The Respiratory Microbiome

Question 1

Define microbiota

Answer 1

The population of micro-organisms in an environment

Question 2

Define Microbiome

Answer 2

All micro-organisms in an environment AND their interactionsbetween each other and the environment)

Question 3

The human microbiome: This is incredibly diverse, containing not only bacteria but _____, _____, _____ and archea. The microbiome is _____ to the host niche. It is _____ to the individual (more similar in twins and families but not identical). There is enormous _____ and _____ potential.

Answer 3

The human microbiome: This is incredibly diverse, containing not only bacteria but viruses, phages, fungi and archea. The microbiome is specific to the host niche. It is unique to the individual (more similar in twins and families but not identical). There is enormous metabolic and genetic potential.

Question 4

The phylogenetic tree is a way of sorting species by _____. There are three main domains: bacteria, _____ and eucarya (Virus are categorised separately). The number of _____ indicates the genetic difference between the species.

Answer 4

The phylogenetic tree is a way of sorting species by genetics. There are three main domains: bacteria, archea and eucarya (Virus are categorised separately). The number of branches indicates the genetic difference between the species.

All organisms are microbial - big things are aberrations

Question 5

What can influence your microbiome? (sources of microbiome)

Answer 5

1. Birth delivery
   
   1. Vaginal - have vaginal bacteria in their gut
   2. Caesarean section - have skin bacteria in their gut
2. Environment
   
   1. Air, surface, other people
   2. Micro-aspirations (gut)
3. Hereditary

Question 6

Microbiomes can be inherited. Our phylogeny tree with great apes similar to the phylogeny of our gut microbiome. Suggest two reaons why this may be?

Answer 6

1. Microbiome evolved with us
   
   1. Suggests strong association and interaction between host and microorganisms
2. Diet

Question 7

Describe the advantages and disadvantages of using a culture-based technique

Answer 7

Advantages

1. Cheap
2. Widely available
3. Provide good indication of microbiome complexity

Disadvantages

1. Most bacterial community is unculturable (only 5% culturable) - Great plate count anomaly
2. Naturally selective
   
   1. No one combination of nutrients, temprature allows all microorganisms in a sample to grow
3. Inconsistent techniques lead to contamination

Question 8

Name two techniques which measure or identify microorganisms.

Answer 8

1. Cell culture
2. PCR (through 16S rRNA)

Question 9

Briefly describe how PCR is used to identify microogranisms by RNA expression.

Answer 9

16 rRNA gene is used (ribosome)

Highly conserved house-keeping genes and present in every cellular organism

Variable regions allow for the identification of microorganism

Question 10

Describe the advantages and disadvantages of using PCR to identify microogranisms

Answer 10

Advantages

1. 16S rRNA present in every cellular organism
2. High throughput screening
3. More defined than biochemistry (as with cultures)

Disadvantages

1. Doesn’t tell you what’s alive or dead

Question 11

Samples have to be taken in order to identify microorganisms. Identify two potential problems with any sampling that occurs

Answer 11

Different sampling techniques between people

Preservation (or lack of) may skew the population (i.e. not representative)

Question 12

Define alpha diversity

Answer 12

The number of different organisms and their proportion in a sample

Question 13

Define Beta diversity

Answer 13

The similarity or difference in diversity between samples and populations

Question 14

Define the Operational Taxonomic Unit (OTU)

Answer 14

Sequence clusters which are simliar to each other by 97% relative to 16S gene (proxy)

Question 15

Define a pathogen

Answer 15

Microorganism that causes disease

Question 16

What is Koch’s Postulates used for and what are they? (4)

Answer 16

Used to establish a causative relationship between organism and disease

1. Found in diseased individuals but not in healthy individuals
2. Isolated and grown in pure culture
3. Isolate causes disease in healthy individuals
4. If you isolate again from the test subject, it should be the same organism

Question 17

Describe the limitations of Koch’s portulates

Answer 17

1. Vibrio cholerae and cholera, Salmonella typhi and typhoid present in healthy individuals
2. Organisms may be difficult or not possible to culture
3. Animal models may not be susceptible (M. leprae)
   
   1. ​Models used as unethical to cause disease in humans
   2. Leprosy only in armadillo
4. May be difficult or not possible to culture

Question 18

What does the pathogenicity of a microorganism depend on?

Answer 18

The interaction of the organism with its host environment

Pathogenicity is an emergent property, they are context dependent

Question 19

Healthy lungs have a microbiota. Bacterial communites are alterd in diseased lungs. They remain individual and may become more individual. The communites present are different between different _____. Though some common bacteria can be _____ with a number of different diseases. There are differences within the topography of the lung.

Answer 19

Healthy lungs have a microbiota. Bacterial communites are alterd in diseased lungs. They remain individual and may become more individual. The communites present are different between different diseases. Though some common bacteria can be associated with a number of different diseases. There are differences within the topography​ of the lung.

Question 20

Name some sampling techniques for sampling the respiratory microbiome

Answer 20

Nasal brushings

Swabs

Sputum culture

Induced Cough

Biopsy

BAL fluid

Whole lung

Question 21

What kind of bacteria are found in the lungs: aerobic or anaerobic?

Answer 21

Anaerobic (especially with CF)

Doesn’t need to depend on oxygen to grow therefore not limited to the surrounding

Question 22

When studying the microbiome of severe asthmatics, what is a possible confounder?

Answer 22

Severe asthmatics will be on steroid inhalers, therefore the microbiome present could have been selected by the steroid.

The current microbiome would not have been representative of the microbiome before (ie the microbiome before potentially causing the asthma)

Question 23

Outline 3 approaches to medicating the microbiome

Answer 23

1. Selective therapies
2. Community manipulation
3. Novel drug targets

Question 24

Describe an advantage and disadvantage with using antibiotics

Answer 24

Pro: Can selectively kill bacteria

Con: Use is a selection pressure towards resistant microogranisms (bacteria can share resistant mechanisms easily)

Question 25

Outline four alternatives to antibiotics

Answer 25

1. Phage therapy
   
   1. Virus that infects specific bacteria
      
      1. Need multiple due to the high specificity
         
         1. Currently only used in salami
2. Vaccination
   
   1. Hard to design vaccines for bacteria (will cause infection)
3. Probiotics
   
   1. Manipulate the community
4. Faceal transplants

Question 26

Describe one way in ehcih Ivacaftor could be beneficial in CF other than its correcting ability

Answer 26

Osmotic pressure and pH difference selects fot bacteria and may have an overall beneficial effect

Question 27

Define metagenomics

Answer 27

Direct sequencing of DNA extract, rather than a PCR product

Distinct from 16S sequencing, but commonly confused

Question 28

Define diversity

Answer 28

Summary statistic for community structure, calculated from:

1. Species richness - number of different organisms present
2. Species evenness - are the microorganisms present at similar levels or is one more dominant