Structure and Function

TWiM #273: The Value of Wiping

Podcast and Annotation Information
  • Annotation by Kendall Conway, Canaan Woldekidan, Madonna DiMartino, Ariana Siddique, Eileen Hotze
  • Podcast audio by TWiM: Listen to TWiM #273 Podcast
  • Podcast transcript by Otter.ai and edited by Kendall Conway, Canaan Woldekidan, Madonna DiMartino, Ariana Siddique, and Gwen Knapp: Access Podcast Transcripts
  • Papers Discussed:
    • Faircloth J, Goulter RM, Manuel CS, Arbogast JW, Escudero-Abarca B, Jaykus LA. 2022. The Efficacy of Commercial Surface Sanitizers against Norovirus on Formica Surfaces with and without Inclusion of a Wiping Step. Appl Environ Microbiol.; 88(17):e0080722. https://doi.org/10.1128/aem.00807-22
    • Huseby DL, Brandis G, Praski Alzrigat L, Hughes D. 2020. Antibiotic resistance by high-level intrinsic suppression of a frameshift mutation in an essential gene. 2020. Proc Natl Acad Sci U S A. 117(6):3185-3191. doi: 10.1073/pnas.1919390117

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 3:01 minutes

The Most Interesting Things (according to students)

When you rub your hands with hand sanitizer, the rubbing does a lot of getting rid of pathogens.

“Commonly used surface sanitizers often lack activity against human noroviruses (hNoVs). The impact of inactivation versus removal when these products are applied via wiping is poorly characterized. The purpose of this work was to assess the anti-hNoV efficacy of various surface sanitizer chemistries, as applied to a laminate material commonly used for restaurant tabletops, using standard surface assays (ASTM E1053-11) and a newly developed wiping protocol. Four commercially available products with different active ingredient(s) (i.e., ethanol [EtOH], acid + anionic surfactant [AAS], quaternary ammonium compound [QAC], and sodium hypochlorite [NaOCl]) and a water control were evaluated against hNoV GII.4 Sydney, hNoV GI.6, and the cultivable surrogate Tulane virus (TuV). Virus concentration was evaluated using RNase-reverse transcriptase (RT)-quantitative PCR (qPCR) (hNoV) and infectivity assay (TuV). Only the EtOH-based product significantly reduced virus concentration (>3.5 log10 reduction [LR]) by surface assay, with all other products producing ≤0.5 LR. The inclusion of a wiping step enhanced the efficacy of all products, producing complete virus elimination for the EtOH-based product and 1.6 to 3.8 LR for the other chemistries. For hNoVs, no detectable residual virus could be recovered from paper towels used to wipe the EtOH-based product, while high concentrations of virus could be recovered from the used paper towel and the wiped coupon (1.5 to 2.5 log10 lower genome equivalent copies [GEC] compared to control) for the QAC- and AAS-based products and for water. These results illustrate the variability in anti-hNoV activity of representative surface sanitizers and highlights the value of wiping, the efficacy of which appears to be driven by a combination of virus inactivation and removal.” (Faircloth et al. 2022)

1.2. Main paper; discussion starts at 31:26 minutes

The Most Interesting Things (according to students)

Loss of function frameshift mutations are typically detrimental, but in the case of the rpoB gene mutation in Mycobacterium tuberculosis, it resulted in advantageous antibiotic resistance.

“A fundamental feature of life is that ribosomes read the genetic code in messenger RNA (mRNA) as triplets of nucleotides in a single reading frame. Mutations that shift the reading frame generally cause gene inactivation and in essential genes cause loss of viability. Here we report and characterize a +1-nt frameshift mutation, centrally located in rpoB, an essential gene encoding the beta-subunit of RNA polymerase. Mutant Escherichia coli carrying this mutation are viable and highly resistant to rifampicin. Genetic and proteomic experiments reveal a very high rate (5%) of spontaneous frameshift suppression occurring on a heptanucleotide sequence downstream of the mutation. Production of active protein is stimulated to 61–71% of wild-type level by a feedback mechanism increasing translation initiation. The phenomenon described here could have broad significance for predictions of phenotype from genotype. Several frameshift mutations have been reported in rpoB in rifampicin-resistant clinical isolates of Mycobacterium tuberculosis (Mtb). These mutations have never been experimentally validated, and no mechanisms of action have been proposed. This work shows that frameshift mutations in rpoB can be a mutational mechanism generating antibiotic resistance. Our analysis further suggests that genetic elements supporting productive frameshifting could rapidly evolve de novo, even in essential genes.” (Huseby et al. 2020)

