Microbial Ecology

TWiM #301: Another Year is Microbial

Podcast and Annotation Information

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 4:00 minutes

The Most Interesting Things (according to students)

Nanopore sequencing is a portable technology that allows genome sequencing using a small, USB-like device. This method makes it possible for researchers to sequence DNA directly in their own labs without large equipment, offering a cost-effective option (though reagent costs can still be high). Its accessibility is changing the landscape of genetic research and diagnostics. Phage-based diagnostics can detect infections like sepsis by identifying E. coli-specific phages in patients’ blood. Since these phages are absent in asymptomatic individuals, their presence offers a promising, non-invasive way to diagnose bacterial infections quickly and accurately. This approach highlights the innovative use of phage diversity and metagenomic sequencing in modern medicine.

The abstract 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 23:37 minutes

The Most Interesting Things (according to students)

The pConj plasmid has no fitness cost. Normally, resistance plasmids slow bacterial growth, so without antibiotic pressure, they are lost over generations. However, pConj does not hurt bacterial growth at all. Because it doesn’t slow bacteria down, it can persist indefinitely, keeping the resistance trait in the population and making infections harder to fight. Neisseria gonorrhoeae keeps the pConj plasmid, even though it produces a toxin called VapD that should be deadly to the cell. Scientists discovered that another plasmid, pCryp, carries a protective antitoxin called VapX. This shows how bacteria sometimes carry multiple plasmids that help each other survive. One plasmid produces a toxin, while another carries the antidote. This teamwork between plasmids reveals how bacteria develop complex ways to keep important genes, even under the pressure of antibiotics, and raises new questions about how plasmid interactions affect long-term antibiotic resistance.

“Plasmids are diverse extrachromosomal elements significantly that contribute to interspecies dissemination of antimicrobial resistance (AMR) genes. However, within clinically important bacteria, plasmids can exhibit unexpected narrow host ranges, a phenomenon that has scarcely been examined. Here we show that pConj is largely restricted to the human-specific pathogen, Neisseria gonorrhoeae. pConj can confer tetracycline resistance and is central to the dissemination of other AMR plasmids. We tracked pConj evolution from the pre-antibiotic era 80 years ago to the modern day and demonstrate that, aside from limited gene acquisition and loss events, pConj is remarkably conserved. Notably, pConj has remained prevalent in gonococcal populations despite cessation of tetracycline use, thereby demonstrating pConj adaptation to its host. Equally, pConj imposes no measurable fitness costs and is stably inherited by the gonococcus. Its maintenance depends on the co-operative activity of plasmid-encoded Toxin:Antitoxin (TA) and partitioning systems rather than host factors. An orphan VapD toxin encoded on pConj forms a split TA with antitoxins expressed from an ancestral co-resident plasmid or a horizontally-acquired chromosomal island, potentially explaining pConj’s limited distribution. Finally, ciprofloxacin can induce loss of this highly stable plasmid, reflecting epidemiological evidence of transient reduction in pConj prevalence when fluoroquinolones were introduced to treat gonorrhoea.” (Yee, et al 2023, no changes).

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

Snippet Main
Vision and Change Topics
  • Information Flow and Genetics (V&C_IFG)
  • Impact of Microorganisms (V&C_IM)
  • Microbial Ecology (V&C_ME)
  • Evolution (V&C_E)
  • Microbial Ecology (V&C_ME)
  • Structure and Function (V&C_SF)
ASM Fundamental Statements
  • Fundamental Statement 15 (ASM_15): Most microbial life is currently unculturable and therefore both cultivation dependent and cultivation-independent techniques are used to identify microbial populations and their potential metabolic pathways.
  • 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.
  • Fundamental Statement 2 (ASM_2): The diversity of microbes has arisen because of processes that include horizontal gene transfer, mutation, reassortment, recombination, and natural selection in varying ecological niches favor the growth and survival of certain variants.
  • Fundamental Statement 7 (ASM_7): Microbes have evolved structures adapted for specific functions that are often associated with a fitness advantage in a particular environment.
  • Fundamental Statement 14 (ASM_14): Extrinsic factors, such as abiotic and biotic interactions in the environment, can impact survival and growth of microbes.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Recall the benefits of using next generation sequencing in bacterial sepsis cases.
  • Identify the advantage of using phage diversity for pathogen identification in clinical settings.
 S L
  • Hypothesize what bacterial species is/are causing infection based on detected phage numbers.
 S H
  • Define plasmids and identify their role in antimicrobial resistance.
  • identify the role of toxin:antitoxin systems in maintenance of plasmids carrying antimicrobial resistance genes.
  • Recall reasons plasmids may be maintained or lost.
 M L
  • Predict the outcome of a specific bacteria-bacteria interaction where each bacterium has a toxin:antitoxin system.
 M H

