Microbial Ecology

TWiM #174: A Gathering Typhoid Storm

Podcast and Annotation Information
  • Annotation by Lauren Ballard, Benjamin Walsh, Kaitlyn Wesselink, Kanwal S. Alvarez, Gwendowlyn S. Knapp, Rebecca Seipelt-Thiemann, and Nancy Boury
  • Podcast audio by TWiM: Listen to TWiM #174 Podcast
  • Podcast transcript by Sarah Morgan: Access TWiM #174 Transcript
  • Papers Discussed:
    • Klemm EJ, Shakoor S, Page AJ, Qamar FN, Judge K, Saeed DK, Wong VK, Dallman TJ, Nair S, Baker SShaheen G, et al. 2018. Emergence of an Extensively Drug-Resistant Salmonella enterica Serovar Typhi Clone Harboring a Promiscuous Plasmid Encoding Resistance to Fluoroquinolones and Third-Generation Cephalosporins. mBio9:10.1128/mbio.00105-18. https://journals.asm.org/doi/10.1128/mbio.00105-18
    • Voyles J, Woodhams DC, Saenz V, Byrne AQ, Perez R, Rios-Sotelo G, Ryan MJ, Bletz MC, Sobell FA, McLetchie et al. 2018.  Shifts in disease dynamics in a tropical amphibian assemblage are not due to pathogen attenuation. Science. 359(6383):1517-1519. doi: 10.1126/science.aao4806.

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 5:04 minutes

The Most Interesting Things (according to students)

Typhoid Mary failed quarantine goals!  In the early 1900s, contracting Typhoid fever was considered a death sentence until the emergence of the antibiotic chloramphenicol.  Also, Salmonella typhi, the causative organism of typhoid fever, has become extremely resistant to a large variety of antibiotics, and may soon become totally drug resistant.

“Antibiotic resistance is a major problem in Salmonella enterica serovar Typhi, the causative agent of typhoid. Multidrug-resistant (MDR) isolates are prevalent in parts of Asia and Africa and are often associated with the dominant H58 haplotype. Reduced susceptibility to fluoroquinolones is also widespread, and sporadic cases of resistance to third-generation cephalosporins or azithromycin have also been reported. Here, we report the first large-scale emergence and spread of a novel S. Typhi clone harboring resistance to three first-line drugs (chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole) as well as fluoroquinolones and third-generation cephalosporins in Sindh, Pakistan, which we classify as extensively drug resistant (XDR). Over 300 XDR typhoid cases have emerged in Sindh, Pakistan, since November 2016. Additionally, a single case of travel-associated XDR typhoid has recently been identified in the United Kingdom. Whole-genome sequencing of over 80 of the XDR isolates revealed remarkable genetic clonality and sequence conservation, identified a large number of resistance determinants, and showed that these isolates were of haplotype H58. The XDR S. typhi clone encodes a chromosomally located resistance region and harbors a plasmid encoding additional resistance elements, including the blaCTX-M-15 extended-spectrum β-lactamase, and carrying the qnrS fluoroquinolone resistance gene. This antibiotic resistance-associated IncY plasmid exhibited high sequence identity to plasmids found in other enteric bacteria isolated from widely distributed geographic locations. This study highlights three concerning problems: the receding antibiotic arsenal for typhoid treatment, the ability of S. typhi to transform from MDR to XDR in a single step by acquisition of a plasmid, and the ability of XDR clones to spread globally.” (Klemm et al. 2018, no changes).

1.2. Main paper; discussion starts at 35:45 minutes

The Most Interesting Things (according to students)

A fungal infection that once wiped out a frog population in now under control following new skin secretions made by the frogs.

The abstract cannot be copied due to licensing restrictions. Please see the article at the journal’s website: https://www.science.org/doi/10.1126/science.aao4806

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

Snippet Main
Vision and Change Topics
  • Information Flow and Genetics (V&C_IFG)
  • Microbial Ecology(V&C_ME)
  • Structure and Function  (V&C_SF)
  • Microbial Ecology (V&C_ME)
ASM Fundamental Statements
  • Fundamental Statement 6 (ASM_6): The distinct structures and processes in microbes can be targets for interspecies competition, antimicrobial treatments, and host immunity.
  • Fundamental Statement 16 (ASM_16): Genetic variation can influence microbial structures and their functions.
  • 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 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
  • Identify nomenclature related to different levels of drug resistance.
  • Identify the symptoms of typhoid fever.
  • Recall what features make Z-Pak drugs so effective, especially against Salmonella
  • Recall the main conclusion of the study’s genomic and antibiotic resistance results.
 S L
  • Predict a possible resistance mechanism given a specific set of strain phenotypes.
 S H
  • Identify the fungal species that caused the chytridiomycosis epidemic in the study.
  • Differentiate between the epidemic phase of an outbreak and the endemic phase of an outbreak
  • Recall the study conclusion about whether the differences in frogs or fungi have allowed the frog population to recover.
 M L
  • Propose an experiment to test a specific hypothesis based on this study.
 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