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

Snippet Main
Vision and Change Topics
  • Structure and Function (V&C_SF)
  • Impact of Microorganisms (V&C_IM)
  • Information Flow and Genetics (V&C_IFG)
  • Structure and Function (V&C_SF)
ASM Fundamental Statements
  • Fundamental Statement 14 (ASM_14): Extrinsic factors, such as abiotic and biotic interactions in the environment, can impact survival and growth of microbes.
  • 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 3 (ASM_3):The evolution of microbes is impacted by their interactions with the environment and a variety of ecological forces, including other microbes, humans, and habitats.
  • Fundamental Statement 12 (ASM_12): Intrinsic factors, such as genotype, metabolism, and cell structures, impact the survival and growth of microbes.
  • Fundamental Statement 16 (ASM_16): Genetic variation can influence microbial structures and their functions.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Identify the difference between a sanitizer and a disinfectant.
  • Recall the features of norovirus.
  • Identify the take-home message of the research.
 S L
  • Design an alternate experiment given a new variable related to this study.
 S H
  • Define frameshift and summarize why it usually results in a loss of function.
  • Identify the difference between frameshift and programmed ribosomal frameshift.
 M L
  • Predict how changes in cellular features would affect programmed ribosomal frameshift-based rifampicin resistance.
 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

  • Surrogate Model Agents (13:59–15:37): These are agents that are used by researchers in place of dangerous pathogens to study pathogen processes.  Usually the agent is an evolutionary relative with similar characteristics, but safer for the researchers to use.  Here, the researchers used a macaque viral surrogate to evaluate cleaning procedures for human norovirus. An acknowledged issue with the surrogate virus was pH sensitivity.

4.2. Main Paper

  • Liquid Chromatography Mass Spectrometry (LCMS) (47:40–49:46): This technique combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. Here, LCMS was used to analyze the proteins produced by the cells with frameshift mutations.  This allowed them to deduce slippage efficiency and confirm the presence of the predicted protein sequences, specifically, RpoB protein levels.
  • Fluorescence Reporter Assays (49:48–50:01): This is a technique for measuring gene expression using a reporter gene that encodes a fluorescent protein. Here, it was used to measure slippage in specific rare codons of interest in this podcast/paper.

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

5.1. Snippet Paper

  • Control of Microbes (8:30–9:00): Disinfection versus sanitization to control pathogens on surfaces was discussed.
  • Chemical Sterilant/Disinfectants (9:13–13:40; 16:00–28:24): Disinfecting food contact surfaces with effective products is extremely difficult due to norovirus’ high threshold concentration for sodium hypochlorite to be useful.  The high concentrations are both bad odor-wise and are too corrosive for use. Quaternary ammonium compounds are another option used in hospitals that are cations binding to negative amino acids on protein structure, but they take too long to dry, so there is a need for alternatives. The researchers compared components of 4 different sterilants and their effectiveness on inactivating the norovirus.  Ethanol and bleach at high concentrations deactivate the norovirus on surfaces but are too damaging to skin. Hand sanitizers are too weak, so only physical agitation of handwashing can remove norovirus from skin.
  • Virus Structure (21:30–21:33): Description of the structure of the norovirus.

5.2. Main Paper

  • Ribosomes (31:26–38:58): The role of ribosomal function and structures in protein synthesis and the role of these in overcoming the frameshift mutation.
  • Frameshift Mutation/Antibiotic Resistance (33:20–50:38): Resistance to rifampicin has been linked to a frameshift mutation in rpoB, which encodes a major subunit in RNA polymerase. Specifically, a “plus-one” frameshift leads to production of rare codons (CCC and AGG) that create pausing in translation, which ultimately increases likelihood of ribosomal shift. Cell growth is slowed but resistant to the drug because of functional RNA polymerase.  The frameshift causes a large section of rpoB to be untranslated unless the ribosome shifts when waiting at the rare codons.  Ribosome shifting/slippage allows RpoB to be translated at 60% levels, causing the slow growth AND the resistance to rifampicin, which acts on RpoB protein.
  • Loss of Function Mutation (36:00–36:18): A mutation that disturbs the original functioning of a protein.
  • Tuberculosis/Slow Growth (52:18  -54:22): Despite a mutation in rpoB, TB is an effective pathogen due to its slow growth. Its generation time is measured in hours, not minutes like E. coli. Mutations affecting transcription enzymes have selective advantages in the case of TB, but not for pathogens such as E. coli. Catalytic protein functions allow some pathogens to survive/operate despite a frameshift mutation because cells don’t need as much of these proteins because enzymes are not “used up” in chemical reactions.