1Papers: Snippet (S) or Main (M)
2Learning 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

  • Metagenomic Sequencing (4:00–14:30): Metagenomic sequencing is a next-generation sequencing (NGS) technique to examine all the species’ DNAs that are present in a mixed sample.  Here, the hosts discuss using metagenomic sequencing to analyze cell-free DNA, enabling the identification of bacterial pathogens in sepsis patients. Sequencing all DNA (human, bacterial, viral, phage) in a blood sample to identify potential infections without needing cultures.
  • Blood Culture (8:33–9:43): This is a laboratory method to identify bacterial infections in sepsis patients by culturing blood samples and determining which bacteria are growing. However, this process takes 1 to 3 days, which is why alternative methods are being explored.

4.2. Main Paper

  • Plasmid Genomic Analysis (23:37–35:20):  This is a comparative DNA analysis method.  The hosts delve into genomic analysis of gonococcal antimicrobial resistance plasmids to understand their evolution and persistence even without antimicrobials present.
  • Transposon Sequencing (Tn-Seq) (30:00–31:11): This is a method of mutating a pool of bacteria using a transposon, identifying a relevant trait, and sequencing the strain to identify the interrupted gene.  Using Tn-Seq, they determined which genes are involved in resistance carrying plasmids by mutating segments of the genome and seeing if selection occurred or not.

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

5.1. Snippet Paper

  • Bacteriophages (4:00–23:37): Bacteriophages (phages) are viruses that infect bacteria. Their diversity in cell-free DNA is analyzed to identify bacterial pathogens in sepsis patients.  Bacteriophages can be used to study microbiome diversity and as a potential tool for research and medical applications.
  • Cell-free DNA (4:00–23:37): Fragments of DNA circulating in the bloodstream, originating from various cells, including bacteria and their phages, are utilized for non-invasive infection detection.
  • Epidemiology & Public Health (13:30–18:00): Epidemiology is the science of tracking disease with the aim of finding ways to control it.  Using sequencing of these phages, we can track the most commonly found bacteria in a patient.  Once we determine the most likely cause of sepsis, it could potentially be used to track infections if there was a common source.
  • Human Microbiome (15:45–20:00): A microbiome is composed of all microbes in a particular environment, and this podcast discusses the gut environment.  In the podcast they discussed that by looking at the phages found in the microbiome we can determine which bacteria are present.  In doing this, we could begin to understand what species are most commonly found in a “healthy” gut and then possibly supplement that by trying to introduce these species to those lacking them.
  • Phage Therapy (19:29–19:38): Bacteriophages (viruses that infect and kill bacteria) can be used as an alternative or supplement to antibiotics for treating bacterial infections.
  • Plasmids (20:29–21:58): Plasmids are small, circular pieces of DNA that exist independently of the chromosome. They often carry genes that provide advantages, such as antibiotic resistance, and can be transferred. The hosts explain that plasmids carry antibiotic resistance genes and can transfer them between bacteria.

5.2. Main Paper

  • Antibiotic Resistance (20:00–45:29): The hosts discuss how some low-copy plasmids (pConj) are maintained even without selection, which is unusual. Plasmids play a role in spreading resistance and inducing resistance in bacterial cells that obtain these plasmids expressing resistance genes. The hosts discuss certain resistance mechanisms, and tetracycline resistance in N. gonorrhoeae can continue after antibiotic treatment stops, influencing how infections are treated.
  • Horizontal Gene Transfer & Plasmid Specificity (24:00–32:30): One of the ways plasmids spread so easily is through horizontal gene transfer (HGT). Although they don’t go into depth about the mechanisms of HGT, this paper demonstrates how plasmids can spread very quickly. The hosts also talked about the specificity of some plasmids and how they cannot be spread to all bacterial species due to differences in either the origin of replication or due to proteins or genes that are required for the integration and usage of these plasmids.
  • DNA Replication & Antibiotic Mechanisms of Action (39:15–42:25): DNA supercoiling occurs when the double-stranded helix becomes overwound, making replication difficult. DNA gyrase, an essential bacterial enzyme, helps relax these supercoils so that replication can proceed. Ciprofloxacin targets DNA gyrase, preventing it from relaxing supercoils, which leads to DNA breaks and cell death. This mechanism explains why ciprofloxacin is effective against N. gonorrhoeae.

