Microbial Ecology

TWiM #247: Therapy with Paleofeces and Phages

Podcast and Annotation Information
  • Annotation by Lauren Farrar, Gabrielle Baca, Benjamin Torres, Enrique Rodriguez, Damaris Hernadez, Suparna Chatterjee, Jovani Catalan-Dibene, and Rebecca Seipelt-Thiemann
  • Podcast audio by TWiM: Listen to TWiM #247 Podcast
  • Podcast transcript by Otter.ai and edited by Nick Bellavia and Isabelle Norris: Access Podcast Transcripts
  • Papers Discussed:
    • Carmody RN, Sarkar A, Reese AT. 2021. Gut microbiota through an evolutionary lens. Science. 372(6541):462-463. doi: 10.1126/science.abf0590.
    • Dedrick RM, Freeman KG, Nguyen JA, Bahadirli-Talbott A, Smith BE, Wu AE, Ong AS, Lin CT, Ruppel LC, Parrish NM, Hatfull GF, Cohen KA. 2021. Potent antibody-mediated neutralization limits bacteriophage treatment of a pulmonary Mycobacterium abscessus infection. Nature medicine. 27(8): 1357-1361. doi: 10.1038/s41591-021-01403-9

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 2:27 minutes

The Most Interesting Things (according to students) 

The idea of returning our microbiome or re-wilding back to its “natural state” pre-industrialization. Though there is not a set definition of what a healthy microbiome is, mimicking the flora to better match the environment of a time when ancient humans were “healthier.” Of course, idiosyncratic factors such as parasites and viral burdens over the course of a lifetime will affect overall fitness.

This article is not licensed for Creative Commons use; see the journal’s website for information. Thus, the abstract and figures cannot be copied here. The paper is behind a paywall but a version of this paper is available on California’s eScholarship repository.

1.2. Main paper; discussion starts at 24:23 minutes

The Most Interesting Things (according to students)

Phage therapy for immunocompetent individuals with a bacterial infection is most likely to not work because their immune system neutralizes the bacteriophage before it gets to the pathogenic bacteria the individual is infected with. However, people who are immunocompromised can successfully go under phage therapy because their immune system is either non-existent or very weak, which means that their antibodies have a harder time neutralizing the phage, or maybe they are incapable of doing so.

This article is not licensed for Creative Commons use; see the article’s copyright information. Thus, the abstract and figures cannot be copied here.  The paper is behind a paywall but a version of this paper is available on PubMed at: https://pubmed.ncbi.nlm.nih.gov/34239133/

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

Snippet Main
Vision and Change Topics
  • Evolution (V&C_E)
  • Microbial Ecology (V&C_ME)
  • Microbial Ecology (V&C_ME)
  • Structure and Function (V&C_SF)
ASM Fundamental Statements
  • 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.
  • Fundamental Statement 24 (ASM_24): Microbes and their communities are essential for supporting all life as we know it.
  • 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 19 (ASM_19): Non-cellular infectious agents, such as viruses, prions, viroids, and satellites, are dependent on host cell processes in order to replicate.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Identify the three inter-related aspects of the authors’ evolutionary perspective on gut microbiome.
  • Define canalization.
 S L
  • n/a
 S H
  • Define bacteriophage.
  • Identify the reason phage therapy was unsuccessful for the patient.
 M L
  • Compare and contrast phage therapy and antibiotic therapy.
  • Draw conclusions based on alternative hypothetical data.
 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

  • Fecal Transplant (3:48): This involves taking fecal microbiota from a healthy person and inoculating the gut of another person.  This was discussed in terms of treatment and reversal to pre-industrialized gut microbiota.

4.2. Main Paper

  • Phage Therapy (25:52): This is a methodology where a specific bacteriophage that kills a pathogen is given to a patient.  Usually, this is used in  patients to help clear antibiotic-resistant bacterial infections.
  • Bacterial Isolation (27:06): This is a culturing method where bacteria are isolated from an infected person.  It is often done to identify the pathogenic bacteria.
  • Phage Preparation (29:22): When phage therapy is performed it is necessary to know the phage concentration or titer, so the correct the amount of phage therapy given to patients.
  • Antibody Neutralization Assay (33:31): This is an assay to determine antibody binding to phage.  This was discussed in terms of testing whether the antibodies were binding and neutralizing the phages that are used in phage therapy.
  • Antibody-Antigen Recognition Assays (34:25): This is an assay to determine antibody binding to an antigen.  This was discussed in terms of testing if antibodies recognize/bind to different antigens of interest.

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

5.1. Snippet Paper

  • Canalization (9:15): This is a measure of the ability of an organism to have the same phenotype regardless of differences in environment and genotype; think of it as phenotype stability.
  • Gut Microbiota (11:00): This is the natural population of microbes in the human gut.
  • Microbial Competition (13:46): Microbes can have many different interactions within an environment (symbiotic relationships), one of which is competition, usually for resources.

