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1. Alavi S, Mitchell JD, Cho JY, Liu R, Macbeth JC, Hsiao A. Interpersonal Gut Microbiome Variation Drives Susceptibility and Resistance to Cholera Infection. Cell. 2020;181(7):1533-1546.e13. https://www.cell.com/cell/fulltext/S0092-8674(20)30631-0?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867420306310%3Fshowall%3Dtrue. doi:https://doi.org/10.1016/j.cell.2020.05.036

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3. Correll R. Symptoms of Cholera. Verywell Health. 2018 Mar 26. https://www.verywellhealth.com/cholera-signs-symptoms-and-complications-4160841

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5. Yoon S hun, Waters CM. Vibrio cholerae. Trends in microbiology. 2019;27(9):806–807. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713289/. doi:https://doi.org/10.1016/j.tim.2019.03.005

6. Zhao W, Caro F, Robins W, Mekalanos JJ. Antagonism toward the intestinal microbiota and its effect onVibrio choleraevirulence. Science. 2018 [accessed 2021 Feb 3];359(6372):210–213. doi:https://doi.org/10.1126/science.aap8775

7. Song Z, Cai Y, Lao X, Wang X, Lin X, Cui Y, Kalavagunta PK, Liao J, Jin L, Shang J, et al. Taxonomic profiling and populational patterns of bacterial bile salt hydrolase (BSH) genes based on worldwide human gut microbiome. Microbiome. 2019;7(1). doi:https://doi.org/10.1186/s40168-019-0628-3

8. Abdullahi Yusuf Muhammad, Amonov M, Chandrika Murugaiah, Atif Amin Baig, Yusoff M. Intestinal colonization against Vibrio cholerae: host and microbial resistance mechanisms. AIMS Microbiology. 2023 [accessed 2023 Jun 7];9(2):346–374. doi:https://doi.org/10.3934/microbiol.2023019

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11. Lawson PA, Finegold SM. Reclassification of Ruminococcus obeum as Blautia obeum comb. nov. International Journal of Systematic and Evolutionary Microbiology. 2015 [accessed 2022 Feb 28];65(Pt_3):789–793. doi:https://doi.org/10.1099/ijs.0.000015

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a mixed CR + DS group demonstrates that CR microbes have a dominant effect and colonization resistance can be restored through microbiome modification

the degree of pathogen colonization resistance and infection outcome is highly dependent on the individual

B. obeum abundance and bsh activity is associated with increased V. cholerae colonization resistance

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bsh levels in human gut microbial communities are correlated to V. cholerae infection outcome 

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bsh levels in human gut microbial communities are correlated to V. cholerae infection outcome 

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Finally, the authors sought to determine the distribution of the bsh phylotypes in healthy and dysbiotic human gut microbiomes

Method: analyzed a previously published shotgun sequencing metagenomic dataset of Bangladeshi cholera patient gut microbiomes

Results:

bsh levels were already affected by diarrhea prior to any treatment (antibiotics)

depleted phylotypes 1, 3, 4, 5

phylotypes highly active against TC: 1, 3, 4

So, a characteristic of healthy human microbiomes that may affect v. cholerae susceptibility is their ability to deconjugate TC into a non-virulence-inducing form (like CA)

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Next, the authors examined genetic determinants of bile acid processing to determine the molecular basis for B. obeum’s effect on TC-dependent virulence induction

Results:

a previous study classified bsh enzymes into different phylotypes based on sequence similarity, showing that different types have various substrate-dependent deconjugation actions7

B. obeum is predicted to encode for bsh type 1 enzymes, which are the most effective at deconjugating TC and other potent activators of tcpA expression in V. cholerae

Additionally, most CR microbes encode for bsh homologs while DS microbes lack bsh genes

B. obeum has such a dominant effect on Vibrio resistance that pure cultures of the strain or its bsh enzyme can overpower the effect of DS microbes like S. salivarius and reduce both tcp activation by TC and TC levels directly

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Weaknesses:

