Lecture #1/2: Bacterial Metabolism & Oral Microbes

Learning Objectives

Compare the structure, physiology, and impact of different classifications of bacteria

Differentiate between aerobic, anaerobic, and facultative metabolism and its possible impact on human health

Explain the biology (including structure, genetics, taxonomy) of major bacterial agents

Provide examples of processes in oral health care that might induce mutations in microbes and humans

Explain the range of therapies available for infectious disease, with the clinical indications for their use and their side effects

What is a microorganism?

Any small organism that can’t be seen w/ naked eye <0.1mm

Some helpful, some cause harm to humans

Why is microbiology important for DHYG students? *EXAM

Microbes bind to hard surfaces and grow in environments with a lot of nutrients

Can bind supra and sub-g, inducing a reaction from the immune system that reacts to the bacteria

Contributes towards caries and periodontal disease, changes oral bacterial ecosystem

What organisms are included under microbes?

Bacteria → Living single celled organisms

Viruses → Non-living particles that need host cell to reproduce, contains genetic viral material

Protozoa → Single cells that have all mechanisms to be independent & alive

Algae → single cell not too relevant to HYG

Fungi → only relevant fungi is candida albica

Where do microbes fit into biological classification system?

Three superkingdoms/domains

1. Archae (Ancient bacteria, archaebacteria)

• Survive and live in extreme environments

2. Prokaryote (BACTERIA)

• Less advanced more ancient and primitive

3. Eukaryote (US)

• More advanced w/ multiple kingdoms

What are 5 kingdoms of eukaryotes?

Protista → Primitive Algae, Protozoa

Fungi → Yeasts, multicellular molds

Plantae → Complex algae & plants

Animalia → Multicellular vertebrate and invertibrates

What is the difference between prokaryotic and eukaryotic cells *EXAM

Prokaryotic

• No nucleus

• No organelles

• Cell wall= peptidoglycan

• Reproduction= binary fission

• 1 circular chromosome

Eukaryotic

• Nucleus

• Organelles

• Cell wall= cellulose/chitin

• Reproduction= mitosis

• Linear chromosome

What are the four unique properties of bacteria?

Size

Metabolically Diverse

Adaptable

Ubiquitous

Explain size as an unique property of bacteria

• Very small, can 2.4×10^11 bacteria /gm of plaque

• Large surface to volume ratio → rapid growth + transport

• Inhabit vv small ecosystems (dentinal tubules)

• Mechanical removal can be difficult

• Many diverse species @ single site

Explain metabolically diverse as an unique property of bacteria

Use full spectrum of nutrients (Carbs, protein, lactate, nylon tires)

Live w or w/out oxygen (aerobic or anaerobic)

Explain adaptable as a unique property of bacteria

• Great opportunity for genetic change

• Short generation time (time it takes to reproduce) → can divide up to every 40 minutes

• Mutation rate 1/106

• Genetic info exchanged rapidly

  • If you give antibiotics, doctors tell you to take them all at once or within a time period because if you don’t, it gives them time to adapt and genetically change so they can become resistant

  • After becoming resistant, the bacteria can also pass on the gene

Explain ubiquitous as a unique property of bacteria

Ubiquitous → present, appearing, found everywhere at the same time

• Present in virtually all environments

  • Hot underwater fissures that are 300 degrees

  • Soil, air

  • Humans → Skin, nose, GI Tract, Genital tract, oral cavity

How are bacteria named?

