UF Bacterial Pathogens MCB4203 Exam 2

5.1 Qualitative methods for profiling microbe communities - Examples

16s rRNA sequencing and metagenomic sequencing (studying dna of all species in an entire community... example is Next-Gen)

5.1 Quantitative methods for profiling microbe communities - Examples

qPCR

5.1 Qualitative methods question

What species are present?

5.1 Quantitative methods question

How abundant is each species in a population?

5.1 16s rRNA pros

common in all bacteria, large enough for discrimination between other species, small enough for sequencing (too large makes sequencing harder), conserved regions while other regions are sequence signature

5.1 16s rRNA sequencing Variable

variable is species specific and used for identification

5.1 16s rRNA sequencing Conserved regions

relatively stable, so is used for PCR amplification

5.1 16s rRNA sequencing process steps

1. get dna of bacteria from host

2. isolate 16s rRna

3. pcr uses primers to amplify conserved regions

4. amplification = get many 16s Rrna amplicants

5. sequence those and analyze variable regoins

6. identify new species

compare to current species and relatives ...

7. update taxonomy

5.1 "NextGen" AKA

"High-Throughput" Sequencing

5.1 NextGen sequencing is both

quant and qual - itative

5.1 NextGen sequencing requires

bioinformatics and statistical software to be able to assemble reads and analyze data

5.1 NextGen technology examples

Illumina, PacBio, Ion Torrent

5.1 NextGen different technologies are unique because

each has unique sequencing chemistry and detection

5.1 NextGen trade off is

between length of sequencing "read" and accuracy

Example--longer reads tend to have a higher error rate compared to shorter reads

5.1 NextGen allows us to analyze

taxonomy and function information of species

5.1 Shotgun metagenomics

allows researchers to comprehensively sample ALL GENES in all organisms present in a given complex sample

5.1 Analyzing all genes is

expensive and time consuming.

5.1 A consensus sequence is

a DNA sequence that represents the most common nucleotides at each position in a group of related sequences. It's determined by comparing multiple sequences and identifying the most frequent residue at each position

5.1 Metagenomics sequencing process steps

1. get bacteria from sample - multiple bacteria means multiple genomes

2. cut up genomes into small fragments

3. create consensus sequencing from the fragments - done by alignment of DNA using computer program

4. gives us larger consensus sequence

5.1 Illumina is the

simultaneous determination of millions of base pairs of DNA sequences in a single reaction run

--creates a consensus sequence

--example of NextGen

5.1 Contigs are

large regions of overlapping sequences

5.1 Bioinformatic

computer analyzes data to rapidly assemble the sequences into contigs

contigs are then mapped into a complete genome

5.1 We amplify DNA with PCR before sequencing it because

most DNA sequencing techniques require a relatively large amount of DNA to generate a reliable signal, and PCR allows you to generate millions of copies of a specific targeted region of DNA from even a very small starting sample, making it possible to sequence even minute amounts of DNA effectively

5.1 Phylogenetic Tree Node

the branching off point representing a common ancestor -- a taxonomic unit, e.g., a taxon

5.1 Phylogenetic Tree Branch

arm of the branching tree itself -- defines the relationship between

the taxa in terms of descent and ancestry

5.1 Phylogenetic Tree Branch length

number of changes that have occurred in that branch -- longer length/distance means further distant relatives and less related

5.1 Phylogenetic Tree Root

the common ancestor of all taxa in that tree

5.1 Phylogenetic Tree Distance Scale

represents number of differences between sequences (e.g., 0.1 means 10% difference between two sequences)

5.1 Dendogram of Phylogenetic Relationships is used

To see differences between species by measuring the length of branches

5.1 Dendrogram distance scales can be used to measure

percent or fraction

5.1 Dendrogram example

add up the numbers on x axis from 2 branches (5+5=10 percent between the top and bottom branch) -- 2 branches at 10% means the 2 species have a 10% difference between their DNA

5.1 Phylogenetic Tree example with Shigella sonnei -- common ancestor

Current S. sonnei strains descended from a common ancestor arising in Europe < 500 years ago

5.1 Shigella sonnei example shows that charts can even show lineages and evolution of

a specific gene like gyrA here because we can see its genetic mutations over time

5.1 qPCR primers are

designed for variable regions, not conserved regions, so bacteria are quantifiable

5.1 In PCR, a "primer" is

a short, single-stranded DNA sequence designed to bind to a specific region on a DNA template, essentially marking the starting point for DNA replication and defining the exact section of the DNA that will be amplified during the PCR process; essentially acting as a guide for the DNA polymerase to copy a specific target sequence within the DNA molecule

5.1 qPCR stands for

Quantitative polymerase chain reaction

5.1 qPCR amplification uses

primer pairs that anneal/bind to 16s rRNA genes of bacteria

5.1 fluorescence in PCR is achieved by

intercalation (insert between layers) of a SYBR green dye into double-stranded DNA of the PCR products

