Metabolic Pathways

TWiM #244: Chewing for Chicha

Podcast and Annotation Information
  • Annotation by Brian Tran, Jeremy Bullock, Santiago Jacob, Rebecca Seipelt-Thiemann, and Roger Greenwell
  • Podcast audio by TWiM: Listen to TWiM #244 Podcast
  • Podcast transcript by Otter.ai and edited by Laurel Thompson and Isabelle Norris: Access Podcast Transcripts
  • Papers Discussed:
    • Freire AL, Zapata S, Mosquera J, Mejia ML, Trueba G. 2016. Bacteria associated with human saliva are major microbial components of Ecuadorian indigenous beers (chicha). PeerJ.4:e1962. doi: 10.7717/peerj.1962.
    • Santos ACM, Fuga B, Esposito F, Cardoso B, Santos FF, Valiatti TB, Santos-Neto JF, Gales AC, Lincopan N, Silva RM, Gomes TAT. 2021. Unveiling the Virulent Genotype and Unusual Biochemical Behavior of Escherichia coli ST59. Appl Environ Microbiol. 87(16):e0074321. doi: 10.1128/AEM.00743-21

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 3:06 minutes

The Most Interesting Things (according to students)

  • One interesting point is that the ExPEC strain of E. coli discussed in this study breaks the traditional understanding of E. coli, as it lacks typical lactose fermentation traits yet remains highly pathogenic, emphasizing the need to look beyond standard biochemical tests for accurate pathogen identification.
  • Another interesting detail is the role of virulence genes within the “virulome,” which are key in enabling the strain to invade and cause disease outside the intestine, highlighting the complex genetic mechanisms that can make certain strains more harmful.
  • Advancements in sequencing DNA has allowed for the discovery of complex E. coli strains that are more aggressive as a contaminant. The next step is to continue to improve on the e coli testing techniques to help humans with complex E. coli infections in the future.

This article is not licensed for Creative Commons use; see article copyright information. Thus, the abstract and figures cannot be copied here.

1.2. Main paper; discussion starts at 20:56 minutes

The Most Interesting Things (according to students)

  • The most interesting part was how human-associated microbes from saliva, like Lactobacilli and Streptococcus, drive the fermentation of chicha, shaping its flavor and safety. This blend of culture and microbiology shows the unique role of microbes in traditional fermentation.
  • Another interesting point was the similarity to sake fermentation, where soybeans are coated with human saliva to provide amylase, breaking down starches and starting fermentation naturally.

“Indigenous beers (chicha) are part of the indigenous culture in Ecuador. The fermentation process of these beers probably relies on microorganisms from fermented substrates, environment and human microbiota. We analyzed the microbiota of artisanal beers (including a type of beer produced after chewing boiled cassava) using bacterial culture and 16S ribosomal RNA (rRNA) gene-based tag-encoded FLX amplicon pyrosequencing (bTEFAP). Surprisingly, we found that Streptococcus salivarius and Streptococcus mutans (part of the human oral microbiota) were among the most abundant bacteria in chewed cassava and in non- chewed cassava beers. We also demonstrated that S. salivarius and S. mutans (isolated from these beers) could proliferate in cassava mush. Lactobacillus sp. was predominantly present in most types of Ecuadorian chicha.” (Freire et al. 2016)

