Microbial Ecology

TWiM #179: Viable but Not Culturable

Podcast and Annotation Information
  • Annotation by Leonardo Baumgartner, Martin Leyhe,Triston Walsh, Nancy Boury, Rebecca Seipelt-Thiemann 
  • Podcast audio by TWiM: Listen to TWiM #179 Podcast
  • Podcast transcript by Sarah Morgan: Access TWiM #179 Transcript
  • Papers Discussed:
    • Michaud JM, Thompson LR, Kaul D, Espinoza JL, Richter RA, Xu ZZ, Lee C, Pham KM, Beall CM, Malfatti F, Azam F, Knight R, Burkart MD, Dupont CL, Prather KA. 2018. Taxon-specific aerosolization of bacteria and viruses in an experimental ocean-atmosphere mesocosm. Nat Commun. 9:2017. https://doi.org/10.1038/s41467-018-04409-z
    • Highmore CJ, Warner JCRothwell SD, Wilks SA, Keevil CW. 2018. Viable-but-Nonculturable Listeria monocytogenes and Salmonella enterica Serovar Thompson Induced by Chlorine Stress Remain Infectious. mBio 9:10.1128/mbio.00540-18. https://doi.org/10.1128/mbio.00540-18

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 3:20 minutes

The Most Interesting Things (according to students)

Microbes in the ocean appear to be preferentially aerosolized. This discovery could help explain the increased presence of bacteria in certain environments and may shed light on possible therapeutics for diseases spread via aerosolization.

“The microbiological safety of fresh produce is monitored almost exclusively by culture-based detection methods. However, bacterial food-borne pathogens are known to enter a viable-but-nonculturable (VBNC) state in response to environmental stresses such as chlorine, which is commonly used for fresh produce decontamination. Here, complete VBNC induction of green fluorescent protein-tagged Listeria monocytogenes and Salmonella enterica serovar Thompson was achieved by exposure to 12 and 3 ppm chlorine, respectively. The pathogens were subjected to chlorine washing following incubation on spinach leaves. Culture data revealed that total viable L. monocytogenes and Salmonella Thompson populations became VBNC by 50 and 100 ppm chlorine, respectively, while enumeration by direct viable counting found that chlorine caused a <1-log reduction in viability. The pathogenicity of chlorine-induced VBNC L. monocytogenes and Salmonella Thompson was assessed by using Caenorhabditis elegans. Ingestion of VBNC pathogens by C. elegans resulted in a significant life span reduction (P = 0.0064 and P < 0.0001), and no significant difference between the life span reductions caused by the VBNC and culturable L. monocytogenes treatments was observed. L. monocytogenes was visualized beyond the nematode intestinal lumen, indicating resuscitation and cell invasion. These data emphasize the risk that VBNC food-borne pathogens could pose to public health should they continue to go undetected.” (Michaud et al 2018)

1.2. Main paper; discussion starts at 29:54 minutes

The Most Interesting Things (according to students)

Don’t eat prepackaged salads!  Dilute bleach treatment of foods can cause pathogens to enter a viable-but-not-culturable state that passes food safety tests, but can still cause disease.

“Ocean-derived, airborne microbes play important roles in Earth’s climate system and human health, yet little is known about factors controlling their transfer from the ocean to the atmosphere. Here, we study microbiomes of isolated sea spray aerosol (SSA) collected in a unique ocean–atmosphere facility and demonstrate taxon-specific aerosolization of bacteria and viruses. These trends are conserved within taxonomic orders and classes, and temporal variation in aerosolization is similarly shared by related taxa. We observe enhanced transfer into SSA of Actinobacteria, certain Gammaproteobacteria, and lipid-enveloped viruses; conversely, Flavobacteriia, some Alphaproteobacteria, and Caudovirales are generally under-represented in SSA. Viruses do not transfer to SSA as efficiently as bacteria. The enrichment of mycolic acid-coated Corynebacteriales and lipid-enveloped viruses (inferred from genomic comparisons) suggests that hydrophobic properties increase transport to the sea surface and SSA. Our results identify taxa relevant to atmospheric processes and a framework to further elucidate aerosolization mechanisms influencing microbial and viral transport pathways.” (Highmore et al 2018)

