1
Q
bacteria associated with periodontal disease
A
- porphyromanas gingivalis
- provotella intermedia
- bacteriodes forsythus
- actinobacillus actinomycetemcomitans
2
Q
bacteria associated with dental caries
A
streptococcus mutans
3
Q
bacteria associated with root canal infections
A
- porphyromanas endodontalis
- fusobacterium nucelatum
4
Q
bacterial detection methods
A
- microbiological culture- grow on agar, isolate and identify
- molecular biological - DNA probes or PCR
5
Q
summary of microbiological culture method
A
- mix sample 30s
- dilute sample
- spiral plate to agar media
- incubate anaerobically for 10 days
6
Q
how to identify bacteria
A
- gram staining - gram positive turn violet due to peptidoglycan layer in cell wall
- anaerobes sensitive to metronidazole so will die out in vicinity
- enzyme activity and sugar fermentation tests
7
Q
DNA probe method summary
A
- label DNA - can be chemiluminescent, radioactive or fluorescent
- prepare sample
- mix probe with sample DNA (hybridisation)
- non binding DNA removed
8
Q
factors for caries
A
- bacteria biofilm + carbohydrate = acid profuction = demineralisation
- demineralisatio caused healthy tooth enamel to become carious tooth enamel
- 4 main factors - saliva, diet, microbiata, tooth (&time)
9
Q
what graph shows drop in pH after sucrose rinse
A
- stephan curve
10
Q
stephan curve key features
A
- between 0-5 mins after sucrose rinse pH falls from above 6.6 - below 5.5
- below pH 5.5 when demineralisation occurs
- approx 5-10 mins after sucrose rinse when demineralisation occurs
- after 10 mins remineralisation begins - takes from 10-20 mins ro return to normal
11
Q
caries progression summary
A
- adhesion
- survival and growth
- biofilm formation
- complex plaque
- acid formation
- caries
12
Q
streptococcus mutans virulence factors
A
- glycolytic system - breaks down glucose
- EPS/sucrose metabolism
- attachment mechanisms such as GTF, Ag I/II - Glucosyltransferase (GTF) is an enzyme synthesized and is responsible for the biosynthesis of extracellular polysaccharides. Glucan formation in dental plaque mediates binding of S. mutans and S.
- ecological competiveness at low pH
- geonomic characteristics
13
Q
factors related to cariogenicity of streptococcus mutans
A
- produces extracelular polysaccharides from sucrose - helps in colonisation of tooth surface by allowing attachment
- initiates and maintains growth at low pH - continues acid production
- rapid metabolism of sugars to lactic acid
- produces intracellular polysaccharides which can act as food store for use when dietary carbohydrate is low
14
Q
why caries does not = streptococcus mutans
A
- s. mutans present in many carious tooth sufaces but not all
- frequency of sugar intake is key factor for caries
- strep mutans also factor but less important
15
Q
other cariogenic foods
A
- not just sucrose that is cariogenic
- other carbohydrates - cooked starches such as crisps and biscuits
- if sticky then more retentive in mouth - this may be crucial
- interaction of sugar and starches some studies find - drives caries
16
Q
what can promote remineralisation of tooth
A
- saliva
- fluoride source
- plaque control
- dietary modification
- allows calcium, phosphate and fluoride ions to help remineralisation
17
Q
modern ways of testing microbiology
A
- bioinformatic tools - human oral microbiome database launched 2010
- microorganisms studied using DNA sequencing 1980s-present
- culture methods and microscopy used since late 1800s
18
Q
oral microbiome development in early childhood and influencing factors
A
- microorgnisms come at very early stage
- in amniotic fluid - streptococcus, fusobacterium, porphyromanas
- influencing factors - human gemes, pregnancy term, delivery method, feeding method
19
Q
endodontic infection from microbiology prospective
A
- microorganisms continue to demineralise enamel and dentine and get into pulp chamber
- can aslo travel down root surface to apex of tooth
- endogenous type of infection - endodontic infections derived from endogenous microflora (our own microflora)
- microorganisms tend to reside in root canals and dentie tubules
- oportunistic infections - occur more often or are more severe in people with weakened immune systems than in people with healthy immune systems - because occurs when RCT damaged or traumatised
20
Q
immune response at apex of endodontic infections
A
- at apex of root is exacerbated immune response
- organisms in root canal space can also be in dentine tubules
- production of LPS by gram negative bacteria
- innate response - macrophages, PMNs arrive due to cell signalling molecules (LPS and cytokines) and migrate to apex
- innate response presents cells to adaptive immune cells such as T-cells and B cells
21
Q
what bacteria do lots of lectures/textbooks mention as important endo pathogen
primary vs secondary infection
A
- enterococcus faecalic
- dont find this as much in primary endodontic infections and most primary are polymicrobial - organisms migrate from carious lesions or periodontal disease
