resident oral microbiota Flashcards
(101 cards)
1
Q
what 3 types of plaque are present in the mouth
A
fissure (1.33 x 10^6 bacteria/fissure)
approximal
sub-gingival (10^3-10^6 bacteria/crevice)
each has a varying bacterial load
as anatomy + biogeography at these sites varies greatly
2
Q
define plaque
A
community of micro-organisms found on the tooth surface as a biofilm embedded in a matrix of polymers of salivary and bacterial origin
3
Q
how many bacterial cells are in 1ml of saliva
A
up to a billion
100 bacteria/cell on tongue dorsum
4
Q
what does saliva under a darkfield microscope show and why
A
shows saliva is v lively / teeming w life + not static
darkfield is
- different to gram staining
- uses light with dark background
- light reflected onto LIVING organism so can view them when alive - for most staining procedures you have to kill the organism you’re trying to view
5
Q
how do we study microbes
A
- grow / culture in lab
- use broth + agar plates
6
Q
what are the types of agar
A
NORMAL = contains all nutrients required for bacterial growth (sugars, minerals, protein in form of yeast / meat extracts)
BLOOD AGAR = contains either horse or sheep blood (found to be rlly nutritious + allow growth of many microorganisms)
7
Q
what is a columbia blood agar
A
- non-selective medium
- allows growth of most bacteria
8
Q
give some examples of selective media
A
MITIS SALIVARIUS BACITRACIN AGAR (selective for S. mutans)
SABOURAUD AGAR (selective for yeasts - yeasts are NOT bacteria but eukaryotic microorganisms + members of fungus kingdom)
9
Q
how do we incubate selective media
A
- anaerobically or in an environment of high CO2 (~5-10%)
- overnight at 37 degreesC
10
Q
how do we incubate columbia blood agar plates (non-selective media)
A
in CO2
11
Q
what is seen on the agar after inocultating overnight / after 10 hours
A
exponential growth of bacteria
colonies visible by naked eye
12
Q
what are some features of bacterium
A
- cytoplasmic membrane (phospholipid bilayer) surrounded by a cell wall made of peptidoglycan
- genetic material floats freely in the cytosplasm + is associated w proteins + is coiled into a nucleoid
- cytoplasm = viscous, packed with ribosomes
- on the outside of the cell there may be
1) polysaccharide capsule
2) flagella (for motility)
13
Q
what does the cell wall structure of bacteria divide them into
A
2 main groups
- gram +ves
- gram -ves
14
Q
why arent all species culturable in the lab
A
- not always able to reproduce the conditions / nutritional requirement that certain species need to grow
15
Q
how many microbes of the world have been cultures in labs
A
less than 1% (huge diversity ie environmental microbes from soil to oceans)
16
Q
how many microbes of the oral microbiota have been cultures in labs
A
70%+
well studied
17
Q
which tools / aspects of the bacterium can microbiologists use to characterise bacteria from a clinical sample
A
- identification using differential characteristics
3) Gram-stain (bacterial cell wall determines outcome)
4) Morphology
5) Haemolysis
6) Pigment
7) Metabolic activities
8) Antigens
9) Cellular composition
10) Nucleic acid
18
Q
what is the difference between gram +ves and gram -ves
A
gram +ve = thick cell wall traversed with lipoteichoic acid molecules
gram -ve = thin peptidoglycan layer surrounded by an outer phospholipid membrane attached to cell wall via lipoproteins
surface of outer membrane displays lipopolysaccharides (O antigens) and lipid A molecules which are v specific to gram -ve bacteria and are recognised by host cells (trigger host defence mechanisms ie inflammation)
19
Q
what are the 5 stages of gram staining (after smearing sample of the grown colony on a glass slide)
A
1) fixation
2) crystal violet
3) iodine
4) decolourisation using acetone
5) counter stain with safaranin
20
Q
explain the fixation stage of gram staining
A
use heat from a Bunsen burner
just enough to stick / seal the cells on the glass slide
this process kills the bacterial cells
21
Q
explain the crystal violet stage of gram staining
A
flood surface of the glass slide w crystal violet (a purple dye) for 1 min
then rinse the slide (tap water)
22
Q
explain the iodine stage of gram staining
A
flood slide with iodine
at this stage = crystal violet dye has been retained on the bacterial cells esp on the gram +ve cells
23
Q
why do gram +ve cells especially retain crystal violet dye
A
peptidoglycan v thick + exposed on the outside as the cell wall absorbs it
iodine seals it in (cannot be washed off)
24
Q
explain the decolourisation using acetone (or methanol or ethanol) stage of gram staining
A
washes crystal violet dye away in case of gram -ve
so we can differentiate…
gram +ves = purple
gram -ves = cannot see
25
explain the counter stain with safaranin stage of gram staining and why is it used
safarin is redy-pink
for gram +ve = remains purple
for gram -ve = turns pink
we can visibly see mixture of the 2 and differentiate them
used because if its all been decolourised + bacterial cells we were studying were gram - (would be all completely decolourised + if viewed under microscope you wouldn’t be able to see anything) but if gram + (would be seen in purple)
