Week 1 Flashcards
(119 cards)
1
Q
Where does the word probiotic come from?
A
Probios = for life
2
Q
What is the definition of probiotics?
A
Live microorganisms which when administered in adequte amounts, confer a benefecial health effect on the host
3
Q
What was the work of Elie Metchikoff?
A
Residing bacteria in the gut have the ability to cause disease.
Gut bacteria can potentially promote health of the host
In 1908 he was awarded the Nobel prize for medicine for his studies on cellular immunity
4
Q
Who played a vital role in the discovery of probiotics?
A
Elie Metchnikoff
5
Q
How many microbes are in a healthy gut?
A
10^14 microbes - weighs around 1kg
6
Q
How many more genes are expressed in gut microbiome compared to human genome?
A
150 times - one of the most metabolically active areas of the body
7
Q
What are bacteria that live in the gut microbiome and are opportunistic parasites?
A
Staphylococci
Proteus sp
Ps aeruginosa
8
Q
What are bacteria that live in the gut microbiome and are purely benefical?
A
Lactobacilli
Bifidobacteria
9
Q
What are bacteria that live in the gut microbiome and are opportunistic parasites and beneficial?
A
Streptococci
Enterococci
Clostridial Cluster IX
10
Q
What is the main gene used for bacterial identification?
A
The 16S ribosomal RNA gene
11
Q
What is the overview of 16S ribosomal RNA gene for bacterial identification?
A
Ribosomal RNA is the most abundant form of cellular RNA (~80% total)
Multi-copy gene
rRNA present in all life forms, slowly evolving, good for molecular taxonomy
Complex secondary structure with conserved and variable regions
Large database of known rRNA sequences
12
Q
What is the bacteria in Yakult?
A
Lactobacillus casei Shirota
13
Q
How big is Yakult company?
A
Serves 26 million people in 31 countries around the world
80,000 Yakult ladies deliever Yakult products directly to their customers
14
Q
What bacteria is used in Actimel?
A
Lactobaxillis casei Immunitas
15
Q
What is example of Lactobacilli in probiotics?
A
Lactobacillus acidophilus
Lactobacillus rhamnosus
Lactobacillus casei
Lactobacillus bulgaricus
Lactobacillus reuter
16
Q
What are examples of Bifidobacteria in probiotics?
A
Bifidobacterium bifidum
Bifidobacterium longum
Bifidobacterium breve
Bifidobacterium lactis
17
Q
What are examples of yeast probiotics?
A
Saccharomyces cerevisiae
Saccharomyces boulardiii
18
Q
What are examples of other bacteria used in probiotics?
A
Streptococcus thermophilus
Bacillus subtilis
Enterococcus faecalis
Escherichia coli Nissle 1917
19
Q
What is requred in probiotics?
A
Contain sufficient live, active ‘good’ bacteria
Be stable and viable for the life of the product
Survive the stomach acids
Survive exposure to bile acids
Provide health benefits
Be safe and amenable for human consumption
20
Q
What are some of the probiotic health claims?
A
Stimulation of host immune system
Aids digestion
Prevention of colon cancer
Reduction of antibiotic associated diarrhoea
Protection against heavy metal toxicity
21
Q
What is an example of probiotic disinfectant?
A
Aggie’s probiotic cleanining products - disinfectant kills bacteria so cant be used in context
22
Q
What is an example of probiotic hygine product?
A
Probiotic aftershave lotion - aftershave needs to be antispetic so cant work
23
Q
What was the practical used to see is probiotics help reduce eczema?
A
Randomised double blind placebo controlled clinical trial –Finland
159 pregnant women with family history of atopic disease given Lactobacillus GG 2-4 weeks prior to birth
Probiotics given to babies until 6 months.
24
Q
What was the impact of using probiotics in reducing eczema on babies?
A
At both 2 and 4 years they were protected from atopic eczema though the effect wore off
As infants atopic eczema increased in the probiotic group but still lower than placebo group
25
What was the experiment for seeing that probiotic and inflammatory bowl disease?
