Structure and Function

TWiM #294: You’ll Scream After Ice Cream

Podcast and Annotation Information
  • Annotation by Rohini Donakonda, Mikayla Mejorada, and Amanda Swedrowski, Rebecca Seipelt-Thiemann, Jeremy T. Ritzert 
  • Podcast audio by TWiM: Listen to TWiM #294 Podcast
  • Podcast transcript by Otter.ai and edited by Rebecca Seipelt-Thiemann, Isabelle Norris, Rohini Donakonda, Mikayla Mejorada, and Amanda Swedrowski: Access Podcast Transcripts
  • Papers Discussed:
    • Valentino V, De Filippis F, Sequino G, Ercolini D. 2023. Psychrotrophic Bacteria Equipped with Virulence and Colonization Traits Populate the Ice Cream Manufacturing Environment. Appl Environ Microbiol. 89(8):e0076523. https://journals.asm.org/doi/10.1128/aem.00765-23 
    • Morton SU, Hehnly C, Burgoine K, Ssentongo P, Ericson JE, Kumar MS, Hagmann C, Fronterre C, Smith J, Movassagh M, et al. 2023. Paenibacillus spp infection among infants with postinfectious hydrocephalus in Uganda: an observational case-control study. Lancet Microbe. 4(8):e601-e611. doi: 10.1016/S2666-5247(23)00106-4.

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 3:21 minutes

The Most Interesting Things (according to students)

Psychotrophic bacteria can harbor antibiotic resistant traits in plasmids and can stimulate biofilm or endospore formation to encourage colonization despite rigorous sanitization practices.

Valentino et al (2023) is not licensed for Creative Commons use, so, the abstract cannot be copied here. Please see the article at the journal’s web page.: https://journals.asm.org/doi/10.1128/aem.00765-23 

1.2. Main paper; discussion starts at 31:58 minutes

The Most Interesting Things (according to students)

In regions that have limited access to surgical intervention, it is important to identify routes of infection in order to improve prevention efforts of lethal infections caused by Paenibacillus in infants.

“Paenibacillus thiaminolyticus is a cause of postinfectious hydrocephalus among Ugandan infants. To determine whether Paenibacillus spp is a pathogen in neonatal sepsis, meningitis, and postinfectious hydrocephalus, we aimed to complete three separate studies of Ugandan infants. The first study was on peripartum prevalence of Paenibacillus in mother–newborn pairs. The second study assessed Paenibacillus in blood and cerebrospinal fluid (CSF) from neonates with sepsis. The third study assessed Paenibacillus in CSF from infants with hydrocephalus.” (Morton et al. 2023)

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

Snippet Main
Vision and Change Topics
  • Microbial Ecology (V&C_ME)
  • Structure and Function (V&C_SF)
  • Impact of Microorganisms (V&C_IM)
  • Structure and Function (V&C_SF)
  • Impact of Microorganisms (V&C_IM)
ASM Fundamental Statements
  • 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 20 (ASM_20): Microbes are ubiquitous, found in diverse and dynamic ecosystems, where they use available resources and often form complex communities.
  • Fundamental Statement 28 (ASM_28): A minority of microbes are pathogens that can cause diseases and harm host organisms, society, and ecosystems.
  • Fundamental Statement 29 (ASM_29): The extent of microbial damage can be minimized by host-derived and external factors, including the microbiome, antibiotics, and immunity.
  • Fundamental Statement 5 (ASM_5): The structure and function of microbes are revealed by the use of microscopy, culture, and metabolic analyses, molecular methods, and bioinformatic tools.
  • 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 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 29 (ASM_29): The extent of microbial damage can be minimized by host-derived and external factors, including the microbiome, antibiotics, and immunity.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Recall how 16S rDNA ribosomal sequencing and how it was used in this study.
  • Identify the most abundant bacterial genera found in the study.
  • Explain how bacteria use certain traits to resist cleaning techniques.
  • Identify the source of the bacterial genera found in the final manufacturing product and its implications.
 S L
  • Propose a source of a new pathogen given a specific scenario.
 S H
  • Identify how Paenibacillus was identified as the pathogen.
  • Compare the features of Legionella to what is now known for Paenibacillus.
 M L
  • Predict how different cultural practices might contribute to disease spread.
 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

  • 16S rDNA Gene Sequencing (11:42- 12:28): This is a technique used to identify species by sequencing the 16S RNA gene, encodes the 16S RNA of the ribosome, and comparing its sequence to a database.  This sequence is commonly used in metagenomic studies.  This sequencing revealed the species present in the study without having to culture them.
  • TORMES Analysis (9:10 -10:05; 23:00–25:45): This is a computational pipeline for bacterial genome sequencing.  Here, the whole bacterial genome analysis identified shared virulent genes across contigs.
  • Bray-Curtis Distance (20:00): This is a type of Principal Component Analysis (PCA).  Here the researchers use this plot to display the similarities and differences among bacterial communities.

