Impact of Microorganisms

TWiM #171: If You Feed a Bee a Fungus

Podcast and Annotation Information
  • Annotation by Lauren Ballard, Ben Walsh, Kaitlyn Wesselink, Amaya Garcia Costas, Rebecca Seipelt-Thiemann, Nancy Boury
  • Podcast audio by TWiM:  Listen to TWiM #171 Podcast
  • Podcast transcript by Sarah Morgan: Access TWiM #171 Transcript
  • Papers Discussed:
    • Paludo CR, Menezes C, Silva-Junior EA, Vollet-Neto A, Andrade-Dominguez A, Pishchany G, Khadempour L, do Nascimento FS, Currie CR, Kolter R, et al. 2018. Stingless Bee Larvae Require Fungal Steroid to Pupate. Sci Rep. 8(1):1122. doi: 10.1038/s41598-018-19583-9.
    • Dejea CM, Fathi P, Craig JM, Boleij A, Taddese R, Geis AL, Wu X, DeStefano Shields CE, Hechenbleikner EM, Huso DL, et al. 2018. Patients with familial adenomatous polyposis harbor colonic biofilms containing tumorigenic bacteria. Science. 359(6375):592-597. doi: 10.1126/science.aah3648.

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 2:00 minutes

The Most Interesting Things (according to students)

Ten of ten stingless bees say that this fungus is a MUST HAVE!! Also, the symbiotic relationship between stingless bees and Zygosaccharomyces is essential for the survival of both organisms.

“The larval stage of the stingless bee Scaptotrigona depilis must consume a specific brood cell fungus in order to continue development. Here we show that this fungus is a member of the genus Zygosaccharomyces and provides essential steroid precursors to the developing bee. Insect pupation requires ecdysteroid hormones, and as insects cannot synthesize sterols de novo, they must obtain steroids in their diet. Larval in vitro culturing assays demonstrated that consuming ergosterol recapitulates the developmental effects on S. depilis as ingestion of Zygosaccharomyces sp. cells. Thus, we determined the molecular underpinning of this intimate mutualistic symbiosis. Phylogenetic analyses showed that similar cases of bee-Zygosaccharomyces symbiosis may exist. This unprecedented case of bee-fungus symbiosis driven by steroid requirement brings new perspectives regarding pollinator-microbiota interaction and preservation.” (Paludo et al 2018, no changes)

1.2. Main paper; discussion starts at 15:35 minutes

The Most Interesting Things (according to students)

Good ol’ roly poly coli is up to no good!  Evidence indicates that colorectal cancer seen in 60-68% FAP patients is associated with the presence of pathogenic Escherichia coli and B. fragilis breaching the mucosal space. Also, two microorganisms act in a concerted manner to breach the mucin layer and induce colonic tumorigenesis. And finally, phage are ubiquitous and are often neglected in microbial ecology, while they play an important role in ecosystem dynamics, controlling bacterial populations (lysis by phage infection) and transferring genes (transduction).

The abstract cannot be copied due to licensing restrictions. Please see the article and copyright information at the article’s web page: https://www.science.org/doi/10.1126/science.aah3648

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

Snippet Main
Vision and Change Topics
  • Impact of Microorganisms (V&C_IM)
  • Microbial Ecology (V&C_ME)
  • Impact of Microorganisms (V&C_IM)
  • Structure and Function (V&C_SF)
  • Microbial Ecology (V&C_ME)
ASM Fundamental Statements
  • Fundamental Statement 21 (ASM_21):Microbes and the environment interact with and affect each other. 
  • Fundamental Statement 24 (ASM_24): Microbes and their communities are essential for supporting all life as we know it. 
  • Fundamental Statement 27 (ASM_27): The extent of microbial diversity is largely unknown, and exploration of this diversity is critical to understanding microbes and their role in the biosphere. 
  • Fundamental Statement 20 (ASM_20): Microbes are ubiquitous, found in diverse and dynamic ecosystems, where they use available resources and often form complex communities. 
  • 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 symbiosis.
  • Identify specific symbiotic benefits as noted in the podcast.
  • Recall results that identify the critical elements of the fungus-bee interaction
 S L
  • Design an experiment to determine the contribution of a symbiotic fungal partner to its host insect.
 S H
  • Describe how bacteria interact with the mucus lining in the colon.
  • Explain the difference between hereditary colon cancer and sporadic colon cancer.
  • Identify the origin of the colibactin toxin.
 M L
  • Evaluate the experimental design to identify how this study does not fully adhere to Koch’s postulates.
 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