- 9 times more likely to contain e.faecalis in secondary infections
- secondary - composed of one or a few bacterial species - predominantly gram positive microorganisms
22
Q
enterococcus faecalis virulence factors
A
- endotoxins
- ahesins
- callagenases
- hyaluronidase
- immune invasion
23
Q
whats so exciting about enterococcus faecalis
A
- gram positive coccus
- facultative anaerobe - grows in both aerobic and anaerobic conditions, preferably anaerobic
- aggregatve, adhesive, biofilm formation
- superoxide formation which protects it
- lipotechoic acid is pro inflammatory
24
Q
bacteria in dentine tubules
A
- can see coccus species
- likely organisms can penetrate down tubules
- albicans also present
- bacteria and yeast cooperating together
25
why its difficult to be certain of bacteria in endo testing
* access cavity to get to root canal so organisms in oral environment have potential to penetrate (integrity of sampling)
* can you ensure correct site
26
microbiology behind endo chemodisinfection methods
* in general 24 hours after endo disinfection microorganisms start to regrow
* more and more growth after time
* EDTA in study shows to be better than NaoCl as less grow back after tx
* coombination of NaOCl and EDTA had best result in terms of limiting regrowth of microorganisms
27
influences on the oral microflora
* host factors - systemic disease, antibiotic use, oral hygiene
* diet - chemical composition, frequency of intake
* saliva - flow rate, pH balance, antimicrobial factors
* gingival crevicular fluid - antimicrobial components
* micorbial interactions - competition and co-operation
* gaseous environment - oxygen concentration
28
types of periodontitis
* chronic periodontitis - adult related
* aggressive periodontitis - cause unclear could be genetic, could be one microorganism causing this
29
what causes change from healthy biofilm to disease
* dietary changes
* salivary flow
* drugs
* smoking
* alcohol
* immunodeficiency
* exacerbated by cytotoxins, proteinases, LPS which are released by bacteria
30
what causes change from healthy biofilm to disease
host factors
* smoking/tobacco use
* genetics
* pregnancy/puberty - hormonal changes
* systemic disease
* nutrition
31
summarise the development of periodontitis microbiology
* healthy tooth is colonised with normal oral flora and harmless bacteria
* in gingivitis proportion of pathogenic bacteria increases and there is mild inflammation at the gumline
* in periodontitis high conc of periodontopathogenic bacteria - severe inflammation and pocket formation plus bone resorption
* community of organisms grow deeper and anaerobic conditions allows for more aggressive biofilm
32
what type of microorganism associated with deep pocket
* gram negative anaerobic cocci associated with oxygen depletion - LPS release
* heslthy microbe associated with gram-positive aerobic
33
biofilm development stages
* adhesion
* colonisation
* accumulation
* complex community
* dispersal
34
important microorganisms
periodontal health
* oral streptococci
* actinomyces
* fusobacterium
* dont cause health as if they were to overgrow could cause disease
35
important microorganisms
gingivitis
* actinomyces
* prevotella intermedium
36
important microorganisms
periodontitis
* porphyromonas gingivalis
* T. forsythia
* T denticola
* prevotella intermedium
* A.a
37
what is socranskys model
* periodontal pathogens are identified and classified by color
* to indicate which bacteria are associated with the onset and progression of periodontal disease
* for example red found in high abunance in pt with periodontal disease
* colour code sheme
38
microbial interactions types
* competition (dominance)
* co-operation (integration)
39
microbial interactions types
competition
* metabolic products - acids, oxidants
* bacteriocins are peptides which kill other bacteria
* receptor antagosism - adhesion competition
40
microbial interactions types
co-operation
* metabolic products - saccharides, peptides, growth factors
* adhesion substrates
* immune avoidance
41
how can commesal community prevent pathogenic microorganism growth
* contraint of pathogens - restricted growth
* exclusion of pathogens - no growth
* because lots of healthy microorganisms prevent pathogen from getting in
* in disease community there is environmental modification which allows for overgrowth of pathogens
42
summarise bacterial colonisation on tooth surface
* colonisation through various adhesion molecules
* acquired pellicle on tooth surface allows adhesion of early colonisers
* late colonisers are pathogenic which bind to intermediate colonisers
43
what is koch's postulate and criteria
* guidelines for establishing that microbes cause specific diseases
* association with disease as increased numbers of pathogen at site of disease
* elimination or decreased numbers results in health
* evidence of host response to pathogen
44
describe porphyromonas gingivalis
* keystone oral pathogen increased numbers in periodontal disease - important pathogen but not structurally important (which is what keystone suggests)
