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BDS3 Microbiology and Oral Pathology > Typing and Diagnostic Methods > Flashcards

Typing and Diagnostic Methods Flashcards

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1
Q

what are the four major microbes associated with periodontal disease

A

p gingivalis
AA
p intermedia
bacteroides forsythus

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2
Q

what bacteria is associated with dental caries

A

strep mutans

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3
Q

what are the two bacteria associated with root canal infections

A

porphyromonas endodontalis
fusobacterium nucleatum

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4
Q

what are two principle methods for bacteria detection

A

microbiological culture
molecular biological

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5
Q

what is the process of microbiological culture

A

culture on suitable agar medium
isolate bacteria
identify by characterisation of enzyme activities, sugar fermentation tests

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6
Q

what are the two classes of molecular biological detection

A

DNA probes
PCR

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7
Q

how do you gather a microbiological culture

A

vortex mix sample for 30 seconds
serial dilutions
spiral plate to agar media
incubate anaerobically for 10 days
obtain total bacteria counts

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8
Q

what is included in the fastidous anaerobe agar media

A

7.5% defibrinated horse blood as the bacteria like to grow on here

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9
Q

what is used sometimes in addition to FAA

A

vancomycin as it is a selective agent for gram-negative anaerobes

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10
Q

what are the two types of agar we can use for microbiological culture

A

fastidious anaerobe agar
fastidious anaerobe agar with vancomycin

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11
Q

what are the three types of biomechanical identification

A

anaerobes noted by sensitivity to metronidazole disc
gram stain
enzymatic tests, sugar fermentation tests

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12
Q

what does gram staining cause

A

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

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13
Q

what is the rapid API 32 A test

A

can identify bacteria on the basis of which enzymatic activities they possess and which sugars they can ferment to produce acid from

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14
Q

what type of bacteria is sensitive to metronidazole

A

gram negative

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15
Q

what occurs in API strips if the cupules contain yellow fluid

A

a variety of sugars have been fermented by the bacterial suspension that was placed into the cupule that would produce acid

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16
Q

what are advantages of culture methods

A

yields bacteria isolated for future testing and study

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17
Q

what are disadvantages to culture methods

A

requires viable cells
insensitive
only small numbers of samples can be analysed at once
inconclusive results
labour intensive

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18
Q

what are DNA probes

A

segments of DNA that have been labelled with chemolumiscent, fluorescent or radioactive agents

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19
Q

what are the different types of DNA probes

A

whole genomic
cloned gene - particular gene that has been cloned from a bacterial genome
oligonucleotide - target a perticular bacterial gene

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20
Q

hoe do DNA probes work

A

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

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21
Q

how do genomic probes work

A

extract and purify DNA from bacteria
DNA is cut into smaller fragments
label the fragments to create a whole genomic probe with label attached

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22
Q

what is the major problem with whole genomic probes

A

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

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23
Q

how do oligonucleotides work

A

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

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24
Q

why are oligonucleotides the DNA probes of choice

A

their high specificity

25
Q

what is hybridisation

A

when the DNA of the bacteria immobilises and reacts to the DNA probe

26
Q

what is the 16S ribosomal RNA gene

A

found in all bacteria
essential for survival
highly variable regions provide unique signatures to any bacterium

27
Q

what are advantages of PCR

A

highly specific
highly sensitive
can be used to directly detect bacteria in clinical specimens

28
Q

what is PCR

A

exponential amplification of DNA sequences

29
Q

what does PCR require

A

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

30
Q

what are the steps of PCR

A

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

31
Q

what occurs after each cycle of PCR

A

the amount of DNA is doubled

32
Q

what do the primers in PCR usually target

A

the 16S rRNA gene

33
Q

what are the three types of PCR primers

A

general bacterial primers
group specific primers
species specific primers

34
Q

what are general bacterial primers

A

target conserved region of 16S rRNA gene

35
Q

what are group specific PCR primers

A

they detect all members of a particular bacterial genus

36
Q

what are species specific primers

A

a single primer pair to detect a single bacterial species

37
Q

what is multiplex PCR

A

when multiple primer pairs are used for a single PCR reaction to detect more than one species at a time

38
Q

what are advantages of DNA probes are PCR

A

less time consuming than culture
very sensitive
can directly detect bacterial DNA
do not require viable cells
can detect uncultivable species

39
Q

what are the disadvantages of DNA probes and PCR

A

may detect dead cells
detect only preselected species

40
Q

what is PCR restriction fragment length polymorphism (RFLP)

A

used for identifying bacterial isolates that have been obtained by culture methods

41
Q

what are the two main reasons its important to subtype bacteria

A

track routes of transmission during disease outbreaks
study pathogenicity of specific strains

42
Q

what are the two traditional methods for subtyping bacteria where you test for one or more phenotypic marker

A

serotyping
biotyping

43
Q

what are the problems with traditional methods for subtyping bacteria

A

limited discriminatory capacity
organism specific methods
specialised reagents required

44
Q

what is an alternative to traditional subtyping methods

A

molecular genetic typing methods which are DNA based

45
Q

what are five examples of molecular genetic typing

A

restriction enzyme analysis
gene probe typing
ribotyping
16S-23S intergenic spacer region
DNA sequencing

46
Q

what is restriction enzyme analysis

A

whole genomic DNA is digested with one or more restriction enzyme

47
Q

why can restriction enzyme analysis not be resolved on an agar gel

A

many thousands of DNA fragments are generated

48
Q

why is it almost impossible to discriminate between different strains in REA

A

DNA fragments tend to merge together producing a smear effect

49
Q

what is gene probe typing

A

can reduce number of DNA fragments generated by REA using a suitable gene probe
number of fragments for analysis are greatly reduced

50
Q

what is ribotyping

A

they use e. coli rRNA operon as a DNA probe following REA
rRNA operon is present in multiple copies in bacterial genomes

51
Q

what is the process of ribotyping

A

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

52
Q

what is 16S-23S intergenic spacer region

A

very variable sequence - different strains of same bacterial species will have a different sequence

53
Q

where does the intergenic spacer region lie

A

between 16S and 23S ribosomal RNA genes

54
Q

what is the ultimate typing method

A

DNA sequencing

55
Q

why is DNA sequencing the ultimate typing method

A

can detect single base differences between strains

56
Q

what occurs in DNA sequencing

A

sequences all the bases in a stretch of DNA and detects even single base changes between different bacterial strains of the same species

57
Q

how can we identify uncultivable or even novel bacteria in clinical samples

A

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

58
Q

what indicates that the clone sequence has originated from a particular bacterial species

A

gene sequence match of 98%

59
Q

what does a gene sequence match of less than 97% with known gene sequences indicate

A

potentially novel bacterial species

BDS3 Microbiology and Oral Pathology (6 decks)

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