  • Culturing Fungi (7:45–9:40): This technique is a way to grow fungi in the lab.  Here, the researchers were unable to grow these fungi, but they were successfully when they cultured fungal cells in high osmolarity medium.
  • Growing Cultures (9:40–11:15): Bee larvae cultures were grown in 96 well plates with the addition of fungus at pupa stage of larvae growth.
  • Mass Spectrometry (11:15–12:30): This is a technique to identify and quantify molecules in a mixture.  Here, the major sterol from the fungus was identified as ergosterol.

4.2. Main Paper

  • Fluorescence in Situ Hybridization (FISH) (24:40–26:00): This is a microscopy technique that can be used to identify the location of molecules inside tissue or cells.  Here, the researchers used 16S rDNA gene probes with fluorescent tags to visually identify organisms in biofilms.
  • Quantitative Reverse Transcriptase–Polymerase Chain Reaction (qRT-PCR) (40:30–41:20): This molecular technique is used to quantify the levels of mRNA for specific genes.  Here the researchers used it to measure mRNA levels of the gene encoding for interleukin-17 (IL-17) to evaluate its role in the development or exacerbation of colorectal cancer.

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

5.1. Snippet Paper

  • Symbiosis/Mutualism (2:10–3:05: 5:40–6:21): The bees eat the fungus, providing it with nutrients and an environment to grow. The fungus in return provides the bees with hormones vital for survival (ergosterol).
  • Microbiome/Mycobiome (13:44–14:27): The environment in which the fungus lives and interacts is the mycobiome.

5.2. Main Paper

  • Exotoxin:(31:40–32:15): Toxin that damages DNA and is produced by coli in colon cancer patients.
  • Cytokines (39:32–43:06): Observing the role of proinflammatory cytokines (i.e. IL-17) in the promotion and exacerbation of colorectal cancer.

