15. Mouth as a Habitat for Microbes Flashcards
Define ‘autochthonous microbiota’
- microorganisms found characteristically at particular site
- they’re adapted to survive and grow here
Define ‘allochthonous microbiota’
- microorganisms transiently present at site
- don’t thrive here but may colonise transiently or if site becomes compromised in disease
Relative sizes of oral microbes and structures from smallest to largest
- individual cells
- chains or filaments of cells
- candida
- neutrophils
- hedgehogs with sweetcorns
- epithelial cells
- rotund aggregates
What’s making detection of viruses easier?
PCR methods
Viruses present in oral microbe
- bacteriophage
- herpes simplex type 1 (cold sores) is most common
- HIV and Hep B can be asymptomatically carried
- SARS-CoV-2 can replicate
Most common oral virus
herpes simplex type 1
Archaea in the mouth
- rarely isolated from healthy individuals
- detected in periodontal disease
Fungi in oral microbiota
- occasional infections like aspergillus
- most common are Candida spp.
- many fungi in low abundance
- most fungi only infect vulnerable like young, old, cancer - generally we are robust against them
The most important fungi do what?
make hyphae
Are viruses in the mouth dangerous?
not usually
- control bacterial population
- imapct dental plaque
Any individual has … to … different bacterial species in mouth from 700 … species
100-200
allochthonous
3 types of bacteria seen in health in mouth
- obligate aerobes like neisseria
- facultative anaerobes e.g streptococcus
- obligate anaerobes e.g veillonella
Bacteria seen in dental caries
- acidogenic, aciduric bacteria
- like streptococcus mutans
Difference between acidogenic and aciduric
- produce acid
- tolerate low pH
Bacteria in periodontitis
- obligate anaerobes
- like treponema denticola
From healthy bacteria in mouth to bacteria in periodontitis, what shift is seen?
from gram pos to gram neg bacteria
Give 4 different microbial habitats in the mouth
- lips, cheeks, palate
- tongue
- teeth
- saliva
Explain lips, cheeks, palate as a habitat
- epithelial cells
- continually shed (desquamation)
Explain tongue as a habitat
- highly papillated
- resevoir for obligate anaerobes (perio. pathogens)
- tonsils can harbour these pathogens too
Explain teeth as a habitat
- non-shedding
- lots of diff surfaces with diff microbial populations
- covered with acquired enamel pellicle
Explain saliva as habitat
- transient presence in the mouth
- carries microbes between locations
- microbes often attached to desquamatised epithelial cells
Do you brush away the acquired pellicle?
- no
- only removed by vigorous acid washing
What would you see from the mouth and nose on a kiss agar plate?
- alpha-haemolysis by oral streptococci producing water to bleach haemoglobin
- gamma haemolysis (no haemolysis) at nose as most bacteria here are staphylococcus spp.
Bacteria on tongue
- strep (salivarius/mitis)
- prevotella
- actinomyces
- veillonella
- haemophilus
Bacteria on cheek
- strep (mitis
- haemophilus
- simonsiella
Bacteria on teeth in supragingival plaque
- strep
- actinomyces
- haemophilus
Bacteria on palate
- strep
- actinomyces
Bacteria on teeth in subgingival plaque
- strep
- actinomyces
- peptostreptococcus
- fusobacterium
- aggregatibacter
- porphyromonas
Saliva is … when secreted
What then happens?
- sterile
- rapidly accumulates around 10^9 bacterial cells per ml
Around 3 times more bacteria is attached to … than are free in saliva
epithelial cells
3 ways bacteria are removed from oral surfaces
- sloughing of epithelial cells
- mechanical debridement
- active release (possible)
How to get a reflection of overall oral microbiome?
sample saliva
Limitations of sampling saliva as a reflection of oral microbiome`
- proportions of microbial species are diff from plaque/soft tissue
- care required when sampling to standardise
How to culture oral microbiota
- isolate bacteria
- culture on agar
- estimate number of diff species by culturing and counting colonies
4 reasons we isolate bacteria
- understand basic physiology
- link organism to disease (Koch’s postulates)
- identify pathogenesis mechanisms
- test antibiotics
Explain the Great Plate Count anomaly
- some bacteria are dormant and not easily reactivated
- some species are fastidious - don’t grow on nutrient agar
- better to culture from humans but will then miss species
Define ‘culture-independent microbial analysis’
- techniques based on comparison of DNA sequences from diff bacteria
In culture-independent microbial analysis, do you isolate the organism?