6. Podcast Questions

  1.  Why is detection using next-generation sequencing (ngs) valuable in bacterial sepsis cases?
    1. NGS identifies bacteria within just a few days but requires a pure culture.
    2. NGS identifies bacteria using genetics and in a mixture, so it is quickly done.
    3. NGS identifies common clinical strains using a patented fast culturing method.
    4. NGS identifies a broader range of microbes than traditional diagnostic methods.
  2. What is a primary advantage of using phage diversity for pathogen identification in clinical settings?
    1. Phages are specific to bacterial strains, making pathogen identification clearer.
    2. Phage diversity is stable, making it easy to track bacterial infections over time.
    3. All phages infect multiple species, making identification more generalizable.
    4. Phage detection is done by genetic methods making sample storage convenient.
  3. Hypothetical phages A, B, C, and D infect bacterial species 1, 2, 3, and 4, respectively.  You gather data on the phages present in a sepsis case and find that phages B and C are present in high numbers, but phages A and D are present in low numbers.  What might you conclude about the pathogen responsible for the patient’s sepsis?
    1. Phages B and C are the pathogens responsible for disease.
    2. Phage A and D are protective, but found in low numbers.
    3. Bacteria 2 and 3 are the pathogens responsible for disease.
    4. Bacteria 1 and 4 are the pathogens responsible for disease.
  4. What is a plasmid?
    1. A protein that aids in antibiotic resistance
    2. A virus that integrates into the host genome
    3. A part of the bacterial CRISPR immune defense
    4. An extrachromosomal, circular piece of DNA
  5. Plasmids play an essential role in antimicrobial resistance by carrying genes that help bacteria survive in the presence of antimicrobials. How do plasmid-encoded toxin-antitoxin systems contribute to the maintenance of plasmids carrying antimicrobial resistance genes?
    1. The toxin-antitoxin system enhances the bacterial cell’s ability to acquire more plasmids.
    2. The antitoxin neutralizes the toxin, preventing cell death so long as the plasmid is retained.
    3. The toxin kills any competing bacteria they encounter that do not also have the plasmid.
    4. The toxin allows bacteria to mutate more quickly, increasing resistance to antibiotics.
  6. Why might a plasmid be lost from a bacterial species over time?
    1. The segregation mechanisms for plasmids do not work as well as for chromosomes.
    2. Plasmids are only retained if there are resistance genes located on the plasmid.
    3. If the fitness cost is greater than the benefits it provides, a plasmid will be lost.
    4. A plasmid will be retained so long as any genes are expressed from the plasmid.
  7. Cell A has no toxin-antitoxin system.  Cell B has a plasmid-encoded toxin-antitoxin system.  You culture them alone and together.  What is the most likely outcome?
    1. Both cell A and cell B can grow independently and in co-culture.
    2. Cell A produces a toxin that kills cell B, but only in the co-culture.
    3. Cell B produces a toxin that kills cell A, but only in the co-culture.
    4. Cell A acquires the plasmid in co-culture so both survive all conditions.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Extended Data Figure 3) and one from the main paper (Figure 2).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to:

  • Identify key features of experimental groups, experimental design, nomenclature used, and violin plot visualizations.
  • Evaluate the data to make conclusions about the abundance of “not annotated” phages in each sample group.
  • Interpret the data to make conclusions about the use of “not annotated” phages as a biomarker for sepsis.
Experimental Background (Haddock et al., Extended Figure 3)