4.2. Main Paper

  • 16S rDNA Gene Sequencing (37:50–38:07): This is a technique used to identify species by sequencing the 16S RNA gene, encodes the 16S RNA of the ribosome, and comparing its sequence to a database.  This sequence is commonly used in metagenomic studies.  This sequencing revealed the species present in the study without having to culture them.
  • Quantitative Polymerase Chain Reaction (qPCR) (38:45–39:11): This technique uses fluorescence to quantify the amount of specific DNA present in a sample by using sequence-specific primers. The researchers used this technique to quantify the amount of Paenibacillus in cerebral spinal fluid samples.
  • Ultrasound Imaging (45:10–45:39): This technique uses sound waves to generate an image of what is inside the body.  The researchers used this to compare the cranium of a healthy infant to an infant with hydrocephalus.
  • Whole Genome Sequence Analysis (47:02–47:26): Using next generation sequencing technologies, whole genomes can be sequenced and compared.  Here, the sequence analysis revealed that clinical isolates of Paenibacillus differed from environmental isolates.

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

5.1. Snippet Paper

  • Curvibacteria (15:52–16:36): The structural characteristics and features of Curvibacteria were described.
  • Rothia (17:51–18:58): Structural characteristics of Rothia including filamentous features were described.
  • Biofilm (18:36–19:22; 24:17–24:44): Some bacteria identified in ice cream manufacturing have biofilm forming capabilities.
  • Shannon and Simpson Analysis (20:52–21:48): These are a measure of species diversity within a community and were compared across regions.

5.2. Main Paper

  • Type IV Pili (47:14–47:56): Pili are linked to the ability of microbes to adhere to surfaces.
  • Microbial Ecology (43:20–44:23): Hydrocephalus cases were higher in regions of Uganda that were wetlands and the number of cases also increased during rainy seasons.
  • Cerebral Spinal Fluid (CSF) (34:19–34:45; 47:56–48:30): CSF was one of the main topics of interest to discover how Paenibacillus was present and affecting the brain.

6. Podcast Questions

  1. What is 16S rDNA sequencing and what did the researchers use it to investigate in this study?
    1. It is a sequencing method used to identify variation in the 16S rDNA sequence and produce a phylogenetic tree.
    2. It is a mass spectroscopy technique used to identify the species present by calculating the total bacterial mass.
    3. It is a spectroscopic technique used to quantify the level of biological metabolism within a specific biofilm.
    4. It is a metagenomic technique used to identify the species present using variation in the 16S rDNA sequence.
  2. Which bacterial genus was most abundant in the ice cream study?
    1. Acetinobacter
    2. Curvibacter
    3. Pseudomonas
    4. Rothii
    5. Streptococci
  3. What properties of Rothia may allow it to thrive on non-food surfaces?
    1. Rothia most likely have filamentous branches that allow them to adhere and stay on surfaces, even if they get cleaned.
    2. Rothia most likely have enzymes that can use lactose from ice cream as a food source which allows them to thrive
    3. Rothia most likely use quorum sensing to communicate with  nearby bacteria that a food source is readily available
    4. Rothia most likely make spores which can survive in more stressful and colder environments that the ice cream is found in.
  4. What was the source of the bacteria present in the final product and what implications does this specific result have for the ice cream manufacturer?
    1. Raw materials; Quality control on ingredients is important
    2. Food contact surfaces; Having operators sanitize equipment is important
    3. Non-food contact surfaces; Having operators sanitize the environment is important
    4. Operators; Having operators taking sanitizing showers is important
    5. Undetermined; Quality control on final product is important
  5. Suppose a new infectious disease was identified in individuals who fish from the shore in local streams.  What could be the possible source(s) of this new pathogen?  Pick all that apply.
    1. Fish
    2. Shore soil
    3. Stream water
    4. The bait used
    5. An upstream factory
    6. The nearest restroom
    7. Practices of eating raw fish
    8. Convenience stores that sell bait
    9. Stores selling outdoor equipment
  6. How was the identity of the hydrocephalus-causing pathogenic bacterium established?
    1. The researchers tested maternal blood, placenta, vaginal swabs, umbilical cord blood, and cerebrospinal fluid of affected infants.
    2. The researchers performed metagenomic 16S rDNA sequencing on DNA from cerebrospinal fluid of affected and non-affected infants.
    3. The researchers used standard culturing techniques to identify specific bacterial colonies in cerebrospinal fluid of affected infants.
    4. The researchers provided different antibiotics to hydrocephalus patients and determined minimal inhibitory concentrations (MIC).
  7. Identify the features as Legionella (L) and/or Paenibacillum (P).
    1. ______ Contracted from an environmental source:
    2. ______ Spread by person to person contact:
    3. ______ Causes pneumonia:
    4. ______ Causes hydrocephalus:
    5. ______ Spread via aerosols:
    6. ______ Spread via direct contact with a biofilm:

  8. Open Response: Different cultural practices can influence disease spread.  Here, Michele mentions the practice of packing the umbilicus.  What other cultural practices have you heard of/read about that might contribute to disease spread?

