Diagnostic and typing methods Flashcards
1
Q
What are the main bacteria associated with periodontal disease?
A
- Porphyromonas gingivalis
- Actinobacillus actinomycetemcomitans
- Prevotella intermedia
- Bacteroides forsythus
2
Q
What are the main bacteria associated with dental caries?
A
- Streptococcus mutans
3
Q
What are the main bacteria associated with root canal infections?
A
- Porphyromonas endodontalis
- Fusobacterium nucleatum
4
Q
What are the two bacterial detection methods?
A
- Microbiological culture
- Molecular biological
5
Q
What is the microbiological culture method?
A
- Vortex mix sample for 30 seconds to get homogenous suspension of bacteria
- Serial dilute samples in FAB (fastidious anaerobe broth) to 10-^6
- Spiral plate to agar media using either;
a - Fastidious anaerobe agar (FAA) + 7.5% defibrinated horse blood
b - same as a but supplemented with vancomycin for gram-negative anaerobes - Incubate anaerobically for 10 days
- Obtain total bacterial counts
- Can then sub culture specific bacteria on separate agar medium
6
Q
What are the two molecular biological methods?
A
- Use DNA probes
- Polymerase chain reaction (PCR)
7
Q
Why might we want to use vancomycin supplement when looking for oral disease?
A
- Vancomycin is a selective agent for gram-negative anaerobes
- Many oral disease are caused by gram- negative anaerobes so look for these spdecifically
8
Q
Why must we conduct serial dilutions in microbiological culture methods?
A
- If not then would be so many bacteria that would not be able to discern individual colonies
9
Q
What colour do genera porphyromonas and prevotella bacteria appear on culture medium?
A
- Appear black pigmented
- Associated with periodontal disease
10
Q
What are the 3 ways we can biochemically identify the isolated bacteria?
A
- Anaerobes noted by their sensitivity to metronidazole disc (5ug/disc)
- Gram stain
- Rapid API 32 A: compare enzymatic activities , sugar fermentation of those bacteria to those held on central database
11
Q
What is the outcome of gram staining?
A
Gram-positive bacteria = violet colour due to thick peptidoglycan layer in cell wall and retains the gram stain
Gram-negative bacteria = won’t stain as only thin peptidoglycan layer in cell wall
12
Q
What does it mean if we see a zone of clearing in agar due to metronidazole disc?
A
- Growing a gram-negative anaerobe
13
Q
What are the advantages of microbiological culture methods?
A
- Yields bacterial isolates
- Can use these for future testing and to study e.g. for antibiotic sensitivities
14
Q
What are the disadvantages to microbiological culture methods?
A
- Requires viable cells (most die off and aren’t detected)
- Insensitive (only show if 10^5-10^6 cells)
- Only small numbers of samples analysed at once
- Inconclusive results
- Labour-intensive
- Expensive process
15
Q
What are DNA probes?
A
- Segments of DNA that have been labelled with chemoluminescent, fluorescent or radioactive agents
16
Q
What are some types of DNA probes?
A
- Whole genomic (entire genome)
- Cloned gene
- Oligonucleotide (20-50 bases)
17
Q
How sensitive are DNA probes compared to culture?
A
- 10^3 cells required to detect species so more sensitive
18
Q
How is the DNA probe prepared?
A
- Probe is DNA double stranded molecule
- Need to pull apart so label can be attached
- Heat is used to denature the DNA and expose bases on both strands
- One strand then labelled with either chemiluminescent, radioactive or fluorescent label
19
Q
How is the sample prepared ready for the DNA probe?
A
- Extract double stranded DNA from sample
- Denature the DNA to single strands using heat
20
Q
What is a hybridisation reaction in DNA probe method?
A
- Mix the single stranded sample and DNA probes
- Probe binds to its complimentary sequence of DNA within sample if that bacterial species is present
- Remove any non-binded DNA
- Leaves use with labelled DNA probe identified within sample
21
Q
How are genomic probes made?
A
- Extract and purify DNA from bacterial species you wish to investigate
- DNA cut into smaller fragments and labelled
22
Q
What are some disadvantages of genomic/cloned gene probes?
A
- Used back in 80’s when we didn’t have genetic info available for different bacterial species
- Extremely non specific
- Lots of cross-reactivity between whole genomic probes which made them unreliable
23
Q
How are cloned gene probes made?
A
- A gene of interest would be cloned into E.coli
- Cloned fragment isolated, purified and label attached
- Gene is specific to bacterial species you are looking for (more specific than genomic)
24
Q
How do oligonucleotides probes work?
