Flashcards in Public Health bacteriology Deck (70):
What are the two typing systems?
Name a method of genotyping which can be used to determine the phylum of the bacteria?
1. Microscopy, Gram stain
Name 4 types of phenotyping used to determine the species of the bacteria?
1. Differential and selective medial
2. Biochemical tests
3. Numerical taxonomy
4. infrared spectroscopy
Name 2 phenotypic tests to determine subspecies?
1. fatty acid analysis
Name 3 phenotypic methods to determine strain of bacteria?
1. 2D PAGE
2. MALDI-TOF MS
Name 1 method used for genotyping the species?
Name 2 genotyping methods used for subspecies?
1. DNA-DNA hybridisation
2. rRNA sequencing
Name 6 methods used to genotypically determine the strain of bacteria?
1. Genetic fingerprinting (PCR)
2. Multilocus Sequence Typing
4. Whole genome sequencing
5. Pulse Field Gel Electrophoresis
Name 5 advantages of using phenotypic typing methods?
2. basic equipment
3. easier to interpret results
4. greater acceptance by clinical community, 'gold standard'
5. based on live isolates
Name 3 disadvantages of using phenotypic typing methods?
2. variable results
3. lower resolution
Name 4 advantages of using genotypic typing methods?
2. highest resolution (e.g. SNPs)
3. Automation, parallel processing
4. lower variability
Name 4 disadvantages of using genotypic typing methods?
2. highly trained staff and complex equipment
3. dead bugs. garbage in = garbage out.
4. acceptance/ understandning
Name 3 methods of genotypic target gene detection?
1. southern blotting
2. DNA sequencing
3. PCR (conventional, real-time)
Name 6 methods of genotypic DNA fingerprinting and molecular subtyping?
1. Pulsed-field gel electrophoresis
3. Amplified fragment length polymorphism
5. Multilocus sequence typing
6. DNA microarray
What are becoming the two preferable methods for genotypic typing?
MLST and DNA microarrays
How are genotype and phenotype linked?
The phenotype is defined by the genotype
What is recommended when tackling an outbreak investigation?
Using a combination of typing methods.
Which legislation states that Listeria should be absent in processed meat?
EC 2073/ 2005
Presence/ absence tests are used a lot in what hygiene?
Meat hygiene - 2-class and 3-class sampling plans.
Give an example of presence/absence tests.
Listeris should not be present therefore not interested in strain therefore selective agar can be used - positive batches rejected.
What does listeria look like on Oxford agar?
Black/ brown colonies with black/ brown halos.
What is the next thing you have to determine when investigating a disease outbreak?
At what % does DEFRA detect disease at and why?
5% prevalence or above - limited sensitivity
What is the equation for working out prevalence?
(100/x) * 3
x = prevalence
Name two methods you can use to limit the number of potential sources of infection (source attribution)?
2. relative risk analysis
Name an incredibly important aspect of epidemiology and issues associated with this?
Source attribution - economic and legal issues associated.
Describe Pulse Field gel electrophoresis?
Whole genomes of bacteria are cut with restriction enzymes called rare cutters which cut infrequently in the genome, yielding large segments. These fragments are separated by PFGE.
Explain why PFGE is very useful?
Because it correctly identifies the strains determining the source of infection.
What is the issue with salmonella enterica?
it contains more than 2, 500 serotypes.
Name 3 salmonella enteric serotypes which are host restricted?
3. Typhi (man - typhoid)
What does it mean that there are lots of phagetypes of salmonella?
Need to identify the phage type AND the strain
How does PCR work and what are the two types?
Selectively amplifies a small, targeted area of the genome (gene or locus).
1. Uniplex PCR (one gene/ locus)
2. Multiplex PCR (multiple genes/ loci)
Name 2 advantages of PCR?
2. relatively quick
Name 4 disadvantages of PCR?
1. needs specialist equipment
2. Difficult to use directly for environmental samples
3. does not distinguish between live and dead cells
4. Only samples a small part of the genome, so may falsely link isolates which have differences elsewhere in their genome.