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

Snippet Main
Vision and Change Topics
  • Metabolic Pathways (V&C_MP)
  • Structure and Function (V&C_SF)
  • Impact of Microorganisms (V&C_IM)
  • Metabolic Pathways (V&C_MP)
  • Microbial Ecology (V&C_ME)
ASM Fundamental Statements
  • 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 13 (ASM_13): Intrinsic factors, such as genotype, metabolism, and cell structures, impact the survival and growth of microbes.
  • Fundamental Statement 28 (ASM_28): A minority of microbes are pathogens that can cause diseases and harm host organisms, society, and ecosystems.
  • Fundamental Statement 7 (ASM_7):The distinct structures and processes in microbes can be targets for interspecies competition, antimicrobial treatments, and host immunity.
  • Fundamental Statement 13 (ASM_13): Intrinsic factors, such as genotype, metabolism, and cell structures, impact the survival and growth of microbes.
  • Fundamental Statement 20 (ASM_20): Microbes are ubiquitous, found in diverse and dynamic ecosystems, where they use available resources and often form complex communities.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Define chicha and the major species present in it.
  • Recall how specific procedures contribute to chicha fermentation.
 S L
  • Predict how changes in the fermentation process reduce or increase health risks of consuming chicha.
 S H
  • List the biochemical characteristics that differentiate the EC141 strain of E. coli from other common strains.
  • Describe the role of biochemical characteristics in bacterial detection methods.
 M L
  • Justify why finding strains with differences in biochemical characteristics has significant implications for public health.
 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 Ribosomal DNA Sequencing Analysis (3:06–3:45; 8:36–9:00): This molecular technique is used to identify bacterial species by comparing the genetic sequence of  the 16S ribosomal RNA gene to a database of known 16S rDNA sequences.  The Trueba lab used this technique to analyze the bacterial composition in chicha by sequencing genetic material from natural samples.
  • Natural Fermentation (4:25–6:45): To create chicha, a traditional fermentation process is used where grains are chewed by the maker to introduce saliva, which contains amylase, an enzyme that helps break down starches. This initiates fermentation by adding natural fermenting organisms from the mouth as well as from the environment.
  • Microbiome Analysis (11:49–12:41): The study includes analysis of the microbiome in both chewed and mashed versions of chicha, examining the microbial communities that make up each.

4.2. Main Paper

  • Chromogenic Agar (27:20–27:35): Chromogenic agar was used in the study to observe an unusual colony color (light cream), which was atypical for E. coli. This medium allows for visual differentiation of bacteria based on metabolic activity.
  • Aquateste/Colilert (34:50–37:15): Aquateste, also known as Colilert, which is an industry standard test, is used to detect E. coli.  This tests for beta-glucuronidase and beta-galactosidase activity, enzymes typical of E. coli.
  • Whole Genome Sequencing (37:10–38:05): Whole genome sequencing is sequencing used to determine the bases of the entire genome of a species.  Here, it was performed to confirm that the strain, despite lacking typical E. coli characteristics, was indeed E. coli.
  • Antibiotic Resistance Screening (43:15–44:05): There are a number of ways to screen for antibiotic resistance, including Kirby-Bauer plate assay and looking for genetic antibiotic resistant markers.  Here the researchers used the genetic approach to identify antibiotic resistance genes.  They identified a pmrB mutation, which conferred resistance to colistin, a last-resort antibiotic for severe infections.

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

5.1. Snippet Paper

  • Microbial Ecology of Alcoholic Fermentation (07:07–09:15): The unique microbial composition of chicha is primarily composed of Lactobacilli, which initiates and controls the fermentation process. This microbial ecology not only shapes the flavor but also enhances the drink’s safety by preventing spoilage. Additionally, Streptococcus species from saliva are present, illustrating a diverse microbial environment.
  • Biofilm Formation (11:49–12:40): The podcast speculates on how bacteria in fermentation containers contribute to biofilm formation, potentially affecting the drink’s microbial makeup.

5.2. Main Paper

  • Bacterial Classification (20:56–21:45): Escherichia coli (E. coli) is described as a gram-negative, facultative anaerobic bacterium, meaning it can grow in both oxygen-rich and oxygen-poor environments. This characteristic allows E. coli to survive in various niches, such as the gut and blood, underscoring its adaptability and relevance as both a commensal and pathogenic organism.
  • Coliforms (22:05–23:10): Coliforms, including E. coli, are used as indicators of water and food sanitation because they are naturally present in feces of warm-blooded animals. The discussion on coliforms illustrates their importance in environmental microbiology, particularly for monitoring public health, by identifying potential fecal contamination in drinking water sources.
  • Enteric Pathogenic Strains of E. coli (24:05–25:30): Different E. coli pathotypes, such as Enteropathogenic (EPEC), Enterotoxigenic (ETEC), and Enterohemorrhagic (EHEC), are categorized based on their mechanisms of infection. ExPEC (Extraintestinal Pathogenic E. coli) is introduced as a strain that causes disease outside the intestine, emphasizing its significance in healthcare as a pathogen capable of causing systemic infections.
  • Virulome (27:35–31:10): The concept of a “virulome” is introduced to discuss how certain genetic factors (e.g., chuA, fyuA, yfcV) contribute to E. coli´s pathogenic potential.
  • Antibiotic Resistance (45:00–46:30): The study examines antibiotic resistance in E. coli, specifically the presence of the pmrB mutation that confers resistance to colistin.

