IMPORTANT

Question 1

What are the pathogenicity factors of C. albicans?

Answer 1

L7

• adhesion
• dimorphism
• secretion hydrolytic enzymes
• interaction with immune system
• toxin production: candidalysin

Question 2

What are the secreted hydrolytic enzymes of C. albicans and how are they pathogenic factors?

Answer 2

L7

• secreted aspartic proteinases: degrade sIgA, promote adhesion + aid tissue penetration/destruction
• phospholipase: degrades phospholipids = destroy cell membranes + aid tissue penetration

Question 3

How is interaction with immune system a pathogenic factor in C. albicans?

Answer 3

L7

• grow out of phagocytes after being engulfed
• secreted proteinases may degrade antibodies
• hyphae secrete a substance that inhibits polymorphonuclear leukcyte (neutrophil, basophil etc.) degranulation (release of cytotoxic molecules + prevents phagocytosis
• supress T-cell proliferation
• bind C3b + C3d complement proteins which masks cell from phagocytes

Question 4

What are some anti-fungal drug types and their sites of action?

Answer 4

L8
5-fluorocytosine: DNA RNA synthesis
polyenes: membrane integrity
azoles: sterol synthesis
echinocandins: wall synthesis

Question 5

What are some anti-viral drugs?

Answer 5

L6

• acyclovir: HSV + VZV
• abacavir: HIV
• lamivudine: HIV
• ritonavir: HIV

Question 6

What is the mechanism of action for abacavir + lamivudine antivirals?

Answer 6

L6
- nucleoside analogues
- inhibit retroviral reverse transcriptase
Abacavir is an antiviral drug. It is a nucleoside analogue and thus competes with viral molecules and is incorporated into viral DNA. Once the drug is incorporated into the viral DNA, it inhibits transcription and HIV reverse transcriptase thus inhibiting viral replication.

Question 7

What are the components of the innate immune system?

Answer 7

L10
- physical barriers: skin
- flushing action: saliva
- biological components: enzymes, phagocytosis, complement, inflammation, 
nutrient privation 
- microbial competition

Question 8

What are the stages involved in plaque development?

Answer 8

L14

• clean enamel surface
• pellicle formation (2 sec)
• pioneer bacteria (2 min)
• microcolonies and extracellular polysaccharide (2 hours)
• biofilm development (2 hours +)
• mature plaque + sealing in bacteria (48 hours)

Question 9

How is difference between supragingival and subgingival plaque?

Answer 9

L14
supragingival:
- >90% gram +ve
- cocci and filaments
- carbohydrate fermentation = acidogenic
- microaerophilic (low oxygen levels)
- bathed by saliva
- dental caries
subgingival:
- >50% gram -ve
- rods and fusiforms 
- saccharolytic and asaccharolytic (proteolytic)
- anaerobic 
- bathed by gingival cervicular fluid
- periodontal disease

Question 10

What are the virulence factors of Streptococcus pneumoniae?

Answer 10

L15

• alpha-haemolytic: partial lysis RBC
• capsule: prevents complement activation
• cell wall adhesins: endothelial cell adhesion
• pneumolysin: cytolytic and cytotoxic
• autolysin: self-destruct protein
• neuroamidase: expose receptors for adhesion
• IgA1 protease: cleaves IgA antibodies + evades mucosal immune system
• hydrogen peroxide: toxic to cells
• competence: ability to take up DNA + antigenic variants

Question 11

What types of virulence factors allow a microbe to be a successful pathogen?

Answer 11

L15

• ability to adhere to and colonise the host
• adapt to the environmental changes within the host
• avoid host defence system
• be able to defeat the host defence system

Question 12

What are the virulence factors of Mycobacterium tuberculosis? Why do they contribute to pathogenicity of M.tb?

Answer 12

L16

• mycolic acid (waxy layer) on cell wall: resists desiccation
• grows slowly: common antibiotics (penicillin) are ineffective
• resistant to lysis agents: can grow inside phagocytes
• produce cord factor: increase survival within the host

Question 13

What are the virulence factors of S. aureus and what do they do?