  • Antibiotic Resistance Survey (7:33): A survey performed in order to identify what antibiotics in what doses an organism is resistant to.
  • Genomic Analysis/Sequencing (7:39): This technique involves sequencing genomes and comparing them.  The researchers used this to identify genomic variations, plasmid features, and antibiotic resistances among the strains.
  • Clinical Trial (14:40–16:00): These are research studies performed on people that are aimed at evaluating a medical treatment’s efficacy and safety.  There are many phases of these.
  • Quarantine (27:09–28:14): This is the isolation of an infected individual in order to prevent them from spreading disease, or the isolation of a healthy individual in order to prevent them contracting a disease.  Typhoid Mary was quarantined for 3 years.
  • Contact Tracing (28:45–29:08): This is a technique to identify contacts and sources of infectious disease. The researchers collected a large number of samples from the outbreak in Asia and analyzed them in order to identify where each microbe came from and where/when each resistance plasmid was acquired.

4.2. Main Paper

  • Polymerase Chain Reaction (PCR) (42:25): This is molecular technique to make copies of specific DNA segments. It has many different uses, but here was used to determine the prevalence of Chytrid fungus before and after the epidemic.
  • Comparative Morphology (42:37–48:46) : This technique involves comparing a variety of features by eye or microscopy. Here, the researchers examined the structures of both the historical isolates and contemporary isolates and found no differences
  • Testing Pathogenicity: (44:23–49:30): These are techniques/assays for examining the virulence of a pathogen. Here, the researchers compare historical and new fungal isolates in two frog species; they found no difference in their ability to infect the frogs.
  • Comparative Genomics (49:29–49:49): This technique involves sequencing genomes and comparing them. The researchers performed whole genome sequencing on both the HI and the CI and examined 25,000 single nucleotide polymorphisms (SNPs) and found no significant differences

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

5.1. Snippet Paper

  • Antibiotic Resistance (8:15–8:42; 17:20–18:31): Salmonella typhi maintains R factor on IncH1 plasmid and encodes resistance to most first-line antimicrobials.  XDR/TDR: XDR is extensive drug resistance; TDR is total drug resistance. Salmonella typhi is now XDR and is developing more resistances, but it may soon be TDR
  • Disease Transmission (9:35–10:10): Salmonella typhi is spread within a population via the fecal-oral route
  • Case Fatality Rate (11:30–11:50; 16:04–16:10): Prior to the introduction of antibiotics, typhoid fever had a CFR of 15%, and after the discovery of chloramphenicol, that CFR plummeted to <1%.
  • Disease-induced Diarrhea (12:35–13:59): There are three distinct types of disease-induced diarrhea: First: ride it out on a toilet, 24-hour bug; Second: More severe, requires antibiotic treatment but recoverable; Third: Infection becomes systemic, leading to bacteremia and sepsis
  • Plasmids (16:50–17:05, 18:20–18:31): Incompatibility group plasmid: plasmid in Salmonella that kept the antibiotic resistance plasmids out. However, that plasmid has been ousted and the resistance plasmids are in.
  • R Factors (17:00–17:32): Resistance traits found on plasmids convey antibiotic resistance to the organism
  • Antibiotic Targets (18:47–20:25): Different antibiotics target different parts of a microbe, which allows a new antibiotic to be utilized against an organism that has become resistant to other treatments
  • Asymptomatic Spread (26:46): Typhoid Mary spread typhoid fever as she was a carrier and shedder of the disease but did not ever present with symptoms

5.2. Main Paper

  • Epidemic Epizootic Phase (38:30–38:49): The epidemic phase of the fungal infection within the frog population caused widespread amphibian death
  • Endemic Enzootic Phase (38:30–38:49): In the post-epidemic phase, the fungal infection was a more common and less devastating disease and the frogs survived the illness