Gionchetti et. al. – randomised double blind placebo controlled clinical trial with pouchitis patients
Pouchitis = inflammation/infection of pouch
40 patients receievd a probiotic mix for 9 months
26
What was in the probiotic mix used as potential treatment for inflammatory bowl disease?
One strain of Streptococcus thermophilus,
Three strains of Bifidobacterium
Four strains of Lactobacillus
Each sachet contains 450 billion live probiotic
27
What were the results for using probiotics as a treatment for inflammatory bowel disease?
100% of the control group suffered relapse
Only 15% of patients given probiotics suffered relapse
28
What is the use of probiotics as a therapeutic treatment of COVID-19?
70 hospitalized COVID patients (aged 50-70) had fever, cough, headache, diarrhoea and required oxygen were enrolled in the study
42 of these were given hydroxychloroquinone and antibiotics alone or in combination
28 received a multistrain probiotic in addition to the standard treatment
Sivomixx (2400 billion/day)
29
What were the results of the probiotics as a therapeutic treatment of COVID-19?
Within 72 hours all patients treated with bacteriotherapy showed remission of diarrhea and other symptoms as compared to less than half of the not supplement group
Repritory rates were eightfold lower in patients recieving probiotics
Lower mortality rates and reduced transffered to ICU
30
What is the believed benefit of probiotic bacteria?
Prevention of cytokine storm
31
What is cytokine storm?
Cytokine storm and cytokine release syndrome are life-threatening systemic inflammatory syndromes involving elevated levels of circulating cytokines and immune-cell hyperactivation
32
What are prebiotics?
A prebiotic is a selectively fermented ingredient that results in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health
33
What is the requirements for prebiotics?
Resists gastric acidity, hydrolysis by mammalian enzymes
and absorption;
Is fermented by the intestinal microbiota
Stimulates selectively the growth and/or activity of intestinal Bacteria associated with health
34
What are examples of prebiotics?
Fructo-oligosaccharides:Inulin and oligofructose
(FOS)
Galacto-oligosaccharides (GOS)
Soybean oligosaccharides
Lactulose, Lactosucrose
Xylo-oligosaccharides
Isomalto-oligosaccharides
35
What are some prebiotic health claims?
Stimulate the growth of ‘good’ bacteria in the gut
Improve the immune system
Reduce intestinal infections
Improvement of bowl function
Reduce gut inflammation
36
What is Bergamot?
Citrus bergmia Risso - peel was hypothesised to be a prebiotic
37
How was the prebiotic effect of Bergamot tested?
Gibson Model of Human colon
38
What was the prebiotic effct of bergamot oligosaccharide?
Increase in symbiotic bifidobacteria and lactobacilli
Decrease in oppotunistic bacteroides
39
How does bergamot oligocaccharide compare to fructo-oligosaccharides?
BOS greater increase in symbiotic bifidobacteria and lactobacilli compared to FOS
BOS greater decrease in bacteriods compared to FOS
Overal higher score on prebioitc index - FOS - 6 and BOS 7
40
What are synbiotics?
Probiotics + Prebiotics
41
How many people does sepsis kill?
Sepsis in early infancy results in 1 million deaths worldwide each year
No effective prevention measures available
42
What was the study parameters to study the impact of synbiotics on reduction in sepsis deaths?
Randomised double blind placebo controlled trial
4556 infants were recruited into the study (with no sign of sepsis)
Synbiotic capsules(L. plantarum 109 + Fructooligosaccharide 150 mg)
Synbiotic or placebo given for the 7 days
Output monitored for 60 days
43
What were the results of using synbiotics in the reduction of infections?
Dramatic decline in death and sepsis
Steep decline in death diarrhaea cases
Also decline in other local infections
44
What is the future of probiotics?
