Typing and Diagnostic Methods Flashcards
what are the four major microbes associated with periodontal disease
p gingivalis
AA
p intermedia
bacteroides forsythus
what bacteria is associated with dental caries
strep mutans
what are the two bacteria associated with root canal infections
porphyromonas endodontalis
fusobacterium nucleatum
what are two principle methods for bacteria detection
microbiological culture
molecular biological
what is the process of microbiological culture
culture on suitable agar medium
isolate bacteria
identify by characterisation of enzyme activities, sugar fermentation tests
what are the two classes of molecular biological detection
DNA probes
PCR
how do you gather a microbiological culture
vortex mix sample for 30 seconds
serial dilutions
spiral plate to agar media
incubate anaerobically for 10 days
obtain total bacteria counts
what is included in the fastidous anaerobe agar media
7.5% defibrinated horse blood as the bacteria like to grow on here
what is used sometimes in addition to FAA
vancomycin as it is a selective agent for gram-negative anaerobes
what are the two types of agar we can use for microbiological culture
fastidious anaerobe agar
fastidious anaerobe agar with vancomycin
what are the three types of biomechanical identification
anaerobes noted by sensitivity to metronidazole disc
gram stain
enzymatic tests, sugar fermentation tests
what does gram staining cause
stains gram positive bacteria a violet colour as they have a very thick peptidoglycan layer in cell wall
gram negative will not stain as they have a very thin peptidoglycan layer
what is the rapid API 32 A test
can identify bacteria on the basis of which enzymatic activities they possess and which sugars they can ferment to produce acid from
what type of bacteria is sensitive to metronidazole
gram negative
what occurs in API strips if the cupules contain yellow fluid
a variety of sugars have been fermented by the bacterial suspension that was placed into the cupule that would produce acid
what are advantages of culture methods
yields bacteria isolated for future testing and study
what are disadvantages to culture methods
requires viable cells
insensitive
only small numbers of samples can be analysed at once
inconclusive results
labour intensive
what are DNA probes
segments of DNA that have been labelled with chemolumiscent, fluorescent or radioactive agents
what are the different types of DNA probes
whole genomic
cloned gene - particular gene that has been cloned from a bacterial genome
oligonucleotide - target a perticular bacterial gene
hoe do DNA probes work
prepare probe and sample
double stranded DNA pulled apart to a single strand by heat denature
mix probe and sample
probe will bind to complementary DNA in the sample if the particular bacterial species is present in the sample
remove any non-binding DNA
the DNA left is the probe left in the sample
how do genomic probes work
extract and purify DNA from bacteria
DNA is cut into smaller fragments
label the fragments to create a whole genomic probe with label attached
what is the major problem with whole genomic probes
there is a lot of cross reactivity between whole genomic probes for one particular bacterial species as they share a lot of the same gene sequences
how do oligonucleotides work
target 16S ribosomal RNA gene
the gene is ideal as it targets the part of the gene that contains the unique DNA sequences that provide specific signatures for each bacterial species
synthesised single stranded oligonucleotide is labelled and used as a probe
why are oligonucleotides the DNA probes of choice
their high specificity
what is hybridisation
when the DNA of the bacteria immobilises and reacts to the DNA probe
what is the 16S ribosomal RNA gene
found in all bacteria
essential for survival
highly variable regions provide unique signatures to any bacterium
what are advantages of PCR
highly specific
highly sensitive
can be used to directly detect bacteria in clinical specimens
what is PCR
exponential amplification of DNA sequences
what does PCR require
double stranded DNA template from sample
DNA primers which are specific for a particular gene
dNTPs - building blocks of DNA
taq DNA polymerase - catalyses synthesis of new DNA strands
what are the steps of PCR
double stranded DNA pulled apart - heat denatured
PCR primers hybridise to their target sequences - annealing primer
Taq DNA polymerase synthesises opposite strand incorporating dNTPs - primer extension
what occurs after each cycle of PCR
the amount of DNA is doubled
what do the primers in PCR usually target
the 16S rRNA gene
what are the three types of PCR primers
general bacterial primers
group specific primers
species specific primers
what are general bacterial primers
target conserved region of 16S rRNA gene
what are group specific PCR primers
they detect all members of a particular bacterial genus
what are species specific primers
a single primer pair to detect a single bacterial species
what is multiplex PCR
when multiple primer pairs are used for a single PCR reaction to detect more than one species at a time
what are advantages of DNA probes are PCR
less time consuming than culture
very sensitive
can directly detect bacterial DNA
do not require viable cells
can detect uncultivable species
what are the disadvantages of DNA probes and PCR
may detect dead cells
detect only preselected species
what is PCR restriction fragment length polymorphism (RFLP)
used for identifying bacterial isolates that have been obtained by culture methods
what are the two main reasons its important to subtype bacteria
track routes of transmission during disease outbreaks
study pathogenicity of specific strains
what are the two traditional methods for subtyping bacteria where you test for one or more phenotypic marker
serotyping
biotyping
what are the problems with traditional methods for subtyping bacteria
limited discriminatory capacity
organism specific methods
specialised reagents required
what is an alternative to traditional subtyping methods
molecular genetic typing methods which are DNA based
what are five examples of molecular genetic typing
restriction enzyme analysis
gene probe typing
ribotyping
16S-23S intergenic spacer region
DNA sequencing
what is restriction enzyme analysis
whole genomic DNA is digested with one or more restriction enzyme
why can restriction enzyme analysis not be resolved on an agar gel
many thousands of DNA fragments are generated
why is it almost impossible to discriminate between different strains in REA
DNA fragments tend to merge together producing a smear effect
what is gene probe typing
can reduce number of DNA fragments generated by REA using a suitable gene probe
number of fragments for analysis are greatly reduced
what is ribotyping
they use e. coli rRNA operon as a DNA probe following REA
rRNA operon is present in multiple copies in bacterial genomes
what is the process of ribotyping
genomic DNA extracted from clinical sample and digested with one or more restriction enzymes to give fragments
DNA fragments are separated to bands by agarose gel electrophoresis - smaller more mobile DNA fragments migrate more quickly
DNA bands in the gel are transferred to a nylon membrane in prep for hybridisation with the labelled DNA probe
DNA probe binds to specific DNA fragments on the membrane
what is 16S-23S intergenic spacer region
very variable sequence - different strains of same bacterial species will have a different sequence
where does the intergenic spacer region lie
between 16S and 23S ribosomal RNA genes
what is the ultimate typing method
DNA sequencing
why is DNA sequencing the ultimate typing method
can detect single base differences between strains
what occurs in DNA sequencing
sequences all the bases in a stretch of DNA and detects even single base changes between different bacterial strains of the same species
how can we identify uncultivable or even novel bacteria in clinical samples
DNA is extracted from clinical sample
general primers used to amplify the 16S rRNA gene
16S rRNA genes are separated by cloning onto suitable plasma vector and introduced to e. coli
50 clones randomly chosen and sequenced using Sanger method to give snapshot of clone library
once clone sequences are obtained 16S rRNA genes of the unkown bacterial species in the samples are analysed using a programme called BLAST which compares it to known bacterial species in public access databases
what indicates that the clone sequence has originated from a particular bacterial species
gene sequence match of 98%
what does a gene sequence match of less than 97% with known gene sequences indicate
potentially novel bacterial species