5.2. Main Paper

  • Bacteriophages (25:52): Bacteriophages are bacterial viruses and they can be used to treat a bacterial infection by lysis.
  • Antibacterial Resistance (27:15): Bacteria have evolved many different mechanisms to combat antibacterial compounds.  With several species of bacteria becoming resistant to antibiotics, such as mycobacterium, we must find other methods of treating bacterial infections, such as bacteriophage.
  • Phage Resistance (29:50): Like resistance to antibiotics, bacteria can also evolve resistant to phage.
  • Antibodies  (32:20): These are host immune proteins that can neutralize a virus, like a bacteriophage and prevent it from infecting the bacterial cells.
  • Passage of Bacteriophage (39:49): Phage is passaged several times through the body in order to isolate a phage that is resistant to immune system clearance of bacteriophage.

6. Podcast Questions

  1. What are the three aspects that the authors of the snippet paper propose as major interacting determinants in gut-related human health?
    1. Gut microbiota; environment; host
    2. Gut structure; gut microbiota; host
    3. Environment; host health; gut structure
    4. Microbiota; gut structure; environment
  2. Canalization is the measure of ________.
    1. whether an organism is able to successfully adapt to changes in environment through horizontal gene transfer.
    2. an organism’s ability to use developmental processes that increase fitness in a particular environment.
    3. how well an organism is able to retain the same phenotype regardless of genotype or environment.
    4. an organism’s success in developing structures in the embryonic stages that are involved in neurogenesis.
  3. Bacteriophage are bacterial _________ .
    1. epibonts
    2. viruses
    3. organelles
    4. metabolites
  4. Which statement is the best description of the conclusions for the main paper?
    1. Phage therapy was unsuccessful for this patient due to antibody neutralization of the phage.
    2. Phage therapy was successful for this patient because the patient’s infection was eliminated.
    3. Phage therapy was unsuccessful for this patient; phage were ineffective against the bacteria.
    4. Phage therapy was successful for this patient; phage were able to bind and kill bacteria in vitro.
  5. Match the statements below with phage (P), antibiotics (A), both (B), or neither (N).
    1. _______ Can be used to kill bacteria
    2. _______ Bacteria have not evolved resistance to this/these
    3. _______ Bacteria can evolve resistance to this/these
    4. _______ Bacterial genes can encode production of this/these
    5. _______ Fungal genes can encode production of this/these
    6. _______ Involves an infection step to kill bacteria
  6. In the discussion, the podcasters mention several specific ways the patient’s outcome could have come about.  What results would have been observed if the patient’s bacteria had been resistant to phage?
    1. The patient’s serum would have neutralized the bacteriophage and the phage would have destroyed the bacteria in vitro, but not in vivo.
    2. The patient’s serum would have not have neutralized the phage and the phage would have destroyed the bacteria in vivo, but not in vitro.
    3. The patient’s serum would have not have neutralized the phage and the phage would not have destroyed the bacteria either in vivo or in vitro.
    4. The patient’s serum would have neutralized the bacteriophage and the phage would have destroyed the bacteria both in vitro and in vivo.

7. Figure Reading Exercises

The following are two figure reading exercises, both from the main paper (Figures 1d and 2c).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify the controls for this experiment.
  • Identify specific phage-bacteria inoculum spot-on-spot locations.
  • Analyze the evidence and draw conclusions about phage-based killing of GD82.
Experimental Background (Dedrick et al., Figure 1d)

With the rise in antibiotic resistance worldwide there is an even greater need for effective alternative therapies to treat antibiotic resistant infections.  One alternative therapy that was used historically, particularly in World War II, was bacteriophage therapy.  Bacteriophage (phage) therapy uses specific bacterial viruses to kill specific bacteria. Because the phage must be shown to be specific for a bacterium and be lytic, the number of phages available is important.  Known phage, and thus phage therapy has been boosted by the SEA-PHAGES project where high school and college students isolate and characterize environmental bacteriophages. Here, Dedrick et al. (2021) report phage therapy treatment outcomes for an 81 year old patient with a recurrent infection of antibiotic-resistant Mycobacterium abscessus (strain GD82). Three SEA-PHAGE phages were used: Muddy (left panel of 1d), BPsΔ (middle panel of 1d), and ZoeJΔ (right panel of 1d). First, the ability of each phage to kill the GD82 was determined using phage spot assays, sometimes called spot-on-spot tests (panel d).  Briefly, a liquid bacterial culture was used to inoculate a spot on the agar with the culture increasingly diluted 10 fold across from left to right.  Each phage was spotted on top of the bacterial spot with the phage inoculum increasingly diluted 10 fold from top to bottom.  The top row of each panel has no phage inoculum.