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

Snippet Main
Vision and Change Topics
  • Structure and Function (V&C_SF)
  • Microbial Ecology (V&C_ME)
  • Impact of Microorganisms (V&C_IM)
  • Structure and Function (V&C_SF)
  • Metabolic Pathways (V&C_MP)
  • Microbial Ecology (V&C_ME)
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 27 (ASM_27): The extent of microbial diversity is largely unknown, and exploration of this diversity is critical to understanding microbes and their role in the biosphere
  • Fundamental Statement 14 (ASM_14): Extrinsic factors, such as abiotic and biotic interactions in the environment, can impact survival and growth of microbes.
  • Fundamental Statement 18 (ASM_18): The regulation of gene expression is influenced by external and internal molecular cues and signals.
  • Fundamental Statement 22 (ASM_22): Most microbes interact with hosts in beneficial or neutral ways, with a minority having a detrimental impact on their host.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Define aerosolization, how it happens for microbes and viruses, and what may contribute to it.
  • Identify some key differences in the laboratory experiment setup and the environment it is mimicking.
 S L
  • Predict aerosolization of microbes and viruses.
 S H
  • Define viable, but not culturable (VBNC).
  • Identify common food pathogens.
  • Explain how green fluorescent protein can be used in microscopy to follow microbe location differences.
 M L
  • Assess the ability of different methods to detect VBNC, culturable, and dead pathogens.
  • Analyze culture and bacterial count data to support or refute induction of the VBNC state.
 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

  • Artificial Algal Blooms (9:00–11:30): Algal blooms, which are when the abundance of environmental algae increases in a particular area and become visible, were induced in the experimental set up with F2 media and metasilicate.
  • Epifluorescence Microscopy (9:00–11:30): This is a type of fluorescent microscopy.  Here the researchers used it for cell counts.
  • Metagenomic Sequencing Analysis (9:00–11:30): This is a method were total DNA is isolated from a sample, amplified using locus-specific primers, and the sequence determined to identify a bacterial profile present in the sample, here in water and aerosol.

4.2. Main Paper

  • C. elegans Lifespan Assay (36:00–36:50; 52:10–54:25): This is a survival assay involving pathogen-based killing of the nematode C. elegans which eats bacteria for nutrition.  It was used to see if C. elegans has lower lifespan after exposure to test foods containing different pathogen treatments.
  • Green Fluorescent Protein (GFP) (41:10–42:10): This is a protein from jellyfish that emits fluorescence all the time.  The fluorescence was used to quantify viable bacteria because GFP is being produced by the living bacteria on spinach and in C. elegans.
  • Viability Assay (41:00–44:00; 45:40): This is an assay to determine bacterial viability.  It was performed after exposure to chlorine stress, and presence of green fluorescent protein indicates viability.
  • Selective and Differential Media (49:20–50:35): Selective media are media that enable growth of only certain bacteria due to the stringency of nutrients while differntial media are media that differentiate bacterial types by some feature such as colony color.  This ensures only the organisms providing data are those being studied; also harsh environments that can maintain VBNC state.

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

5.1. Snippet Paper

  • Microbial Impact (4:25–6:30): Microbes aerosolize in the ocean sea spray, travel long distances, and have environmental impacts.
  • Disproportionate Rate of Microbial Aerosolization (7:30–7:50): Past studies on ocean conditions suggest specificity in bacterial aerosolization, (20:13 – 23:10) Data shows genomes in the same order tends to aerosolize at similar rates, (15:50–18:20) Cell surface (hydrophobicity) may influence aerosolization, Non-enveloped viruses are less enriched in aerosols than lipid-rich, (20:13–22:00) constitutive and random bacterial aerosolization.
  • Microbial Structure and Function (12:42–13:35): Alphaproteobacteria, Flavobacteria, and Gammaproteobacteria are found in sea spray aerosols, (15:30 – 15:50) Bacteriophages were the most common viruses in sea spray aerosols.

5.2. Main Paper

  • Food Safety (33:30–34:10; 34:50–36:00): Food safety testing protocols use culture-based methods to determine if food is safe for consumption, so VBNC contaminants would produce false negatives (i.e. test would say the food is not contaminated when it actually is). Past studies on the topic had been dismissed due to the perception that lab conditions are too dissimilar to the real world (Petri dish =/= chicken).
  • Viable but Nonculturable (33:30–35:50; 48:00–51:55): VBNC bacteria are alive but will not grow and form colonies; may be rescued and will grow when put into better conditions (in this case, C. elegans). (39:00–40:25) VBNC state is induced by environmental stress; Dilute chlorine induces VBNC state in Salmonella.
  • Biofilms (44:00–47:30; 51:00): Microbes form biofilms on food, can enhance chlorine tolerance (epiphytes).