Discrepancies between U.S. and Bangladesh samples

U.S. cohort had extensive qualifications (thorough inclusion/exclusion criteria)

e.g. participants could not have active GI disorders, systematic antibiotic use, acute illness, etc.¹

Bangladesh sample was from pre-sequenced data from a 2014 study ⁹

reduced experimental manipulations for cholera-endemic regions may cause other confounding variables to be overlooked (e.g. additional health concerns, genetic predispositions), providing an inaccurate representation of the population

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Locations of samples from Alavi et al. study: Riverside, California (left) and Bangladesh (right). Image Source: Sara Sampaio, created using Google Slides

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Enough distance is maintained to ensure interpersonal variation in microbiome and diet, but not so drastically that external factors become significant confounding variables!

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Redesign experiment to analyze samples from more geographically-similar places

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Redesign experiment to analyze samples from more geographically-similar places

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For example,

Compare the US cohort to one from the Dominican Republic, a country with a (recent) history of cholera outbreaks²

Compare Bangladesh citizens to a non-endemic country in South-East Asia like Singapore

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Concept has not been well studied and becomes a confounding variable for Alavi et al.

While interpersonal variation is the focus of this paper, the distance and therefore dramatic differences between the lifestyles of each cohort must be taken into consideration

Factors such as climate, agriculture, medical practices, political regimes, and cultural differences all contribute to the composition of the microbiome

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the most common bile compound

conjugated to the amino acid taurine

can induce tcp gene expression - induces virulence

modulates upstream virulence activator in V. cholerae TcpP

activates P tcpA

de-conjugated compound is cholic acid (CA)

neutral - doesn't activate P tcpA

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Introduction and Cholera Background Information

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Introduction and Cholera Background Information

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Studies have transferred R. obeum to the genus Blautia, meaning these strains are virtually the same in function¹¹

Moreover, the study used samples from Bangladesh children

Children's microbiomes are neither fully developed nor stable, meaning the findings of this paper may not be fully indicative of infection outcomes in the general Bangladeshi public

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heat resistant

spend a long time in the SI - where B. obeum is!

human bile acids are commonly conjugated to amino acids like taurine and glycine

these conjugated acids like Taurocholate (TC) can induce tcp virulence gene expression

commensal microbes encode for bile salt hydrolase (bsh) enzymes which are critical for bile acid metabolism

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Members of the human gut microbiota involved in recovery from Vibrio cholerae infection (2014)

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Members of the human gut microbiota involved in recovery from Vibrio cholerae infection (2014)

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Using similar methods, the authors recognized a pattern of taxa in recovering cholera patients⁹

Identified R. obeum as a primary candidate for virulence suppression and colonization resistance instead of B. obeum

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Investigate quorum sensing systems that modulate TcpP activity

These pathways disrupt the expression of virulence factors that encode for TCP and are hypothesized to involve AI-2⁹

Determine whether AI-2 and B. obeum synergize to mediate colonization in vivo (AI-2 studies were mimicked in vivo using ex vivo studies)

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This was from an isolated study to determine the effects of B. obeum on V. cholerae

A 1 log drop in colonization with B. obeum vs 2 log-fold difference in the colonization of CR, DS microbiome suggests that additive effects are at play¹

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B. obeum was sufficient to ablate tcpA expression without AI-2

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B. obeum was sufficient to ablate tcpA expression without AI-2

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Are there additive effects on pathogenesis?

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Are there additive effects on pathogenesis?

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No significant additive effects were observed on V. cholerae abundance when B. vulgatus or C. scindens were added to pure cultures containing B. obeum

AI-2 could play a role in suppressing colonization via downregulation of TCP expression (↓ TCP biogenesis)

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Alavi et al then used cultured human isolates to create two defined gut communities:

1. CR (the "healthy state") – characterized by a high taxonomic diversity, consists of Bacteroides, Clostridium, and Blautia.

2. DS (the "dysbiotic state") – characterized by a low taxonomic diversity, consists of Streptococci, E.coli, Enterococcus faecalis.

