a: Water)

• 98-99% of saliva

• makes saliva hypotonic

• aids in lubrication

• cleansing teeth and oral cavity

• taste

• remineralization via dissolved Ca and other minerals

Saliva Components (1b: Electrolytes)

• osmolarity: Na, K, Cl, HCO3

• Buffering of pH between 5.75-7.05 (HCO3 and HPO4)

• remineralization: Ca, F

Salivary Components (2a salivary glycoproteins - i. Mucin)

• tissue coating; branches of oligosaccharides that play a role in forming the pellicle

• lubrication

Salivary Components (2a salivary glycoproteins - i. Proline-rich proteins)

• 40% of protein in saliva

• high affinity for hydroxyapatite

• lines tooth surface and allow bacteria to bind to tooth surface

  • also has negative charge that recruits bacteria

• slows down loss of dissolved Ca and PO4 ions

Salivary Components (2a salivary glycoproteins - i. Alpha-amylase)

• makes up 40-50% of all salivary proteins

  • 80% of amylase is produced in the parotid glands

• encoded by gene ‘Amyl’ on chromosome 1

• is an endoglyco-hydrolase

• digests starch into maltose

• only active at low pH

Salivary Components (2a salivary glycoproteins - i. Lingual Lipase)

• secreted by von ebner’s glands (in the tongue)

• digests fat

  • breaks down medium-long-chain triglycerides

  • digests milk fat in newborns

• highly hydrophobic and enter fat globules

Salivary Components (2b anti-microbial proteins - i. Lactoferrin)

• a transferrin family protein that transfers to the cell

  • found in milk

• binds to free iron in saliva and deprives bacteria of iron need for growth

Salivary Components (2b anti-microbial proteins - i. Lysozyme)

• damages bacteria by degrading peptidoglycan cell wall (effective against gram +)

• part of innate immune sys

• derived from:

  • major/minor salivary glands

  • phagocytic cells

  • gingival crevicular fluid (GCF)

Salivary Components (2b anti-microbial proteins - i. Growth factors)

• epidermal growth factors (EGF)

• transforming growth factors alpha/beta (TGF-a, TGF-b)

• fibroblast growth factor (FGF)

• insulin-like growth factor (IGF-I, IGF-II)

• nerve growth factor (NGF)

Function:

• interact with oral epithelium for;

  • wound healing

  • regulation of epithelial growth

  • epithelial lining homeostasis

Exocrine Glands

glands that release secretions via a duct directly to the surface

Endocrine glands

release secretions (generally hormones) into extracellular space (usually the bloodstream)

Structure of salivary glands

1. Secretory acinar cells

   • serous, mixed, mucous

   • lined by myoepithelial cells which contract to release secretions into collecting ducts

2. Intercalated ducts

   • transitional cells between the excretory ducts and the acinar cells

3. Striated ducts

4. Main secretory ducts

   • empties into excretory duct

5. Main excretory ducts

   • empties into oral cavity

Stages of the formation of saliva

1. Primary secretion

2. Secondary secretion (ductal secretion)

Primary secretion

• Na-K-ATPase and Na-K-Cl symporter is establish due to conc. gradient in acini lumens

• Na + water travels into lumen via conc. gradient

  • moves through leaky tight junctions between acini cells