bacterial pathogens exam 2

16S rRNA sequencing

technique used to identify and classify microorganisms by analyzing the DNA sequence of the 16S ribosomal RNA gene

NextGen or high-throughput sequencing

can be quantitative and identify mutations; requires bioinformatics and statistical software to assemble reads and analyze data; many different technologies available

metagenomic sequencing

the collective analysis of all genetic material from a sample

phylogenetic tree

a diagram that illustrates the evolutionary relationships between different species or other groups of organisms, showing how they have branched from common ancestors over time

node

a taxonomic unit

branch

defines the relationship between the taxa in terms of descent and ancestry

branch length

proportional to evolutionary distance, representing the amount of genetic change or time since divergence

root

the common ancestor of all taxa

distance scale of phylogenetic tree

represents number of differences between sequences

dendrogram

a tree-like diagram that visualizes hierarchical relationships; to see the differences between species, measure the length of branches and a scale indicates the percentage of difference between the branches

Shigella sonnei phylogenetic tree

dysentery pathogen; current strains descended from a common ancestor arising in Europe < 500 years ago

qPCR

a purely quantitative method used to amplify and quantify specific DNA or RNA sequences; employs primer pairs that anneal specifically to 16S rRNA genes of bacterial species of interest in a population; less time to achieve fluorescence = higher level of gene abundance; specific threshold called Ct is used to indicate the point at which fluorescence appeared

Ct

threshold cycle; refers to the number of PCR cycles required to amplify a specific target gene to a detectable level in qPCR

higher gene abundance

lower Ct value indicates:

increases

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

metagenomics

the study of the genetic material of an entire community of microorganisms in a specific environment

transcriptomics

the study of the transcriptome, which is the complete set of RNA transcripts produced by a cell, tissue, or organism at a given time

proteomics

field of study that focuses on the analysis of proteins

metabolomics

the large-scaled study of metabolites, which are the small molecules produced during metabolism

systems biology

integrates multiple biological levels to create comprehensive predictive models; the approach provides a detailed understanding of cellular complexity and investigates the phenotype; multiple omics techniques provide insight into molecular mechanisms, analyzed using advanced bioinformatics tools

RNA-sequencing

a technique to measure gene expression levels

process of RNAseq:

1. RNA isolation

2. RNA selection/depletion to isolate only mRNA from the sample

3. cDNA synthesis to convert mRNA into cDNA

4. sequencing of the cDNA library to obtain transcript information

16S rRNA seq

what technique is commonly used for the qualitative method in microbiome studies?

it is large enough to enable discrimination between species

what is the main advantage of 16S rRNA gene for bacterial identification?

the conserved regions

which regions of the rRNA molecule are used for PCR amplification?

the larger regions of overlapping sequences in metagenomic sequencing

what are contigs?

the relationship between the taxa in terms of descent and ancestry

what do branches in phylogenetic trees represent?

the cycle number at which fluorescence begins to appear

what does the Ct value in qPCR represent?

higher gene abundance

what does a lower Ct value in qPCR indicate?

microbiome

the collective genomes of the microbes (composed of bacteria, bacteriophage, fungi, protozoa, and viruses) that live inside and on the human body

microbiome importance:

number of microbes in our body far outweighs the number of human of human cells; bacteria help digest our food, producing short-chain fatty acids; microbiome synthesizes essential vitamins such as K, B12, and other B vitamins; controls pathogen colonization and trains our immune system; maintains integrity of our gut barrier; helps prevent harmful substances from crossing into bloodstream; produces a variety of metabolites that can influence overall health

less desirable functions of the microbiome:

1. bad smells

2. formation of plaque 

3. various pathologies/opportunistic infections

Lactobacillus rhamnosus

bacteria found in the gut that activates GABA, the main CNS inhibitory neurotransmitter

Prevotella histicola

used as a probiotic and reduces muscular dystrophy in EAE in mouse models for muscular dystrophy

vagus nerve

connects the gut and brain

probiotics

live cultures/beneficial bacteria in fermented foods; should be incorporated regularly into one’s diet

prebiotics

specific types of fiber such as fructo-oligosaccharides; do not contain live cultures, serve as nourishment for microbiomes

high-fiber diets

promote the growth of Bacteroidetes bacteria; short-chain fatty acid-generating bacteria lower gut pH, making it less hospitable to potentially harmful bacteria

high-fat diets

shift the gut microbiota towards the growth of Firmicutes and Enterobacteria; negatively impact overall health

exposome

sum of all external and internal environmental factors that a person is exposed to throughout their lifetime