6. Podcast Questions

  1. Aerosolization of oceanic bacteria and viruses occurs when ocean water ___________.
    1. evaporates and the water becomes very high in salinity affecting microbe survival.
    2. from waves is aerated through wave motion and also surface bubbles/foam popping.
    3. hits the sand surface and churns around diatom-derived particles of silicon dioxide.
    4. of higher temperatures displaces ocean water of lower temperatures creating eddies.
  2. The researchers used 13,000 liters of water in a experimental set up to mimic ocean movement and aerosolization. What factors do the podcasters note as being possible ways the experimental setup is different and might contribute to findings that are not entirely accurate for the ocean? Pick all that apply.
    1. The seeded microbes in the experimental setup are only a subset of the oceanic microbes and viruses.
    2. The viscosity of ocean water in nature was different from the ocean water in the experimental setup.
    3. The water in the experiment  is a modified salt solution, while the ocean water has more components.
    4. The experimental temperature is a set temperature that might influence selection of certain microbes.
  3. If a bacterium is in the same family as another bacterium that has a high aerosolization factor, what would you predict for its aerosolization and why?
    1. The same aerosolization because microbes in a family share cell surface properties.
    2. You wouldn’t be able to predict aerosolization factors because bacteria are all different.
    3. More aerosolization because most microbes are highly able to aerosolize in the ocean.
    4. Less aerosolization because most microbes are resistant to aerosolize in the ocean.
  4. What common food pathogens are discussed in the podcast?
    1. Norovirus
    2. Listeria
    3. Salmonella
    4. Hepatitis A
    5. Campylobacter
    6. E. coli
  5. What does viable but not culturable mean and is this state permanent or dynamic?
    1. It is permanent; it means that a bacterium is alive but is now unable to divide and will soon die out.
    2. It is dynamic, it means that a bacterium is in stasis and will recover when iron levels reach a minimum.
    3. It is permanent for that bacterium, but when it divides it recovers it ability to be cultured into colonies.
    4. It is dynamic; it means that a bacterium is alive, but is not actively dividing and unable to form colonies.
  6. The researchers were able to follow the division and location of bacteria being eaten by nematodes. What statement is true about this experiment?
    1. Green fluorescent protein bleaching was monitored as as loss of fluorescence when bacteria were ingested.
    2. Green fluorescent protein was expressed in the bacteria and ingestion visualized using fluorescent microscopy.
    3. Green fluorescent protein was produced in nematodes and bleaching visualized using phase-contast microscopy.
    4. Green fluorescent protein was tagged using antibodies and visualized by immunohistochemical techniques.
  7. Common ways to monitor bacteria are culture plating, optical density measurements over time to produce growth curves, and microscopic counts.  Which of the following results are consistent each method and bacterial state? [VC = viable and culturable ; VBNC = viable but not culturable ; D = dead bacterial cells]
    1. ______ colonies present on culture plates
    2. ______ colonies are not present on culture plates
    3. ______ optical density growth curve increases over time
    4. ______ optical density growth curve shows no change over time
    5. ______ phase-contrast microscopic images of bacterial cells
    6. ______ fluorescent microscopic images of GFP-producing bacterial cells
  8. You are testing the ability of three different compounds (A, B, and C) to induce the VBNC state in a GFP-tagged bacterium.  The following data are observed (Table).  Match the compound effect and the evidence.
Treatment Colony counts OD at 0 hours OD at 10 hours Phase contrast microscopy Fluorescent microscopy
Untreated 100 0.101 0.651 cells present cells are green
Compound A 95 0.099 0.588 cells present cells are green
Compound B 2 0.105 0.109 cells present cells are green
Compound C 3 0.103 0.103 cells present cells are not green
  1. induced VBNC because no growth is observed in colony counts or OD measures, but cells are green showing viability, but inability to be cultured.
  2. killed the bacterial cells because no growth is observed in colony counts or OD measures and cells are present, but not green showing inviability.
  3. did not induced VBNC because growth is observed in colony counts and OD measures and cells are green in fluorescent microscopy showing viability.
    1. _____ Compound A
    2. _____ Compound B
    3. _____ Compound C

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 3) and one from the main paper (Figure 6).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Explain the difference between the two aerosolization factors (A:B and A: S)
  • Analyze aerosolization heat map data to identify taxa with enhanced and reduced aerosolization.
  • Design a research plan to identify factors responsible for enhanced aerosolization.
  • Predict a bacterium’s transmissibility based on aerosolization
Experimental Background (Michaud et al., Figure 3)