- not required
- but this can be used to aid isolation and identification
2 approaches within culture-independent microbial analysis
- community sampling
- environmental genomics
Explain community sampling in culture-independent microbial analysis
- amplify single gene for example gene encoding 16S rRNA or for specific function
- sequence and generate a tree
- get a single gene phylogenetic tree
- snapshot of most members of a community and diversity of microbial guild
Explain environmental genetics for culture-independent microbial analysis
- restriction digest total DNA and shotgun sequence
- or do it directly using high-throughput DNA sequencer
- assemble and annotate a total gene pool of community
- identifies all gene categories, discovers new genes and links genes to phenotype
We are holobionts. What does that mean?
- superorganisms of our own cells and our microbiota
Define ‘16S ribosomal RNA’
- an RNA molecule around 1500 nucleotides long
- part of small subunit of ribosome in prokaryotes
Define ‘biofilm’
- sessile community of microbes characterized by cells attached to surface or to each other
- embedded in a extracellular polymeric substances
Define ‘bioinformatics’
application of computational techniques to analyse complex biological data like genetic codes
Define ‘coevolution’
parallel evolution of interacting species
Define ‘community profiling’
- characterisation of complex microbial communities
- by their 16S rRNA gene sequences
Define ‘dysbiosis’
condition in which normal microbiome population structure is disturbed
- often by external burdens like disease or medication
Define ‘epigenetics’
the study of heritable changes in gene expression not caused by changes in DNA sequence
Define ‘gene amplification’
- an increase in gene copies by lab methods for research purposes
- produces enough copies of a gene to allow gene sequencing
Define ‘holobiont’
host organism and all its symbiotic microbial residents
Define ‘metagenomics’
- analysis of genetic info of complex population typically from microbes in environmental or host sample
- metagenome consists of genomes of many individual microbes
Define ‘metatranscriptomics’
analysis of active genes and species of microbiome
Define ‘microbiome’
- sum of microbes, their genetic info and environment they interact in
Define ‘microbiota’
all living microbial organisms in the microbiome
Define ‘next-generation sequencing’
- umbrella term for number of different modern high throughput sequencing technologies
Define ‘operational taxonomic unit’
- grouping of bacterial 16S rRNA sequences by similarity
- typically grouped at 97% to 99%
Define ‘phenotype’
observable physical characteristics of organism e.g behaviour or appearance
Define ‘phylogenetics’
study of evolutionary relationships among groups of organisms
Define ‘phylotype’
type of bacterium defined by placement in phylogenetic tree on basis of 16S rRNA gene sequence
Define ‘species’
coherent and distinct groups of bacteria that have been isolated, cultured and named
Define ‘symbiosis’
two or more species living closely together in long term relationship
Difference in core and peripheral microbiome for saliva
- core is the same 13 phyla that is present in everyone
- peripheral is species present in some and not others
Microbiomes of diff individuals form … and these are associated with …
clusters
diffs in the metabolome
What questions do saliva differences between people raise?
- do the types correlate with disease susceptibility?
- can they be used as a marker for estimating disease risk?
Baacteriophage role in mouth
- control bacterial population in mouth
- reduce levels of invading pathogens as commensal bacteria have to co-exist with phage and they carry genes between bacteria
Do bacteriophage communties vary lots or little between mouth sites?
little
- very different between individuals but similar in same mouth
- couples have more similar than twins however
2 main types of oral mycobiome
- candida-dominated
- low-level mixed species
Role of acquired enamel pellicle
protect enamel
Salivary compounds help to control plaque accumulation how?
