Information Flow and Genetics

TWiM #102: Happiness is the spore formers in your gut

Podcast and Annotation Information
  • Annotation by Gwendalyn Krekeler, Rutuja Deoskar, Henry Davis, Dylan Bjorn, and Blythe Janowiak.
  • Podcast audio by TWiM: Listen to TWiM #102 Podcast
  • Papers Discussed:
    • Thulin E, Sundqvist M, Andersson DI. 2015. Amdinocillin (Mecillinam) resistance mutations in clinical isolates and laboratory-selected mutants of Escherichia coli. Antimicrob Agents Chemother. 59(3):1718-27. doi: 10.1128/AAC.04819-14.
    • Yano JM, Yu K, Donaldson GP, Shastri GG, Ann P, Ma L, Nagler CR, Ismagilov RF, Mazmanian SK, Hsiao EY. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis. Cell. 2015 Apr 9;161(2):264-76. doi: 10.1016/j.cell.2015.02.047

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 03:14 minutes

The Most Interesting Things (according to students)

  • A lot of potentially useful drugs may not be used or tested further because they are known to cause a high rate of resistance. It would be more beneficial to measure antibiotic drug resistance with a combination of factors including physiology of the pathogen at its level of fitness, drug exposure levels, and host response mechanisms such as the innate immune system.
  • The mutation that occurred in vitro with microbes that share similar fitness costs was rare, meaning it is extremely unlikely to occur in vivo in the environment of an infected bladder. Thus, when this antibiotic is used, it has a low likelihood of a mutant being present and able to have a growth rate high enough to create resistance.

The abstract for this paper cannot be copied due to licensing restrictions. Please see licensing information and links to the article at the journal’s web page and/or PubMed in Section 8.1.

1.2. Main paper; discussion starts at 19:04 minutes

The Most Interesting Things (according to students)

  • Eighty percent of serotonin is made in the GI tract. In the gut, the neurotransmitter is responsible for regulating the movement of the track. In the rest of the body, it is responsible alertness, mood, appetite, regulating immune responses, regulating bone development and more.
  • Microbes are involved to produce serotonin via enterochromaffin cells. Germ-free mice have lower levels of serotonin because they do not have the microbes necessary to produce adequate amounts of the neurotransmitter.

The abstract for this paper cannot be copied due to licensing restrictions. Please see licensing information and links to the article at the journal’s web page and/or PubMed in Section 8.2.

2. Vision and Change Core Concepts and 2024 ASM Fundamental Statements

Snippet Main
Vision and Change Topics
  • Information Flow (V&C_IFG)
  • Evolution (V&C_E)
  • Information Flow and Genetics (V&C_IFG)
  • Impact of Microorganisms (V&C_IM)
ASM Fundamental Statements
  • Fundamental Statement 3 (ASM_3): The evolution of microbes is impacted by their interactions with the environment and a variety of ecological forces, including other microbes, humans, and habitats.
  • Fundamental Statement 14 (ASM_14): Extrinsic factors, such as abiotic and biotic interactions in the environment, can impact survival and growth of microbes.
  • Fundamental Statement 21 (ASM_21): Microbes and the environment interact with and affect each other.
  • Fundamental Statement 22 (ASM_22):Most microbes interact with hosts in beneficial or neutral ways, with a minority having a detrimental impact on their host.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Define antibiotic resistance and how it may differ in various environments.
 S L
  • Defend the idea that an antibiotic that shows antibiotic resistance can form easily in vitro may still be a viable clinical drug choice.
 S H
  • Identify the source of serotonin found in the human gut.
 M L
  • Predict changes to hormones under when the microbiome is changed.
 M H

1 Papers: Snippet (S) or Main (M)

2 Learning Objectives: Lower Order or Higher Order (H)

4. Techniques Described (with Time Stamps)

Here is a link to a bio-dictionary that has many, but not all definitions if you need a definition: Explore Biology Bio-Dictionary

4.1. Snippet Paper

  • Genetic Sequencing (7:15–7:41): DNA sequencing is a technique used to identify the bases of the genome or specific DNA fragments. They used genetic sequencing to identity changes in the genome of resistant strains and determined what genes these changes impacted.
  • Recombinant DNA (16:49–17:02): Recombinant DNA technologies are those where genetic material is engineered by researchers.  They describe using plasmid vectors to add genes into the bacteria.

4.2. Main Paper

  • Germ Free Mouse Model (23:50–24:15): Germ-free (GF) mice do not have microbiota.  Here they were used as one of the control mice compared to mice whose guts were colonized with specific bacteria or bacteria groups.
  • Tail Bleed Assay (25:23–36:30): This is an assay for blood clotting.
  • Red Dye Transit Time Assay (33:58–34:52): Red dyes are used to assess the transit time through the mouse digestive tract based on microbiome colonization.