Answer 13

L17
enzymes:
- coagulase: clots blood
- hyaluronidase: breaks down hyaluronic acid
- staphylokinase: breaks down blood clots
- lipases: break down lipids
- beta-lactamase: destroys beta-lactam antibiotics (penicillin)
anti-phagocytic defences:
- polysaccharide slime layer (capsule): prevents phagocytosis + enables adherence
- protein A: binds to antibody stems = prevents phagocytosis + complement activation
toxins:
- cytolytic toxin: disrupts cell membranes
- leukocidin: kills leukocytes (WBC)
- exfoliative toxin: breaks down desmosomes
- Toxic Shock Syndrome toxin: superantigen

Question 14

why dental caries spreads laterally at the dentino-enamel junction?

Answer 14

Towards the DEJ odontoblasts have lots of processes and less towards the base of the odontoblast. At first there is more than one process at the point where the odontoblast contacts the enamel matrix but as more dentine is laid down, the cells recede, leaving a single process called a Tomes fibre.
There is less mineralisation at the DEJ because there are more spaces due to more branching of the odontoblast.
Caries spreads along the sides in the DEJ in mantle dentine because it is less mineralised than the rest of the dentine and thus caries is able to spread more quickly in lateral direction at the DEJ

Question 15

Discuss the condition Molar Incisor Hypomineralisation. In your answer, state what teeth may be affected, describe the characteristics of the affected teeth, and the advice that should be given to a patient (or parent of a patient) with this condition about its possible causes and treatment?

Answer 15

Hypomineralisation occurs when there is a disruption in tooth development during the mineralisation stage and this results in enamel with a higher than normal protein content. In the case of molar incisor hypomineralisation, the central incisors and 1 or more of the molars are affected. These teeth have a yellow/brown appearance but no associated thickness alteration (thus it is not hyperplasia)
Treatment options for MIH are preventive or operative treatment, or extraction.
Possible causes for MIH are systemic conditions, these include but are not limited to:
• Prolonged breast feeding
• Low infant birth weight and oxygen starvation
• Environmental conditions
• Childhood diseases
• antibiotics

Question 16

What are the functions of the periodontal ligament?

Answer 16

Periodontal ligament is a soft, specialised, fibrous connective tissue located between the cementum and alveolar bone. It functions as a support for the tooth within the socket. The development of periodontal ligament accompanies tooth eruption and root development, and the arrangement of the collagen it produces into fibres is related to the stages of eruption and function of the mature tooth.

Question 17

Briefly discuss the differences between primary, secondary, and tertiary dentine

Answer 17

Primary dentine
• Formed before root formation is completed
• Forms most of the tooth
• Outlines the pulp chamber
• Mantle dentine is the outer layer of primary dentine
• Produced by odontoblasts
Secondary dentine
• Develops after root formation is complete
• Preprogrammed age change and not a response to external stimuli
• Slower than the formation of primary dentine
• Same tubular structure and composition as primary dentine
• Bulbous layer of dentine interproximally
• Deposition continues through life
• Produced by odontoblasts
Tertiary dentine
• Formed as a result of pathological response, may be reactionary or reparative
• Produced by cells affected by the stimulus
• Architecture depends on intensity and duration of the stimulus
• Very rapid deposition
• Irregular arrangement
• Demarcated line from primary and secondary dentine by a calciotraumatic line = a band of unmineralised predentine and a band of hypermineralised dentine that forms when the cells recover and form tertiary dentine
• Produced by newly differentiated cells from the dental pulp (pulp mesenchymal cells)