6. Podcast Questions

  1. A disinfectant kills/inactivates _______ while a sanitizer kills/inactivates ________ .
    1. Bacteria and viruses; bacteria
    2. Fungi and bacteria; viruses
    3. Viruses; bacteria and viruses
    4. Fungi; fungi and viruses
  2. Which of the following are characteristics of norovirus? [Pick all that apply]
    1. Causes diarrhea
    2. Causes vomiting
    3. Causes facial rashes
    4. Single-stranded RNA genome
    5. Double-stranded DNA genome
    6. Enveloped virus
    7. Non-enveloped virus
    8. Remains infectious on surfaces for weeks
    9. Remains infectious on surfaces for only hours
    10. Has a 90 nanometer diameter
  3. What is a reasonable take-home from the snippet paper and podcaster discussion regarding hand hygiene?
    1. Bleach-based hand sanitizer and rubbing your hands is the best defense!
    2. Make sure your hand sanitizer has at least 90% ethanol for effectiveness.
    3. Vigorously rub your hands when you wash them with soap and water!
    4. Household bleach and alcohol work equally well for disinfection purposes.
  4. The researchers for the snippet paper were focused on surface decontamination of Tulane virus and norovirus. To measure decontamination, they quantified norovirus using genome equivalents and Tulane virus using a plaque assay.  If they were next interested in surface decontamination of bacteria (such as E. coli O antigen 157), what assay should they perform instead?
    1. Assay for population growth using spectroscopy
    2. Assay for living bacteria using colony counts
    3. Assay for bacterial morphology using microscopy
    4. Assay for dead bacteria using IR colorimetry
  5. What is a frameshift and what are the typical consequences?
    1. It is a change in the DNA that results in elongated RNAs being transcribed.
    2. It is a change in transcription that causes high levels of RNA to be produced.
    3. It is a change in translation that results in a reduction in protein synthesis.
    4. It is a change in the DNA that alters translation, producing a shortened protein.
  6. A frameshift and a programmed ribosomal frameshift are different in that a frameshift is a change in _____ while a programmed ribosomal frameshift is a change in _______ .
    1. DNA; codon usage
    2. Codon usage; RNA
    3. DNA; translation
    4. Translation; RNA
  7. The rifampicin resistance the researchers identified involves ribosome pausing at rare codons CCC and AGG.  What would be the most likely effect of adding additional tRNAs for these codons to the rifampicin-resistant strains the researchers isolated? Why?
    1. They would become rifampicin-sensitive because the ribosome would no longer pause and slip at the rare codons.
    2. They would be equally-resistant as before because tRNAs are not required for rifampicin resistance, only RpoB.
    3. They would become ultra-resistant because the ribosome would no longer pause, encoding the full length RpoB.
    4. They would be equally resistant, but grow faster since they now have a full complement of translation machinery.

7. Figure Reading Exercises

The following are two figure reading exercises, both from the snippet paper (Figures 3 and 4).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify key features of bar charts related to these experiments.
  • Explain how you can tell whether a decontamination agent is effective given how the data are represented.
  • Predict how results would appear if the data were displayed differently.
  • Evaluate the data and draw conclusions on the effectiveness of decontamination regimes.
  • Predict the effects on microbial survival when the properties of the antimicrobial are altered.
Experimental Background (Faircloth et al., Figure 3)

