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Graduate College Dissertations and Theses Dissertations and Theses

2023

The Effect Of Tryptamine Producing Bacteria On Gut Motility In

Mice
Emilia Sofia Norberg
University of Vermont

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Norberg, Emilia Sofia, "The Effect Of Tryptamine Producing Bacteria On Gut Motility In Mice" (2023).
Graduate College Dissertations and Theses. 1762.
https://scholarworks.uvm.edu/graddis/1762

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THE EFFECT OF TRYPTAMINE PRODUCING BACTERIA ON GUT MOTILITY IN
MICE

A Thesis Presented

by

Emilia Norberg

to

The Faculty of the Graduate College

of

The University of Vermont

In Partial Fulfillment of the Requirements

for the Degree of Master of Science
Specializing in Microbiology and Molecular Genetics

August, 2023

Defense Date: July 17, 2023

Thesis Examination Committee:

Gary Mawe, Ph.D., Advisor

Margaret Vizzard, Ph. D., Chairperson
Brigitte Lavoie, Ph.D.
Matthew Wargo, Ph.D.
Cynthia J. Forehand, Ph.D., Dean of the Graduate College
 ABSTRACT

Research within the last 20 years has revealed GI bacterial metabolites to have a
significant effect on gut motility, and some of these actions involve influencing serotonin
signaling in the epithelial layer of the intestines. Serotonin (5-HT) has many functions
within the gut including propulsive and segmentation motility, vasodilation, and epithelial
cell secretion. There are several bacterial species that have been discovered to synthesize
one of 5-HT’s precursory molecules, including Bacillus subtilis, which produces
tryptophan. Prior data obtained from the Mawe lab has suggested that administering a
short daily course of B. subtilis strain R0179 spores to mice promotes GI motility.

The effects of another metabolite, tryptamine, on 5-HT signaling have also been
investigated. Tryptamine is a derivative of tryptophan and has been found to stimulate the
release of 5-HT in the mammalian GI tract. Moreover, it has recently been discovered
that tryptamine may also bind to and activate 5-HT receptors in the gut, primarily 5-
HT4Rs. Activation of these receptors has been associated with increased prokinetic
effects in the GI tract. Bacterial species have been identified that can produce tryptamine
in the gut via tryptophan decarboxylase. Kashyap and colleagues have recently
determined that administering a modified Bacteroides thetaiotomicron strain capable of
synthesizing tryptophan decarboxylase (Trp D+) to germ-free mice accelerated GI transit.
However, the prokinetic effects of B. thetaiotomicron Trp D+ on conventional mice with
normal gut microbiota are currently unknown as well as its effects in probiotic
formulations. The primary aim of this study was to further understand how administering
B. thetaiotomicron Trp D+ in combination to B. subtilis in mice may affect GI motility by
modulating 5-HT signaling.

In this study, C57/BL6 mice approximately 8 weeks of age were used to measure
whole GI transit time, colonic transit time, and fecal water content (FWC). Mice were
either administered two treatments of B. thetaiotomicron Trp D+ one week apart followed
by a one-week daily course of B. subtilis R0179, two treatments of B. thetaiotomicron
WT one week apart, followed by a one-week daily course of B. subtilis R0179, B.
thetaiotomicron Trp D+ alone, B. subtilis R0179 alone, or a vehicle control. While the
mice receiving B. thetaiotomicron Trp D+ alone and B. thetaiotomicron WT with B.
subtilis R0179 did not have any significant changes in while GI or colonic transit times,
they did have a lower FWC. The mice receiving both B. thetaiotomicron Trp D+ and B.
subtilis R0179 similarly demonstrated lower FWC. Interestingly, mice receiving both
bacterial strains had significantly slower GI transit times yet faster colonic transit times.
However, notably the mice receiving both bacteria did not have significantly faster
colonic transit times than the mice receiving B. subtilis R0179 alone.

In conclusion, this study revealed that B. thetaiotomicron Trp D+ administration

may not be an effective method for enhancing GI motility. As probiotics are becoming
more widely considered as a treatment for motility disorders including IBS, it is
important to understand how various bacterial strains may impact the gut and serotonin
signaling within. Overall, this study provides valuable insight in how to possibly improve
probiotic formulations.
 ACKNOWLEDGEMENTS