5. Connections to General Microbiology Processes/Concepts (with Time Stamps)

5.1. Snippet Paper

  • Antibiotic Resistance (5:00–8:00): Bacteria develop antibiotic resistance over time as resistant bacteria will have greater fitness. However, this resistance can also harm the bacteria and make them less fit compared to wild-type bacteria, thus conferring a disadvantage
  • Stress Response (8:00–10:00): Stress responses in bacteria typically occur in times of starvation and shock. If a bacteria is induced into a stress response than it is much less likely to divide and will be less fit overall.

5.2. Main Paper

  • Human Microbiome (19:04–57:00): The human microbiome plays a significant role in shaping the person’s health. It aids in digestion, but also helps control and direct the immune system, modify drug metabolism, and can even produce changes in the nervous system.
  • Gut-Brain Axis (19:04–57:00): Many recent studies have shown a connection between the gut microbiome and the nervous system. One such way, as the paper highlights, is the regulation of serotonin which can modulate a variety of effects, but most notably regulate alertness and change mood.

6. Podcast Questions 

  1. An antibiotic is tested for a bacterial species using a rich nutrient medium and a poor nutrient medium.  While both show some resistance, the results regarding the dose do not agree.  Why might the growth be different on different media?
    1. The antibiotic resistance has a fitness cost
    2. The bacteria are more fit after gaining resistance
    3. The bacteria have lost resistance to the antibiotic
    4. The bacteria are defective in DNA repair enzymes
  2. Experiments with a new antibiotic indicate that bacteria quickly acquire resistance in the laboratory  What are some reasons why or scenarios in which it still be a useful drug?
    1. The antibiotic could be used in conjunction with other antibiotics.
    2. Laboratory and clinical conditions may show different results.
    3. The antibiotic can still limit bacterial growth, so could be useful.
    4. Bacteria in the body do not mutate, so it is good for clinical use.
  3. Which cells typically produce the serotonin that is found in the gut?
    1. T-regulatory cells
    2. Gut epithelium
    3. Spore-Forming Bacteria
    4. Pathogenic Bacteria
  4. A mouse with no bacteria undergoes a microbiome transfer from a healthy mouse what is likely to happen?
    1. Serotonin decrease
    2. Serotonin increase
    3. Tryptophan increase
    4. Tryptophan decrease
  5. If you were to take a mouse with a healthy gut microbiome and destroy half of its microbiome abundance, what would you expect to happen?
    1. Serotonin decrease
    2. Serotonin increase
    3. Tryptophan increase
    4. Tryptophan decrease

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 4), and one from the main paper (Figure 3)

7.1. First Figure Reading Exercise 

7.1.1. Learning Objectives

Students will be able to: 

  • Identify patterns of resistance in growth assays using different antibiotic concentrations.
  • Describe how resistance and growth rate affect fitness.
  • Calculate the expected cell number given specific growth variables.
Experimental Background (Thulin et al., Figure 4)

With antibiotic resistance becoming ever more prevalent, Thulin et al. (2015) wanted to identify genes involved in resistance to the antibiotic amdinocillin, which is commonly used to treat urinary tract infections.  They were also interested in determining why amdinocillin resistance is rare in clinical settings as compared to the laboratory. They hypothesize that if genetic mutations are the reason for the differences in antibiotic resistance in vitro vs in vivo then there will be mutations in E. coli strains from laboratory settings when compared to E. coli isolates from clinical settings. To test this, they measured the growth of different laboratory generated amdinocillin-resistant E. coli strains and clinical isolates at different concentrations of amdinocillin, using absorbance measurements.  The strain identifications as engineered or clinically-derived are noted in the figure legend.

  • The figure cannot be copied due to licensing restrictions. Please see licensing information and links to the article at the journal’s web page and/or PubMed in Section 8.1.

7.1.2. Questions

  1. What strain has the highest growth without amdinocillin present?
    1. DA14719
    2. DA28432
    3. DA5438
    4. DA29712
  2. What strain exhibits the greatest resistance to amdinocillin?
    1. DA28432
    2. DA28436
    3. DA5438
    4. DA24686
  3. A new E. coli strain has amdinocillin resistance but has a relative growth rate equal to wild-type E. coli. If amdinocillin continues to be used as a treatment for E. coli, which strain has the greatest fitness and what is most likely to happen?
    1. The wild-type strain; the wild-type strain will outcompete the new strain because it is wild-type.
    2. The new strain ; the new strain will be the dominant strain because it had advantage of resistance.
    3. The wild-type strain; the new strain will mutate, lose resistance to  amdinocillin, and die off
    4. Neither; the new strain and wild-type strain will be found in equal abundances when found together.
  4. The wild-type strain (DA5438) has a relative growth rate of 1 without amdinocillin. DA28434, a mutant strain, has a relative growth rate of 0.6 without amdinocillin. Assuming that the number of DA5438 E. coli doubles every hour, how much of DA28434 is there expected to be after 4 hours if there are initially 100 bacteria?
    1. 0
    2. 400
    3. 655
    4. 800