7.1.2. Questions

  1. Where would the most concentrated bacterial culture treated with the most concentrated phage inoculum be located?  Please exclude the no phage row noted with “-” at the top left.
    1. Top right of each panel
    2. Top left of each panel
    3. Bottom right of each panel
    4. Bottom right of each panel
  2. Where would the most concentrated bacterial culture treated with the most dilute phage inoculum be located?  Please exclude the no phage row noted with “-” at the top left.
    1. Top right of each panel
    2. Top left of each panel
    3. Bottom right of each panel
    4. Bottom left of each panel
  3. In this experiment, _________ is the positive control and _________ is the negative control.
    1. No positive control; No phage
    2. No phage; No bacteria
    3. No phage; No negative control
    4. No bacteria; No phage
  4. Which phage is most effective at killing strain GD82? What is your evidence?
    1. Muddy; there are not bacteria growing except on the negative control
    2. BPsΔ; the bacteria grow only at low phage inoculum and high bacteria
    3. ZoeJΔ; there are not bacteria growing except on the negative control
    4. All are equally effective

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify expected results for different levels of antibody neutralization.
  • Identify key features of the bar charts.
  • Analyze the evidence and draw conclusions about antibody neutralization of each phage.
Experimental Background (Dedrick et al., Figure 2c)

With the rise in antibiotic resistance worldwide there is an even greater need for effective alternative therapies to treat antibiotic resistant infections.  One alternative therapy that was used historically, particularly in World War II, was bacteriophage therapy.  Bacteriophage (phage) therapy uses specific bacterial viruses to kill specific bacteria. Because the phage must be shown to be specific for a bacterium and be lytic, the number of phages available is important.  Known phage, and thus phage therapy has been boosted by the SEA-PHAGES project where high school and college students isolate and characterize environmental bacteriophages. Here, Dedrick et al. (2021) report phage therapy treatment outcomes for an 81 year old patient with a recurrent infection of antibiotic-resistant Mycobacterium abscessus (strain GD82). Three SEA-PHAGE phages were used to treat the patient (Muddy, BPsΔ, and ZoeJΔ). Initially, the therapy reduced the bacterial load in patient sputum, but at 2 months of treatment the bacterial load began to increase, suggesting that phage therapy had become ineffective.  To investigate the cause of this outcome, the researchers examined whether the patient had begun to produce phage-specific antibodies which would destroy the phage.  To test this, they pre-incubated each phage with patient serum that had been taken pre-treatment and at each month for 6 months.  The phage-serum mixture was incubated for 2, 4, or 24 hours and then serial dilutions were made before being plated onto lawns of bacteria.  The results were quantified at efficiency of plaquing (EOP) for each phage, each serum, and each time point (panel c).

7.2.2. Questions

  1. What color bar represents the data for antibody neutralization for ZoeJΔ phage and which panel would show the data for a 24 hour phage-serum incubation?
    1. Red; left panel
    2. Blue; center panel
    3. Purple; left panel
    4. Red; center panel
    5. Blue; right panel
    6. Purple; right panel
  2. In an antibody neutralization assay such as this, the assay is quantified as efficiency of plaquing (EOP) based on plaques formed on bacterial lawns.  What would you expect to see on the growth plates and EOP graph if the patient produced high affinity antibodies to the phage?
    1. There would be so many bacteria, so the bar would be high.
    2. There would be few or no plaques, so the bar would be low.
    3. There would be many plaques, so the bar would be very high.
    4. There would be some bacteria, so there would be a non-zero bar.
  3. Antibody neutralization assays for the two hour pre-treatment of serum and phage are shown in the left panel.   For this experiment, _________ is the positive control and _________ is the negative control.
    1. No bacteria and no phage; No negative control
    2. No phage incubation; Month 6 serum
    3. No positive control; pre-treatment serum
    4. Pre-treatment serum; Month 6 serum
  4. Phage therapy became ineffective at 2 months of treatment.  Which phage, if any, shows neutralization with 2 hours of pre-treatment using the patient serum taken at 2 months? What is your evidence?
    1. Muddy; there is little plaque formation at 2 months
    2. BPsΔ; there is significant plaque formation at 2 months
    3. ZoeJΔ;there is significant plaque formation at 2 months
    4. All are equally ineffective at plaque formation at 2 months
  5. Phage therapy became ineffective at 2 months of treatment, but they continued the experiment for 6 months.  Based on the entire patient serum collections, categorize the phage neutralization level and when it reached its maximum level.

Serum month: 1, 2, 3, 4, 5, 6

Neutralization level: completely neutralized (A), somewhat neutralized (B), not neutralized (C)

    1. ______ Muddy
    2. ______ BPsΔ
    3. ______ ZoeJΔ

8. Paper Information and Licensing

8.1. Snippet paper

8.2. Main paper

  • Dedrick RM, Freeman KG, Nguyen JA, Bahadirli-Talbott A, Smith BE, Wu AE, Ong AS, Lin CT, Ruppel LC, Parrish NM, Hatfull GF, Cohen KA. 2021. Potent antibody-mediated neutralization limits bacteriophage treatment of a pulmonary Mycobacterium abscessus infection. Nature medicine. 27(8): 1357-1361. https://doi.org/10.1038/s41591-021-01403-9
  • This article is not licensed for Creative Commons use; see the article’s copyright information. Thus, the abstract and figures cannot be copied here. The paper is behind a paywall but a version of this paper is available on PubMed at: https://pubmed.ncbi.nlm.nih.gov/34239133/

License

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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.

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