* gram negative non-motile rod
* strict anaerobe
* black pigmented due to iron accumulation from hemin
45
why is porphyromonas gingivalis a keystone pathogen for perio
* increased numbers in PD
* suppressed or undetectable in successfully treated lesions
* increased serum of gingival crevicular fluid in PD
* an array of virulence factors
46
porphyromonas gingivalis
virulence factors
* fimbrae - allow adherence and invasion
* proteases which are degradative enzymes - collagenase, fibrinolysin, phospholipase
* endotoxin (LPS) which is pro-inflammatory
* capsular polysaccharide and outer membrane vesicles
* metabolic by-products which are tissue toxic - fatty acids, ammonia
47
porphyromonas gingivalis
adhesion factor
* fimbriae - long and short
* bind to cellular integrins
* long fiimbriae (FimA) - initial attachment and biofilm organisation
* short fimbriae (Mfa1) - cell to cell autoaggregation and microcolony formation
48
porphyromonas gingivalis
gingipains
* a group of arginine or lysine specific cysteine proteinases
* provide nutrition by providing peptides from heme
* have attachment component - haemaglutanin
* activates MMPs - signals tissue destruction
49
A.a periodontal disease pathogen virulence factors
* leukotoxin
* cytotoxin
* LPS
* fimbriae
* glycoprotein matrix
* subvert host cell immunity
50
periodontal disease systemic implications
* increased risk cardiovascular disease
* suggested increased risk of rheumatoid arthritis
* increased risk diabetes up to 6 fold
51
clinical management options for perio microorganisms
* mechanical disruption - toothbrushing, PMPR, RSD etc
* chemotherapeutic
* probiotics - rebalance oral microbiome
* surgery
52
53
54
where is fungi found in body
* mouth
* gut
* vagina
* skin
55
size of candida
* yeast cells approx 25-50 X the biovolume of bacteria
* high physical volume
* provide ohysical scaffold for bacteria "micofilms"
* create protective environment
56
risk factors for developing candidiasis
* immuno-comprimised patients
* long term corticosteroid use
* biggest risk factors ^
* others include - HIV infection, immunosuppressive drugs, dialysis, intra-abdominal surgery
57
candidaemia
* 4th most common bloodstream infection in the ICU setting
* due to candida albicans
* predisposing factors - broad spectrum antibiotic tx, corticosteroid tx, parenteral nutrition, malignancy, neutropenia
58
spectrum of candidal disease
* denture stomatitis
* ventilator associated pneumonia
* UTI
* sutures
* catheters
* chronic wounds
59
classification of oral candidosis
* pseudomembranous - thrush
* erythematous - atrophic or denture related
* hyperplastic - candida leukoplakia
* angular chelitis
* generalised candidosis with oral manifestations - chronic mucocutaneous
60
describe pseudomembranous candidosis
* thrush
* white patches on surface of mucosa
* easily scraped off
61
describe chronic hyperplastic candidosis
* goes into tissue
* cant be scraped off
* need to test for premalignancy so do biopsy
62
erythematous denture induced stomatitis summary
* candida can live in cracks and crevices of denture surface
* cause inflammation of mucosa
* classified by newtons type I, II or III from least to most severe
63
newtons types of denture induced stomatitis
* newtons type I - localised inflammation
* type II - diffuse inflammation
* type III - granular inflammation
64
denture induced stomatitis signs and symptoms
* inflamed mucosa
* burning sensation
* discomfort
* bad taste
* in most cases pt unaware of problem
* if aspirated can cause pneumonia
65
main candida species
* opportunistic pathogenic yeasts
* candida albicans
* candida glabrata
66
fluconadole prescription and candida type
* candida albicans sensitive to fluconazole antifungals
* candida glabrata is not - if given you will suppress other candida and let galbrata grow
67
candida albican summary
sites/kingdom/size/reproduce by/morphogenesis
* present in most healthy individuals 71%
* variable sites - oral cavity, vagina, gut
* kingdom - fungi
* small yeast 4-6um
* reproduce by budding
* can undergo morphogenesis changing from yeast to hyphae form
68
morphogenesis of candida albicans and implications
* can change from yeast to hyphae
* hyphae allows it to adhere to surface and also invade through tissue
* can go through tight junctions so potential to get into bloodstream
* galbrata doesnt form this hyphae so key characteristic for albicans
69
candida albicans enzyme interaction
* within extracelliular matrix produced it can also produce hyrdolysis enzymes
* these are enzymes that catalyse the hydrolysis of a substrate through the addition of water
* phospholipase
* haemolysin
* proteinase
70
hydrolytic enzymes produced by candida albicans and their contribution to infection
* phospholipase - hydrolysis of lipid - host cell penetration
* haemolysin - hydrolysis of RBC - fascilitate hyphae invasion
* proteinase - hydrolysis of protein - adhesion to epithelial cells
71
candida and oral cancer link
* chronic candida in mouth can drive oral cancer
* candida can produce acetaldehyde enzymes
* ADH genes key to biofilm formation