These cultures were administered into germ-free mice.

Alavi et al observed the mice receiving CR cultures were resistant to V. cholerae colonization in the small intestine.

They also made a third “mixed group” that involved the administration of a 1:1 mixture of CR and DS cultures into germ-free mice.

This mixed group experienced lower V. cholerae levels than the DS group, and dropped even lower than the CR group two days after infection.

These two experiments highlighted the significance of interspecies and inter-individual microbiome variation in pathogen susceptibility.

The mixed group demonstrates that colonization resistance can be restored through microbiome modification.

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Bernice Liu: Major Findings (minus "Dysbiotic Microbiomes in response to V. cholerae", and "Microbiome Modification restores Colonization Resistance").

Rachel Igbokwe: Introduction and Cholera Background Information, Major Findings (only "Dysbiotic Microbiomes in response to V. cholerae", and "Microbiome Modification restores Colonization Resistance").

Sara Sampaio: Article Critiques and Future Directions

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Dysbiotic Microbiomes in response to V. cholerae

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Dysbiotic Microbiomes in response to V. cholerae

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Alavi et al. studied microbiome variation on pathogen resistance by creating two methods through reconstituting the human gut microbiome into V. cholerae animal models:

1. Complete human fecal microbiomes.

2. Constructed defined gut communities using cultured human isolates.

The use of cultured microbes is the big difference between these two methods --> ensures that the microbes’ effect was factual and takes the natural context into consideration.

Complete fecal microbiomes from healthy American adults (contol) and previously published Bangladeshi (disease) samples were compared.

Alavi et al noticed that their microbial compositions were distinct, in which the Bangladeshi disease state was highly dysbiotic, had low-diversity microbial content, and dominated by the Streptococci strain

Even the microbiomes of healthy Bangladeshi adults had a microbial community composition similar to cholera-infected communities.

Diarrhea, dehydration, and malnutrition are common in developing countries, and may also push one’s microbiome to a dysbiotic state.

Therefore, they hypothesized that the distinct microbiome dysbiosis during diarrhea recovery may be a recurring window of vulnerability to cholera.

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Interaction of the gut microbiome with environmental signalling during V. cholerae life cycle. Angel Hsiao and Jun Zhu, Nature, 2014 

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Cholera Toxin Mode of Action, Ryan Abbott (2017)

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After the identification of B. obeum as a potential driver of V. cholerae colonization resistance, the authors strove to determine the molecular mechanisms underlying its action

Previous studies identified that the AI-2 autoinducer produced by B. obeum downregulates the expression of TCP biogenesis genes during infection, and cues from both host and commensal species can regulate V. cholerae gene expression in vivo

With this in mind, the authors looked to see if there was a connection between the molecular mechanism and B. obeum-induced colonization resistance

Method: mimic in vivo conditions for virulence gene regulation via anaerobic incubation of suckling mice intestinal tissue with a V. cholerae resistance reporter ex vivo

Result: ablated tcpA expression in samples containing B. obeum

However!

A control experiment involving boiling samples of B. obeum-incubated to remove the heat-labile AI-2 still remained unable to induce tcpA --> there must be another heat-resistant mechanism at play here

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Fig 6C: Effects of CR and DS pure cultures on TC activation of virulence in vitro 

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Since TC is the most common bile acid and has several relevant characteristics regarding V. cholerae virulence, the authors wanted to see the effect CR and DS communities had on it

Method: measured each species' ability to induce PtcpA by incubating TC solutions with pure CR and DS cultures

Results: CR (and SR) species were better at preventing TCP gene activation because they efficiently processed TC to CA, maintaining a low TC:CA ratio

B. obeum was found to be a particularly important species involved in this processing

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Interpersonal Variation - the microbiome is not black and white

The authors emphasize differences among and within host microbiomes that can dictate infection outcomes

Avoids using a dichotomous lens on the microbiome, i.e. "healthy" vs. "unhealthy", treated with antibiotics or untreated, etc. that is common in the field

These novel perspectives demonstrate the clinical applications of host uniqueness, and better the journey towards personalized medicine

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Previously published scientific papers have studied the gut microbiome in response to V. cholerae infection. Other papers also show how the gut microbiome differs in people with Cholera versus those that don’t experience cholera symptoms.¹

But the effect of interpersonal microbiome variation on pathogen resistance and susceptibility has yet to be explored.

