Diagnostics and Typing Flashcards
Which bacteria are commonly associated with periodontal disease?
Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia, Bacteroides forsythus.
What is the primary bacterial cause of dental caries?
Streptococcus mutans.
Name bacteria commonly found in root canal infections.
2
Porphyromonas endodontalis, Fusobacterium nucleatum.
What are the two main types of bacterial detection methods?
Microbiological culture
Molecular biological methods (DNA probes, PCR)
How are bacteria identified using microbiological culture?
By isolating bacteria on agar and characterising enzyme activity and sugar fermentation.
What are the steps in microbiological culture of oral bacteria?
Vortex sample for 30s
Serial dilutions to 10⁻⁶ in FAB
Spiral plate on FAA + 7.5% horse blood
Use selective FAA with vancomycin
Incubate anaerobically for 10 days
Obtain total bacterial counts
How are anaerobic bacteria identified biochemically?
Sensitivity to metronidazole disc (5μg/disc)
Gram stain
Rapid API 32A tests (enzyme activity, sugar fermentation).
Metronidazole Sensitivity: Determines the bacterium’s anaerobic nature by assessing its response to the drug.
Gram Stain: Helps distinguish between Gram-positive and Gram-negative anaerobic bacteria, providing insight into their basic morphology.
API 32A Tests: Analyzes enzyme activity and sugar fermentation to generate a metabolic profile for identifying the species of the anaerobe.
What are the advantages and disadvantages of culture methods?
pro - Yields bacterial isolates for future testing, e.g. antibiotic sensitivities.
con - Requires viable cells, low sensitivity (10⁵–10⁶ cells), time-consuming, labour-intensive, inconclusive, limited samples.
What are DNA probes and how are they labelled?
DNA segments labelled with chemoluminescent, fluorescent, or radioactive agents.
What types of DNA probes exist?
Whole genome
Cloned gene
Oligonucleotide probes (20–50 bases).
How do DNA probes compare to culture methods in sensitivity?
More sensitive—can detect as few as 10³ cells.
Why is the 16S rRNA gene commonly used in bacterial identification?
Present in all bacteria, essential for survival, contains variable regions for species-specific probes, and conserved regions for broad-range detection.
What are the key features of PCR in bacterial detection?
Highly specific and sensitive; detects bacteria directly from clinical specimens.
What are the types of primers used in PCR?
General bacterial primers
Group-specific primers
Species-specific primers (can be single or multiplex)
What is multiplex PCR?
PCR using multiple primer pairs to detect several species simultaneously.
What are the advantages of DNA probes and PCR over culture methods?
Faster
More sensitive (PCR: 10 cells)
Direct detection
Doesn’t require viable cells
Detects uncultivable species
What are the disadvantages of DNA probes and PCR?
May detect dead cells
Limited to pre-selected species
What is PCR-RFLP and its purpose?
Restriction digestion of PCR-amplified 16S rRNA to create species-specific fingerprints. It’s rapid, cheap, and specific.
Why is subtyping of bacterial isolates important?
Tracks disease transmission
Studies strain-specific pathogenicity
What traditional methods are used for subtyping bacteria?
Serotyping: Identifies bacteria based on surface antigens.
Biotyping: Characterizes bacteria based on biochemical properties.
What are the limitations of traditional typing?
Low discrimination: Doesn’t always differentiate closely related strains.
Organism-specific: Methods may be limited to specific species.
Need for specialized reagents: Requires specific tests and chemicals.
What is REA and its limitation?
Digestion of genomic DNA using restriction enzymes to create patterns for analysis
. Limitation: too many fragments, making analysis difficult.
How does gene probe typing improve REA?
uses specific probes to target certain DNA fragments, simplifying the interpretation by reducing the number of fragments.
What is ribotyping?
Uses the rRNA operon (commonly from E. coli) as a probe after REA to detect variation in DNA fragments that hybridise with rRNA.
Why is ribotyping effective?
They exist in multiple copies in bacteria, allowing for detection of strain-specific variations.
What is the 16S–23S intergenic spacer (IGS) region and how is it used in typing?
A highly variable region amplified by PCR and digested with restriction enzymes to produce strain-specific fingerprints.
What makes DNA sequencing the ultimate typing method?
It can detect even single base differences between bacterial strains.
How are uncultivable and novel bacteria identified molecularly?
Extract DNA
PCR amplify 16S rRNA
Clone genes
Randomly sequence 50 clones
Analyse sequences (e.g., via BLAST)