Using the taxonomy and binomial system

Taxonomy → Study of classifying & naming organisms based on similarities & differences

Binomial system → system used to give organisms their specific names

What is the old classification system for bacteria? *EXAM

1. Staining

• Gram stain (Gram + or -)

• Ziehl Neelsen → stain used to ID acid-fast bacteria like Mycobacteria tuberculosis

• Silver Impregnation staining → Treponema pallidum

2. Shape

• Microscopic

  • Spherical (cocci)

  • Rod (baccili)

• Macroscopic

  • Colony

3. Nutritional Characteristics

• What nutrients they consume

• Bacteriodes melanogenicus asaccharolyticus → does not ferment carbohydrates

• Bacteriodes melanogenicus intermedius → ferments carbohydrates

4. Fermentation end product

• Lactobaccilus species ferments glucose to lactic acid

5. Cell Wall Comoposition

• Ex. Streptococci subdivided based on cell-wall antigens (A-H) w/ the Lancefield Serotypes

6. DNA Base Composition

• Molar Fractions G+C/G+C+A+T

• Calculating the % of DNA ratios

7. Bacteriophage Bacterial virus susceptibility

• Classifying bacteria based on whether they are susceptible to certain viruses

8. Plasmid transfer

• Plasmids= small sections of DNA, can be transferred b/w similar grps of bacteria

• This is the reason for why bacteria can become resistant to antibiotics

• Ex. Enterobacteriaceae bcs bacteria in this family carry plasmids

What was the major method used to classify bacteria based on the old classification system?

Culturing (Growing on a petri dish)

What is the limitation of the culturing method

No sensitivity & poor specificity

1. Does not ID huge uncultivated majority of microbes

• Location: Difficulties in sampling due to physical environment

• Organisms mb not capable of adapting in laboratory culture

  • Lack of essential nutrients, physiological needs/growth factors in culture media. overfeeding, toxicity in culture, inhibitory substances produced by other bacteria, metabolic dependance on other species, disruption of quorum-sensing systems (comm system b/w bacteria to track population density) induced by separation of bacteria, cell in low metabolic state (dormant)

• KEY idea is that when you take a sample somewhere, there is thousands of species of bacteria, but you don’t capture all of them, then when you grow them some of them are unable to grow in standard laboratory conditions

2. Even after successful cultivation, may not be successfully identified

• In the traditional method, identification mb heavily based on phenotypic (observable) characteristics, which you compare to reference slides but its so subjective, it might be done incorrectly → Misidentification

What is the new method of classifiying bacteria?

Using genes & molecular biology → 16S Ribosomal RNA sequencing

What is 16sRNA?

16sRNA=part of machinary that bacteria uses to make proteins from amino acids

16sRNA when bound to ribosomal proteins make 30S subunit of ribosome

Gene that codes for 16sRNA found in all bacteria, part of sequence = conserved, so once the scientist find the conserved part, they can amplify it and look at the variable gene sections to classify the bacteria

Why is 16sRNA used?

• Stable + present in all organisms

• Sequence must be conserved sufficiently that regions of homology can be clearly established

• Must be sufficiently large that info is adequate to make phylogenetic inferences

Can be used for phylogenetic reclassification (classifying organism based on how evolutionary related they are)

Similar 16sRNA= more similar

Classifying microbes based on evolutionary relationship = more stable, less likely to need additional revision

16sRNA= documents evolutionary history >2000 sequences determined for bacteria but 16sRNA makes up only 0/05% of the genome

How is 16sRNA used?

16S rRNA is isolated & sequenced

Using 16S rRNA to determine phylogeny of bacteria (relationship of organisms based on evolution)

Can be used for accurate & rapid identification of known bacterial species (PCR)

16S rRNA to differentiate, quantify, classify unknown species (esp those that cannot be cultivated)

What is PCR (Polymerase Chain Reaction)?

Laboratory technique to amplify specific DNA sequence, produce millions of copies from small starting amnt

Rxn → thermal cycler, changes temp w/ 3 main steps

What does PCR require?

• Template DNA → DNA w/ target sequence

• Primers → short DNA sequence that defines region to be amplified

• DNA polymerase → heat-stable enzyme like Taq polymerase

• Nucleotides (dNTPs) → building block for new DNA

• Buffers + ions like Mg3+

What are the 3 steps for PCR?