6. Podcast Questions 

  1. Microbiologic and genomic data presented by Klemm et al. suggests that the serovar salmonella is considered an XDR.  What is XDR and how is it different from TDR?
    1. XDR refers to organisms that are “totally resistant to the available antimicrobials” whereas TDR refers to “extensively resistant organisms”
    2. XDR refers to organisms that are “extensively resistant to available antimicrobials” and TDR refers to “totally resistant to available antimicrobials.”
    3. XDR and TDR are different ways of naming strains according to whether they have an extensively flat cell morphology or a tall cell morphology.
    4. XDR and TDR refer to different groups of drugs and their synthetic derivatives that are no longer effective against specific fungal infections.
  2. Symptoms of Typhoid fever include fever, malaise, headache, nausea, abdominal pain and _______________.
    1. Constipation with rash
    2. Diarrhea with rash
    3. Diarrhea only
    4. A or B
  3. Which one of the following statement(s) is true regarding the genomic and resistance data for new Salmonella strain?
    1. This analysis revealed that it is resistant to chloramphenicol, amoxicillin, trimethoprim sulfate caused by a single mutation in the gene encoding gyrase.
    2. This analysis revealed that the strain is resistant to chloramphenicol, trimethoprim sulfate caused by a single mutation in the gene encoding DNA polymerase.
    3. This analysis revealed that the strain is resistant to all available drugs that were tested in this study due to mutations in multiple plasmid-encoded genes.
    4. This analysis revealed that it is only resistant to azithromycin, but not amoxicillin or trimethoprim sulfate.  The resistance was due to R plasmid genes.
  4. The podcasters refer to azithromycin or Z-Pak as a “wonder drug.” Which of the following statement(s) is true about Z-Pak and S.typhi? [Pick all that apply]
    1. The drug concentrates in the pancreas where the Salmonella forms cyst-like, drug resistant structures.
    2. The drug is acid labile so it can be readily processed in the acidic acid environment of the stomach.
    3. The drug is acid stable so it can survive and persist past the acidic acid environment of the stomach.
    4. The drug accumulates in phagocytes where the bacteria inhabit as a facultative, intracellular parasite.
  5. You discover a new strain of Salmonella that is sensitive to azithromycin in in vitro assays, such as disk diffusion assays.  However, the strain infects mice very well (in vivo).  What might be a mechanism to explain this result?
    1. The strain might produce a compound that induces host phagocytes to pump the drug out.
    2. The strain might have acquired a plasmid that encodes a protein that degrades the drug directly.
    3. The strain might have lost an R factor plasmid that enabled resistance to all known drugs.
    4. The strain might have acquired a plasmid that encodes a protein killing all phagocytic cells.
  6. What was the causative agent of the frog epidemic chytridiomycosis in the main paper’s study?
    1. Boletus albisulphureus
    2. Batrachochytrium dendrobatidis
    3. Allomyces macrogynus
    4. Albatrellus avellaneus
  7. What is the difference between epidemic and endemic phase of an outbreak?
    1. Epidemic is the phase when disease is present at high levels and endemic is the phase when disease is not present at all.
    2. Epidemic is the phase when disease is present at low levels and endemic is the phase when disease is present at high levels.
    3. Epidemic is the phase when disease is present at high levels and endemic is the phase when disease is present at low levels.
    4. Epidemic is the phase when disease is present at low levels and endemic is the phase when disease is not present at all.
  8. What did the researchers conclude about the differences in wild and captive frogs, as well as historical and current fungal isolates, as it relates to this epidemic?
    1. The now avoid areas of fungal contamination, which have allowed the population to recover.
    2. All wild sensitive frogs have died and only the resistance frogs remain, so they recovered.
    3. The fungi are less virulent now than in the past, which allowed the frog population to recover.
    4. The wild frogs have antifungal compounds, which allowed the wild frog population to recover.
  9. Among the many hypotheses suggested by the podcasters, was the possibility that the frog skin microbiome plays a role.  How could you test whether the frog skin microbiome produces a fungal inhibitor?
    1. Isolate frog skin microbiome bacteria; test for production of fungal inhibitors using a growth inhibition assay.
    2. Use scanning electron microscopy to examine sections of frog skin for areas of fungal growth and inhibition.
    3. Use mass spectrometry to identify the fungal species present on the frog skin and identify genomic regions.
    4. Isolate different strains of the chytrid fungus and evolve strains that are resistant to the frog secretions.