     

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 5).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify key features in stacked bar charts that are relevant for this experiment.
  • Interpret abundance bar graphs from 16S rDNA sequencing taxonomic diversity data.
  • Analyze abundance graphs to identify bacteria that are different between different samples (line, surface, final product, etc).
  • Analyze abundance graphs to identify the likely origin source of bacteria in the final product for each line.
Experimental Background (Valentino et al., Figure 1)

Food quality and safety are important for maintaining consumer health and satisfaction.  Despite stringent sanitization protocols there are numerous microbial-related recalls each year because food itself has microbes and food-contact surfaces and equipment can resist decontamination. Here, Valentino et al. (2023) investigate the possible origins and biodiversity of bacterial species for two different lines of an Italian ice cream manufacturer.  Each line (Line C or wafer line at top; Line M or almond line at bottom) processes different ice cream types, and thus includes some different ingredients.  To compare the bacterial species present, they isolated total DNA from the raw materials, food contact surfaces, non-food contact surfaces, operators, and final products for both lines.  They then used a 16S rDNA metagenomic sequencing pipeline to identify and compare the bacterial species present and their relative abundances from all the different source materials and locations.

7.1.2. Questions

  1. What is the main question that can be answered using the abundance plots in this figure?
    1. What is the effectiveness of cleaning procedures on different surfaces?
    2. What are the specific temperature and surface preferences of microbial genera?.
    3. What is the prevalence of microbes in the ice cream production environment?
    4. What is the impact of different ice cream ingredients on microbial growth at 7C?
  2. For Line C’s final product, which genus is represented by the bright red bar?
    1. Comamonadaceae; Other
    2. Enterobactacease; Enterobacter
    3. Nocardiaceae; Rhodococcus
    4. Pseudomonaceae; Pseudomonas
  3. Across both lines, which named genus is consistently most abundant on non-food contact surfaces?
    1. Lactobacillaceae; Lactobacillus
    2. Moraxelleaceae; Acinetobacter
    3. Streptococcaceae; Streptococcus
    4. Thermaceae; Thermus
  4. Which type of surfaces show a higher abundance of the genus Curvibacter?
    1. Food contact surfaces in Line C
    2. Non-food contact surfaces in Line C
    3. Food contact surfaces in Line M
    4. Non-food contact surfaces in Line M
  5. What is the primary source of the bacteria present in the final product and what implications does this specific result have for the ice cream manufacturer?
    1. Raw materials; Quality control on ingredients is important.
    2. Food contact surfaces; Having operators sanitize equipment is important.
    3. Non-food contact surfaces; Having operators sanitize the environment is important.
    4. Operators; Having operators taking sanitizing showers is important.
    5. Undetermined; Quality control on final product is important.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify the methodology used in this study and its purpose.
  • Identify structural features in scanning imagery.
  • Evaluate scanning imagery to identify clinically-relevant differences.
  • Analyze scanning imagery as diagnostic tool to determine whether clinical symptoms have progressed or receded.
  • Predict long term consequences of untreated infection.
Experimental Background (Morton et al., Figure 5)

Emerging infectious diseases are particularly destructive in world regions with little healthcare infrastructure.  Morton et al. (2023) identified Paenibacillus thiaminolyticus as the causative agent of neonatal meningitis and subsequent post-infection hydrocephalus in a region of Uganda.  To better characterize clinical evidence of disease and how the disease progressed in the brain, they performed ultrasound (three left images of panels A, B, and C) and computed tomography (CT; right-most image of panels A, B, and C) scans on healthy (panel A) and affected infants (Panels B and C). A variety of features are noted in the scan images and are notated: arrow = cyst with abscess; star = white matter lesion; S symbol = fluid and thickened tissue; circle = hyperechogenic (high echo intensity) regions.