Pathogen detection in the clinical setting must be fast and accurate to provide timely treatment options, particularly in cases of sepsis where mere hours can make the difference in life and death.  In this study Haddock et al.  (2023) investigate the possibility of using next-generation sequencing to identify pathogens from cell-free DNA present in serum and other body fluids.  The additional twist to their investigation is that they propose to use the mixture of bacteriophage DNA (the phageome) present in patient serum rather than the pathogen DNA for their detection.  Recall that bacteriophages, also called phages, are viruses that infect bacteria.  To begin the study, the researchers isolated and sequenced cell-free DNA from 10 asymptomatic people and 61 septic patients (Stanford Sepsis Cohort), used bioinformatics methods to remove human DNA, and searched the remaining sequences using a curated phage DNA database that also contains metadata about the phage host. In these data they found no difference in the phage composition between healthy and septic patients for known phages. However, they found a surprisingly large proportion of phage sequences were not annotated in the database but were previously found in the gut.  To determine whether these “not annotated” gut-originating phages might be a biomarker for sepsis, they investigated the proportions of “not annotated” gut phages in their sample set (panel A) and another published data set (SepSeq study; Blauwkamp et al, 2019) that they processed in the same way.  The SepSeq data set included 224 septic and 167 asymptomatic patients (Panel B). Violin plots display the distribution of individual “not annotated” gut-associated phage proportions for each group with each individual data point being represented by a dot within the violin shape. Unpaired two-sided t tests (ns = not significant, ** = p < 0.01) were used to compare groups within the same study.

  • Blauwkamp TA, Thair S, Rosen MJ, et al. 2019. Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease. Nat Microbiol 4(4): 663-674. doi: 10.1038/s41564-018-0349-6
  • The abstract and figures 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.  The PubMed version of this figure can be found here: https://pmc.ncbi.nlm.nih.gov/articles/PMC10911932/#F7

7.1.2. Questions

  1. Which of the following best describes the two groups compared in each panel?
    1. Healthy individuals and those with chronic gastrointestinal disorders.
    2. Asymptomatic healthy individuals and those diagnosed with sepsis.
    3. Patients with bacterial infections and those with viral infections.
    4. Healthy and septic individuals before and after antibiotic treatment.
  2. What does the term “Not Annotated” phages refer to in the context of these data?
    1. Lab-engineered phages for antimicrobial therapy.
    2. Phages with known bacterial hosts/targets.
    3. Phages with as yet unknown function or hosts.
    4. Phages are artificially introduced into the gut.
  3. In violin plots, the data point that is represented most often (mode) is the widest part of the “violin” shape.  Which group across both studies had the highest mode proportion of “Not annotated” gut-associated phages?
    1. SepSeq cohort, asymptomatic group
    2. Stanford Sepsis Cohort, asymptomatic group
    3. Stanford Sepsis Cohort, sepsis group
    4. SepSeq cohort, sepsis group
  4. Which group has the most consistent proportions of “not annotated” phage when you consider both panels A and B?
    1. All groups are highly variable as indicated by the wide width of each of the violin shapes.
    2. The SeqSep sepsis group, which has the shape with the highest phage proportion value.
    3. The Stanford asymptomatic group has a small shape with most values at a single point.
    4. The SeqSep sepsis group has the tallest shape from top to bottom, according to the y-axis.
  5. Which sepsis study, if any, have a significant difference in “Not Annotated” gut phage proportions between asymptomatic and septic individuals? What is your evidence?
    1. SepSeq study; The larger sample size and difference in violin/data shape indicates this.
    2. Stanford Cohort; The difference in “Not Annotated” proportions is statistically significant.
    3. Both studies; There is a difference in phage composition between both of the groups.
    4. Neither; There is too much variability in the data for both studies to draw any conclusion.
  6. Based on these data, would the proportion of “not annotated” gut-associated phage be a good biomarker (indicator) for sepsis? [pick all that apply]
    1. No, there is a difference but the data for all groups for both studies overlap.
    2. Yes, there is a statistical difference so it would be considered a good indicator.
    3. No, the data represented for the two studies do not agree on the conclusion.
    4. Yes, the modes are different for each sample pair but are consistently different.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key feature in stacked bar charts.
  • Analyze the data to make conclusions about trends in tetM+ pConj persistence across different geographic locations and time periods.
  • Evaluate the data to make conclusions about how the discontinuation of doxycycline treatment impacted the abundance of tetM+ pConj plasmids.
  • Predict plasmid maintenance rates for future studies.
  • Justify how these results have clinical significance.
Experimental Background (Yee et al., Figure 2)