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To tackle this gap in knowledge, Alavi et al used different methods and models to assess how microbial diversity affects resistance or susceptibility to intestinal pathogens, with its main focus on V. cholerae.

The results from these experiments prove that a specific microbial species shape the gut’s environment via bile salt hydrolase to reduce Cholera infection risk.

To consider individualized microbiome variation, Alavi et al steered away from a “dichotomous” comparison of a “normal” versus “dysbiotic” microbiome.

These findings can aid in creating Cholera prevention strategies that are unique to the individual.

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Cycle of Malnutrition - Khan Academy Medicine (2013)

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What does B. obeum do?

its presence statistically reduces V. cholerae colonization, but doesn't reduce the amount of the pathogen present

these effects are dominant and generalizable across various gut microbiomes

How does B. obeum do this?

it depletes virulence-activating signals by producing AI-2, which downregulates TCP

it produces a bile salt hydrolase (bsh) enzyme that degrades virulence-activating signals in the gut

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Targeted Microbiome Modification

When the authors mixed CR and DS communities, resistance colonization was restored

Even in severely dysbiotic microbiomes, administration of SR microbial species rescued most inhibitory functions¹

The results suggest that modifying the microbiome could be a promising approach to preventing cholera colonization or alleviating associated symptoms, as opposed to common antibiotic treatments for relapsing patients

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Why randomization?

fecal sampling is the most obvious, but the authors found that it masks critical features of the species that could affect disease pathogenesis

importantly, the species found in the small intestine (SI) and feces are different for CR microbes

How they did it:

take five species randomly from both CR and DS strains

inject into suckling mice (either affected or unaffected with cholera)

see which species correlate the most against V. cholerae infection via a combinatorial strategy

identify good targets for intervention

Findings:

the only CR species that consistently correlated against V. cholerae infection was Blautia obeum (B. obeum)

all DS microbes correlated positively with V. cholerae levels (Fig 5D)

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Now that we've laid out what's been done before and which overall directions we need to go in, the next step is to identify commensal microbe species that are correlated with V. cholerae infection

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Malnutrition and Dysbiosis

The microbiomes of the Bangladesh cohort showed limited diversity and significant dysbiosis

Besides cholera, this could be due to malnutrition, a condition common in cholera-endemic regions¹

Individuals are predisposed to heightened vulnerability, emphasizing that dysbiotic microbiomes increase susceptibility to infection

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Virulence during early infection

Cholera patients first experience diarrhea, which rids the intestine of crucial commensal species and bile metabolites

Subsequent bile production will result in increased TC levels, which influence virulence8

Identification of B. obeum as a key antagonist of colonization emphasizes its required presence before V. cholerae can diminish bsh activity

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Intestinal colonization against Vibrio cholerae: host and microbial resistance mechanisms (2023)

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Intestinal colonization against Vibrio cholerae: host and microbial resistance mechanisms (2023)

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“The observations have shown that V. cholerae is highly tolerant of conjugated bile toxicity and that in the presence of conjugated bile acids and its amino acid component, the pathogen activates several virulence genes.”

