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Y1 - Microbio Exam 2 > 6_Intro to Microbiota > Flashcards

6_Intro to Microbiota Flashcards

1
Q

human microbiome:

define, locations

A

bacteria that:

  • colonize surfaces including:
    • Mucosal surfaces
      • Oral Cavity/Tooth Surface
      • Nares
      • Oropharynx
      • GI tract
      • Vagina
    • Skin
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2
Q

human microbiome:

variability and issue/challenge

A
  • variability:
    • are highly variable person to person
  • issues:
    • many bacteria present in the microbiota cannot be cultured
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3
Q

microbiota vs microbiome

A
  • Microbiota are the bacteria present; a mixed species biofilm
  • Microbiome is genetic analysis of the genomes present.
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4
Q

what conditions vary within a biofilm?

A

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)
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5
Q

what allows for differences in transcriptional regulation?

A
  • differences in environment (diff’t phenotypic states in biofilms);
  • phenotypes also differ due to mutation and seletion and biphasic switches
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6
Q

evolutionary relatedness of 16S rRNA

(phylogeny)

how is this tested?

A
  • 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
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7
Q

which regions show closely-related bacteria?

which regions show distantly-related bacteria?

A
  • highly variable regions allow distinguish closely related bacteria
  • highly conserved to distinguish distantly related bacteria
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8
Q

what are the 3 kingdoms of life?

A
  • bacteria
  • archaea
  • eucaryota
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9
Q

archaea:

define

A
  • 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
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10
Q

bacterial taxonomy:

how are bacteria typically referred?

A
  • 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.
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11
Q

Numerous types of samples can be used to characterize the microbiome and microbiota.

What are the 3 types of specimens?

A
  1. tissue
    • animal model tissues
    • human surgical removed (e.g. bowel resection)
    • post-mortem
  2. swab
    • normal flora that can be removed w/ a swab
  3. 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
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12
Q

why is the use of culture limited in its ability to characterize microbiota?

A

because only a small percent of the bacteria can be cultured and many different kinds of growth medium are used

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13
Q

what are culture techniques useful for?

A

screening for carriage of pathogens as part of the microbiota (e.g. MRSA)

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14
Q

advantages/ disadvantages of

culturing

A
  • 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
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15
Q

how to study microbiota?

A
  1. once the specimen is obtained –> the DNA can be characterized
  2. depending on the type of specimen, PCR reactions can be performed either:
    • directly on the sample, or
    • purified DNA
  3. DNA has to be extracted for whole genome sequencing (WGS)
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16
Q

describe the steps in processing of DNA:

A

D, L, R, P, C

  1. disrupt the tissue
  2. lyse bacterial cells
  3. remove cellular debri (e.g. lipids and proteins)
  4. remove proteins from DNA (protease)
  5. concentrate DNA by precipitation or affinity chromatography
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17
Q

what is 454 pyrosequencing?

A
  • 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.
18
Q

what is 16S rRNA sequencing used for?

A
  • results are used to generate phylogenic trees; can group the organisms
19
Q

what happens when an organism is identified by 16S rRNA that has not yet been studied?

A
  • 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
20
Q

why are so many bacteria not culturable?

A
  • 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
21
Q

culture conditions are often hard to establish.

what happens to an uncultured bacteria after the growth requirements are established?

A

can be cultured,

characterized,

and named

22
Q

why are co-culture techniques needed?

A
  • since many of these organisms are adapted to live in mixed species biofilms.
23
Q

what is the purpose of culturing?

A

allows for characterization of new pathogens and use of consortia of microbiota to manipulate the microbiota

24
Q

how are populations compared?

A
  • relative abundance of different bacteria
  • diversity of the population
25
Q

what is the metabolic pathway programs KEGG?

A
  • 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
26
Q

metagenome:

define

A

all the genes in the genome

27
Q

bacteria can use multiple carbon sources;

what are some metabolizable sugars?

A
  • hexoses
    • glucose
    • mannitol
    • sorbitol
    • inositol
    • rhamnose
    • melibiose
    • amygdalin
    • arabinose
  • complex sugars
    • raffinose
    • starch
    • sucrose
    • lactose
28
Q

describe the bacteria in the colon

(amount, content, types)

A
  • 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
29
Q

bacteria in the colon produces which enzymes and why?

A
  • produce hydrolases, esterases, and lyases
  • these degrade complex polysaccharides (eg. starch, inulin, pectin, cellulose, arabinoxylan) –> into fermentable sugars
30
Q

what can fermentable sugars be used for?

A
  • the sugars can be fermented to form –> short chain fatty acids (scfa) incl. acetate, propionate and butyrate
31
Q

short chain fatty acids (SFCAs)

are thought to have the following effects?

A
  • 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.
32
Q

how can PCR be used when studying microbiota?

A
  • 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
33
Q

what can whole genome sequencing be used for, with regards to studying microbiota?

A
  • 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
34
Q

how can 16S rRNA be used to determine species that are present and their relative abundance?

A

16S rRNA alone can be amplified by PCR and sequenced to determine the species present and their relative abundance

35
Q

what is the difference between the 2 types of microbiota?

(resident and transient)

A
  • 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)
36
Q

benefits of the GI tract microbiota - overview

A
  • has a number of effects on the human host.
  • It affects:
    • metabolism
    • development of the immune response, and
    • protects the GI tract from pathogens
37
Q

dysbiosis:

define and potential sequelae

A
  • imbalance in the microbiota
  • can lead to a # of health problems
    • chronic inflammation
    • metabolic dysfunctio
38
Q

pathogens can be part of the microbiota.

define opportunistic pathogens

A
  • normal flora (part of the microbiota) that can cause disease in immunocompromised patients or if they are introduced into sterile sites.
39
Q

commensal bacteria:

define

A
  • do NOT cause disease
  • members of the microbiota
40
Q

pathogens:

define

A
  • 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)

Y1 - Microbio Exam 2 (18 decks)

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