6. Podcast Questions

  1. What is chicha and which microorganism is the major contributor to its fermentation?
    1. A highly alcoholic Peruvian drink fermented using ancient red wheat grains; Clostridium
    2. A Chilean drink with psychedelic properties fermented from mushrooms; Salmonella
    3. A Nicaraguan drink made by fermenting fruit pulp with aged honey; Staphylococcus
    4. A low alcoholic Ecuadorian drink fermented using corn grains and saliva; Lactobacillus
  2. Why is the practice of chewing the grains important in chicha preparation?
    1. The saliva contributes proteases, which break down proteins necessary for respiration.
    2. The saliva contributes lipases, which allow fats to be distributed for a smooth texture.
    3. The saliva contributes amylase, which breaks down starches into sugars for fermentation.
    4. The saliva contributes cellulases, which aid in releasing the endosperm of grain kernels.
  3. If a particular batch of chicha had an unsuccessful fermentation, what would be the health risk and why?
    1. The lowered pH due to fermentation would not be present to inhibit growth of harmful bacteria.
    2. The high alcohol content normally consumed in fermentation would cause alcohol poisoning.
    3. The chemicals normally neutralized by the alcohol produced by fermentation would still be present.
    4. The bacteria present on the raw ingredients do not become balanced out by the fermentation.
  4. Which of the following is a distinguishing biochemical characteristic of the ST59  strain of E. coli (EC141) compared to typical E. coli?
    1. Lacks beta-galactosidase and beta-glucuronidase enzymes.
    2. Produces beta-galactosidase in response to oxidative stress.
    3. Has an unusually high ability to ferment lactose anaerobically.
    4. Has no antibiotic resistance gene clusters and no plasmids.
  5. How do biochemical characteristics aid in detecting microbes of public health concern?
    1. These tests are used to characterize flagella and other motility involved in disease spread.
    2. These tests involve using genomic sequencing to detect family level bacterial taxonomies.
    3. These tests involve using chromogenic plates to quickly monitor the presence of bacteria.
    4. These tests are used to determine when atmospheric oxygen concentrations are too low.
  6. Which of the following is a limitation of using only biochemical tests for detecting E. coli strains? What implication does this have for public health?
    1. They are more costly and time-intensive; the tests are not performed as often as they should be.
    2. They do not detect strains lacking classic markers; atypical virulent strains may escape detection.
    3. They are not as accurate as fluorescent microscopy; the false positives affect public confidence.
    4. They do not detect antibiotic resistance or virulence; highly virulent strains may escape detection.

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 1).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to:

  • Identify key experimental design features, such as the dependent and independent variables in the experiment and method choice.
  • Calculate the number of living bacteria in an aliquot and/or total volume of the culture at a particular time.
  • Analyze the data to make conclusions about bacterial growth.
  • Predict the safety of beverages fermented for different lengths of time.
Experimental Background (Friere et al., Figure 1)

Fermentation products have been produced by humans longer than animals have been domesticated. One of these fermentation products is a slightly alcoholic, fermented drink called chicha, that originated in South America. This drink, an indigenous beer, is produced by human chewing of corn or particular fruits that are subsequently fermented. To characterize the fermentation process and its constituents, Friere et al. (2016) first identified the species present in the fermentation of different chicha drinks using 16S rDNA sequencing.  In addition to many environmental-source species, they found two bacteria common in the human oral microbiome that are also pathogenic to humans, Streptococcus salivarius and S. mutans.  Knowing these bacteria were pathogens, that chicha fermentation has existed for a long time without significant outbreak infections, and that fermentation produces an antimicrobial environment, the researchers wanted to investigate the dynamics of bacterial growth of these pathogens during the typical chicha fermentation process.  To do this, they cultured  S. salivarius in sterile chewed cassava solution over a 72-hour period, with living bacteria in the culture being quantified as colony forming units (CFU) at 0, 24, 48 and 72 hours.

