Quiz 4 Content Flashcards
What does phenotypic subtyping of pathogens include? [3]
- Serotyping
- Phage typing
- Antimicrobial resistance profiling
Phenotypic - can be observed with the eyes
These have some utility for characterizing bacteria but are not adequate for outbreak detection principally due to low diversity. Plus, some require a high amount of specialization and the reagents are not available to all public health labs.
Why is the methodology of phenotypic subtyping of pathogens not adequate for outbreak detection?
Principally due to low diversity, but also because some require a high amount of specialization and the reagents are not available to all public health labs.
Phenotypic subtyping methods are useful in terms of initial characterization, but they are not very specific.
What are the criteria for effective subtyping methods? [4]
- Markers must be (1) stable, (2) consistent, and (3) present in all isolates
- Method must have (1) a high discriminatory power with (2) the ability to assign a designation for all isolates
- Must be reproducible over time
- Convenience criteria: (1) Be flexible and (2) be technically uncomplicated
What do molecular subtyping methods generally rely on?
Detecting differences in the nucleic acid sequence of organisms
Name two molecular subtyping methods.
- Fragment-based genotyping; older approach (e.g., PFGE)
- Nucleic acid sequencing-based genotyping; newer approach (e.g., MLST; WGS)
What does fragment-based genotyping rely on?
- Comparison of one or many DNA fragments produced from an isolate
e.g., pulsed-field gel electrophoresis (PFGE)
Give two examples of nucleic acid sequencing-based genotyping.
- Multiple-locus sequence typing (MLST)
- Whole genome sequencing (WGS)
Describe pulsed-field gel electrophoresis.
- Gold standard for bacterial subtyping
- Produces a genetic ‘fingerprint’ of a bacterial isolate
- Achieved through breaking of chromosomal DNA into segments by restriction enzymes
- Creates and visualizes segments of DNA from a bacterial sample to be compared with other samples
- Advanced method of gel electrophoresis
Describe bacterial chromosomal DNA.
- Circular DNA molecule within bacterial cell
- Carries all ‘normal’ genes employed for growth
- Sequence specific for each species and even each strain
How does PFGE work?
- Uses molecular scissors, called restriction enzymes, to cut bacterial DNA at the restriction sites
- These molecular scissors are selected to generate a small number of DNA pieces that can be separated based on size
- Usually these DNA pieces, or restriction fragments, are large and need to be specially treated and separated to generate a DNA fingerprint
How does PFGE differ from regular gel electrophoresis?
- Regular gel electrophoresis cannot separate large size DNA molecules that are more than 50 KB - genome of foodborne bacterial pathogens is usually 3-5 MB
Describe the PFGE process [5].
- Bacterial cells are taken from an agar plate
- Cells are mixed with melted agarose and poured into plug mold
- Cells are broken open with biochemicals, or lysed, so that the DNA is free in the agarose plugs
- DNA gelatin plug is loaded into a gel, and an electric field is applied that separates the DNA fragments according to their size
- The gel is stained so that the DNA can be seen under UV light; a digital camera takes a photo and stores it on a computer.
How are gels visualized in PFGE?
- Similar to PCR product visualization, following electrophoresis, PFGE gels are:
- Stained with ethidium bromide
- Visualized through exposure to UV light
Describe the analysis of PFGE patterns.
- Comparison of patterns using software
- Statistical measure of similarity
- Can produce dendograms
What is PulseNet?
A global network of PFGE results
What are the advantages of PFGE? [3]
- Easily applied to different species
- Generally yields a high amount of pattern diversity that provides good discriminatory power and other measures of subtyping validity
- DNA restriction patterns are stable and reproducible
What are the limitations of PFGE? [8]
- Time consuming
- Requires trained and skilled technician
- Does not discriminate between all unrelated isolates
- Pattern results vary slightly between technicians
- Don’t really know if bands of the same size are the same pieces of DNA
- Change in one restriction site can mean more than one band change
- “Relatedness” should be used as a guide, not true phylogenic measure
- Some strains cannot be typed by PFGE
What are the advantages of multiple-locus sequence typing (MLST)? [4]
- Uses sequence data and can therefore detect changes at the DNA level
- A generic technique that can be readily reproduced and does not require access to specialized reagents or training
- Based on PCR, do not require direct access to live bacterial isolates or high-quality genomic DNA
- The data generated are fully portable among laboratories and can be shared throughout the world
What are the limitations of multiple-locus sequence typing? [2]
- Uses only seven loci (for the housekeeping genes), which limits the ability to detect some switches
- Requires performing PCR and sequencing of the PCR products using an automated sequencer, which is not always available
Briefly introduce whole-genome sequencing.
- Bacterial genomes are roughly 1000 times smaller than the human genome (e.g., Listeria monocytogenes genome is ~3 MB)
- WGS platforms involve many different technologies all of which generate large, genome-scale datasets
How does whole genome sequencing work?
- Specimen - selection and cultivation
- Bacterial culture - DNA isolation
- Genomic DNA - quality control and DNA processing
- Sequencing library - sequencing
- Raw sequence data - data processing and analysis
- Analysis results - central data repository; summarize
- Diagnostic report
What is surveillance? [4]
- Collection
- Analysis
- Interpretation
- Dissemination
… that leads to action to prevent and control disease.
List the steps in an outbreak investigation.
- Detect possible outbreak
- Find cases in outbreak
- Generate hypotheses through interviews
- Test hypotheses through analytic studies and laboratory testing
- Solve point of contamination and original source of outbreak vehicle
- Control outbreak through recalls, facility improvements, and industry collaboration
If cases stop, decide outbreak is over.
If cases continue, continue to generate hypotheses for testing.
How does public surveillance help in outbreak detection?
- Reportable diseases via public health surveillance
- Cluster: a larger number of people than expected appear to have the same illness in a given time period and area
How are the effects of recalls tracked in outbreak investigation?
- Investigators use an epidemic curve or epi curve to track the number of illnesses over time.
How is odds ratio calculated?
Example:
Exposed & Diseased: 7
Exposed & No disease: 10
Non-exposed & Diseased: 6
Non-exposed & no disease: 56
OR = (7/10) / (6/56) = 6.5
or
OR = (7/10) * (56/6) = 6.5
OR > 0, so exposure is associated with higher odds of disease.
