6_Intro to Microbiota Flashcards
human microbiome:
define, locations
bacteria that:
- colonize surfaces including:
- Mucosal surfaces
- Oral Cavity/Tooth Surface
- Nares
- Oropharynx
- GI tract
- Vagina
- Skin
- Mucosal surfaces
human microbiome:
variability and issue/challenge
- variability:
- are highly variable person to person
- issues:
- many bacteria present in the microbiota cannot be cultured
microbiota vs microbiome
- Microbiota are the bacteria present; a mixed species biofilm
- Microbiome is genetic analysis of the genomes present.
what conditions vary within a biofilm?
All of the following vary throughout the biofilm
- nutrients and oxygen concentrations
- The availability of metabolites (host vs. byproducts from different bacterial species)
- cell-cell signaling (quorum sensing)
what allows for differences in transcriptional regulation?
- differences in environment (diff’t phenotypic states in biofilms);
- phenotypes also differ due to mutation and seletion and biphasic switches
evolutionary relatedness of 16S rRNA
(phylogeny)
how is this tested?
- 16S rRNA sequencing is a method used to compare the evolutionary relatedness of bacteria
- present in all cells
- non-transferrable
- large enough
- has appropriate level of conservation
- functional stability so under the same selective pressure in all bacteria
which regions show closely-related bacteria?
which regions show distantly-related bacteria?
- highly variable regions allow distinguish closely related bacteria
- highly conserved to distinguish distantly related bacteria
what are the 3 kingdoms of life?
- bacteria
- archaea
- eucaryota
archaea:
define
- single celled organisms originally considered bacteria,
- but on the basis of evolutionary relatedness and key differences in structure and physiology are now considered a distinct kingdom
bacterial taxonomy:
how are bacteria typically referred?
- when discussing bacteria involved in disease, bacteria are referred to by their genus and species
- e.g. streptococcus pyogenes
- Occasionally pathogens are referred to by subspecies
- When analyzing the microbiota, bacteria are often grouped by phylum and/or families.
Numerous types of samples can be used to characterize the microbiome and microbiota.
What are the 3 types of specimens?
-
tissue
- animal model tissues
- human surgical removed (e.g. bowel resection)
- post-mortem
-
swab
- normal flora that can be removed w/ a swab
-
fecal sample to study GI microbiota
- bacteria can be found:
- attached to epithelium
- in the mucosal layer
- can be rapidly growing as attached cells or as on the outer layer of the biofilm
- these cells are physically sheared away and exit the body with the feces
- many human GI tract studies are done on fecal specimens
- bacteria can be found:
why is the use of culture limited in its ability to characterize microbiota?
because only a small percent of the bacteria can be cultured and many different kinds of growth medium are used
what are culture techniques useful for?
screening for carriage of pathogens as part of the microbiota (e.g. MRSA)
advantages/ disadvantages of
culturing
- advantages:
- many types of media
- useful for screening for particular bacteria
- disadvantages:
- yields only culturable bacteria
- different species can be hard to distinguish
- non-quantitative
how to study microbiota?
- once the specimen is obtained –> the DNA can be characterized
- depending on the type of specimen, PCR reactions can be performed either:
- directly on the sample, or
- purified DNA
- DNA has to be extracted for whole genome sequencing (WGS)
describe the steps in processing of DNA:
D, L, R, P, C
- disrupt the tissue
- lyse bacterial cells
- remove cellular debri (e.g. lipids and proteins)
- remove proteins from DNA (protease)
- concentrate DNA by precipitation or affinity chromatography
what is 454 pyrosequencing?
- For pyrosequencing (deep sequencing):
- single fragments of DNA are attached to beads, PCR amplified and sequenced.
- Fxn: can also be used to determine relative abundance of the genes.
what is 16S rRNA sequencing used for?
- results are used to generate phylogenic trees; can group the organisms
what happens when an organism is identified by 16S rRNA that has not yet been studied?
- Often organisms are identified that have not been studied.
- They are assigned OTU numbers
- OTU = operational taxonomic unit
- In this case, the OTUs are often unidentified bacterial species
why are so many bacteria not culturable?