6. Podcast Questions

  1. Which of the following describes symbiosis? [pick all that apply]
    1. The interaction is beneficial for one organism and neutral for the other.
    2. The interaction is beneficial for both of the organisms involved.
    3. The interaction is neither harmful nor beneficial for both organisms.
    4. The interaction is beneficial for one organism and harmful for the other.
  2. This TWIM podcast mentions a classic well-studied symbiosis of insect, fungus, and bacterium in addition to the one described in the research paper. The role of the bacterium in that case was to:
    1. contribute genes for biosynthesis of steroids such as ecdysone.
    2. contribute food for the symbiotic fungi so they can survive better.
    3. contribute food for the host ants so they can survive better.
    4. prevent parasitic fungi from contaminating the ant’s fungal food.
    5. prevent pathogenic bacteria from infecting the host/partner ants.
  3. Order the steps for a study that evaluates the contribution of a fungal species to the development of stingless bees, starting with 1 as first.
    1. ______ Identify exudates via mass spectrometry
    2. ______ Culture the fungal species in an axenic culture
    3. ______ Grow the larvae in the presence of the isolated fungus
    4. ______ Grow the larvae in the presence of the identified exudates
    5. ______ Identify the fungal species via 18S and 26S sequencing
    6. ______ Extract exudates from isolated species
  4. Which experimental result convinced the researchers that they had identified the symbiotic element of the bee-fungus interaction?  The percentage of stingless bee larvae that pupated was similar to natural levels when larvae were grown with ___________.
    1. No additional components
    2. Fungal cells of the genus Zygosaccharomyces
    3. Purified versions of ergosterol alone
    4. Fungal cells of the genus Zygosaccharomyces and ergosterol
    5. Fungal cells of the genus Zygosaccharomyces or ergosterol
  5. The podcasters discuss/present the interaction of bacteria with the mucus layer in the colon as it relates to colon cancer.  Which of the following statements are true?
    1. Bacteria can use gastric mucin as carbon and nitrogen sources.
    2. Bacteria in the colon can produce mutagenic or carcinogenic toxins.
    3. Bacteria can breach the mucin layer and cause inflammation.
    4. Bacteria are solely responsible for mucin destruction in colon cancer.
  6. What is the difference between sporadic and hereditary colon cancer?
    1. Sporadic cases are caused by bacteria destroying the epithelial lining, but hereditary cases are caused by inheriting mutant versions of genes.
    2. Sporadic cases are caused by pathogenic bacteria in the gut, but hereditary cases are caused by acquiring mutant genes from the bacteria.
    3. Sporadic cases are caused by distorted ratios of specific bacteria in the gut, but hereditary cases are caused by acquiring mutant viral genes.
    4. Sporadic cases are those where the individual is not genetically predisposed, but hereditary cases are where the individual is genetically predisposed.
  7. The gene encoding colibactin toxin is ________.
    1. present in all E.coli strains, but it is highly regulated and only some strains express it under specific conditions.
    2. a mutant version of a normal E. coli gene where an amino acid change alters the protein product to be a toxin.
    3. found in a genotoxic (pathogenicity) island which some, but not all, E. coli strains have in their genomes.
    4. found in only in enterohaemorrhagic E. coli strains associated with specific enteric and diarrheal diseases.
  8. How does the finding that enterotoxigenic B. fragilis and pks-containing E. coli act cooperatively to induce colonic tumor formation appear to violate Koch’s postulates?
    1. Those organisms cannot be grown in pure culture, therefore we are unable to satisfy the second of Koch’s postulates.
    2. Those organisms are not found in all instances of the disease, therefore we cannot satisfy the first of Koch’s postulates.
    3. Both organisms, not a single organism, is responsible for the disease, so we can’t satisfy the second of Koch’s postulates.
    4. There is not a good animal model for disease caused by these organisms, therefore we can’t satisfy the third postulate.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 4abde) and one from the main paper (Figure 1).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify key aspects of experimental design including controls and variable types in this experiment.
  • Analyze fluorescent microscopy images to make conclusions about the localization of specific molecules/structures.
  • Evaluate the data to make conclusions about the impact of fungus strain and ergosterol on bee pupation.
  • Evaluate the data to make conclusions about whether a symbiotic relationship is mutualistic, commensal, and  parasitic.
Experimental Background (Paludo et al., Figure 4abde)

Symbiotic relationships with microbes are prevalent and complex in the natural world and typically fall into one of three general categories: mutualism, commensalism, and parasitism.  In this study, Paludo et al (2018) investigated the symbiotic relationship between a fungus, Zygosaccharomyces, and an insect, the stingless bee, Scaptotrigona depilis. In prior experiments in this study, they identified a strain of the fungus was present in brood cells during bee development.  In their characterization of the laboratory strain compared to the brood cell strain, they found lipid droplets inside the fungus growing in laboratory conditions and wanted to determine whether this was also true for fungus isolated from its native insect environment of the bee brood cell, so they used a lipid stain (Nile Red) to stain fungi grown in each condition and examined each using fluorescent microscopy (panels a and b).  Next, knowing lipids are nutrients and also have the capability of carrying important lipid-soluble hormones, they hypothesized that the fungus was providing something essential for bee development.  So, they performed two experiments in succession.  First, they grew bee larvae without the fungus, with the fungus isolated from the brood cells, and with a laboratory strain of the fungus.  They quantified successful bee development as percent completing pupation (panel d).  Next, they grew bee larvae without the fungus, with the fungus isolated from the bee brood cells, and with a purified hormone, ergosterol.  They quantified successful bee development as percent completing pupation (panel e).

Multipart figure including microscopic photos, bat charts, and figures of chemicals.