1. Denaturation ~95C

• Double stranded DNA separate into 2 single strands

2. Annealing ~50-65C

• Primer bind to complementary sequence on DNA strands

3. Extension ~72C

• DNA polymerase extends the primers and synthesizes new DNA strands

3 steps repeated 25-40 cycles, bcs DNA approx doubles each cycle, target sequence increases exponentially, generated millions of copies

How is PCR used in gel electrophoresis?

PCR makes million of copies of DNA, after put amplified DNA in gel, electrical current moves DNA thru gel

Smaller DNA → move farther

Larger DNA → move less far

DNA then become fragment bars

used to visualize DNA

Gel electrophoresis used to check if PCR copied the DNA data it wanted

Name bacterias in Phylum 1-Proteobacteria and what they cause

Beta Division

• Genus: Eikenella → Periodontal disease

Gamma Division

• Genus: Actinobacillus → Periodontal disease

Epsilon Division

• Genus: Campylobacter → Progressive periodontal sites & root canals w/ necrotic pulp

Name bacterias in Phylum 2-Gram + and what they cause

Fusobacteria Division

• Genus: Fusobacterium → associated with periodontal infections, found in root canals w/ teeth having necrotic pulp

SSV Division

• Genus: Selenomonas → Aggressive forms of PD

Firmicutes

• Genus: Clostridium → SubG plaque in pt w/ aggressive forms of PD

• Genus: Eubacterium → PD + root canals w/ necrotic pulp

• Genus: Lactobacilli spp → Root canals w/ necrotic pulp

• Genus: Enterococcus → Root canals w/ necrotic pulp

• Genus: Streptococcus → Root canals w/ necrotic pulp

• Genus: Peptostreptococcus → Root canals w/ necrotic pulp

Name bacterias in Phylum 3-Actinobacterium and what they cause

Genus: Actinomyces spp → Root canals w/ necrotic pulp

Genus: Propionobacterium → Root canals w/ necrotic pulp

Name bacterias in Phylum 5-Spirochetes and what they cause

Genus: Treponema → Anaerobic, seen in pt w/ moderate-severe periodontal pockets

***Spirochetes are motile, have a flagella that allows it to go into the sulcus area and hide itself, brings other bacteria in with it

Name baceterias in phylum 6-Bacteriodes and what they cause

Bacteriodes Division

• Genus: Porphyromonas. Species: P. gingivalis → Most known species associated w/ periodontal disease + in teeth with necrotic pulp

• Genus: Prevotella. Species: P. intermedia, P.negrescens → associated w/ PD + found in root canals w/ necrotic pulp

Flavobacter Division

• Genus: Capnocytophaga → Gingivitis

Name a few bacteria involved in periodontal disease or necrotic pulp

Gingivitis

• Capnocytophaga

Periodontal Disease

• Eikenella

• Actinobacillus

• Fusobacterium

• Eubacterium

• Porphyromonas gingivalis

• Prevotella intermedia

• Prevotella nigrescens

Aggressive Periodontal Disease

• Selenomonas

• Clostridium

Moderate–Severe Periodontal Pockets

• Treponema

Root Canals with Necrotic Pulp

• Campylobacter

• Fusobacterium

• Eubacterium

• Lactobacilli

• Enterococcus

• Streptococcus

• Peptostreptococcus

• Actinomyces

• Propionibacterium

• Porphyromonas gingivalis

• Prevotella intermedia

• Prevotella nigrescens

What is Koch’s postulate?

1. Microorganism must be demonstrable in all cases of disease

2. Microorganism must be isolated from diseased animal + grown in pure culture

3. Microorganism from pure culture must cause disease when inoculated into healthy animal

4. Experimentally infected animal must contain microorganism

What are limitations to Koch’s postulate

Not all diseases are caused by microbes!!!