- aggregate bacteria which are then swallowed
- antimicrobial effects
Salivary molecules that bind to bacteria
What do they do?
- MG2, salivary agglutinin (gp340), PRPs, statherin
- binds to bacteria and teeth to agglutinate or inhbiit bacteria and exhibit specificity
Explain aggregation/agglutination in saliva
- occurs in fluid phase
- results in large clumps
- these adhere poorly and are removed with swallowing
Explain adhesion in saliva
- proteins in saliva pellicle
- interaction with bacteria causes adhesion of bacteria to teeth
Some proteins exhibit different bacteria binding proporties when in … than when on a …
solution
surface
Role of immunoglobins in aggregation
- present in saliva
- agglutinate bacteria (removal by swallowing)
- secretory IgA (33mg/100ml resting flow, 6mg/100ml stimulated flow)
- immunoglobins present in gingival fluid (IgG, IgM to activate complement and opsonization)
MG2 is a … gene product and secreted by …
It is relatively … and a … (explain)
It binds to …
It’s not/found in pellicle
The non-glycosylated peptide domain can be … - explain
- muc7
- serous acini of submandibular and sublingual and minor glands
- small
- glycoprotein (70% carb, sialyated di-tri-saccharides, lacks ABO blood group determinants)
- various bacteria inc. streptococcus sanguinis
- not
- bactericidal (histamine rich N-term domain can disturb cell membranes)
Salivary agglutinin is a … gene product secreted by …
Size?
It’s highly .. and binds to …
It’s identical to … and present in …
- DMBT1
- all salivary glands
- 340kDa
- glycosylated
- original strep. mutans salivary aggregator
- gp340
- pellicle
gp340’s role is …
- a glycoprotein in lung washings
- binds to surfactant protein-D
- enhances phagocytosis and killing microorganisms by neutrophils and macrophages
Explain proline-rich proteins
- can be acidic, basic, glycosylated
- high concs found in parotid and submandibular saliva (70% of total protein)
- for calcium phosphate stabilisation
- C-term is a cryptitope
Functional domains of PRPs
- N-term binds to hydroxyapatite
- C-term binds
- to actinomyces spp, strep mutans and sanguinis
Explain statherin
- low molecular weight
- secreted by parotid and submandibular gland
- for calcium phosphate stabilisation with PRPs
- binds to hydroxyapatite and porphyromonas gingivalis and actinomyces spp
Statherin doesn’t bind bacteria when …
in soluble state
3 broad spectrum antibacterials in saliva
- lysozymes
- lactoperoxidase/thiocyanate/hydrogen peroxide system
- lactoferrin
Lysozyme effect in saliva
- broad distribution through tears, saliva, sweat
- not equally effective against all bacteria - oral bacteria more tolerant
- reduces viability of oral bacteria by cleaving the cell wall peptidoglycan
- can cause non-enzymatic cell degradation
Reactions and role of lactoperoxidase in saliva
- thiocyanate in saliva as KSCN
- lactoperoxidase is produced by host and bacteria
- H2O2 is a metabolic by-product of many oral streptococci
- reaction product is hypothiocyanite plus cyanosulphurous acid and cyanosulphuric acid (all toxic)
- pK for HOSCN/OSCN is 5.3, more acid favours HOSCN
- due to uncharged nature, HOSCN penetrates bacterial cell envelope better
Role of lactoferrin in saliva
- nearly all life forms have requirement for iron
- enzyme co-factor
- lactoferrin binds iron and makes it unavailable
- some bacteria produce iron-binding proteins called siderophores to compete for host iron
Toothbrushing doesn’t eliminate plaque but what kinds of markets can it be specialised to?
- pets
- dry mouth
- children
Examples of toothpases and their antibacterial enzymes
- Biotene (dual enzyme system)
- BioXtra (contains lysozyme, lactoperoxidase and lactoferrin)