Ultrasound and CT scan imagery for hydrocephalus and healthy infants.
Figure 5. “Imaging of neonatal Paenibacillus thiaminolyticus sepsis cases that subsequently developed postinfectious hydrocephalus.  1 month=28 days. Representative cranial ultrasound from coronal, parasagittal and coronal views, and axial CT images, shown from left to right. A) Cranial ultrasound of a healthy neonate and CT image of a healthy 2-month-old infant are shown (reproduced from https://radiopaedia.org).20 B) 8-day old neonate (patient 1) with P thiaminolyticus CNS infection. Cranial ultrasound shows abnormal white matter and cortex echogenicity, extensive cortical and white matter injury from frontal to occipital lobes bilaterally, periventricular white matter injury, and a left frontal cortex and subcortical cystic lesion (green arrow). CT image of the same patient at 1 month before surgery for postinfectious hydrocephalus shows enlargement of the cyst with abscess formation (green arrow). C) 12-day-old neonate (patient 2) with P thiaminolyticus CNS infection. Cranial ultrasound shows cortical and subcortical white matter lesions with cystic evolution (white star), ventricular fluid with loculation, thickened hyperechogenic ventricular ependymal lining (S symbol), hyperechogenic gyri and sulci, and hyperechogenic extracerebral space (green circle). CT image of the same patient at 2 months of age before surgery for postinfectious hydrocephalus shows destruction of the bilateral frontal lobes, and residual ventricular debris.” (Morton et al. 2023)

 

7.2.2. Questions

  1. What technology was used to create the images on the right of each panel and what was the researchers’ purpose of using this methodology?
    1. Positron emission tomography; determine how different types of the bacteria alter structural brain tissue
    2. Cranial ultrasound; determine effects of P. thiaminolyticus-based injury for certain parts of the neonatal brain
    3. fMRI imaging; determine deterioration level in distinct regions of the brain and which functions were affected
    4. Computed tomography (CT) scan; determine the effects of P. thiaminolyticus-based injury for brain regions
  2. How do the researchers represent the subcortical cystic lesion and cyst with abscess formation, and in which infant(s) is/are this observed?
    1. The bright white areas in the ultrasound images; both patients 1 and 2
    2. The large dark areas observed in the aged 2 month CT scan; patient 2
    3. The green arrows throughout the panel B row of images; only patient 1
    4. The green stars throughout the panel C row of images; only patient 2
  3. What are the key differences between a healthy neonate’s ultrasound and CT images and those of a neonate with Paenibacillus postinfectious hydrocephalus?
    1. Both showed hyperechogenicity (intense sound echo in the ultrasound), but only the infected neonates show enlarged ventricles.
    2. Healthy and infected infants showed abscess-related cysts in the ultrasound, but the CT scan shows enlarged ventricles for affected only.
    3. Healthy neonates showed normal ventricle size and the infected neonates showed reduced ventricles, lack of structure, and hypoechogenicity.
    4. Healthy neonates showed normal ventricle size and the infected neonates showed enlarged ventricles, cyst formation, and hyperechogenicity.
  4. Compare the CT scan images for the healthy infant (panel A), post-infection patient 1 at 1 month (panel B), and post-infection patient 2at 2 months (panel C), what can be inferred about the progression of hydrocephalus in the affected neonates?
    1. There is no significant change in the ventricle size at any of the timepoints between healthy and either post-infection infant.
    2. Ventricle enlargement increases with time in affected infants indicating progression of hydrocephalus and brain structure damage.
    3. Following surgery, structural damage has resolved in both affected infants indicating hydrocephalus  progression has ceased.
    4. Ventricle enlargement is seen in early infection infants but the hydrocephalus and structure damage resolve by two months.
  5. What are the likely long-term neurological implications for infants with untreated post-infection hydrocephalus?
    1. Potential long-term neurological deficits, such as cognitive impairments, due to observed brain damage.
    2. Likely development of enhanced sensory perception due to brain plasticity, due to observed brain changes.
    3. Potential for improved motor skills and cognitive function with age due to observed brain structure changes.
    4. No long-term implications; infants typically recover without any deficits or prolonged effects on function.

8. Paper Information and Licensing

8.1. Snippet paper

  • Valentino V, De Filippis F, Sequino G, Ercolini D. 2023. Psychrotrophic Bacteria Equipped with Virulence and Colonization Traits Populate the Ice Cream Manufacturing Environment. Appl Environ Microbiol. 89(8):e0076523. doi: 10.1128/aem.00765-23 
  • This article is not licensed for Creative Commons use; see https://journals.asm.org/doi/10.1128/aem.00765-23. Thus, the abstract and figures cannot be copied here. Please see the article at the journal’s web page.   

8.2. Main paper

  • Morton SU, Hehnly C, Burgoine K, Ssentongo P, Ericson JE, Kumar MS, Hagmann C, Fronterre C, Smith J, Movassagh M, et al. 2023. Paenibacillus spp infection among infants with postinfectious hydrocephalus in Uganda: an observational case-control study. Lancet Microbe. 4(8):e601-e611. doi: 10.1016/S2666-5247(23)00106-4.
  • This article is licensed for Creative Commons use using CC BY-NC-ND 4.0, which allows non-commercial re-use with no adaptation. see https://www.sciencedirect.com/science/article/pii/S2666524723001064?via%3Dihub

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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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