7. Figure Reading Exercises

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

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify key features of phylogenetic trees and associated data.
  • Interpret a phylogenetic tree to determine patterns of similarity between organisms.
  • Predict sources of antibiotic resistance genes when given differential strain genomic data.
Experimental Background (Klemm et al., FIgure 1)

Typhoid fever is caused by Salmonella enterica serovar Typhi, and similar to many pathogenic microbes, is developing significant resistance to antibiotics.  Klemm et al. (2018) have characterized a newly emergent, extensively drug-resistant strain (XDR) from a recent outbreak.  They used whole genome sequencing to characterize and compare the  genome and  large plasmids of isolates of this new strain to other extensively drug-resistant strains.  The relationship of all the strains is displayed as an unrooted phylogenetic tree (panel a) and the relationship of a subset including the new XDR isolates is rooted and displayed with detailed data regarding the country  of origin, plasmids present, AMR cassette information, and QRDR features in columns (panel b).

phylogenetic tree and heat map
Figure 1.“XDR isolates from Sindh, Pakistan, form a distinct cluster within the H58 phylogeny. (a) An unrooted maximum-likelihood phylogenetic tree of 98 Sindh, Pakistan, H58 isolates; 1 United Kingdom traveler isolate; and 853 global H58 isolates inferred from 1,920 SNPs using 22420_1_10_Pak60006_2016 as a reference. The XDR samples (red branches) are separated by 6 SNPs from the rest of the branch (blue), which is separated by 17 SNPs from the other H58 isolates. (b) A higher-resolution diagram of the branch from panel a rooted on nearest neighbor 10060_5_62_Fij107364_2012 displaying data for each isolate: country, sample collection (XDR or contextual organisms collected in Sindh, Pakistan, from 2016 to 2017; XDR organism from United Kingdom traveler to Pakistan; or organism from the global collection), plasmid content (p60006, described in this study, or pHCM2 cryptic plasmid), presence of chromosomal AMR cassette (integrated composite transposon), and quinolone resistance-determining region (QRDR) mutations according to the color key.” (Klemm et al. 2018, no changes).

7.1.2. Questions

  1. A phylogenetic tree, like that shown in Figure 1, panel A, is a visual comparison to show similarities and differences of features.  Here we are looking at the similarities/differences in bacterial genomes and plasmid DNA sequences taken from different bacterial isolates.  How many genomes are compared in this analysis?
    1. 98
    2. 1920
    3. 952
    4. 17
  2. The red/blue region of the phylogenetic tree shown in panel A is shown in greater detail in panel B.  The left section of the tree shows 17 single nucleotide polymorphisms (SNPs) distinguish these strains (red) from the larger tree (black) and that 6 additional SNPs distinguish the “red” strains from the blue strains.  What does this tell us about the similarity and differences between the “red” strains and “blue” strains?
    1. Blue strains have 6 genetic differences that distinguish them from the black strains.
    2. Red strains have more genetic changes than the blue strains or the black strains.
    3. Blue strains are more dissimilar to each other than red strains are similar to each other.
    4. Red strains are more similar to each other than red strains are to blue strains.
  3. What is the country of origin for the new XDR strains?
    1. Pakistan
    2. Bangladesh
    3. Iraq
    4. India
    5. Palestine
    6. UK
  4. Based on the data in panel b, including the extended part, which plasmid does the XDR strain isolated in Pakistan carry?
    1. p60006
    2. pHCM2
    3. none
  5. Based on the additional genomic context information provided at the right of panel B, where should investigators look for genetic changes related to the XDR strain’s extensive antibiotic resistance?
    1. plasmid p60006
    2. plasmid pHCM2
    3. chromosomal AMR cassette
    4. quinolone resistance-determining region (QRDR)

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features of bar charts and box plots.
  • Analyze specific host measures to make conclusions about whether host adaptation to fungal infection has occurred.
  • Predict how pathogen and host characteristics might change relative to each other from pre-disease to post-outbreak.
Experimental Background (Voyales et al., Figure 4)

The ability to examine changes in both the host and infectious agent before, during, and after an outbreak is critical to understanding outbreaks.  In this paper, the authors have been able to conduct such a study in frogs.  Frog species at three sites in Panama were studied before, during, and after an outbreak of chytridiomycosis, which is caused by Batrachochytrium dendrobatidis (Bd).  Surveys of the areas included nine frog species as well as the fungus. Voyales et al. (2018) found that while all species are recovering, two species were particularly susceptible.  They saw no detectable no differences between historic and contemporary isolates in fungal features such as growth rate, zoosporangium size, and ability to inhibit specific immune cell activities.  To test for changes in the frog host response pre- and post-outbreak, they exposed historic and contemporary isolates of Bd to skin secretion samples of six frog species taken either pre- or post-outbreak.  They report their data as inhibition of Bd growth, inhibitory effectiveness (Figure 4, panel A).  Further, they investigated whether this was an adaptation by comparing Bd growth in the presence of skin secretions taken from captive frogs (that could not have been exposed to the outbreak) to skin secretions taken from post-outbreak wild frogs (Figure 4, panel B).