What does phage typing do?
Determines sensitivity of a bacterium to infection with a panel of different bacterial viruses (phage)
Name 2 advantages of phage typing?
2. no specialist equipment needed
Name 4 disadvantages of phage typing?
1. phage sensitivity may change
2. needs skilled person to interpret
3. labour intensive
4. needs live bacteria which can be easily grown to produce a 'lawn' onto which the phage are spotted.
What is a bacteriophage?
A virus which parasitizes a bacterium by infecting it and reproducing inside it.
name two ways sensitivity to a phage can change?
Either strain infected with new phage or they acquire antibiotic resistance
Name 3 advantages of PFGE?
2. samples the whole genome, rather than a small part e.g. PCR
3. Can be standardised across multiple laboratories
Name 2 disadvantages of PFGE?
1. expensive equipment and software needed
What is serotyping?
Bacteria mixed with antisera which has been raised against specific serotypes of pathogens. When there are antibodies present, cause agglutination of cells (positive result)
Name 2 advantages of serotyping?
1, specific and robust
2. easy to perform and interpret
Name 2 disadvantages of serotyping?
1. very expensive if lots of serotypes involved e.g. >2,500 for salmonella
2. can ID to species and serotype/serovar level, but not strain level.
What is bacterial cell culture used for?
Selective and non-selective media to isolate a pathogen from background contamination.
What is an advantage of enrichment media?
Can recover low levels of sub-lethally injured cells before subjecting them to selective media which may otherwise kill them.
What if large numbers of bacteria are suspected?
Direct plating onto selective agar.
What bacteria is selective agar frequently used for?
Salmonella and campylobacter
Does PCR require isolation of live bacteria?
What is PCR commonly used for?
Identification of poultry and pig viruses
How do salmonella and E. coli colonies differentiate on XLD agar?
Salmonella: Black colonies
E. coli: yellow colonies
What is the function of MacConkey Agar?
Differentiates lactose-fermenting (e.g. E.coli) from non-lactose fermenting e.g. (Salmonella) enterobacteria.
What is the function of Brilliant Green Agar?
Differentiates salmonella (red colonies) from other types of bacteria e.g. (E.coli = yellow colonies)
What colour is E.coli generally on selective media?
What colour is salmonella generally?
red or black.
Name 4 advantages of bacterial cell culture?
1. you have an isolate which can be processed for further testing (phenotypic and genotypic)
2. Easy to perform, can be more difficult to interpret results
3. possible to enumerate bacteria in a sample
4. often seen as a 'gold standard'
name 4 disadvantages of bacterial cell culture?
1. labour intensive
2. may not recover sub-lethally injured cells
3. some bacteria difficult to grow or very slow (Mycoplasma/mycobacteria)
4. There may be multiple strains of a pathogen present in a sample, and you might only recover one of them.
What is IMViC tests?
A series of biochemical tests which distinguish between different coliform bacteria.
What tests does IMViC involve?
2. Methyl Red
Name two advantages of IMViC tests?
1. easy to perform
2. Robust results
Name two disadvantages of IMViC tests?
1. does not distinguish between strains of the same bacterium
2. labour intensive
Name 5 features of coliform bacteria?
2. Gram -ve
3. Non-spore forming
4. Motile or non-motile
5. Can ferment lactose producing acid and gas when incubated 35-37 degrees C
What is the E.coli result of the IMViC test?
Positive Methyl Red
Negative Voges- Proskauer
What is the Salmonella result of the IMViC test?
Indole - negative
Methyl red - positive
Voges-Proskauer - negative
Citrate - positive
What is whole genome sequencing?
The ultimate level of discrimination
-computers compare genomes and detect similarities/ differences.
Name 2 disadvantages of whole genome sequencing?
1, requires expensive machinery, software and technical staff.
What is the cost of WGS?
Used to be £1 million, not £30
What is a general problem with WGS?
How many differences to tolerate before you consider bacteria different strains?
What can be constructred following WGS?
Phylogenetic trees showing similarities between isolates.