Stricter regulation on probiotic claims
Evidence based market product
Probiotics designed for specific benefit- engineered -‘SMART probiotics’ or next generation probiotics
Understanding of the probiotic function
45
What is taxonomy?
The science of grouping together organisms that share properties
Taxonomy tools are important for understanding prokaryotic and eukaryotic microbial diversity.
46
What is classical taxonomy?
Taxonomy that uses the phenotype e.g. shape, metabolic activity, pathogenicity and nutritional requirements.
47
What is molecular taxonomy?
Molecular taxonomy (Modern)-uses the genotype e.g. rRNA and genome information.
48
What are the 3 main parts of taxonomy?
classification - arrangement of organisms based on similarity
nomenclature - naming of the organisms
Identification - determining whether an organism belongs to the group under which it is classified and named
49
What is the method for classical taxonomy?
A binary code of classification
Look at a large number of characteristics/phenotypes, that are scored. Positive is 1, or negative is 0
E.g. Gram stain (+ = 1, - = 0) , requirements for pH, oxygen, temperature, salt, cell wall composition, protein analysis, membrane lipids
50
What is the assumption made when using classical taxonomy?
The more characteristics the organisms share, the closer their evolutionary relationship is assumed.
51
What is a modern quick approach to identification?
Analytical Process Index (API) strips
52
What does Analytical process index use to identify bacteria?
Strips have little ‘wells’ where you can add an unknown bacterial suspension.
Each ‘well’ can test a different biochemical property, ie whether the bacteria can utilise lactose
53
How can Analytical Process Index identify bacteria?
Each ‘well’ can test a different biochemical property, ie whether the bacteria can utilise lactose
A positive result will give a colour change. The results can be represented as a number (0 for no reaction) and (1 for a reaction). This then gives a binary code (of 0s and 1s) which can be matched to a database.
54
What are the pros of API?
Strips can be designed for specific bacteria, beneficial for disease diagnosis
Quick & easy
55
What are the cons of API?
Not as detailed as molecular analysis
Cultures must be axenic (pure)
56
What is a dendrogram?
Dendrogram a tree diagram, especially one showing taxonomic relationships
57
What can an dendrogram show?
Data - %S of previous 10 strains arranged into a dendogram
Vertical lines S and G indicate %S levels at which higher categories may be established
>85% similarity - same species
>65% similarity - same genus
58
What is molecular taxonomy based on?
Molecular Taxonomy is classification based on hereditary molecular differences in DNA/RNA or even proteins.
59
What is the overview of how molecular taxonomy work?
Analysis of whole genomes can be challenging and smaller segments – conserved gene regions may be used as a proxy
60
What is main function of molecular taxonomy?
Molecular taxonomy is also known as cladistic taxonomy
From these analyses it is possible to assess evolutionary or phylogenetic relationships – when species shared common ancestors
61
What is the overview of the ribosome?
The ribosome is a complex molecule made of ribosomal RNA molecules and proteins that form a factory for protein synthesis
Comprised of two subunits - large and small
62
What is the Svedberg unit?
The Svedberg unit (Symbol S) is a measure of the sedimentation rate of a particle when centrifuged. More precisely, it is a measure of time and is equal to the value of 100 femtoseconds (10-13 seconds).
63
What is the sedimentation rate?
The sedimentation rate indicates the size of the molecule, with the larger molecules having a larger sedimentation coefficient. It is dependent on the particle's volume, shape and molecular mass. If a particle is heavier with a more compact shape, its Svedberg value will be greater than lighter particles with a less compact shape
64
Who is the Svedburg unit names after?
It is named after Theodor Svedberg (1884-1971), a Swedish chemist awarded the Nobel Prize in 1926 for the discovery of the ultracentrifuge.
65
What is the overview of the prokaryote ribosome?
The 70S ribosome, which is key in protein translation, is made up of 50S and 30S ribosomes.
66
What are the subunits and structure of prokaryotic ribosome?