I would like to thank several incredible people who have made this experience so

meaningful. First, thank you so much Gary Mawe, for first welcoming me to the lab as an

undergraduate MMG student and for allowing me to continue in the lab to complete my

master’s degree. When I began thinking about my undergraduate thesis in microbiology, I

never would have imagined myself in a neuroscience lab focusing on the enteric nervous

system, but you have made my research experience so enjoyable. I have learned so much

from you, thank you so much for your support and advice throughout the years! Thank

you so much Brigitte Lavoie, for always being willing to help with my project, for

answering my millions of questions, and for proofreading this document. I appreciate

your dedication to the lab and your patience so incredibly much! Thank you Matt

Kwasnik, for completing all of the mouse gavages and helping with mouse care. Special

thank you to all of the members of the Mawe lab, past and present. The lab would not be

the same without you all, thank you for the laughter and words of encouragement over

the years. I would like to thank Theresa Montgomery for her expertise and help with all

things bacteria. Thank you to my thesis defense committee for your support during this

process. Lastly, I would like to thank my family, who have helped me so much. Thank

you so much for listening to me talk about school and my research, even if you don’t

fully understand what it means. I would like to especially thank my mom for being my

biggest supporter, for comforting me when an experiment didn’t work out but for also

telling me to suck it up when I needed to hear it. All of these people have made my time

in the lab so wonderful and I couldn’t have done it without you! Thank you so much.

ii
 TABLE OF CONTENTS
ACKNOWLEDGEMENTS ................................................................................................ II
LIST OF FIGURES .......................................................................................................... IV
CHAPTER I: COMPREHENSIVE LITERATURE REVIEW .......................................... 1
I. Gastrointestinal Tract Structure and Function ........................................................ 1
II. The Enteric Nervous System and Serotonin Signaling in the Gastrointestinal
Tract 5
III. The Gut Microbiome........................................................................................... 9
Gut Bacterial Biogeography ....................................................................................... 9
Factors Affecting Gut Biodiversity ............................................................................... 14
IV. GI Bacterial Metabolites and the Host .............................................................. 16
V. Microbial Effects on Serotonin Signaling and Gastrointestinal Motility ............. 17
Tryptophan Modulating Bacteria .............................................................................. 17
Tryptamine Modulating Bacteria .............................................................................. 22
VI. Specific Aims .................................................................................................... 25
VII. Works Cited....................................................................................................... 26
CHAPTER II: ASSESSING PROKINETIC EFFECTS OF TRYPTOPHAN AND
TRYPTAMINE PRODUCING BACTERIA IN MICE ................................................... 33
I. Introduction ........................................................................................................... 33
II. Materials and Methods ...................................................................................... 35
III. Results ............................................................................................................... 42
IV. Discussion ......................................................................................................... 56
V. Works Cited........................................................................................................... 60
CHAPTER III: FINAL CONCLUSIONS AND FUTURE DIRECTIONS .............. 62
I. Summary and Conclusions ................................................................................... 62
II. Future Directions .............................................................................................. 63
III. Concluding Remarks ......................................................................................... 67
IV. Works Cited....................................................................................................... 68
COMPREHENSIVE BIBLIOGRAPHY .................................................................. 69

iii
 LIST OF FIGURES

Figure 1……………………………………………………………………………………4

Figure 2……………………………………………………………………………………7

Figure 3……………………………………………………………………………………8

Figure 4…………………………………………………………………………………..14

Figure 5…………………………………………………………………………………..19

Figure 6…………………………………………………………………………………..21

Figure 7…………………………………………………………………………………..22

Figure 8…………………………………………………………………………………..23

Figure 9…………………………………………………………………………………..23

Figure 10…………………………………………………………………………………38

Figure 11…………………………………………………………………………………39

Figure 12…………………………………………………………………………………41

Figure 13…………………………………………………………………………………46

Figure 14…………………………………………………………………………………47

Figure 15…………………………………………………………………………………48

Figure 16…………………………………………………………………………………49

Figure 17a………………………………………………………………………………..50

Figure 17b………………………………………………………………………………..51

Figure 18…………………………………………………………………………………52

Figure 19…………………………………………………………………………………53

Figure 20…………………………………………………………………………………54

iv
Figure 21…………………………………………………………………………………55

Figure 22…………………………………………………………………………………57

Figure 23…………………………………………………………………………………66

Figure 24…………………………………………………………………………………67

v
CHAPTER I: COMPREHENSIVE LITERATURE REVIEW

I. Gastrointestinal Tract Structure and Function

The mammalian gastrointestinal (GI) tract is a highly complex organ system

comprised of several functionally diverse regions, responsible primarily for food

digestion, nutrient absorption, and waste excretion. The GI tract is tightly regulated and

requires the coordination of multiple different physiological processes and cell types,

including the host microbial population (Saffrey, 2014). There are several components

within the GI system. The human GI tract is an approximately 10-meter-long continuous

pathway of hollow, tube-like organs. From rostral to caudal ends, it is comprised of the

oral cavity, pharynx, esophagus, stomach, small intestine, large intestine, and anal canal.

Accessory organs, including teeth, tongue, salivary glands, liver, gallbladder, and

pancreas are also parts of the GI system and assist in digestion (Ogobuiro et al., 2023).