5.1 the faster fluorescence is achieved in PCR sample

the higher level of gene abundance

5.1 cycle threshold is

the number of cycles needed for a fluorescent signal to exceed a threshold level

symbol is Ct

5.1 a lower Ct value indicates _ because _

a higher gene abundance because fewer cycles were needed to detect more DNA (more DNA means stronger signal so faster result)

5.1 Ct graph

x axis=cycle number

y axis=fluorescence level

Ct=horizontal line across the curves (meaning Ct value is taken on the x axis aka cycle number)

5.1 Alternative to Ct is

Single probe multiplex

5.1 Single probe steps

1. PCR → sequence-specific DNA probes labeled with fluorescent dye at one end and a fluorescence quencher moiety at the other

2. DNA detection after hybridization//binding of the labeled probe with complementary sequence

3. Quencher removed by 5′ to 3′ exonuclease activity of the thermostable DNA polymerase

4. Detection of fluorescence from the probe bound to its complementary DNA

5.1 Single probe multiplex is a technique that

allows for the detection of multiple targets in a single detection channel of a closed-tube PCR

5.1 quencher is

suppressor, so dna sequence only lights up/shows fluorescence when it goes thru single probe process steps

5.1 Ct inverse relationship

As the number of DNA molecules in a sample decreases, the time it takes to reach the fluorescence threshold (the Ct value) increases.

5.1 Ct limits

The Ct value is affected by factors like the efficiency of the PCR reaction and the quality of the DNA template. Also, qPCR can only detect specifically targeted DNA sequences (because we make specific primers where PCR will occur)

--cannot detect diff or unknown DNA in a sample

----so it is undercounting actual amount of DNA in sample

5.1 Metagenomics in the body - pros and cons

Genes present at a given body site -- does not tell if gene is expressed

5.1 Transcriptomics

Genes expressed at a given body site

5.1 Proteomics -- pros and cons

Proteins expressed at a given body site and protein interactions; does not tell if protein is functional

5.1 Metabolomics

Metabolites present at a given body site

5.1 Multi Omics is

combining all 4 -omics -- very useful and impactful

5.1 Transcriptomics steps

1. rna extract/isolate

2. get mrna only by selecting/depleting others out

3. make cDNA from mRNA

4. sequence cDNA to compare to known cDNA library

5. see what mRNA is being expressed

5.1 Transcriptomics pros

study gene expression, see effects of microbiome on gene expression, see how microbiome influences health, see complexity of microbiome

--develop treatments

5.1 Proteomics steps

1. isolate the protein

2. cut it up using Trypsin

3. detract using chromatography like HPLC

4. detector makes a graph

5. can see which proteins exist in the sample

5.1 metabolites are

functional proteins bc working in reactions

--helpful bc can see cell process and regulation information

5.1 metabola

all metabolites in one organism

5.1 Metabolomics extraction + analytical techniques (2)

diff extraction methods with diff types of metabolites

Ex. analyze lipid metabolites, use hexane petrol as an extraction method

--analyze with chromatography and mass spec to see chemical structure

--Nuclear magnetic resonance (NMR) spectroscopy is a key analytical technique in metabolomics

--general mass spec

5.1 Multi Omics example

use multi omics to see biomarkers for IBD because can see dysbiotic microbiota (dysbiosis=imbalance in the community of microorganisms that live in the body)

--biomarkers are metabolites that are unique to IBD

5.2 define Human Microbiota

collective GENOMES of the microbes (composed of bacteria, bacteriophage, fungi, protozoa and viruses) that live INside AND on the human body.

5.2 Dr. Brett Finlay, Professor of microbiology and

immunology at the University of British Columbia:

--Provides practical advice on how we can cultivate a healthy microbiome

--Sheds light on how microbiome changes with age

and the implications for health in the elderly

5.2 Microbiota function, makes

breaks down food - FIBER specifically, produces short-chain fatty acids bc breaking down into simple form

synthesizes essential vitamins

5.2 Microbiota makes which vitamins

K, B12, and other B vitamins

5.2 Microbiota immune defense

Helps to prevent harmful substances and pathogens from crossing into the bloodstream

--bc maintain mucus and thickness

5.2 Microbiota metabolism function

Produces a variety of metabolites that can influence our overall health

--short chain fatty acids SCFA made during fiber digest, anti-inflammatory!!