Commonly used surface disinfectants often lack activity against human noroviruses (hHoV). The purpose of this work by Faircloth et al. (2022) was to assess the anti-hHoV efficacy of various surface disinfectant chemistries with or without physical wiping afterward for the food service industry, such as restaurants. Four commercially available products with different active ingredients were used. The first was ethanol [EtOH] at a pH of 12.4. The second was an acid + anionic surfactant [AAS] at a pH of 2.7. The third was a quaternary ammonium compound [QAC] at a neutral pH. The fourth sodium hypochlorite [NaOCl]) (bleach) at a pH of 10.  Water served as a control.  The efficacy of each treatment regime was evaluated using the cultivable surrogate, the macaque Tulane virus (TuV) with 30 seconds (panels a, c) or 60 seconds (panel b, d) of exposure, with wiping (panels a, b) or without (panel c, d).

bar charts
Figure 3. “Log10 reduction of Tulane virus (TuV) PFU by various sanitizing products and contact times (30 and 60 s) on Formica coupons without (a, b) and with (c, d) a wiping step with paper towel. The dotted line for each panel represents the limit of detection of the assay. Different letters within a panel represent significant statistical differences (P < 0.05) in the log10 reduction of TuV PFU when comparing products within that panel. Asterisks used in the margin above panels c and d indicate situations where a statistically significant (P < 0.05) increase in log10 reduction of TuV PFU occurred as a result of incorporating a wiping step into the process. Error bars represent the standard deviation. EtOH, ethanol; AAS, acid + anionic surfactant; NaOCl, sodium hypochlorite; QAC, quaternary ammonium compound.” (Faircloth et al. 2022, no changes).

7.1.2. Questions

  1. The data are displayed in bar charts with each treatment regime as a bar within one of four panels.  Where would you find the data for the effectiveness of sodium hypochlorite (bleach treatment) and a wiped surface?
    1. Panel a; light blue bar
    2. Panel a and b; pink bar
    3. Panel a and c; dark blue bar
    4. Panel c and d; green bar
  2. What would an effective decontamination treatment show if you quantified the data as log reduction, as these researchers have done?
    1. The reduction should be lower (smaller bar) than the water control.
    2. The reduction should be higher (larger bar) than the water control.
    3. The reduction should be lower (smaller bar) than the ethanol control.
    4. The reduction should be higher (larger bar) than the ethanol control.
  3. If the researchers had decided to display their data as log recovery instead of log reduction, what results would you expect to see for the water control, an ineffective treatment, and an effective treatment?
    1. The ethanol control and effective treatments would show high recovery and the ineffective treatments would show low recovery.
    2. They would show: water =  low recovery, ineffective treatments  = high recovery,  and the effective treatments = low recovery.
    3. The water control and all ineffective treatments would show high recovery and the effective treatments would show low recovery.
    4. They would show: water =  high recovery, ineffective treatments  = high recovery,  and the effective treatments = low recovery.
  4. In the experiment where the surfaces were decontaminated for 60 seconds and not wiped, which treatment is most effective at decontamination?  What is your evidence?
    1. AAS; it has smallest variability
    2. EtOH; it has the highest reduction
    3. NaOCl; it has the largest whiskers
    4. QAC; it has the smallest recovery
  5. Based on the results presented, how does wiping the surface with a paper towel affect the efficacy of the disinfectant tested?
    1. Wiping the surface decreases the efficacy of the disinfectant.
    2. Wiping the surface has no effect on disinfectant efficacy.
    3. Wiping the surface has variable effects on disinfectant efficacy.
    4. Wiping the surface increases the efficacy of the disinfectant.
  6. If the virus was found to be most inactivated at an acidic pH, which antimicrobial would be the most effective?
    1. EtOH
    2. AAS
    3. QAC
    4. NaOCl

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features of horizontal bar charts.
  • Identify key aspects of experimental design, such as the dependent and independent variables in an experiment.
  • Explain how you can tell whether contamination is present.
  • Draw conclusions on the effectiveness of decontamination regimes based on the given data.
  • Make a real life recommendation for keeping restaurant staff safe based on these data.
Experimental Background (Faircloth et al., Figure 4)

Commonly used surface disinfectants often lack activity against human noroviruses (hHoV). In the earlier part of this study Faircloth et al. (2022) assessed the efficacy of various surface disinfectant chemistries with or without physical wiping afterward for the food service industry, such as restaurants. Four commercially available products with different active ingredients were used: ethanol [EtOH], an acid + anionic surfactant [AAS], a quaternary ammonium compound [QAC], and sodium hypochlorite [NaOCl]) (bleach).  Water served as a control.  The efficacy of each treatment regime was evaluated using two human noroviruses (hNoV GII.4 and hNoV GI.6), as well as the cultivable surrogate, the macaque Tulane virus (TuV) with no wiping and with wiping.  Having identified the most effective decontaminant, the researchers next investigated whether the cleaning paper used in the wiping step was now contaminated.  They used no treatment control, 30 seconds, or 60 seconds of treatment with the compounds which are identified at the right side of each row.  The experimental virus is noted at the top of each panel.  The amount of recovered virus for each treatment is displayed as a horizontal bar chart.