Auto-immune

Cancer

Asymptomatic

What is Evan’s postulate Not on exam jus know overall

Updated epidemiology criteria from Koch

In exposed/diseased groups, higher prevalence, greater exposure frequency, and higher incidence of the microbe

Preventing/eliminating microbes should decrease incidence of disease and presence of it

What is Frederick and Relman’s postulate?

Also modern nuclear version of Koch’s postulate

Since some pathogens cannot be cultured, instead used criteria of nucelic acid sequences being present

Nucleic acid sequence of pathogen should be present in areas w/ disease

Nucleic acid sequence should be absent in tissues w/out disease

What is the issue of applying Kochs postulate in periodontal disease?

1. Inability to culture all organisms

2. Difficulties defining + culturing sites of active disease

3. Lack of animal model system

What is Sigmund Socransky’s criteria

Criteria for whether a microbe can be considered a periodontal pathogen

1. Must be associated w/ disease → increase in # of organisms at diseased site

2. Must be eliminated/decreased in sites that demonstrate clinical resolution of disease w/ treatment (mechanical or antibiotics)

3. Must demonstrate host response (alteration in host cellular or humoral immune response)

4. Must be capable of causing disease in experimental animal model

5. Must demonstrate virulence factors responsible to enable microbe to cause destruction of periodontal tissue

What size are prokaryotic cells?

Length: 2u → 8u

Diameter: 2u → 0.2u

What is the morphology of prokaryotic cells?

Rods → bacilli

• Diplobacilli

• Streptobacilli

• Coccobacilli

Spheres → cocci

• Diplococci

• Streptococci

• Tetrads

• Sarcinae

• Staphylococci

Spirals

• Vibrio (curbed bacilli)

• Spiralla

• Spirochete

How motile are prokaryotic cells depending on their shape?

Almost all spirals = motile

½ of bacilli = motile

Almost all cocci = non-motile

What are the sources of energy and carbon for chemoheterotrophs

Chemoheterotrophs include animals, fungi, and most other bacteria

Use oxidation-reduction reactions to produce ATP

Use organic molecules to supply itself with oxygen

What other factors impact bacterial growth?

Temperature

pH (7.2-7.4, some caries: 5)

CO2/O2

• Facultative Anaerobes → Respire in O2, ferment otherwise (Can switch)

• Obligate Anaerobes → Killed by O2

• Obligate Aerobes → Require O2

• Aerotolerant Anaerobes → Always ferment BUT have limited capacity to detoxify partial reduction products of O2

Why is oxygen important for bacterial growth?

For bacteria that uses oxygen, it enhances metabolism + growth of bacteria

Helps bacteria make ATP, oxygen is the final electron/hydrogen acceptor in aerobic metabolism BUT using oxygen creates H2O2 and free radical superoxide (O2), which is toxic and needs to be removed

What type of bacteria uses respiration and how many ATP does it produce?

Supra-gingival plaque

1 glucose → 38 ATP

What type of bacteria uses fermentation

Sub-gingival plaque

1 glucose → 2 ATP + Lactic acid

How is the oral cavity related to oral microbiology?

Oral cavity= continuous culture system w/ nutrients supplied in form of

• saliva

• gingival fluid

• desquamated epithelial cells

• initial despository of “incoming nutrients”

Describe the presence of microbes in the oral cavity

avg person 20-100mg plaque, equal to 10¹⁰ bacteria

1-5mm pocket = 1 million bacteria

6-10mm pocket= 1000 million bacteria

Up to 1000 diff species, 100-200 species per person

100 million bacteria/mg of plaque

Instrument full of plaque= 100 million bacteria

Only 70% of bacteria= identified & cultured

Are bacteria motile in suprag or subg plaque?

Suprag plaque=visible + adherent

Subg plaque=not visible, more difficult to demonstrate, loose flora (motile)

What is the composition of oral plaque?

Bacteria, epithelial cells, PMNs, yeast

(PMNs=polymorphonuclear leukocytes, most abudant WBC that are in blood circulation, leak out of capillaries and fight infection)

What makes the oral cavity a unique ecosystem for microbes?