26
why cant gram -ve cells retain crystal violet dye
thin peptidoglycan cell wall surrounded by a phospholipid membrane
27
how can we further look at cell morphology after gram staining
- microscope to see if rods or cocci (round shape cells)
| - really helps their identification
28
how do bacteria reproduce
binary fission
| 1 cell splits equally into 2 identical daughter cells
29
how do streptococci and staphylococci divide/grow differently (both gram +ves)
```
streptococci = divide on one plane into chains
staphylococci = divide on multiple planes into clusters
```
30
which shapes and forms can bacteria come in
```
rods / bacilli
cocci
coccobacilli
vibrio / curved rods
spirilla
filamentous (filament like cells are typical of fusobacteria)
```
31
what factors make up colony morphology and when is this used
shape
pigment
haemolysis
- 1st characteristic used to differentiate bacteria when grown on an agar plate
32
what does the size of a bacterium range by
1-10um
| invisible to eye
33
what are some bacterial species classified base on when cultured on blood agar plates
what 3 categories is this divided into
haemolytic properties
1) alpha haemolytic
2) beta haemolytic
3) gamma haemolytic
34
what do alpha haemolytic species do when cultured on blood agar plates
- cause oxidation of iron in haemoglobin contained in the agar (contain/posses genes to do this)
- leads to greenish colour / shine on blood agar plates around the bacterial colonies
35
what do beta haemolytic species do when cultured on blood agar plates
- cause complete rupture of red blood cells contained in the agar medium
- appears as clear areas surrounding the colonies
36
what do gamma haemolytic species do when cultured on blood agar plates
dont cause any haemolysis
37
which 4 differential characteristics can be used to further differentiate bacteria
1) metabolic activities
2) antigens
3) cellular composition
4) DNA
38
how can metabolic activities be used to further differentiate bacteria
certain species have specific metabolic activities
- fermentation +/- acid or gas production
- preformed enzyme production
- use specific carbs or proteins (use in selective media for their growth)
39
how can antigens be used to further differentiate bacteria
find specific antibodies that bind to antigens that certain bacteria may produce
use these as a way to identify these bacteria
40
how can cellular composition be used to further differentiate bacteria
peptidoglycan = used in gram staining method to differentiate between gram +ve and -ve
similarly other specific components (lipids / amino acids etc) may be used / targeted in differentiationo between specific species
41
how can DNA be used to further differentiate bacteria
extract it from bacterial samples
determine nucleotide sequence
compare it to a database of know sequences for homology
can also use probes
42
list GRAM POSITIVE COCCI genus names (found in mouth)
streptococcus
43
list GRAM POSITIVE RODS genus names (found in mouth)
actinomyces
eubacterium
lactobacillus
44
list GRAM NEGATIVE COCCI genus names (found in mouth)
neisseria
| veillonella
45
list GRAM NEGATIVE RODS genus names (found in mouth)
```
aggregatibacter (actinobacillus)
fusobacterium
porphyromonas
prevotella
treponemma
```
46
which GRAM POSITIVE COCCI are found in HEALTH
streptococcus oralis
streptococcus mitis
streptococcus sanguinis
47
which GRAM POSITIVE COCCI are found in ABSCESSES
streptococcus intermedius
48
which GRAM POSITIVE COCCI are found in CARIES
streptococcus mutans
| streptococcus sobrinus
49
which gram positive cocci are the early colonisers of the mouth (1st pioneer colonisers of a babys mouth as soon as they are born)
- streptococcus salivarius
- streptococcus oralis
- streptococcus mitis
because we are born edentulous
50
which gram positive cocci are found in low abundance in the mouth
what changes when caries lesions are present
streptococcus sanguinis
streptococcus mutans
streptococcus sobrinus
in a case of caries legions they are found mostly around these lesions in HIGH abundance
51
# define the main characteristic of
a) S. salivarius
b) S. oralis and S. mitis
c) S. sanguinis
d) S. mutans and S. sobrinus
a) early coloniser of mouth
b) ubiquitous (found everywhere)
c) colonises teeth
d) caries
52
GRAM POSITIVE RODS:
| describe actinomyces
- branched rods
- common in plaque
- implicated in root surface caries
- can be considered opportunistic pathogens
53
GRAM POSITIVE RODS:
| describe lactobacillus
- Implicated in advanced caries
| - some species/strains of lactobacillus (genus) are contained in certain probiotic yoghurts
54
GRAM POSITIVE RODS:
| describe eubacterium
- gram positive rods AND FILAMENTS
- difficult to grow in lab
- found mostly in plaque biofilm
55
GRAM NEGATIVE COCCI:
| describe neisseria
- aerobic
| - early coloniser of tooth
56
GRAM NEGATIVE COCCI:
| describe veillonella
- anaerobic
- GOOD to have in the mouth = it utilises lactic acid in plaque as nutrients metabolising it into weaker acids (ie propionic acid)