50S subunit 23S rRNA
5S rRNA
34 proteins
30S subunit 16S rRNA
21 proteins
67
What is the sedimentation times of prokaryote ribosome and its subunits?
These subunits have sedimentation times of 70 x 10^-13, 50 x 10^-13 and 30 x 10^-13 seconds respectively
The 70S ribosome, the largest, and so will sediment fastest, whereas 30S will sediment more slowly as it is the smallest subunit
68
What is the overview eukaryote ribosome?
80s with 60S and 40S
69
What is the overview eukaryote ribosome subunits?
60S subunit - 28S rRNA
5.8S rRNA
5S rRNA
50 proteins
40S subunit - 18S rRNA
33 proteins
70
How frequent is the 16S rRNA?
Every cell has a 16S rRNA gene as the ribosomal RNA (rRNA) is an integral part of the ribosome, which is responsible for making cellular proteins
71
How big is the 16S rRNA?
This gene is only about 1550 bp in length in prokaryotes and every cell has from one to more than 25 copies of this gene
72
What is the structure of 16S rRNA?
The 16S rRNA molecule folds itself into a shape that must fit like a puzzle piece with other molecules and consequently, some structural elements must be maintained
Therefore, some sections of the sequence may vary and others may not.
73
What are the base regions of varible regions 3 and 4?
V3 - 342 to 529
V4 - 515 to 806
74
Why are the amplicon targets of V3 and V4 the start and end base sequences?
They are highly conserved making them prime amplicon targets for primers which can then build SBSR for forward and reverse so that the v region can be sequenced
75
What is the use of 16S ribosomal gene for taxonomy?
The ribosome gene acts as a molecular clock.
Sequence differences can tell us the time since the divergence of two species.
The rRNA sequence is very useful as its structure and function is similar across all organisms.
76
What is the method for using 16S for taxonomy?
1 Alignment of 16S rRNA gene sequences (gene is ~ 1500 base pairs long
2 Similarity matrix (%) for entire 16S rRNA gene sequences
3 Computer algorithms used to construct phylogenetic trees
77
What was the origin of molecular taxonomy?
Modern biosystematics includes phylogeny as an integral part of the classification process - Molecular taxonomy originated from Darwin’s evolutionary explanation (1860).
78
What is the issue be with relying on a single gene
to build phylogeny trees/cladograms?
Single genes may have been transferred over by horizontal transfer or convergent evolution makes distantly related bacteria seem more closely related
79
What are the two ways to generate phylogenetic trees?
Maximum parsimony or maximum likelihood
80
What is the overview maximum parsimony?
Evolutionary distances are computed for all pairs of taxa based on the number of nucleotide changes between them. e.g. 100 seqs each compared to each other
A proposed common ancestor is then generated
Many possible trees are calculated by comparing relative time of divergence from the common ancestor.
81
What are the limitations of maximum parsimony?
A limitation = more than one tree may produce results consistent with the data.
Requires academic judgement
82
With maximum parsimony what is the most likely phylogenetic tree?
Under the maximum-parsimony criterion, the optimal tree will minimize the amount of homoplasy (i.e., convergent evolution, parallel evolution, and evolutionary reversals). In other words, under this criterion, the shortest possible tree that explains the data is considered best
83
What is the maximum likelihood method?
This is based on statistical calculations. For each possible tree the program will calculate the likelihood (probability) that such a tree would have produced the observed data. The advantage of this method is that it will produce just one tree, but a disadvantage is that it needs a large amount of computation (not a problem)
84
How does the maximum likelihood require?
The method requires a substitution model to assess the probability of particular mutations; roughly, a tree that requires more mutations at interior nodes to explain the observed phylogeny will be assessed as having a lower probability
85
What is a difference between maximum likelihood and maximum-parsimony methods?
This is broadly similar to the maximum-parsimony method, but maximum likelihood allows additional statistical flexibility by permitting varying rates of evolution across both lineages and sites
86
What is phylotyping?