bar charts
Figure 4. “Log10 recoveries of GII.4 hNoV GEC (a), GI.6 hNoV GEC (b), and TuV PFU (c) from paper towels following treatment of virus-inoculated Formica surfaces with various surface sanitizers applied with a paper towel wiping step. Controls represent the viral titer of dried inocula on untreated coupons. The dotted line represents the limit of detection of the assay. Different letters within a column of panels represent significant statistical differences (P < 0.05) in the log10 recovery of virus when comparing sanitizing products, with lowercase letters being used for 30 s and uppercase letters being used for 60 s. EtOH, ethanol; AAS, acid + anionic surfactant; NaOCl, sodium hypochlorite; QAC, quaternary ammonium compound.” (Faircloth et al. 2022)

7.2.2. Questions

  1. The data are displayed in horizontal bar charts with each treatment regime as a bar within one of the panels.  Where would you find the data for the hNoV GII.4 paper towel contamination after 30 seconds of decontamination using quaternary ammonium compound?
    1. Panel a, row 1; light blue bar
    2. Panel c, row 2; dark yellow bar
    3. Panel b, row 3; medium green bar
    4. Panel a, row 4; medium pink bar
  2. Let’s consider the experiment looking at recovery of hNoV GII.4 virus on the paper towel after decontamination with ethanol (panel a, row 1).  The dependent variable for this experiment is ______ and the independent variable for this experiment is ______.
    1. Viral recovery; length of EtOH decontamination
    2. Type of decontaminant used; viral recovery
    3. Viral reduction; length of wiping procedure
    4. Wiping procedure time; viral recovery time
  3. What should a decontamination treatment that also effectively decontaminates the cleaning towel show if you quantified the data as log recovery, as these researchers have done?
    1. The recovery should be lower (smaller bar) than the water control.
    2. The recovery should be higher (larger bar) than the water control.
    3. The recovery should be lower (smaller bar) than the untreated control.
    4. The recovery should be higher (larger bar) than the untreated control.
  4. A high log recovery of virus on the paper towel surface post-wipe can be attributed to the fact that:
    1. The disinfectant did not deactivate the virus that was transferred to the paper towel.
    2. The viruses replicated on the paper towel after encountering a eukaryotic host cell.
    3. The virus was deactivated on the surface and then reactivated on the paper towel.
    4. The disinfectant deactivated the virus; dead viruses were detected on the paper towel.
  5. Based on the figure, which compounds were not at all effective at deactivating viruses on the paper towel used for the wiping step?
    1. EtOH
    2. AAS
    3. NaOCl
    4. QAC
    5. Water
    6. None of the antimicrobials deactivated viruses effectively.
  6. To prevent contraction of viral illness for employees responsible for wiping down tables at a restaurant, which two compounds would be very effective to use?
    1. EtOH
    2. AAS
    3. NaOCl
    4. QAC
    5. Water

8. Paper Information and Licensing

8.1. Snippet paper

  • Faircloth J, Goulter RM, Manuel CS, Arbogast JW, Escudero-Abarca B, Jaykus LA. 2022. The Efficacy of Commercial Surface Sanitizers against Norovirus on Formica Surfaces with and without Inclusion of a Wiping Step. Appl Environ Microbiol. 2022 Sep 13;88(17):e0080722. https://doi.org/10.1128/aem.00807-22
  • 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/aem.00807-22

8.2. Main paper

  • Huseby DL, Brandis G, Praski Alzrigat L, Hughes D. Antibiotic resistance by high-level intrinsic suppression of a frameshift mutation in an essential gene. 2020. Proc Natl Acad Sci U S A. 2020 Feb 11;117(6):3185-3191. doi: 10.1073/pnas.1919390117
  • 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://www.pnas.org/doi/full/10.1073/pnas.1919390117

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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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