Figure 4. “Zygosaccharomyces sp. lipid droplets and S. depilis pupation experiments. (a) Fluorescence microscopy of Zygosaccharomyces sp. fixed cells stained by Nile Red to show the presence of cytoplasmic lipid droplets under laboratory conditions and (b) natural conditions (cells of the fungus collected directly from brood cells of S. depilis). (c) Different stages of larval development: (i) 1–2 days-old egg, (ii) 3–4 day-old egg, (iii) 1 day-old larva, (iv) 3–4 day-old larvae (Zygosaccharomyces sp. growth observed), (v) 6–10 day-old larva, (vi) 15–18 day-old pre-pupa, (vii) 20–25 day-old pupa, (viii) 30–34 day-old pupa, (ix) 35–40 day-old emerging bee. (d) Percentage of larvae that completed metamorphosis in vitro without microorganism inoculation (first bar), with fungus collected directly from brood cells (second bar) and with isolated Zygosaccharomyces sp. SDBC30G1, cultivated in laboratory (third bar), in three different experiments (N = 48 per treatment, Cochran-Mantel-Haenszel, P < 0.0001 compared with uninoculated control). (e) Percentage of larvae that completed metamorphosis in vitro without fungal or sterol inoculation (first bar), with fungus collected directly from brood cells (second bar) and with ergosterol at 2.5 µM added in the larval food (third bar), in three different experiments (N = 48 per treatment, Cochran-Mantel-Haenszel, P < 0.0001 compared with untreated control). (f) Chemical structures of ergosterol, cholesterol, 24-epi-makisterone A and 20-hydroxyecdysone.” (Paluda et al 2018, no changes)

7.1.2. Questions

  1. Fungal cells were stained with Nile Red and visualized with fluorescent microscopy (panels a and b).  What are the green structures within the fungal cells and what does the comparison of panel a and panel b tell us?
    1. The nucleus; Cultured Zygosaccharomyces and Zygosaccharomyces isolated from bee pupa show the same 16S rDNA genes.
    2. Mitochondria; Cultured Zygosaccharomyces and Zygosaccharomyces isolated from bee pupa show the same mitochondrial localization.
    3. Chloroplasts; Cultured Zygosaccharomyces and Zygosaccharomyces isolated from bee pupa show the same chloroplast localization
    4. Carbon storage structures; Cultured Zygosaccharomyces and Zygosaccharomyces isolated from bee pupa show carbon storage structures.
    5. Lipid droplets; Cultured Zygosaccharomyces and Zygosaccharomyces isolated from bee pupa show the same location of lipid droplets.

Use this text for the next three questions (2, 3, and 4).

To test how fungi affect pupation, the researchers grew larvae without fungus, with the brood cell fungus, and with a laboratory fungal strain (panel d).

  1. The dependent variable in this experiment is ____ and the independent variable in this experiment is _____.
    1. Percent pupation; fungus type
    2. Percent pupation; pupa length
    3. Fungus type; percent pupation
    4. Fungus type; pupa color score
  2. In this experiment __________ is the positive control.
    1. Bee larvae grown without any fungus
    2. Bee larvae grown with a laboratory fungal strain
    3. Bee larvae grown with the brood cell fungus
    4. There is no negative control for this experiment.
  3. Which conditions support successful bee pupation, and what do these results indicate? [pick all that apply]
    1. Only bee larvae grown without any fungus successfully pupate. The fungus harms the bee pupa.
    2. Bee larvae grown with a laboratory fungus successfully pupate. The lab strain aids the bee pupa.
    3. Bee larvae grown with the brood strain fungus successfully pupate. The brood strain aids the bee pupa.
    4. Only bee larvae grown with a fungus successfully pupate, so a fungus is required for successful pupation.
  4. To test whether ergosterol was the compound affecting pupation, the researchers tested pupation without fungus/sterol, with the brood cell fungus, and with ergosterol. The positive control for this experiment is _________.
    1. Bee larvae grown without fungus/sterol.
    2. Bee larvae grown with the steroid ergosterol.
    3. Bee larvae grown with the brood cell fungus.
    4. There is no positive control for this experiment.
  5. Based on the results shown in panels d and e, the percentage of stingless bee larvae that pupated was similar to natural levels when the larvae were grown with:
    1. No additional components
    2. Fungal cells of the genus Zygosaccharomyces
    3. The steroid ergosterol alone
    4. Fungal cells of the genus Zygosaccharomyces and ergosterol
    5. Fungal cells of the genus Zygosaccharomyces or ergosterol
  6. Based on the results in panels d and e, what symbiotic relationship exists between the bee and fungus?
    1. commensal
    2. mutualistic
    3. parasitic