- because bacteria are adapted to live in a mixed species environment –> therefore many species cannot be cultured in pure culture using current techniques
- mixed species culture techniques can be used and once growth requirements are established pure cultures can be grown and studied
culture conditions are often hard to establish.
what happens to an uncultured bacteria after the growth requirements are established?
can be cultured,
characterized,
and named
why are co-culture techniques needed?
- since many of these organisms are adapted to live in mixed species biofilms.
what is the purpose of culturing?
allows for characterization of new pathogens and use of consortia of microbiota to manipulate the microbiota
how are populations compared?
- relative abundance of different bacteria
- diversity of the population
what is the metabolic pathway programs KEGG?
- the activity of the genes can be monitored by reverse transcriptase quantitative PCR (RT qPCR)
- relative abundance of the genes can be determined by number of hits
- the number of different bacteria can be assessed by the number of homologs present
- the genes are not associated with a specific bacterium unless the bacteria has already been sequenced
metagenome:
define
all the genes in the genome
bacteria can use multiple carbon sources;
what are some metabolizable sugars?
- hexoses
- glucose
- mannitol
- sorbitol
- inositol
- rhamnose
- melibiose
- amygdalin
- arabinose
- complex sugars
- raffinose
- starch
- sucrose
- lactose
describe the bacteria in the colon
(amount, content, types)
- has a large amount of bacteria in the colon (1011-1012 bacteria)
- by the time food reaches the colon it doesn’t have any sugars in it
- bacteriodetes (phylum) and bacteriodes (genera) are generally associated w/ intestinal health
bacteria in the colon produces which enzymes and why?
- produce hydrolases, esterases, and lyases
- these degrade complex polysaccharides (eg. starch, inulin, pectin, cellulose, arabinoxylan) –> into fermentable sugars
what can fermentable sugars be used for?
- the sugars can be fermented to form –> short chain fatty acids (scfa) incl. acetate, propionate and butyrate
short chain fatty acids (SFCAs)
are thought to have the following effects?
-
scfas are proposed to affect a number of different systems:
- gut-brain axis, obesity, cancer, arthritis, bowel disease
- current research focuses on the types of microbiota associated with disease states and determining the signaling pathways of the fatty acids.
how can PCR be used when studying microbiota?
- species present (16S rRNA sequencing)
- presence of specific genes
- reverse transcriptase PCR can be used to determine transcription of specific genes of interest
- quantitative PCR (qPCR) can be used to determine abundance
what can whole genome sequencing be used for, with regards to studying microbiota?
- species present by 16S rRNA
- metagenome- assembled genome of all the bacterial genes present in the microbiota
- currently the genes cannot be directly related to specific species present in the microbiota
- certain sequencing methods allow the relative abundance of sequences to be determined
- abundance can be determined by the number of hits
how can 16S rRNA be used to determine species that are present and their relative abundance?
16S rRNA alone can be amplified by PCR and sequenced to determine the species present and their relative abundance
what is the difference between the 2 types of microbiota?
(resident and transient)
-
resident:
- life-long members of an individual’s microbiota
- composition can be influenced by early events (e.g. breast milk vs. formula)
-
transient:
- •competition from resident flora
- immune system elimination
- nutrient availability (e.g. changes in diet)
benefits of the GI tract microbiota - overview
- has a number of effects on the human host.
- It affects:
- metabolism
- development of the immune response, and
- protects the GI tract from pathogens
dysbiosis:
define and potential sequelae
- imbalance in the microbiota
- can lead to a # of health problems
- chronic inflammation
- metabolic dysfunctio
pathogens can be part of the microbiota.
define opportunistic pathogens
- normal flora (part of the microbiota) that can cause disease in immunocompromised patients or if they are introduced into sterile sites.
commensal bacteria:
define
- do NOT cause disease
- members of the microbiota
pathogens:
define
- cause disease in healthy individual
- can be carried as part of the microbiota and remain controlled so they are carried asymptomatically (e.g. Clostridium difficile or Methicillin resistance Staphylococcus aureus MRSA)