The movements of water across the planet promote aerosolization of bacteria and viruses, which plays an important role in microbe and viral transmissions.  Michaud et al (2018) investigated this phenomenon over a period of 34 days by isolating DNA from bulk ocean water (called SSB), the upper layer of ocean water (called SSML), and aerosols (called SSA). They sequenced the DNA and identified its species origin and prevalence in all three types of water (SSB, SSML, and SSA).  By comparing the amount of each genome in the aerosol to either the bulk (SSA/SSB) or the aerosol to the upper layer (SSA/SSML), they were able to calculate an aerosolization factor, which they called AF.  To identify whether related bacteria or related viruses had similar aerosolization patterns, they next performed a phylogenetic analysis of the bacteria and viruses found in the study.  They then visualized the data as a combined phylogenetic tree and heatmap of the two AF ratios. The bacterial or viral genetic relationships are shown in the phylogenetic tree at the center of each figure while the heat map of AF ratios for days 0, 8, 19, 24, and 34 are color-coded surrounds the tree.  Red is more abundant in aerosol compared to bulk sea water (A:B) or aerosol compared up upper layer (A:S), while blue is less abundant in aerosol compared to bulk sea water (A:B) or aerosol compared up upper layer (A:S).  Yellow shows no difference.  The final outer layer of each figure indicates the taxon of the species noted in the tree.  The roman numerals identify outliers among the taxon/family, that is where the species does not behave like its relatives.

Figure on concentric circles

Figure 3. “The taxonomic basis of bacterial and viral aerosolization. Genome-based phylogenetic trees from read-based annotations trimmed of genomes with low spatial coverage: a 76 bacterial genomes and b 30 viral genomes. Aerosolization factors (AF), A:B and A:S, on different days are indicated (Blue = Diminished aerosolization; Yellow = Neutral aerosolization; Red = Enhanced aerosolization). Blanks indicate samples below threshold limits. a Bacteria class is indicated on outer ring to further indicate species and shading of species names indicate orders of interest. In b, the outer ring denotes viral family and shading indicates presence or lack of a viral envelope. i, ii, and iii indicate genomes with aerosolization patterns that differ from their closest relatives. Trees generated using phyloT (http://phylot.biobyte.de/) and iTOL (http://itol.embl.de/)”  (Michaud, et al 2018, no changes).

7.1.2. Questions

  1. If there was no difference in abundance for a particular bacterium between bulk sea water and aerosol, what aerosolization factor (AF) ratio and what color bar would you see in the figure for that species?
    1. A:B blue
    2. A:B yellow
    3. A:B red
    4. A:S blue
    5. A:S yellow
    6. A:S red
  2. If smaller amounts of a particular virus were found in aerosol compared to upper layer sea water, what AF ratio and color would you expect to see in the figure for that virus?
    1. A:S blue
    2. A:S yellow
    3. A:S red
    4. A:B blue
    5. A:B yellow
    6. A:B red
  3. If a bacterial species had an AF A:B value that showed dark red, what could you conclude about its aerosolization?
    1. Its aerosolization was enhanced compared to upper layer sea water
    2. Its aerosolization was reduced compared to upper layer sea water
    3. Its aerosolization was enhanced compared to bulk sea water
    4. Its aerosolization was reduced compared to bulk sea water
  4. Which bacterial taxa have an enhanced ability to be aerosolized from either bulk or upper layer sea water?
    1. Alphaproteobacter and Gammaproteobacter
    2. Betaproteobacter and Epsilonproteobacter
    3. Actinobacteria and Bacillus
    4. Cyanobacteria and Flavobacteria
    5. Fusobacteriia, Bacteriodia, and Cytpphagia
  5. Which bacterial taxon is consistently reduced in aerosols compared to either bulk or upper layer sea water?
    1. Alphaproteobacter
    2. Gammaproteobacter
    3. Actinobacter
    4. Flavobacteriia
    5. Fusobacteria
  6. Which Alphaproteobacter family is aerosolized more often than you would expect based on random transfer from bulk or upper layer sea water?
    1. Cornebacteriales
    2. Alteromonadales
    3. Pseudomonadales
    4. Rhizobiales
    5. Sphingomonadales
    6. Rhodobacterales
  7. Based on the patterns you see for aerosolization, how might you investigate bacterial factors that enhance aerosolization?
    1. Compare the protein synthesis machinery of the enhanced, no difference, and reduced aerosol species to identify factors common to each group, but different than the others.
    2. Compare the membranes of the enhanced, no difference, and reduced aerosol species to identify factors common to each group, but different than the others.
    3. Compare the preferred temperature ranges of the enhanced, no difference, and reduced aerosol species to identify factors common to each group, but different than the others.
    4. Compare the preferred salinity profiles of the enhanced, no difference, and reduced aerosol species to identify factors common to each group, but different than the others.
  8. If a bacterium acquired genes that increased aerosolization, what effect would you expect to see on its transmissibility?
    1. Decreased transmission
    2. Increase transmission
    3. No change in transmission