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features of experimental design, including  positive and negative controls in the experiments
  • Evaluate fluorescent microscopy images to make conclusions about the abundance and localization of specific molecules/structures and differences between tissues.
  • Analyze tabular data to make conclusions about the correlation of specific bacteria and tumor formation.
  • Analyze the polymerase chain reaction (PCR) data to make conclusions about the correlation of specific genes on tumor formation.
  • Predict the production of specific oncotoxins based on DNA-based results.
Experimental Background (Dejea et al., Figure 1)

Familial adenomatous polyposis (FAP) is an inherited condition that can lead to colon cancer if left undiagnosed and untreated.  To gain a better understanding of the relationship of bacteria and early tumor formation, Dejea et al. (2018) investigated bacterial biofilm presence and abundance in the mucosal surface of FAP polyps and matched normal colon tissue using fluorescent staining for bacterial biofilms and DNA (panel A, top images).  They next wanted to localize specific bacterial species that have a known relationship to oncogenesis to determine any abundance differences between the polyp and normal tissue.  They used species-specific probes in fluorescent in situ hybridization to detect both Bacterioides fragilis and E. coli (panel A, bottom images).  Next, to get a better idea of where early bacterial colonization of the mucosa was occurring, the researchers wanted to localize bacteria in FAP colon tissue with a normal appearance. To do this, they used fluorescent probes to Enterobacteriaceae (yellow, panel B) and E. coli (red, panel B) and performed fluorescent microscopy on this tissue (panel B).  Having localized bacteria in these tissues, they next investigated the prevalence of different bacteria in individuals with and without FAP.  These data are quantified as percentages of patients with and without the presence of specific bacteria (panel C). Having identified specific strains of bacteria and their prevalence in patients and healthy controls, the researchers next investigated the prevalence of specific tumor-related bacterial genes in the biofilms of FAP patients and controls. To do this, they isolated DNA from these tissues and used polymerase chain reaction (PCR) to amplify segments of the clbB (a gene in the pks pathogencity island) and bft (a gene implicated in B. subtilitis oncogenicity).  Their data are presented as agarose gel electrophoresis and include several controls, as noted in the figure legend.

  • The figure from the Main paper cannot be copied due to licensing restrictions.  Please see the article and copyright information at the article’s web page.

7.2.2. Questions

  1. To compare the biofilms at FAP polyps and normal colon tissue, the researchers first used fluorescent in situ hybridization to identify bacterial biofilms in polyps and normal tissue (panel A top images). Which tissue has a larger biofilm and what is your evidence?
    1. paired normal; a larger section of blue staining is observed
    2. polyp; more swirling patterns of microbe growth are present
    3. paired normal; blue staining is observed in both mucosal areas
    4. polyp; a larger section of red staining is observed for this tissue
  2. To identify the bacterial composition of biofilms at FAP polyps and normal colon tissue, the researchers again used fluorescent in situ hybridization, but this time with species-specific probes for B. fragilis  and E. coli (panel A bottom images). What conclusion can you make from these data?
    1. There is more B. fragilis than E. coli in biofilms at both of these tissues.
    2. There is more E. coli and B. fragilis in polyp biofilm than normal tissue biofilm.
    3. There is more B. fragilis in polyp biofilm but more E. coli in normal tissue biofilm.
    4. Neither B. fragilis nor E. coli are present in the biofilm of normal colon tissue.
  3. What statement would be most accurate in describing how the polyp and normal colon images in panel A are different?
    1. Biofilms in patients with a FAP polyp are more developed and have higher numbers of both species of bacteria than biofilms in healthy samples.
    2. Biofilms in patients with a FAP polyp appear red and purple because the bacteria have induced mutations for pigments that survival in the colonic environment.
    3. Biofilms in patients with a FAP polyp are larger and filled with more fluid than the biofilms from the normal paired colon tissue of the FAP patients.
    4. Biofilms in patients with a cancerous polyp share a lot of similarities with biofilms in healthy tissues, so we cannot really tell them apart using these methods.
  4. To investigate the location of the bacteria in FAP colon tissue with a normal appearance, fluorescent probes to Enterobacteriaceae (yellow, panel B) and E. coli (red, panel B) were used to localize each bacterium.  The fluorescent staining of both bacteria at the base of colonic crypts (arrows, panel B) is a good indicator of what?
    1. Enterobacteriaceae and E. coli  are present in distinct areas of the mucin
    2. Enterobacteriaceae alone is present in biofilms within the mucin layer
    3. E. coli, but not Enterobacteriaceae, is present in biofilms in the mucin layer
    4. Enterobacteriaceae and E. coli  are present and invading the colonic epithelia
  5. The authors next investigated the prevalence of different bacteria in individuals with and without FAP and reported these data in a tabular format (panel C).  What statement would be most accurate in describing how the frequencies of bacteria in FAP and normal colon are different?
    1. The FAP polyps were more likely to harbor pks-containing E.coli, but less likely to contain enterotoxigenic B. fragilis than healthy colonic tissue.
    2. Sixty-eight percent of all FAP polyp bacteria were pks-containing E.coli and 30% of all normal colonic bacteria were enterotoxigenic B. fragilis.
    3. Twenty-four percent of FAP polyps did not have either bacterial strains and 70% of healthy colonic tissue did not have either of those strains.
    4. The presence of pks-containing E.coli and enterotoxigenic B. fragilis in FAP polyps is not statistically different in polyp and  healthy colonic tissue.