Growth graph. At 24 hours, bacterial count is much higher than at any other point.
Figure 1. “Growth of S. salivarius in sterile chewed cassava solution. There is a significant increase in CFU (Mann-Whitney U test) at the 24 h of incubation compared with those at inoculation time (0 h).” (Friere et al. 2016, no changes)

7.1.2. Questions

  1. In this experiment the dependent variable is _______ and the independent variable is _______ .
    1. CFU/mL; species in culture
    2. Time in culture; CFU/mL
    3. species in culture; CFU/mL
    4. CFU/mL; time in culture
  2. Colony forming units per milliliter (CFU/mL) is a measure of bacterial growth. The number of colonies is noted on the y-axis. If we were to take 100 microliters of the culture at 72 hours, where there are about 104/mL, and put it all on a plate, how colonies would we expect to see?
    1. 10,000
    2. 1,000
    3. 100
    4. 10
  3. If the culture volume was one liter (1L), how many bacteria would be present in the entire culture volume at 24 hours, where the CFU/mL is about 9 x 109?
    1. 9 × 1012 
    2. 9 × 106
    3. 9 × 1010 
    4. 9 × 1036
  4. Bacterial growth is often measured by either light spectroscopy (OD600) or colony-forming units (CFU).  What is the benefit of quantifying growth using CFU/mL over OD600?
    1. CFU is independent of the chemical composition of the growth medium; OD is dependent.
    2. CFU quantifies the metabolic rate of bacteria; OD quantifies the respiration rate of bacteria.
    3. CFU quantifies the number of viable bacteria; OD quantifies the number of all bacteria.
    4. CFU quantifies the number of viable bacteria; OD quantifies the number of viable fungi.
  5. At what time point did Streptococcus salivarius show the peak cell number? What is your evidence?
    1. 24 hours; this timepoint has the smallest error bars.
    2. 24 hours; this timepoint has the highest CFU/mL.
    3. 48 hours; this timepoint shows the most change.
    4. 72 hours; this timepoint has the longest growth.
  6. Which statement best describes the pattern of growth for S. salivarius over the 72 hour fermentation?
    1. Growth increased from 0 to 24 hours, then decreased.
    2. Growth remained constant until 48 hours then decreased.
    3. Growth increased for the entire experimental culture time.
    4. Growth lagged from 0 to 24 hours then increased steadily.
  7. Based on the growth of S. salivarius, which fermentation time should be the safest for consuming traditionally-fermented chicha? What is your evidence?
    1. 0 hours; this is the least fermented, least alcoholic chicha.
    2. 24 hours; this has the most number of beneficial bacteria.
    3. 48 hours; this has the largest decrease in living pathogens.
    4. 72 hours; this has the smallest number of living pathogens.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to: 

  • Identify experimental design aspect of this experiment, including positive and negative controls and use of specific methods.
  • Evaluate the data and identify visual results that represent the statement given.
  • Hypothesize the implications of the results for diagnostic test efficacy and also public health.
Experimental Background (Santos et al., Figure 1)