In the most straightforward approach, the reads resulting from the high-throughput sequencing platforms are grouped and classified based on their sequence similarity to a reference taxonomic dataset
87
What is the disadvantage of phylotyping?
A binning procedure will be biased towards the existing taxonomic classification—including its classification errors—which is largely based on cultivable organisms
88
What are alternative methods that can be used
Therefore, the most widely used bioinformatics pipelines like mothur, QIIME and UPARSE implemented an alternative approach, where all sequencing reads are grouped together based on their sequence similarity to each other rather than a reference taxonomic dataset, resulting in cluster of reads, referred to as operational taxonomic units (OTUs).
89
What is the similarity threshold for OTUs?
For clustering those reads into OTUs, a similarity threshold of 97% is frequently used for pragmatic reasons, as it offers a compromise between the potential inflation of the number of OTUs due to sequencing errors and the cut-off used for taxonomic classification.
90
What can be an issue with OTUs?
That organisms that share more than 98.65% of their full 16S rRNA gene sequences may or may not represent the same species, potential for underestimating bacterial diversity through 16S rRNA amplicon sequencing is intrinsic to the molecule studied and cannot be avoided
91
How can percent sequence similarity can oversite the evolutionary similarity?
Sequence similarity computed from pairwise alignments underestimates the number of substitutions compared with similarity computed from MSAs
In addition, the percent similarity is a nonevolutionary-based distance metric; it fails to take into account that multiple substitutions can occur at the same site
92
What is seen as a better alternative method for computing similarity?
These studies suggest that the best practice for computing similarity between sequences is to use evolutionarily corrected distances based upon a MSA; however, typical analyses use uncorrected distances based upon pairwise sequence alignments.
93
What is the disadvantage of the 97% threshold?
Second, the 97% 16S rRNA sequence similarity threshold used to delineate species is only a rough approximation
94
What is a case study of the disadvantage threshold value of 97%?
Two different species may have 99% similar 16S sequences (such as Bacillus globisporus and B. psychrophilus11) or the same strain may have multiple copies of the 16S rRNA gene that differ by 5% for some regions (such as Escherichia coli K12)
95
What does a graph showing microbial community look like?
A graph with relative abundances of the Y axis with the organisms on the x axis. With a stacked bar chart showing the relative frequencies of the different bacteria groups therefore can compare the different groups eg different diet
96
What are key genuses in the Firmicutes?
The Firmicutes include the endospore forming rods such as the genus Bacillus and Clostridium. Non-spore forming Firmicutes include the Lactic acid bacteria.
97
What are a key genus of the Actinobacteria?
The Actinobacteria include the Streptomyces, which are filamentous spore forming bacteria. They play a major role in the soil ecosystem as well as giving us the majority of our clinically useful antibiotics.
98
What are the two main groups of gram postive bacteria?
All Gram positive bacteria were once classified as Firmicutes. Today though, we have Firmicutes, which have a low ‘GC’ content and Actinobacteria which have a high ‘GC’ content.
99
What are the overview of Alpha Proteobacteria?
The Alpha Proteobacteria contain the Rickettsiales which are obligate intracellular parasites. Rickettsia prowazekii is the causative agent of typhus.
Contains the Rhizobiaceae, important agriculturally, can live symbiotically, induce and occupy root nodules on legume plants, can fix atmospheric nitrogen. E.g. Rhizobium leguminosarum which nodulates peas, beans, clover and vetch.
100
What are the overview of Beta Proteobacteria?
The Beta Proteobacteria contain three important human pathogens.
Neisseria meningitidis, causes meningococcal meningitis,
Neisseria gonorrhoeae, causes gonorrhea, a sexually transmitted disease and Bordetella pertussis which causes whooping cough.
101
What is the overview of gamma proteobacteria?
The Gamma Proteobacteria are the largest and most diverse group.