Question 18

Discuss the structure and composition of the oral mucosa

Answer 18

The oral mucosa is a mucosal lining of the oral cavity that lines surfaces that are communicating with the exterior. It consists of oral epithelium and superficial connective tissue (lamina propria), with the submucosa located underneath.
The oral epithelium is stratified squamous epithelium made up of 3 layers: basal cell layer, prickle cell layer, and keratin layer.
• The basal layer contains progenitor cells for the continuous renewal of cells, it also contained other non-keratinocytes: melanocytes, Langerhan’s cells, Merkel cells, and inflammatory cells. These cells do not have desmosomes connecting adjacent cells together.
• The prickle cell layer are where cells from the basal layer mature and migrate to the surface. The cells are flattened off and lose their nuclei to form part of the protective keratin layer. This layer contains desmosomes connecting the adjacent cells (form prickles between the cells)
• The keratin layer can be of two types: orthokeratin or parakeratin. The orthokeratin layer contains cells with no nuclei - usually a granular cell layer (thin layer of cells). The parakeratin layer contains cells with retained shrunken pyknotic (irreversibly condensed) nuclei. This layer is considered normal in the mouth but pathogenic in the skin.
The junction between the epithelium and connective tissue is an undulating interface consisting of interdigitation of connective tissue papillae and epithelial rete ridges. This great surface area of contact means the junction has greater resistance to forces
The connective tissue layer consists of lamina propria. This layers consists of cells (fibroblasts, macrophages, inflammatory cells), blood supply, nerves, fibres and ground substance.
The oral mucosa is comprised for three types of mucosa:
• Lining mucosa forms 60% and is located on the: lip, buccal, alveolar, soft palate, ventral tongue, and floor of mouth. It is not keratinised (although in many people it is keratinised due to exposure to trauma), has a flatter epithelium/connective tissue junction and contains a higher elastin content in the connective tissue. It is highly mobile and flexible.
• Masticatory mucosa forms 25% and it is a keratined mucosa with highly convoluted epithelium/connective tissue junction. It is an immobile surface that is tightly bound to underlying bone, and inflexible tough and resistant to abrasion.
• Specialised mucosa forms 15% and comprises of a thick keratin layer. It is primarily located on the dorsum of the tongue and is a highly extensible masticatory mucosa containing papillae and taste buds.

Question 19

Briefly discuss the differences between lamellar bone and cementum

Answer 19

Although bone is similar in chemistry to bone, bone is vasularised (blood supply) and innervated (nerves) whereas cementum is avascular and has no innervation.
Cementum is also deposited as a thin layer on the surface of the tooth root whereas bone is arranged into basic structural units called osteons which make up the structure of the bone.
The function of cementum is to structural support (primary cementum) and for maintenance (secondary cementum) whereas bone has several functions: support framework of the body, movement in conjunction with muscles, protection of delicate organs, haematopoiesis production of RBC from red bone marrow, and mineral storage of calcium and phosphate.

Question 20

Dentists aiming to regenerate the periodontium should understand the tissues involved. Describe in detail the difference types of cementum, their characteristics, and functions

Answer 20

There are two types of cementum: acellular and cellular.
Acellular cementum is first formed primary cementum and has a support function. Cellular cementum is secondary cementum which is formed throughout life and has a maintenance function. This maintenance function is carried out by the cementocytes embedded within lacuni.
The first type of cementum produced by cementocytes is primary (acellular) cementum. It is laid down on top of a layer of predentine and has a structural function. The cementoblasts deposit collagen fibrils that mingle with collagen fibrils in the predentine, this leads to the formation of the dentinocemental junction after mineralisation. Cementoblasts continue to secrete collagen until 15-20 micrometres of cementum is formed, this remaining fibre fringe is then stitched with the collagen fibres from the periodontal ligament.
Secondary (cellular) cementum is found in multirooted teeth, it is a more rapid less mineralised cementum. This cellular cementum, although still produces collagen fibres that mingle with predentine collagen, is less involved in tooth attachment and has more of a maintenance role. An unmineralised matrix called cementoid is produced and cementoblasts that get trapped within this become cementocytes. The secondary cementum continues to be deposited around fibres of periodontal ligament - results in mixed cementum.