A
- Very small in size
- Target 16S ribosomal RNA gene (which all bacteria possess)
- Possible to synthesise species specific probes that target one or more hypervariable regions
- Synthesised single stranded oligonucleotides labelled and used as probe
- Best DNA probe due to specificty
25
Why is the 16S ribosomal RNA gene ideal for probes?
- All bacteria possess it
- Approx 1500 base pairs in length
- Possesses nice hypervariable regions (V1 to V9) that contain DNA sequences that provide specific signature for each bacterial species
- Conserved regions give a very broad range and useful as probes or PCR primers
26
Why is Polymerase chain reaction good for detecting bacteria in clinical specimens?
- Highly specific
- Highly sensitive
- Can be used to directly detect bacteria in clinical specimens
27
What is required for PCR to work?
- Double stranded DNA template from sample
- Primers specififc to particular gene e.g. 16S ribosomal RNA of gene wanting to detect
- Deoxynucleoside trisphospates
- Enzyme Taq DNA polymerase which catalyses synthesis of new DNA strands
28
How does PCR work?
- Double stranded DNA from sample heat denatures at 94C (aka denaturation step)
- PCR primers hybridise target sequences on each DNA strand (aka primer annealing step)
- Taq DNA polymerase synthesises opposite strand incorporating dNTPs (aka primer extension step)
- After each cycle of this the DNA is doubled
- Cycle repeated up to 35 cycles
29
What different types of primers are there?
- General bacterial primers
- Group-specific primers
- Species-specific primers (either single primer pair or more than one primer pair)
- All usually target 16S ribosomal RNA gene
30
What does lanes with no PCR product mean and large amount of PCR?
No PCR = no bacteria you're interested in
Large amount PCR = Greater the amount of bacteria interested in
31
What are the advantages of DNA probes and PCR over microbiological culture?
- Less time-consuming than culture methods
- Very sensitive (DNA probes, 103 cells; PCR, 10 cells)
- Can directly detect bacterial DNA within clinical samples
- Do not require viable cells, samples do not have to be analysed immediately
- Can detect uncultivable species
32
What are the disadvantages of DNA probes and PCR over microbiological culture?
- May detect dead cells
- Detect only pre-selected species
33
What is PCR-RFLP?
- PCR - restriction fragment length polymorphisms
- Identification of bacterial isolates
- Digests PCR-amplified bacterial 16s rRNA gene with restriction enzymes
- Yields specific patterns (fingerprints) for individual species
34
What are the benefits of PCR-RFLP?
- Rapid
- Cheaper
- More specific alternative to biochemical tests for identifying bacterial isolates from clinical samples
35
Why is it important to subtype bacteria?
- Track rotes of transmission during disease outbreaks
- Study pathogenicity of specific strains
36
What is subtyping?
- Sufficient genetic diversity exists to allow identification of different clones (strains) among isolates of same species
37
What are the traditional methods for subtyping bacteria?
- Serotyping
- Biotyping
38
What are some problems with serotyping and biotyping?
- Limited discriminatory capacity
- Organism-specific methods
- Specialised reagents required that are costly
39
What is an alternative to traditional subtyping methods?
- Molecular (genetic) typing methods
40
What is restriction enzyme analysis (REA)?
- Type of molecular typing method
- Digest whole genomic DNA with restriction enzymes
- But too many DNA fragments obtained so makes interpretation difficult
41
What is gene probe typing?
- Reduces number of DNA fragments generated by REA using suitable gene probe
42
What is ribotyping?
- Type of gene probe typing
- Use E.coli rRNA operon as DNA probe following REA
-Variation in number and size of fragments in bacterial DNA digest are complimentary with rRNA
43
What is the procedure for any gene probe typing method?
- Genomic DNA extracted from bacterial isolate or clinical sample
- Digested with one or more restriction enzymes to give many DNA fragments
- DNA fragments separated into distinct bands by agarose gel electrophoresis
- Smaller ones migrate quicker
- DNA bands transferred to nylon membrane
- Hybridisation to labelled DNA probe
- Probe is visualised and DNA fragments compared
44
What is 16S-23S intergenic spacer region (IGS)?
- Simple typing method is PCR-RFLP analysis of 16S-23S IGS
- Very variable sequence
- Amplify region by PCR using consensus primers
- Digest PCR product with restriction enzymes to obtains stain-specific DNA finger prints
45
What is DNA sequencing?
- Sequences all the bases in stretch of DNA and detects even single base changes between different bacterial strains of same species
- Best typing method
46
How can we detect uncultivable and novel bacteria using DNA-based methods?
- Genomic DNA extracted from sample
- PCR amplifies 16S rRNA gene from bacterial DNA in clinical specimen
- 16S rRNA genes cloned
- 50 clones randomly chosen and sequenced
- Clone sequences analysed to determine bacterial identity (BLAST)
