lecture 6 microbiota

Question 1

How does S. aureus cause infections and intoxication?

Answer 1

Causes infection due to the bacteria and intoxication due to toxins secreted by the bacteria.

Question 2

How is most microbiota present?

Answer 2

as a mixed species biofilm

Question 3

Bacteria colonizes surfaces such as:

Answer 3

mucosal (nares, surfaces, GI, vagina), skin, are highly variable from person to person, many bacteria present in the microbiota cannot be cultured**

Question 4

What is the microbiota?

Answer 4

the bacteria present , many cannot be cultured, is a mixed species biofilm, highly variable from person to person

Question 5

Microbiome

Answer 5

genetic analysis of the genomes present

Question 6

What varies throughout the biofilm

Answer 6

nutrients and oxygen concentration

Question 7

What varies depending on the location of the biofilm?

Answer 7

metabolic cooperativity and cell-cell signaling (quorum sensing) The availability of metabolites and quorum sensing are different throughout the biofilm

Question 8

Why do phenotypes in the biofilm differ?

Answer 8

mutation and selection , and biophasic switches . Differences also lead to transcriptional regulation

Question 9

What is 16S rRNA sequencing? “phylogeny”

Answer 9

Method used to compare the evolutionary relatedness of bacteria. Present in ALL cells, non-transferable, large, appropriate conservation, variable regions allow distinguish closely related bacteria, highly conserved to distinguish distantly related bacteria, functional stability so under the same selective pressure in all bacteria.

Question 10

What are the three kingdoms of life?

Answer 10

bacteria, archaea, eucaryota archaea are single celled organisms originally considered bacteria, but on the basis of evol relatedness and key differences in structure, they are now considered a distinct kingdom.

Question 11

How are bacteria referred to?

Answer 11

by their genus and species. strep pyogenes. Occasionally pathogens are referred to by subspecies . Bacterial names are always in italics. When analyzing the microbiota, bacteria are often grouped by phylum/families.

Question 12

How can one characterize the microbiome and microbiota?

Answer 12

various samples including: tissues-animal models, surgically removed/post-mortem, swab- mucosal surfaces, fecal samples can be used to study GI microbiota. Fecal specimens will have bacteria shed from the surface usually pieces of biofilms, may or may not fully represent the entire microbiota

Question 13

Why is the use of culture to characterize microbiota limited?

Answer 13

only a small percent of the bacteria can be cultured and many different kinds of growth medium are used. Yields only culturable bacteria. **Culture tech. are USEFUL for screening for carriage of pathogens as part of the microbiota (MRSA)

Question 14

How does the DNA get characterized?

Answer 14

After specimen is obtained, PCR reactions can be performed directly on sample or purified DNA. DNA has to be extracted for whole genome sequencing. (WGS)

Question 15

What does processing of DNA include?

Answer 15

disrupting the tissue, lysing the bac cells, removing lip/pro, removing proteins from DNA -proteases, concentration of the DNA by precipitation or using affinity chromatography.

Question 16

What is pyrosequencing?

Answer 16

single fragments of DNA are attached to beads, PCR amplified and sequenced. This also can be used to determine relative abundance of genes.

Question 17

How are phylogenic trees sequenced?

Answer 17

16S rRNA sequences can be used to group the organisms. Often organisms are identified that have not been studied and they are assigned OTU numbers.

Question 18

How do bacteria get cultured?

Answer 18

many bacteria cannot be cultured because bacteria are adopted to live in a mixed species environment many species cannot be cultured in pure culture using current techniques.

Question 19

What techniques are used to culture bacteria?

Answer 19

conditions are often hard to establish. New tech involve the use of co-culture since many organisms are adapted to live in mixed species biofilms. special req. for culture of the bacteria can be determined and the new species can be grown and characterized. Culture allows for characterization of new pathogens and use of consortia of microbiota

Question 20

How are populations compared?

Answer 20

relative abundance of different bacteria and diversity of the population

Question 21

What is KEGG and other metabolic pathway programs?

Answer 21

metabolic pathways can be assesed using KEGG. reverse transcriptase can monitor quantitative PCR . 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 they have already been sequenced.

Question 22

Can bacteria use multiple carbon sources?

Answer 22

YES

Question 23

Discuss bacteria within the colon

Answer 23

colon has large numbers of bacteria 10 11 - 10 12. By the time food reaches the colon, it does not have any sugars in it. Bacteroidetes (phylum) and Bacteriodes (genera) are generally associated with intestinal health

Question 24

Bacteria in the colon produce what?

Answer 24

hydrolases , esterases, and lyases to degrade complex polysaccharides (starch , inulin, pectin, cellulose) into fermentable sugars.

Question 25

Sugars can be fermented to produce what ?

Answer 25

short chain fatty acids (SCFA) including acetate , propionate, and butyrate.

Question 26

How do SCFA’s affect the body?

Answer 26

they are proposed to affect a number of different systems , gut brain axis, obesity, cancer. Current research focuses on the types of microbiota associated with disease states and determining the signaling pathways of the fatty acids.

Question 27

How can PCR be used to determine bacteria?

Answer 27

can determine species present (16SrRNA sequencing) , presence of specific genes, reverse transcriptase PCR,

Question 28

Whole genome sequencing/ metagenome can determine what?

Answer 28

species present by 16S rRNA , metagenome- all genes present, abundance can be determined by the number of hits.

Question 29

When 16SrRNA is amplified by PCR what can occur?

Answer 29

It can be sequenced to determine the species present and their relative abundance

Question 30

What are resident microbiota

Answer 30

life long members of an individual’s microbiota . Composition can be influenced by early events.

Question 31

What is transient microbiota

Answer 31

competition from resident flora, immune system elimination, nutrient availability

Question 32

What does GI tract microbiota affect?

Answer 32

on the human host, it can affect metabolism, development of the immune response, and protects the GI tract from pathogens. Dysbiosis of the microbiota can lead to a number of health problems

Question 33

Pathogens

Answer 33

can be part of the microbiota and remain controlled (c dif, MRSA) asymptomatic

Question 34

Opportunistic pathogens

Answer 34

normal flora that can cause disease in immunocompromised patients or if they are introduced into sterile sites. Microbiota is disrupted by antibiotic use.

Question 35

Commensals

Answer 35

do not cause disease, are members of the microbiota