this increases pH in the mouth and prevents demineralisation
57
```
GRAM NEGATIVE RODS:
describe treponema (spirochaetes)
```
- strictly anaerobic
- found most likely in sub-gingival plaque (less O2 molecules)
- implicated in periodontal disease
58
GRAM NEGATIVE RODS:
| describe fusobacterium
- gram negative FILAMENT
- anaerobic
- common in plaque
- mesh like structure makes it a bridging organism
- KEY bridging organism between early and late colonisers (helps binding, making a more rich, complex biofilm )
59
what is the method of choice for bacterial identification if we cannot isolate in pure culture in the lab and biochemical properties are unknown
16S RIBOSOMAL RNA GENE (16SRRNA) SEQUENCE ANALYSIS
60
why has the 16SRRNA been chosen as one of the best genes to sequence out of the 3-4 thousand present in bacterial genome
- codes for the small subunit of the bacterial ribosome
- best bc it contains conserved regions AND highly variable regions that make it useful to distinguish between bacterial species
61
what are conserved regions in a gene and why are they helpful
- regions made of nucleotide seq’s that are identical when compared between several organisms
- probes / primers can be designed to hybridise , anneal to these regions and amplify the variable regions
62
what does amplifying certain regions of the gene using pcr enable
us to study them further if we manage to sequence them at a future stage
with the seq of dna we can then compare between other samples or to databases already set up by previous research
63
what can be determined by comparing our dna sequence to whats already identified in databases
- compare bioinformatically
| - determine closeness in terms of relatedness and infer whether the sample is a close match
64
what are variable regions
nucleotide sequences that vary greatly between bacterial species BUT remain similar in members of the same species
65
how is 16S RIBOSOMAL RNA GENE (16SRRNA) SEQUENCE ANALYSIS carried out and used to study genetic relatedness
1) extract genomic dna from sample
2) process dna fragments through a lab process to determine the sequences from the 16SRNA gene
3) search for homology in databases (find what these dna sequences are close to)
4) construct phylogenetic dendrogram (computer algorithms help do this faster)
66
what do dots on phylogenetic dendrogram trees represent
- a different sample
- where dots cluster together = highly related in terms of sequence, almost identical except 1 or 2 bases
- where dots are further away = more different
67
how many phylotypes types have been identified in the human mouth using 16SRNA gene sequencing
700 (now likely to be around 1000 maybe more as new findings every year)
- 400 in subgingival domain
- 35 not yet culturable
68
what do people do now which makes 16SRNA gene sequencing old
try seq the whole genome of dna rather than just a gene
69
which not yet culturable (hard to culture in labs) species were found using 16SRNA gene sequencing
- 47 uncultivated spirochaetes
- TM7 phylum
- Synergistetes
recently. .. ways to culture these in combination with other species that would provide required nutrients has been published
70
using 16SRNA gene sequencing, what has been achieved from studying unculturable bacteria and what has this enabled
- fragments of their genome successfully sequenced enough for probes to be designed
- use fluorescent in situ hybridisation (FISH) techniques to identify some members of the TM7 phylum Synergistetes species using a combo of microscopy
71
why is fluorescent in situ hybridisation FISH used in some research labs
to define bacterial populations
72
what happens in fish
fluorescent probes designed to specifically bind to the dna or rna of specific species if part of their genome sequence is known
73
what are the advantages of FISH
clinical samples of interest do not have to be destroyed and dna extracted
74
what is the FISH technique often used to examine
- structure of biofilm
| - determine where some species are located in the structure
75
what molecular techniques other than 16SRNA gene sequencing and FISH are used in research labs
DENATURING GRADIENT GEL ELECTROPHORESIS (DGGE) - community profiling
DNA-DNA CHECKERBOARD - detect pre-selected organisms
HUMAN ORAL MICROBE IDENTIFICATION MICROARRAY (HOMIM)
NEXT GENERATION SEQUENCING [METAGENOMICS]
76
which of the 4 molecular techniques are not used anymore with advances in technology and price of dna sequencing becoming much cheaper
DGGE and DNA-DNA checkerboard
popular + widely used 10 years ago
77
which molecular technique is most used in modern research and investigations into microbial profiling
next generation sequencing methods
78
what is DGGE
- uses dna fingerprinting methodology based on migration of dna molecules through a gradient of a conc of denaturing agents (these unwind the dna molecules / melt them)
79
what is dna-dna hybridisation (checkerboard) used for
detect preselected organisms for which gene seq’s are already known
AND