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Analyze abundance data to identify bacterial species involved in 5-HT production.
  • Identify and defend the use of different controls for these experiments.
  • Predict the effects of antibiotic use and/or mutation on  5-HT production, Tph1 expression, and mood based on these data.
Experimental Background (Yano et al., Figure 3)

Serotonin is a hormone that plays important roles in sleep, mood, and digestion.  Serotonin, also known as 5-hydroxytryptamine (5-HT) is largely found in the gut, where its metabolism is not clearly understood.  Yano et al. (2015) was interested in the microbiome’s role in serotonin metabolism, that is which types of bacteria impact serotonin production/levels in the gut and how they accomplish this. They hypothesize that if a certain type of bacteria helps with serotonin production, then bacterial abundance will correlate with high levels of 5-HT mRNA and protein in the colon. To test this, they inoculated germ-free (GF) or specific pathogen free (SPF) mice in the gut with specific mono or polycultures of different bacteria and then quantified serum 5-HT (panel A) and colon 5-HT (panel B). SPF (specific pathogen free) mice serve as the control as they are populated with a typical microbiome.   The researchers next investigated whether treatment with PCPA, an antibiotic that halts the production of 5-HT in spore forming bacteria., affected 5-HT levels (panel C) and/or expression of the Tph1 gene, which encodes the protein that is the rate limiting step of 5-HT production (panel D).

  • The figure cannot be copied due to licensing restrictions. Please see licensing information and links to the article at the journal’s web page and/or PubMed in Section 8.2.

7.2.2. Questions

  1. Colonization with which bacterial group shows the highest 5-HT as measured in both from both the serum (panel A) and colon (panel B)?
    1. B. fragilis
    2. Spore-forming bacteria
    3. B. uniformis
    4. Altered Schaedler Flora
  2. What property of specific pathogen free (SPF) mice makes them a good control group for these experiments?
    1. They are pathogen free and have healthy microbiota
    2. They have no microbiome bacteria in their gut
    3. They contain a fully human microbiota composition
    4. They have a disrupted (dysbiotic) microbiome composition
  3. During the course of a bacterial infection, antibiotics are typically used which kill all bacteria. Based on the data in this figure, what is likely to occur because of this?
    1. TPH1 expression decreases, 5-HT production increases, and mood increases
    2. TPH1 expression increases, 5-HT production stays the same, and mood stays the same
    3. TPH1 expression decreases, 5-HT production decreases, and mood decreases
    4. TPH1 expression stops, 5-HT production halts entirely, and mood increases
  4. Why is the expression of GAPDH compared to the expression of TPH1?
    1. GAPDH is only expressed in gut microbiota so it is useful to show the RNA is not degraded.
    2. GAPDH is a “housekeeping” gene that is uniformly expressed and used to normalize the data.
    3. GAPDH is controlled by TPH1, so it shows the relationship of signaling in this system.
    4. TPH1 inhibits GAPDH under certain conditions so it is used as a control for dysbiosis.
  5. If a person had a mutant TPH1 gene that encoded a defective TPH1 protein and normal gut microbiome, what would you expect to see for their 5-HT levels based on the data in this figure?
    1. Low 5-HT levels; disturbed microbiome
    2. High 5-HT levels; disturbed microbiome
    3. Low 5-HT levels; normal microbiome
    4. High 5-HT levels; normal microbiome

8. Paper Information and Licensing

8.1. Snippet paper

  • Thulin E, Sundqvist M, Andersson DI. 2015. Amdinocillin (Mecillinam) resistance mutations in clinical isolates and laboratory-selected mutants of Escherichia coli. Antimicrob Agents Chemother. 59(3):1718-27. doi: 10.1128/aac.04819-14
  • This article is not licensed for Creative Commons use. Thus, the abstract and figures cannot be copied here.  Please see the article on the journal’s web page or see the full paper at PubMed: https://pmc.ncbi.nlm.nih.gov/articles/PMC4325821/

8.2. Main paper

  • Yano JM, Yu K, Donaldson GP, Shastri GG, Ann P, Ma L, Nagler CR, Ismagilov RF, Mazmanian SK, Hsiao EY. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis. Cell. 2015 Apr 9;161(2):264-76. doi: 10.1016/j.cell.2015.02.047
  • This article is not licensed for Creative Commons use. Thus, the abstract and figures cannot be copied here.  Please see the article at the journal’s web page: on the journal’s web page or the full paper at PubMed: https://pmc.ncbi.nlm.nih.gov/articles/PMC4393509/

License

Icon for the Creative Commons Attribution 4.0 International License

Podcast Annotation and Resources in Microbiology Copyright © 2025 by Rebecca Seipelt-Thiemann; Nancy Boury; Gwendowlyn S. Knapp; Amaya Garcia Costas; and Patrick Armstrong is licensed under a Creative Commons Attribution 4.0 International License, except where otherwise noted.

Share This Book