Escherichia coli is a gram-negative rod that has strains that are non-pathogenic as well as highly pathogenic.  One pathogenic strain group for this species is extraintestinal pathogenic E. coli (ExPEC), which causes serious gastrointestinal infections worldwide and is commonly found in water, sediment, food, and the gut.  Being able to accurately detect pathogenic E. coli is therefore a serious health issue.  Current detection methods are based on E.coli’s typical characteristics, such as expression of beta-galactosidase and beta-glucuronidase enzymes. In this study Santos et al. (2021) report the characterization of an unusual ExPEC E. coli strain, EC141 which was cultured from blood.  To begin their characterization they utilize two different types of chromogenic media typical in E. coli detection: 1) CHROMagar Orientation medium, which uses colony color to distinguish a number of different bacteria* (panels A, B) and 2) ONPG-MUG broth (Aquateste), which detects beta-glucuronidase activity via production of a fluorescent product (panel C) and beta-galactosidase activity via production of a yellow color product (panel D). CHROMagar Orientation results are shown for the new EC141 strain (panel A), as well as a typical E. coli strain (ATCC 25922; panel B, bottom of plate) and another water-based, gram-negative, pathogenic rod-shaped species, Aeromonas hydrophila (panel B, top of plate). ONPG-MUG broth results are shown for these same three bacteria: new strain EC141 (panel C and D, row A), E. coli ATCC 25922 (panels C and D; row C), and A. hydrophila (panels C and D, row B).

*Please see the product manual for a full description of expected colony and halo colors: https://www.chromagar.com/wp-content/uploads/2021/11/NT-EXT-002-V13.1.pdf ).

7.2.2. Questions

  1. In this experiment the negative control is _______ and the positive control is _______ .
    1. CHROMagar orientation; ONPG-MUG broth
    2. Aeromonas hydrophila; E. coli ATCC 25922
    3. ONPG-MUG broth; CHROMagar orientation
    4. E. coli ATCC 25922; Aeromonas hydrophila
  2. What is the primary use of ONPG-MUG broth (Aquateste) in this analysis?
    1. To determine the pH level as the bacterial cultures move through log phase.
    2. To detect the activity of beta-galactosidase and beta-glucuronidase enzymes.
    3. To quantify the cell doubling rate of the bacteria under anaerobic conditions.
    4. To identify the presence or absence of distinct antibiotic resistance genes.
  3.  E. coli ATCC 25922 is positive for beta-galactosidase and beta-glucuronidase, but EC141 is negative for both. What results in the figure indicate this?
    1. E. coli ATCC 25922 has fluorescent and yellow products in ONPG-MUG broth, but EC141 has neither fluorescent nor yellow products.
    2. E. coli ATCC 25922 produces fluorescent and blue products in ONPG-MUG broth; EC141 produces non-fluorescent and pink products.
    3. E. coli ATCC 25922 produces non-fluorescent and pink products in ONPG-MUG broth; EC141 produces fluorescent and yellow products.
    4. E. coli ATCC 25922 produces non-fluorescent and white products in ONPG-MUG broth; EC141 produces non-fluorescent and pink products.
  4. Why would the biochemical properties of E. coli EC141 lead to diagnostic challenges?
    1. It is highly antibiotic-resistant and would not be conventionally treatable.
    2. Its atypical enzyme activity would result in species misidentification.
    3. It does not grow on standard media, so it would escape detection.
    4. It produces atypical colonies, so would be considered contamination.
  5. What are the potential public health consequences of the results of this experiment?
    1. Increased resistance to a number of different antibiotics in general populations.
    2. These results suggest that we need fewer diagnostic tests in clinical practice.
    3. Failure to detect and report pathogenic strains, impacting outbreak management.
    4. Inaccurate diagnosis of E. coli infections leading to antibiotic overuse in patients.

8. Paper Information and Licensing

8.1. Snippet paper

  • Freire AL, Zapata S, Mosquera J, Mejia ML, Trueba G. 2016. Bacteria associated with human saliva are major microbial components of Ecuadorian indigenous beers (chicha). PeerJ.4:e1962. doi: 10.7717/peerj.1962.
  • 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://peerj.com/articles/1962/

8.2. Main paper

  • Santos ACM, Fuga B, Esposito F, Cardoso B, Santos FF, Valiatti TB, Santos-Neto JF, Gales AC, Lincopan N, Silva RM, Gomes TAT. 2021. Unveiling the Virulent Genotype and Unusual Biochemical Behavior of Escherichia coli ST59. Appl Environ Microbiol. 87(16):e0074321. doi: 10.1128/AEM.00743-21
  • This article is not licensed for Creative Commons use; see the article’s copyright information. Thus, the abstract and figures cannot be copied here.

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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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