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify features of the viable, but not culturable (VBNC) state.
  • Describe how survival outcome data can be used to determine whether a treatment affects bacterial pathogenicity.
  • Predict whether a bacterial species has evolved to have a VBNC state based on survival data.
Experimental Background (Highmore et al., Figure 6)

Highmore et al (2018) were interested in studying a group of pathogens that are viable, but cannot be cultured, called Viable But Not Culturable (VBNC).  These are of particular importance to food safety where safety is determined by culturing microbes and counting the resulting colonies. Chlorine is typically used to decontaminate produce, so the authors tested the ability of bacteria to enter a viable, but non-culturable state when exposed to chlorine.  In this experiment, Listeria (green lines) or Salmonella (red lines) were treated with chlorine (broken lines) or control solvent (solid lines) and used to to infect nematodes.  A control of E. coli is shown by a black solid).  They monitored the survival of the nematodes over 25 days.

Figure 6 “Survival of C. elegans exposed to culturable (solid line) and VBNC (broken line) L. monocytogenes (green) and Salmonella Thompson (red). E. coli Op50 (black) was used as a nonpathogenic control.” (Highmore, et. al., 2018, no changes)

7.2.2. Questions

  1. If you were to place an environmental sample of a VBNC bacterium onto agar plates and grow them at 37°C, 25°C, and room temperature, what would you expect to see?
    1. Distinct bacterial colonies on the plate grown at room temperature
    2. A bacterial lawn on the plate grown at 37°C
    3. No growth on any plates regardless of temperature
    4. A bacterial lawn on the plates grown at 37°C and 25°C
    5. Distinct bacterial colonies on the plates grown at room temperature and 25°C
  2. If Listeria did not have enter a VBNC state when exposed to chlorine which data line would change?
    1. Green solid (Listeria, no Cl) would look like black solid (E. coli).
    2. Green dotted (Listeria+Cl) would look like green solid (Listeria, no Cl).
    3. Green dotted (Listeria+Cl) would look like black solid (E. coli)
    4. Green solid (Listeria, no Cl) would look like green dotted (Listeria+Cl)
  3. Which data in the figure show that Salmonella has a VBNC state?
    1. The red solid and red dotted lines are alike and survival is low.
    2. The red solid and red dotted lines are alike and survival is high.
    3. The green solid and red solid lines are alike and survival is low.
    4. The solid red line is far from the control (E. coli).
  4. If another bacterium was treated with an antibacterial compound (compound X) that did not induce a VBNC state, what survival results would you expect to see? Let’s say that solvent-treated survival results were graphed with solid purple lines and compound X-treated survival results were graphed with dotted purple lines.
    1. Purple solid (no X) would look like black solid (E. coli)
    2. Purple solid (no X) would look like green dotted (Listeria+Cl)
    3. Purple dotted (+ X) would look like purple solid (no X)
    4. Purple dotted (+ X) would look like black solid (E. coli)

8. Paper Information and Licensing

8.1. Snippet paper

  • Michaud JM, Thompson LR, Kaul D, Espinoza JL, Richter RA, Xu ZZ, Lee C, Pham KM, Beall CM, Malfatti F, Azam F, Knight R, Burkart MD, Dupont CL, Prather KA. 2018. Taxon-specific aerosolization of bacteria and viruses in an experimental ocean-atmosphere mesocosm. Nat Commun. 9: 2017. https://doi.org/10.1038/s41467-018-04409-z
  • This article is licensed for Creative Commons use using CC BY 4.0 International license, which allows re-use and adaptation with proper attribution and notation of any changes. See the article’s copyright information.

8.2. Main paper

License

Icon for the Creative Commons Attribution 4.0 International License

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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