Lips, cheek, palate, tongue, gum, teeth → change during life of individual!

Teeth= nonshedding surface

Specialized mucosal surfaces= KT and nonKT

Affected by chemical habitat

Saliva → Buffer host defense

GCF → “Serum like” host defense

What are the 7 factors that affect microbial growth in the oral cavity?

1. Temperature

• Human mouth is 35-36C affects bacterial metabolism, enzyme activity, habitat

2. Redox potential (anaerobiosis)

• Gradients of O2 [ ] and Eh(redox potential) exist in plaque in oral cavity

  • Some areas in plaque high in O2, deeper= lower

• Affects type of microbes that can be found

3. pH

• Most microbes require pH ~7 (Regulated by saliva)

4. Nutrients

• Endogenous saliva → Can be both + and -, helps bring in WBCs but also helps microbes grow by regulating the environment for them

• Exogenous saliva → What we eat

5. Adherence

• Chewing + saliva will detach

• Soft tissue desquamation limits bacterial load

• Hard tissue=high biofilm

6. Antimicrobial agents & inhibitors

• Dentifrices

• Mouth rinses

• Antibiotics

7. Host defences

• Innate immunity

• Non-specific immunity

• Specific immunity

Is the oral microflora stable of unstable?

As humans, we’ve come to develop an oral community that is stable/balanced but sometimes there can be disruptions that cause microbial growth

Why is it difficult to study oral flora?

High species diversity

Surface attachment/Coaggregation

Obligate Anaerobes

• Do not grow well in lab cuz they die in O2 but collecting it=exposing it to O2

Fastidious nutrition

• specific nutritional requirements

Generation time

• Some microbes take a very long time to divide

Identification problems

Difficulties w/ sampling environment

What are the methods of studying oral flora?

Collect sample and either direct examination under microscope OR bring the sample to a lab and culture it

How does the oral microflora come to be? (6 stages)

6 Stages

1. Transmission → saliva from mother, food, milk, water

2. Acquisition → microbes enter mouth and colonize surfaces (epithelial & hard surfaces)

3. Pioneer species → first organisms to colonize usually (Streptococci, staphylococci, neisseria, veilonella)

• Grow until they meet environmental resistance

  • Physical forces (epithelial shedding, shear forces)

  • Chemical factors (saliva, Eh <redox>, pH, nutritional requirements

4. Succession → Metabolic activity of pioneer community modifies environment, makes it suitable for new bacterial populations to colonize

• Allogenic factors:

  • Physical environement

    • Eruption of teeth

    • Acrylic obturators for cleft palate (used in pts to mimic palate, provides hard surface for bacteria)

• Autogenic factors

  • Toxic end products

  • Cryptotypes (receotors)

    • Receptors/binding sites where pioneer species bacteria attaches and modifies it so other bacteria can stick on it too

  • Metabolism/Eh

    • Ex. pioneer bacteria consume o2, Eh decreases, environment becomes more anaerobic and anaerobic bacteria can grow there

5. Increasing species diveristy

6. Climax community

• Not static (highly dynamic)

• Stable due to complexity

What are the factors responsible for stability in climax community

1. Competition for receptors/ligands for adhesion

• Bacteria need to attach to surfaces/other bacteria

• Established bacteria=limits ability of other bacteria to colonize

2. Competition for essential endogenous nutrients/cofactors

• Bacteria need nutrients + other substance to grow, established bacteria can use up resources, limit whats available

3. Production of inhibitory substances

• Some bacteria produce substance that inhibit/kill other bacteria

4. Creation of micro-environment that discourage growth of endogenous specieis

• Established species can change local environment and make it difficult for other bacteria to grow like pH, O2 [ ]

What are factors that interrupt the climax community?