Similarly, Muhammad et al. (2023) concluded that V. cholerae pathogenesis is mediated by bile acids

They propose that virulence is induced by lowering the permeability of cell membranes to bile salts ⁸

In recovering cholera patients, autoinducer-2 (AI-2) is also noted to prevent further colonization by V. cholerae

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microbiome variation and infection resistance

the suckling mouse model

microbe randomization pipeline

intestinal signals and virulence gene activation

B. obeum bile salt hydrolase

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Bile Salt Hydrolases: At the Crossroads of Microbiota and Human Health (2021)

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Bile Salt Hydrolases: At the Crossroads of Microbiota and Human Health (2021)

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Patients with inflammatory bowel disease (IBD) and type 2 diabetes showed reduced bsh enzyme expression

Patients with relapsing C. difficile infection (CDI) had increased taurocholic acid (TCA) levels

Consistent with Alavi et al. (2020): reduced TC levels were associated with "healthy" human microbiomes, whereas high TC levels induced virulence of V. cholerae

These patients also exhibited decreased BSH-producing species ¹⁰

Alavi et al. (2020): B. obeum (encodes for bile salt hydrolases) abundance was also reduced in colonized microbiomes

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Cholera requires a coordinated regulation of environmental fit genes and V.cholerae virulence genes – which includes a toxin-coregulated pilus (TCP) and cholera toxin (CT).¹

TCP helps in the adherence and colony formation of V.cholerae onto the intestine.⁵

Once V. cholerae is attached, CT release soon follows and enters the intestinal epithelial cells.

This toxin release aids in the V. cholerae colonization, and the pathogenesis of Cholera.

A thorough description and visualization of cholera toxin mode of action is provided in the attached YouTube video.

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People suffering from Cholera typically experience watery diarrhea and dehydration, but symptoms may become severe if not treated.³

Severe symptoms include frequent vomiting, low blood pressure, and muscle cramping.

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Incidence of Cholera cases (including estimated cases of acute watery diarrhea (AWD)) per 100,000 population reported to WHO from 1 January to 30 November 2022.

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Howard L., Vibrio cholerae under microscope, 2023

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Vibrio Cholerae, or V. cholerae, is a gram-negative bacteria that resides in aquatic reservoirs, and thrives in brackish, salt, and fresh waters.⁴

V. cholerae gets introduced into the body through drinking water or consuming fish and seafood.

As mentioned before, Cholera is a notable health concern in developing countries experiencing contaminated drinking water systems, poor sanitation, and reduced access to healthcare.³

V. cholerae is abundant is such contaminated regions, which often results in annual Cholera outbreaks.

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Mouse microbes illustration. Kelly Servick, Science, 2016 

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Strengths:

1. Both male and female subjects used

prevents the risk of generalizing sex-related differences

2. Compared fecal and gastrointestinal (GI) samples

fecal sampling can mask proximal GI tract differences for pathogenesis¹; including both ensures specificity

3. Implemented human microbiota in murine samples

demonstrated that microbial variation (between and within) hosts influences pathogen susceptibility

4. Practised in vivo and in vitro

certain host cues are only observed in V. cholerae gene expression in vivo

5. Multi-omics approach

allows for taxa identification (B. obeum) in animal models

6. Randomized trials

correlations between species abundance and V. cholerae colonization in random microbiome combinations increase reliability¹

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Cholera is a widespread disease in the intestine caused by V. cholerae infection.¹

Although Cholera hasn't been considered a "common" disease in most industrialized countries for a long time, it still is very prominent in developing countries like Bangladesh, Pakistan, Syria, and so on.²

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Why did the authors decide to switch to suckling mice? Well, they:

Better represent the human pathology of cholera

Better represents gene expression of cholera virulence factors during infection

What did they find?

CR communities are dominant over DS

introducing a small amount of CR microbes into a DS animal was enough to restore a nearly CR phenotype

V. cholerae infection resistance is independent of the T6SS molecular machinery

the T6SS was previously identified in the literature as a system that could kill murine E. coli and increase infection virulence⁶

however, the authors found that T6SS didn't alter levels of the E. coli strain in their suckling mice, so concluded that the CR microbes must manipulate another aspect of V. cholerae virulence

The degree of pathogen colonization resistance and infection outcome is highly dependent on the individual

since the Bangladeshi sample had higher inter-personal variation (i.e., higher beta diversity), endemic areas may contain higher levels of infection resistance variation