to design labelled probes (v shot fragments of nucleotides)
the probes = deposited on nitrocellulose membrane
extracted dna (from a clinical sample) is applied to the membrane at 90 degree angle from the probes
complementary fragments of sample dna + probes = hybridise + because probes are chemically labelled hybridised probes emit some fluorescence when excited visually observed as dots on the membrane
80
from dots on the membrane of dna checkerboard, how can we determine what species are present in unknown oral sample
because origin of dna probe is already known
81
how do we determine species present from the output of this technique
- each horizontal line on membrane corresponds to a different bacterium (previously already isolated) + a genome used to design the probes using this technique
- vertical lines = the samples being investigated
- sites of the dots = correspond to their density
82
what is the advantage and disadvantage of dna-dna hybridisation (checkerboard)
a = bypass culturing steps
b = unable to identify unculturable species (wouldnt have their genome data to design a probe)
83
what is the human oral microbe identification microarray (homim) based on and how
dna-dna hybridisation (checkerboard)
- same principle but slightly improved because many more species can be detected (up to 272 preselected ones that can be identified from dna extracts from a clinical sample)
- done without the need for culturing bacteria
84
which companies have come up with different methods of next gen sequencing (NGS)
what do they all have in common
illumina. rosch, specific bioscience, oxford nanopore technologies
ALL enable samples of dna to be fully sequenced at once
85
what does NGS give rise to
deluge of data
- clinical or environmental samples containing millions of microbes from 100s of diff species can all be seq’ed at once
- generates data output ranging in the billions of fragments per sample
86
what is used to deal with the database of known species and why
bioinformatics
- their corresponding genome seq is continually expanding
- our knowledge of their genes + metabolic capabilities also expanding
87
what does metagenomics correspond to
analysis of the totality of genomes present in a particular sample
these genomes come from all the microbes containing that sample if we consider a microbial sample
88
what is the size of the sequencing part of an illumina machine
- v small (almost usb stick size)
89
what organisms would we expect to see on hard palate, buccal mucosa and lips
mainly gram +ve facultatives
- streptococcus spp
- actinomyces spp
90
what organisms would we expect to see on dorsum of tongue
gram +ve
- 40% streptococci
- rods
gram -ve anaerobes
91
what organisms would we expect to see in subgingival plaque
```
gram +ve cocci
- esp streptococci
gram +ve rods
- esp actinomyces
gram -ve anaerobic rods
- prevotella spp
- fusobacterium spp
spirochaetes
```
92
what organisms would we expect to see in supragingival plaque
```
gram +ve cocci
- esp streptococci
gram +ve rods
- esp actinomyces
anaerobes
```
93
list microorganisms thought to be associated with health (from studies investigating microbial population of those w NO oral diseases)
```
Neisseria
Corynebacterium
Rothia
Actinomyces
Haemophilus
Veillonella
Streptococcus
Fusobacterium Capnocytophaga Granulicatella
```
94
which organisms are associated positively with periodontal disease AND lack of caries
Campylobacter rectus
Selenomoas noxia
Fusobacterium nucleatum
95
which caries associated bacteria are associated with good periodontal health
Streptococcus mutans
Lactobacillus fermentum
Atopobium sp.
96
what happens as soon as we are born
- colonised by microbes
| - from birth canal OR in C section microbes from health professionals handling the baby (skin microbes)
97
which bacteria colonise once the teeth (new non-shedding surfaces and pockets) appear
```
Streptococcus sanguinis
Prevotella spp.
Fusobacterium nucleatum
Neisseria spp.
Veillonella spp.
Actinomyces spp.
Lactobacillus spp.
Rothia spp.
```
98
what happens to the oral microbiota over time
when would this NOT happen
stabilises
if we take a lot of antibiotics (disrupts microbiome everywhere on/within our bodies)
after taking it takes time for the microbiome to return to normal
99
what happens when we get old
become edentulous again
lose species adapted to tooth surfaces, subgingival area / pockets (S mutans + sanguinis, strict anaerobes)
BUT they return with dentures
- also colonisation by non-oral bacteria due to decline of host responses
100
what have dna based technologies and improved culture enabled
- detection + identification of not yet culturable organisms
- elucidation of metabolic capabilities
- new associations with disease (bifidobacteria + caries; filifactor alocis + periodontitis)
- greater definition of populations associated w disease and health
- development of high throughput methodologies
101
discoveries of powerful dna based technologies will help devise
new efficient approaches to personalise medicine and dentistry (paradigm shift)