7.2.2. Questions

  1. Fungal growth inhibition was quantified for pre-disease and enzootic strains of fungi in the presence of skin secretions from different frogs.  The data are displayed by as a bar graph. Match the bar plot feature with its description. [1 = mean; 2 = median; 3 = standard deviation; 4 = standard error of the mean; 5 = sample size; 6 = fungal strain origin]
    1. bar height =
    2. bar color =
    3. Whiskers =
    4. numbers in parentheses =
  2. Growth of Bd in the presence of pre-disease and post-outbreak (enzootic) skin secretion samples is shown for six frog species (panel A).  Which species’ skin secretions shows the most growth, that is the least inhibition when you consider both pre-disease and post-outbreak?
    1. Craugastor cassidigitus
    2. Bolyitoglossa schizodactyla
    3. Espadarana proseblepon
    4. Hyalinobatrachium colymbiphyllum
    5. Diasporus diastema
    6. Colostethus panamansis
  3. Looking at the Bd growth inhibition for pre-disease and post-outbreak (panel A), which species’ skin secretions shows the most inhibition when you consider both pre-disease and post-outbreak?
    1. Craugastor cassidigitus
    2. Bolyitoglossa schizodactyla
    3. Espadarana proseblepon
    4. Hyalinobatrachium colymbiphyllum
    5. Diasporus diastema
    6. Colostethus panamansis
  4. Looking at the Bd growth inhibition for pre-disease and post-outbreak (panel A), which frog species shows the greatest adaptation to fungal infection between pre-disease and post-outbreak?
    1. Craugastor cassidigitus
    2. Bolyitoglossa schizodactyla
    3. Espadarana proseblepon
    4. Hyalinobatrachium colymbiphyllum
    5. Diasporus diastema
    6. Colostethus panamansis
  5. Box and whisker plots (panel b) are used to show visual differences in sample statistics.  What statistical value do the vertical lines that extend above and below each box represent?
    1. The range for all values for that sample.
    2. The outliers found for that sample.
    3. The standard error for that sample.
    4. The standard deviation for that sample.
  6. The ability of skin secretions from wild (previously infected) and captive-bred Atelopus varius frogs to inhibit fungal growth was tested (panel b). Box and whisker plots can show visual differences in sample statistics such as variability in the sample.  Which sample has the largest variability among the sample values (panel b)?  What feature tells you this?
    1. Wild; the whiskers’ range (top to bottom)
    2. Captive; the box’s median (horizontal line)
    3. Wild; the distance of the box from the x-axis
    4. Captive; the box’s height (top to bottom)
  7. What can you conclude from the experiment where Bd growth was compared in the presence of skin secretions from captive (naive) frogs and wild frogs (panel B)?
    1. Captive/naive frogs will all die if they become infected with Bd, but all the wild frogs will survive.
    2. There is considerably more Bd growth with skin secretions from wild frogs than captive frogs.
    3. Wild frogs that survived now show a skin-secretion-related adaptation to limit Bd infection.
    4. Captive frogs have a reduced growth environment for fungal growth so less is observed.
  8. Based on the results of panel B, which box plot would most resemble the results you would expect if you examined skin secretions from a wild frog at the very beginning of the outbreak? Why?
    1. Captive; these frogs are not infected yet
    2. Wild; all wild frogs will have the same response
    3. Captive; these frogs have not adapted to Bd
    4. Wild; wild frogs have symbionts that protect

8. Paper Information and Licensing

8.1. Snippet paper

  • Klemm EJ, Shakoor S, Page AJ, Qamar FN, Judge K, Saeed DK, Wong VK, Dallman TJ, Nair S, Baker SShaheen G, et al. 2018. Emergence of an Extensively Drug-Resistant Salmonella enterica Serovar Typhi Clone Harboring a Promiscuous Plasmid Encoding Resistance to Fluoroquinolones and Third-Generation Cephalosporins. mBio9:10.1128/mbio.00105-18. https://journals.asm.org/doi/10.1128/mbio.00105-18 
  • 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 https://journals.asm.org/doi/10.1128/mbio.00105-18 

8.2. Main paper

  • Voyles J, Woodhams DC, Saenz V, Byrne AQ, Perez R, Rios-Sotelo G, Ryan MJ, Bletz MC, Sobell FA, McLetchie et al. 2018.  Shifts in disease dynamics in a tropical amphibian assemblage are not due to pathogen attenuation. Science. 359(6383):1517-1519. https://www.science.org/doi/10.1126/science.aao4806
  • This article is not licensed for Creative Commons use, but is freely available at the journal’s website. Thus, the abstract and figures cannot be copied here. Please see the 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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