Question 21

Discuss how innate immune defences protect humans against oral microbial disease

Answer 21

Physical barrier: skin
The skin is a physical barrier that prevents potential pathogens from invading into the body.
Flushing action: saliva + cough/sneeze reflex
The saliva flows over the surfaces of the oral cavity and acts to flush away any contaminated secretions and pathogens. It also delivers the biological components of the immune system such as enzymes into the oral cavity. Saliva is a protective component of the immune system as saliva flow dysfunction leading to dry mouth is associated with increased incidence of caries and candidosis.
Biological components:
• Enzymes
There are various enzymes that play a role in the innate immune system. Lysozyme is an enzyme that cleaves the peptidoglycans in the bacterial cell walls. Lipases break down the lipids that are found in the bacterial plasma membranes. Proteases break down proteins. Nucleases break down nucleic acids.
• Phagocytosis
The professional phagocytes: neutrophils, macrophages and monocytes, protect the body by engulfing pathogens and killing them by releasing the contents of lysosomes into the vesicles carrying the pathogens. Their degradation products are then released from the phagocyte.
• Inflammation
There is an increase in the blood supply and capillary permeability at the site of infection or tissue damage which leads to the concentration of components of the immune system.
• Complement
The complement proteins have various functions that contribute to the innate immune system. C3b enhances phagocytosis and recruits other complement to form the MAC. C5b-C9 complement proteins form the MAC which is a complex that phagocytoses bacteria. C5a promotes chemotaxis in the inflammatory response. C3b and other proteins are also involved in opsonisation which increases the efficiency of phagocytosis.
• Nutrient privation
Lactoferrin and transferrin are iron-binding proteins that bind iron to reduce its availability for bacterial pathogens. Iron is required for the pathogens to survive so by depriving them of this essential nutrient, they are unable to survive in the body.
Microbial competition
The presence of indigenous microbes within the body is a protective factor that prevents disease. This is because the indigenous microflora, which comprise of mainly non-pathogenic organisms, prevent pathogens from colonising due to their utilisation of nutrients.

Question 22

Discuss the major differences between the micro-organisms involved in dental caries and those involved in generalised periodontal disease.

Answer 22

Dental caries
	• Gram +ve
	• Acidogenic and aciduric
Periodontal disease
	• Gram -ve
	• Proteolytic
	• Asaccharolytic or saccharolytic
The microorganisms involved in dental caries are gram +ve acidogenic bacteria. They are able to withstand pH as low as 4.0 and ferment carbohydrates to produce lactic acid. This lactic acid is what lowers the pH of the supragingival plaque which leads to the development of dental caries. The bacteria that are involved in dental caries are directly responsible for the development and progression of caries.

The microorganisms that are involved in periodontal disease are gram -ve proteolytic bacteria. They are not associated with acid production and thus the subgingival plaque has a neutral pH. The bacterial in the subgingival plaque are either able or unable to ferment carbohydrates but they are proteolytic and break down proteins to release the amino acids which they ferment. The microorganisms involved in periodontal disease are not directly associated with causing periodontal disease.

Question 23

Bacteria are not known to express bone-resorbing enzymes and yet a major clinical feature of periodontal disease, which is unquestionably of bacterial aetiology, is loss of alveolar bone. Explain how this might occur.

Answer 23

Porphyromonas gingivalis is a gram -ve bacteria that is associated with adult periodontal disease. It does not produce acid but is a proteolytic bacteria that metabolises amino acids. The disease is due to the indirect effect of gram -ve bacteria but mainly the direct effect of the immuno-inflammatory response of the host. The host releases signalling molecules such as interleukins (IL-1 IL-8) to recruit neutrophils to the site where the bacteria are breaking down proteins. These endogenous signalling molecules also have an effect on bone remodelling. The IL-1, PGE2 thromboxane inhibit osteoblast activity and inhibit matrix production and calcification. In addition to this, the signalling molecules have the effect of increasing osteoclast differentiation and activity. This overall leads to an imbalance in the bone remodelling process as there is osteoclast overactivity and reduced osteoblast activity and this results in bone breakdown and the bone loss that is characteristic of periodontal disease.

Question 24

With the help of diagrams, explain how supragingival dental plaque forms and how it might subsequently become cariogenic

Answer 24

Clean enamel surface develops a pellicle within 2 seconds. The pellicle is a protein film derived from saliva by selective adsorption of certain substances.
Pioneer bacteria adhere to the pellicle within 2 minutes, this includes bacteria such as Streptococcus sanguis. Pioneer bacteria are hardy bacteria that are the first to colonise a clean surface.
Bacteria, such as Streptococcus mutans binds to the surface of the enamel at 2 hours and form microcolonies. These bacteria produce extracellular polysaccharide and so form local concentrations of this extracellular matrix. After two hours, these microcolonies grow over the entire tooth surface and consist of aerobic bacteria and extracellular polysaccharide matrix. This is plaque.
After 48 hours, this plaque matures and the amount of bacteria increases. This creates a seal for the bacteria that are deepest within the plaque and closest to the tooth surface.
Mature plaque formation can lead to the plaque becoming cariogenic because the polysaccharides secreted by the bacteria include glucans which are secreted by S. mutans and this firmly adheres to the tooth surface and prevents diffusion of substances into and out of the plaque. This prevents the acid produced by the bacteria from diffusing out of the plaque and also prevents buffering agents present in the saliva such as HCO3 from penetrating through the plaque to neutralise this acid. This acid build up on the tooth surface can lead to dental caries.