--indoles and others health effects like prevent cancer

5.2 Microbiota cons

1. bad smell

2. forms plaque/biofilm (teeth, gum disease)

3. various pathologies/opportunistic infections

5.2 Microbiota bad smells

Produced by bacterial metabolism due to the breakdown of proteins/other compounds by certain types of bacteria on our skin/gut

5.2 Microbiota opportunistic infection problem

Disruption/elimination of microbiota can cause overgrowth of harmful microbes like Candida and other infections, like yeast infections or diarrhea, microbial shifts

5.2 Microbiota opportunistic infection process

1. infection=inflammation

2. inflammation of GI=microbiota shift

3. shift=chronic inflammation, hard to self-heal

4. immunological scarring, chronic inflammation, and dysbiosis

5.2 Microbiome-Gut- Brain Axis - Antibiotics example

Antibiotics disrupt the microbiome, reducing Proteobacteria and Bacteroides (good bacteria) but increasing Actinobacteria (decrease diversity!). BAD

5.2 Microbiome-Gut- Brain Axis - Probiotic example

Prevotella histicola used as a probiotic reduces MD (muscular dystrophy) in EAE (Experimental autoimmune encephalomyelitis) in mouse models for muscular dystrophy. GOOD

5.2 Microbiome-Gut- Brain Axis - Lactobacillus rhamnosus example

Lactobacillus rhamnosus activates GABA, the main CNS inhibitory neurotransmitter

5.2 Microbiome-Gut- Brain Axis - Behavior example

Gut microbiota impact emotional behavior (anxiety, depression, and pain).

5.2 Probiotics in therapy

Probiotics may have therapeutic potential in psychological treatments.

5.2 The Vagus nerve connects

the gut and brain

5.2 SCFAs made by microbiota have

immunomodulatory properties, cross the BBB (blood brain barrier) and is antiinflammatory, and regulate microglia

--stimulate vagus nerve

--stimualte gut hormones

5.2 Tryptophan metabolism example

is influenced by microbiota and is required for serotonin production

**5.2 Probiotics

--Live cultures/beneficial bacteria in fermented foods

--yes in diet is good

*5.2 Prebiotics

--Specific types of fiber such as fructo-oligosaccharides

--Do not contain NOT live cultures, serve as nourishment for microbiomes good bacteria's food!

5.2 High-Fiber Diets

--Promote the growth of Bacteroidetes bacteria

--SCFA-generating bacteria lower gut pH → less

hospitable to potentially harmful bacteria - BAD

5.2 precision medicine

considers individual differences in genes, environment, and lifestyle.

5.2 exposome

microbiome + infectome + foodome

5.2 Human Microbiome Project (HMP)

by NIH in 2007... Aims at sequencing collective genomes of all microbes

--Pros: reference for microbe sequences + clarifies healthy microbiota baseline

5.2 metagenomics pros

1. Assembles sequences into contigs to complete reference genomes and annotate → open reading frames (ORFs), small RNAs

2. Predicts metabolic and physiological potential

3. Identifies point mutations, new species

5.2 Gnotobiosis

State in which all the forms of life present within an organism can be accounted for → organism completely germ free or colonized with known set of microorganisms

5.2 mice with antibiotics treatment AKA

Secondary abiotic mice

5.2 Secondary abiotic pros + cons

pro=cheap

con=still have some bacteria, may select for resistant bacteria

5.2 Germ free mice pros + cons

pro=completely free of microbes

cons=expensive, special equipment, developmental defects in mice

5.2 4 primary techniques to study the role of microbiota in host physiology and disease:

1. Monoassociation

2. Human Microbiota-Associated (HMA)

3. Culturable Isolates

4. Replacement of Lost Taxa

5.2 Monoassociation + pros+cons

germ free mice, add in ONE microbe

pros=precise investigation, see functions without normal mask of complex communities, see gene function

cons=hard to get proper controls, not seeing true complexity

5.2 Xenotransplantation hurdles

species loss due to oxygen sensitivity

--in HMA

5.2 Human Microbiota-Associated (HMA) + pros+cons

germ free mice with entire gut microbe community from person with specific disease

pros=causation of phenotype

cons=species loss, some human microbes cannot colonize rodents

5.2 Culturable Isolates + pros+cons

germ free mice with cultural isolates from human gut - see specific microbe groups effect on host

pros=defined microbe subsets used, study in vitro and in vivo

cons=difficult to culture gut microbes

5.2 Replacement of Lost Taxa + pros+cons

Involves testing the ability of beneficial species to correct dysbiosis and prevent disease in conventional or HMA mice with dysbiosis OR microbiota-driven disease

pros+cons=same ones from HMA + Culturable isolates

5.2 Germ free cons for the mice

-Decreased nutrient absorption

-Less-developed intestines

-Vitamin deficiencies

-Underdeveloped immune system

-Susceptibility to infections and diseases

-Heightened sensitivity to pathogens

5.3 Sterile body parts

--Blood, cerebral fluid, brain, CNS

--Bone, bone marrow, joint fluid, internal organs like lymph nodes, heart, liver, spleen, vitreous (in eyeballs), kidney, pancreas, ovary, and vascular tissue

--Fetus and placenta

5.3 Microbiota in infants

--vagina birth=more lactobacillus in baby

--c section=baby has mom skin microbiota

--milk also has diff microbiota

5.3 community microbiota

families have similar microbiota

-microbiota influenced by diet, lifestyle

-seen via PCA principal component analysis plot

5.3 Microbiota predominant microbes present include

bacteria, phages, viruses, (less common) archaea, and fungi(less common)