In adults, resident microflora is usually relatively stable & co-exists in relative harmony w/ host

EXCEPTIONS

• Pregnancy → hormonal changes alter environment

• Tooth eruption in children → creates new surfaces

• Disease (cancer/immunosuppression) → change in host defense

• Denture wearing → artificial surface that changes oral environment

What is dental plaque

general term for complex microbial community found on tooth surface

Embedded in matrix of polymers of bacterial & salivary origin

Plaque being calcified is called calculus/tartar

Bacterial members cooperate + signal to each other

• Fluid channels carry nutrients + remove waste

How does plaque develop?

Immediately after cleaning, on clean tooth surface, an acquired enamel pellcile forms

Enamel pellicle= thin, protein rich film from saliva & GCF

Bacteria than adheres to the enamel pellicle, colonize & multiply creating biofilm, if left alone, mineralizes and turns into calculus

Mineral alba is different from biofilm and pellicle, it is soft loosely attached containing bacteria, salivary proteins, food debris, epithelial cells

Can use disclosing solution like erythrosin to highlight presence of plaque

What is the enamel pellicle?

Uniform thick deposition of salivary proteins between enakel and layer of immature bacterial plaque

Allows for adherence and coaggregation of bacteria

• Pellicle contains receptors and binding sites for bacteria then once first bacterias are attached, they provide receptors for other bacteria to attach → coaggregation

What interactions are involved in adherence and coaggregation of bacteria

Electrostatic interactions

• Divalent cations

• Hydogen bonding

• Ionic interaction

• Hydrophobic bonding

What are the contents in saliva that coat teeth and provide host ligands for bacteria attachment

Acidic proline rich (S. mutans)

Amylase

Lysozyme

Immunoglobulins

Albumin

What is the mechanism of adherence and colonization of bacteria for supragingival plaque

Development of Supragingival plaque

After tooth is clean, acquired dental pellicle is formed, saliva deposits host proteins & glycoproteins on enamel, creating acquired enamel pellicle. Pionner species bacteria bind to host ligands on dental pellicle (adherence)

Once bacteria attach → begin dividing and aggregate

THEN allow diff bacterial species to attach and COaggregate

What is the difference b/w aggregation and coaggregation

Aggregation= clumping with themselves

Co-aggregation= clumping w/ others

What is the mechanism of adherence and colonization for subgingival plaque?

Forms when supragingival plaque migrates into sulcus then inlammation creates subgingival environment

Wide variation of flora from site → site

• Serotypes (dinstinct subgrp of species) w/ diff pathogenicities

Mostly anaerobic bacteria

• 90% anaerobic

• 75% gram (-)

• up to 50% motile spirochaetes

Motile bacteria carry non-motile bacteria in

What part of gram (-) bacteria is bad?

Outer membranes consist of lipopolysaccharides, which stimulates immune response

What are the contributing factors for caries?

Host → have to be susceptible, saliva, tooth anatomy, OH, other host factors

Time → the other factors need to interact in sufficient duration

Microflora → need to have the bacteria that causes caries

Diet → fermentable carbohydrates (which lowers pH of mouth)

What are the bacteria that causes caries (Name 5) and how

S. mutans

Lactobacilli

S. sobrinus

S. sanguis

S. Mitis

Actinomyces naeslundii

A. casei

***Metabolize fermentable carbohydrates, produces lactic acid which causes demineralization

What is an important characteristic of streptococcus mutans and lactobacilli?

Aciduric

• Able to survive + grow in acidic environments

• Most bacteria can’t tolerate it well but these can and thrive in them

Describe the clinical development of caries

1. Tooth surface w/out caries

2. First sign of demineralization “white spot/incipient caries”, surface still hard and caries process can bbe halted/reversed here

3. Enamel surface= broken down, lesion w/ soft floor

4. Filling made to stop it but demineralization did not stop and lesion continues on in filling

5. Demineralization proceeds, undermines tooth

6. Tooth fractured