Question 25

What is the difference between a selective bacterial growth medium and a differential growth medium? Describe the characteristics of one example of a selective medium and one example of a differential medium.

Answer 25

A selective bacterial growth medium is one which contains additives that selects for the growth of a specific species or related species. Mutans selective agar is an example of a selective medium. It contains high sucrose concentrations (20%) and antibacterial bacitracin which enables the plate to selectively grow Streptococcus mutans and other related species.

A differential growth medium is one which distinguishes species on the basis of their utilisation of substances such as amino acids or carbohydrates. Blood agar is a differential growth medium, it contains 4-5% sheep, human, or horse blood and differentiates species on their ability to cause haemolysis: alpha greening = partial haemolysis, beta = haemolysis, gamma - no haemolysi

Question 26

Briefly describe the role of the 16S ribosomal RNA (16S rRNA) gene in studying oral microbial diversity

Answer 26

rRNA genes are highly conserved across all organisms, the 16S rRNA gene is conserved in all bacteria and archaebacteria. This gene also contains 8-9 hypervariable regions which vary between species and subspecies. This gene can be used to study oral microbial diversity by sequencing the 16S RNA genes obtained from PCR amplification. Comparison of the sequence with entire in a database can enable the identification of the different species that are present within the oral microbiota sample.

Question 27

Why is boiling surgical instruments in water inadequate for sterilisation?

Answer 27

Because boiling instruments in water does not remove bacterial spores. The process of sterilisation removes or kills all types of microbes including spores.

Question 28

Briefly describe three roles of complement proteins in immune responses

Answer 28

Opsonisation, they bind to phagocytes to increase the efficiency of phagocytosis
C3b recruits C5b and other complement proteins to from the MAC (C5b-C9) which is a complex that lyses bacterial cells
C5a promotes chemotaxis in inflammatory response, recruiting neutrophils + macrophages to the site where complement is being generated.

Question 29

List the features of an “ideal” antimicrobial drug

Answer 29

• Cidal activity
  
  • Minimal toxicity to the host
  • Selective toxicity to the target
  • Good tissue distribution
  • Low binding to plasma proteins
  • Long plasma half-life
  • Oral and parenteral preparations
  • No adverse drug interactions

Question 30

“The oral cavity is estimated to comprise 700 microbial species”. What laboratory-based techniques would you use to identify bacteria from an oral sample?

Answer 30

There are a large number of bacteria involved in the oral cavity. However, carrying out individual biochemical testing to identify each bacteria is extremely labor intensive and there is a high probability that some slow growing bacteria will get overgrown by faster growing bacteria, meaning inaccurate identification of bacteria may be given. It is also important to maintain anaerobic conditions during and after sampling for anaerobic bacterias in the oral cavity. Therefore the traditional culturing methods to culture the bacteria on a medium is unable to give an accurate indication of all the bacterial species present in the oral cavity. To identify bacteria which cannot be cultured, 16s rRNA sequencing technology is used.

Firstly a sample of the cultures in the oral cavity can be taken by a swab at different sites with cotton buds. Such sites would include pockets, gingiva, tongue, buccal mucosa and also the teeth surfaces. These bacterias are extracted using DNA extraction technology.

16s rRNA is a region that is present in all types of prokaryotes that are able to synthesise protein as it is the component of the 30S small subunit or prokaryotic ribosome that bines to shine Dalgarno sequence. It is composed of a highly conserved region and hypervariable regions that can provide species specific identification. There are nine hypervariable regions.

Sequencing of the 16sRNA gene is done by amplifying the 16sRNA genes in the sample taken with PCR, and comparing the sequenced data against the data base. This allows identification of bacteria in the oral cavity to be possible.