Antimicrobial resistance is a growing concern with a position in the top ten health threats, according to the World Health Organization.  Indeed, a new report in 2025 showed 40% of global monitored samples have some resistance. Resistance-encoding genes are easily transferred/transmitted using plasmids, extrachromosomal circular DNAs.  Therefore, understanding how bacteria maintain and spread plasmids is an important area of research. One system that provides a good model for studying this is investigating antibiotic resistance in Neisseria gonorrhoeae, the causative agent of gonorrhoea. Two similar antibiotics were commonly to treat N. gonorrhoeae. Tetracycline was used in the United Kingdom (UK) until 2004, and doxycycline, a tetracycline drug, in the United States (USA) until 2014. Antibiotic-resistant N. gonorrhoeae frequently contains the pConj plasmid carrying tetM, a gene encoding tetracycline resistance.  Since antimicrobial use provides selective pressure for the maintenance of resistance plasmids and antibiotic use has shifted, the study of historical and modern strains can provide valuable information on plasmid maintenance in the absence of selection.  To investigate this, Yee et al. (2023) examined the prevalence of the pConj plasmid in strains isolated before and after antibiotic usage changed in both the US (panel A) and UK (panel B).  Plasmids were identified as having the resistance-encoding gene tetM (tetM+) or not (markerless).

 

Two bar charts of plasmid prevalence from 2007 to 2019.
Figure 2: “pConj has persisted in the absence of tetracycline. Prevalence of pConj in WGS of isolates from (A) USA and (B) UK. Doxycycline was recommended as part of a dual therapy for treatment before 2014 in USA; pConj carriage (including markerless plasmids) remained constant during/after doxycycline treatment (n = 2,517 isolates), as determined by unpaired t-test. pConj has remained prevalent in the UK after tetracycline treatment (2007–2016, n = 3,240 isolates). ns, p > 0.05.” (Yee, et al 2023, no changes)

 

7.2.2. Questions

  1. What is the difference in the designations tetM+ and markerless that are used in the graphs?
    1. One has the pConj plasmid and the other does not.
    2. One has the resistance gene and the other does not.
    3. One is for resistant bacteria and the other for sensitive.
    4. One is for historical strains and the other for modern.
  2. Which bars in the graphs show the abundance of pConj plasmids that carry the antibiotic resistance gene when selection was occurring in the US and the UK?
    1. 2010-2016 for the USA and 2007-2010 for the UK
    2. 2010-2014 for the USA and 2007-2014 for the UK
    3. 2010-2014 for the USA; not shown for the UK
    4. not shown for the USA; 2007-2014 for the UK
  3. What statistical measures were used in this study and how is statistical significance noted in the graph?
    1. unpaired t-test; by asterisks and ns
    2. ANOVA; by asterisks and ns
    3. chi-square test; by bar heights
    4. Mann-Whitney U test; by bar heights
  4. In what timespan, and which region, is tetM more prevalent?
    1. USA; pre-selection years
    2. UK; pre-selection years
    3. USA; post-selection years
    4. USA; post-selection years
    5. It is not more prevalent at any time
  5. What can you conclude about the relationship of tetM+ pConj plasmid persistence in N. gonorrhoeae and tetracycline/doxycycline use?
    1. The tetM+ is lost completely after tetracycline use stops.
    2. The tetM+ becomes more prevalent only in the UK post-selection.
    3. The tetM+ remains prevalent despite changes in antibiotic treatment.
    4. The tetM+ appears in both tetracycline-resistant and -sensitive strains.
  6. What would you predict for the prevalence of the tetM+ containing pConj plasmid if the study were repeated in 2050?
    1. The pConj plasmid with tetM+ will be observed at the same abundance.
    2. The pConj plasmid will be lost entirely so no plasmids will be found.
    3. The pConj plasmid will acquire a new resistance gene in place of tetM.
    4. The pConj plasmid will continue to lose the tetM gene at the same rate.
  7. Why are these results regarding the abundance of the tetM+ pConj plasmid clinically significant?
    1. The gene encoding resistance is causing increased virulence in N. gonorrhoeae so more infected individuals will die.
    2. The loss of tetM+ from the plasmid improves tetracycline treatment outcomes, so it has become easier to treat.
    3. The loss of the gene encoding resistance makes dosage smaller so it reduces the overall costs of antibiotics.
    4. The gene encoding resistance continue to be present in clinic settings where it could be transmitted to other bacteria.

8. Paper Information and Licensing

8.1. Snippet paper

8.2. Main paper

  • Yee W-X, Yasir M, Turner AK, Baker DJ, Cehovin A, Tang CM. 2023. Evolution, persistence, and host adaptation of a gonococcal AMR plasmid that emerged in the pre-antibiotic era. PLoS Gen. 19(5): e1010743. doi: 10.1371/journal.pgen.1010743
  • 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 article on the journal’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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