* ADH is carcinogen - causes DNA damage
72
lab processes for diagnoses of candida infections
* oral rinse and swab tend to be go to for collecting sample
* microscopy - if can see hyphae then pretty certain its candida albicans
* culture in agar
* histology
73
types of agar for candida testing
* sabourad agar only tells you youve got candida
* chromogenic agar better as shows different colours of colonies
74
types of antifungals and examples
* azoles - fluconazole
* polyenes - nystatin
* echinocandins - caspofungin
75
antifungals work in 3 different ways
* azoles work on egostrol patheways - inhibit synthesis pathways or egostrol
* polyenes act directly on egostrol - bind with it and cause pores which results in leakage of cytoplasmic content leading to cell death and lysis
* echinocandins act on b1 3 glucans enzymes - inhibit this and destabilises cell wall causing cell death
76
nystatin preparations/activity
* topical suspension, ointment
* all candida sensitive
* resistance rare
* no antibacterial activity
77
miconazole preparations/activity
* topical oral gel or cream
* candida have variable sensitivity
* posessed anti-staphylococcal activity
78
fluconazole preparations/activity
* systemic capsules
* C. albicans generally sensitive but resistant strains can develop
* C. galbrata naturally resistant
79
chlorhexidine preparations/activity
* topical solution
* antibacterial and anticandidal
80
what can prevent antifungals working
* ECM prevents penetration of antifungals
* stress
* density
* efflux
* over expressed targets
81
appropriate specimen for identifying cariogenic microorganisms children aged 5
* general swab of saliva or tooth surface
* minimal invasiveness as young children involved
82
methods for isolating bacteria
* streaking method
* pouring method
* spreading method
* serial dilution
* next-generation sequencing
* microbiome analysis of 16s rDNA sequences
83
what preventative measures could be implemented to minimise carriage of cariogenic microorganisms
* improved diet advice
* behaviour advice
* public health measures
84
what specimins could you investigate to understand microbial basis for gingivitis
* subgingival plaque biofilm
* taken using paper point from gingival crevice on site of inflammation
85
methods used to identify culprit organism
* selective agars
* PCR
* next-generation sequencing NGS
* ideally grown on plates and undertake sensitivity testing
86
how to determine cause and effect of microorganism
removal of culprit organism leads to resolution of disease
87
what types of specimen to isolate microorganisms associated with denture stomatitis
* oral rinse
* swab from localised area
* foam pad for imprint
88
systemic indications of denture stomatitis
* possibility of aspiration pneumonia
* possible (though low) links to cardiovascular disease
89
clinical specimen to take for pt with abscess/what microorganisms are you looking for
* look to obtain pus sample
* needle aspirate?
* possibility of oral anaerobes
* must have correct anaerobic transport media for collection/transport
90
techniques to identify microorganism in abscess
* standard plate culture
* possibility of microscopy
91
bacterial detection methods
* microbiological culture: culture on suitable agar medium; isolate bacteria; identify by characteristic of enzyme activities; sugar fermentation tests etc
* molecular biological: DNA probes; polymerase chain reaction PCR
92
biochemical identification (ID) of microorganisms
* anaerobes identified by their sensitivity to metronidazole dic
* gram stain
* enzyme activities/sugar fermentation
93
culture methods advantages and disadvantages
* A: yields bacterial isolates for future testing and study eg antibiotic sensitivities
* D: requires viable cells; only small number of samples can be analysed at once; inconclusive results; labour-intensive
94
describe DNA probes/types
* segments of DNA that have been labelled with chemoluminescent, fluorescent or radioactive agents
* types: whole genomic; cloned gene; oligonucleotide (20-50 bases)
95
16s rRNA gene overview
* found in all bacteria
* essential for survival
* gene sequences for all known bacteria and highly variable regions provide unique signatures to any bacterium - species-specific probes or primers
96
PCR testing overview
* highly specific and sensitive
* can be used to directly detect bacteria in clinical specimens
* ususally target 16s rRNA gene
* general bacterial primers, group specific primers or species-specific primers used
* primers hybridise to target sequence
* polymerase synthesises other strand
97
DNA probes and PCR advantages and disadvantages
* A: less time consuming than culture; very sensitive; can directly detect bacterial DNA in clinical samples; do not require viable cells; can detect uncultivated species
* D: may detect dead cells; detect only pre-selected species
98
why is it important to subtype bacteria
* subtype = different strains among isolates of the same species
* track routes of transmission during disease outbreaks
* study pathogenicity of specific strains