They contain the Enterobacteriaceae, e.g. the well known Escherichia coli, ‘Work horse’ for molecular biology. Inhabits human colon, some strains, e.g. 0157 extremely pathogenic.
Vibrio cholerae -causes cholera, an intestinal disease mostly due to contaminated water.
Pseudomonadaceae, e.g.
Pseudomonas aeruginosa-inhabits soil and water, opportunistic pathogen, important in cystic fibrosis and serious burns. Commonly acquired hospital infection
102
What is the delta proteobacteria?
The Delta Proteobacteria important in nutrient cycling e.g. iron and sulphur reducing bacteria, such as Geobacter metallireduscens. It also contains the Myxobacteria. These are free living soil bacteria which can come together to form differentiated reproductive multicellular structures. Myxobacteria have a major role in the decay of organic matter. They don’t have flagella, but move along a surface by gliding.
103
What is metagenomics?
Metagenomics involves sequencing all the genomic material from many different taxa whose bulk DNA was extracted directly from environmental samples such as soil, biofilms, water, sediments, benthos and air
104
What is metagenomics also called?
This approach is also known as “shotgun sequencing,” “environmental genomics,” “ecogenomics” or “community genomics.”
105
What is an overview of the method of metagenomics?
Typically, this technique samples genes from across the genome, not just DNA barcode regions, so the functional potential of a sample can be explored
106
What is the use of comparative metagenomics?
Comparative metagenomics can be used to compare the metabolic potential of organisms from different environmental samples and even determine taxonomic and functional profiles from the thousands of gene markers from communities of organisms
107
Why was comparative metagenomics proposed?
This method has been proposed as a way to detect uncultured organisms that are difficult to identify by traditional means. Genes and gene families can be identified from metagenomic sequences
108
What is a problem with identifying taxa?
Identifying the taxa that these genes belong to, however, can be challenging. As signature DNA regions suitable for taxonomic analysis will also be present in the sample, these can be used to identify individual taxa
109
What does reconstruction of individual genomes?
Possible depending on the sequencing depth, taxonomic complexity and size of organismal genomes in the sample.
110
How many prokaryote genomes have been assembeled?
Nearly 8,000 metagenome‐derived prokaryote genomes were assembled from 1,500 public metagenomes
111
Can metagenomic be done on eukaroyote genomes?
This type of achievement is not yet possible for eukaryotes due to the size and complexity of their genomes, but it may be in future as sequencing technologies and bioinformatics methods progress
112
What has been the application of metagenomics?
Metagenomics has found application in ancient DNA studies looking at the evolution of antibiotic resistance, studies of the microbes involved in honey bee colony collapse disorder
Metagenomics is a widely used technique to explore microbiomes on a small scale and can be scaled upwards for broad‐scale ecological surveys
113
What is an advantage to metagenomics?
An advantage of this method is that amplification‐free metagenomic sample preparation avoids the PCR bias that other methods may otherwise be subject to
114
What is a disadvantage of this metagenomics?
A challenge with this method is that with sequencing effort spread over all genomic regions, not just the signature DNA regions suitable for taxonomic assignment, there may be a reduced set of taxa that can be identified with confidence
115
How is metagenomics biased?
Taxonomic assignment of nonsignature DNA regions may be biased towards organisms whose whole genomes are present in databases (false positives)
116
How can metagenomic become amplication free?
HTS and single‐molecule sequencing technologies advance, output grows, and costs decrease (Box 5), this method is likely to be even more widely used as amplification‐free methods are very appealing to many investigators hoping to circumvent the many known issues with mixed template PCR in PCR‐coupled DNA metabarcoding
117
What is a cladogram?
A cladogram only represents a branching pattern; i.e., its branch lengths do not represent time or relative amount of character change, and its internal nodes do not represent ancestors.
118
What is phylogram?
A phylogram is a phylogenetic tree that has branch lengths proportional to the amount of character change.
119
What is a chronogram?
A chronogram is a phylogenetic tree that explicitly represents time through its branch length