6–8. Please use this paragraph for the next three questions.

While looking for connections between colon cancer and bacteria, the authors found evidence that E. coli that carry genes that encode colibactin synthesis, such as clbB, and Bacteroides that carry bft  were implicated in colon cancer.  So, they next used polymerase chain reaction to amplify these genes from FAP patient biofilms.  The results for six patient samples (lanes 2-7), purified clbB DNA (lane 8) and purified bft  DNA (lane 9) are shown in panel D.

  1. What are the controls for this experiment?
    1. There is one positive control for this experiment, purified clbB, and one negative control for this experiment, purified bft.
    2. There is one positive control for this experiment, purified bft, and one negative control for this experiment, purified clbB.
    3. There are two positive controls, purified clbB and purified bft, and no negative controls for this experiment.
    4. There are two negative controls, purified clbB and purified bft, and there are no positive controls for this experiment.
  2. What can you infer from the results shown in panel D?
    1. Bacteria in FAP mucosal biofilms likely produce colibacter, but not bft.
    2. Bacteria in FAP mucosal biofilms likely produce colibacter and bft.
    3. Bacteria in FAP mucosal biofilms likely produce bft, but not colibacter.
    4. Bacteria in FAP mucosal biofilms produce neither colibacter nor bft.
  3. Which patient’s biofilm (panel D) is unlikely to produce colibacter and bft?
    1. Patient 3730
    2. Patient 3760
    3. Patient NC101
    4. None

8. Paper Information and Licensing

8.1. Snippet paper

  • Paludo CR, Menezes C, Silva-Junior EA, Vollet-Neto A, Andrade-Dominguez A, Pishchany G, Khadempour L, do Nascimento FS, Currie CR, Kolter R, et al. 2018. Stingless Bee Larvae Require Fungal Steroid to Pupate. Sci Rep. 8(1):1122. doi: 10.1038/s41598-018-19583-9.
  • This article is licensed for Creative Commons use using CC BY 4.0, which allows re-use and adaptation with proper attribution and notation of any changes. See the article on the journal’s website.

8.2. Main paper

  • Dejea CM, Fathi P, Craig JM, Boleij A, Taddese R, Geis AL, Wu X, DeStefano Shields CE, Hechenbleikner EM, Huso DL, et al. 2018. Patients with familial adenomatous polyposis harbor colonic biofilms containing tumorigenic bacteria. Science. 359(6375):592-597. doi: 10.1126/science.aah3648.
  • This article is not licensed for Creative Commons use. Thus, the abstract and figures cannot be copied here. Please see the article and copyright information at the article’s web page.

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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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