Digestion of food begins when it is first placed in the mouth, where it is mixed

with secreted saliva. Saliva contains several enzymes including amylase and lipase which

are responsible for catabolizing starch and fats, respectively, and thus beginning the

chemical breakdown of food (Peyrot des Gachons & Breslin, 2016). The tongue pushes

the food around the mouth into saliva and teeth, allowing for food chewing and softening.

This allows the formation of a food bolus which moves down via muscular contractions

into the esophagus, through the cardiac sphincter, into the stomach. The cardiac sphincter

will open and close in one direction, preventing food reflux from the stomach back into

the esophagus.

1
 Within the stomach, food will be mechanically churned through gastric enzymes

including pepsin and lipase, and hydrochloric acid. After approximately 2-4 hours, the

partially digested food bolus referred to as chyme passes through the small intestine via

the pyloric sphincter into the small intestine (Jones, 2015).

The small intestine is subdivided into three distinct regions, termed duodenum,

jejunum, and ileum, which collectively absorb a significant amount of nutrients and

water. Projecting from the small intestinal mucosa are villi, which are finger-like

structures enabling for increased surface area and therefore absorption. The duodenum,

the shortest of the three subsections, spans approximately 20 to 25 cm in length and is

where food absorption begins. The duodenum is connected to the pancreas via the

hepatopancreatic ampulla, allowing it to receive pancreatic enzymes to digest the chyme

as well as bicarbonate to neutralize gastric acids prior to entrance into the jejunum. The

liver is also connected to the duodenum via the common hepatic duct, where it will

secrete bile which aids in lipid absorption and digestion. The jejunum, which measures

approximately 2.5 meters in length, is the primary site of sugar, amino acid, and fatty acid

absorption. Food then enters the ileum, which absorbs remaining nutrients (primarily

vitamin B12 and bile acids) (Collins et al., 2023).

Following the small intestine, food is propagated through the ileocecal valve into

the large intestine. The large intestine, spanning approximately 1.5 meters, is involved in

very little nutrient absorption. Instead, it mainly functions in the remainder of water

absorption which aids in the formation and expulsion of stool. Notably, the large intestine

contains a diverse population of gut microbiota, which can ferment remaining food

products and release vitamins B and K for host absorption. The large intestine is

2
subdivided into distinct regions, termed the ascending, transverse, descending, and

sigmoid colon. Through propulsive motility, the sigmoid colon will contract and therefore

increase pressure, encouraging feces to pass into the rectum (Azzouz & Sharma, 2023;

Sarna, 2010). Within the rectum, fecal content is lubricated with mucus allowing for

elimination via voluntary relaxation of the anal sphincter (Jones, 2015).

While histology may differ across the length of the GI tract, it can largely be

defined by having four distinct concentric layers – from lumen outwards, the mucosa, the

submucosa, the muscularis externa, and the serosa (Figure 1). The mucosa, the most inner

histologic layer, is comprised of a single layer of epithelial cells. The mucosa is highly

folded, increasing surface area and aiding in its function of absorption and secretion.

Across the mucosa there are invaginations which allow for the secretion of digestive

enzymes, water, mucus, and electrolytes as well as important GI endocrine glands. The

mucosa is supported by a connective tissue layer termed the lamina propria. The next

outer layer is the submucosa, which is a complex connective tissue network containing

blood and lymphatic vessels. Within the submucosa is the submucosal (Meissner) plexus,

which is a neural network responsible primarily for regulating blood flow within the GI

tract and epithelial secretions (Ogobuiro et al., 2023; Nezami & Srinivasan, 2010).

The following histologic layer of the GI tract is the muscularis externa, which is

defined by two major smooth muscle layers (longitudinal and circular). Between these

two layers is the myenteric (Auerbach) plexus, another significant neural group in the GI

tract. The myenteric plexus is responsible for peristalsis, the alternating process of distal

relaxation and proximal contraction which encourages the passage of food products down

the GI tract (Shahrestani & Das, 2023). Notably, the stomach also contains a third muscle

3
layer within the muscularis externa, termed the inner oblique, which facilitates churning

movements. The outermost histologic layer of the GI tract is the serosa (adventitia). The

serosa is a thin layer of epithelial cells and connective tissue which functions in secreting

a serous fluid that lubricates the GI wall, preventing friction (Ogobuiro et al., 2023).

Figure 1:

Histological organization of the GI tract. From the lumen outwards, the GI tract is

organized into four main concentric layers: the mucosa, the submucosa, the muscularis

externa, and the serosa. Also included in this diagram are the submucosal plexus and

myenteric plexus, which are neuronal networks comprising the enteric nervous system

(Kulkarni et al., 2018).

4
 II. The Enteric Nervous System and Serotonin Signaling in the
Gastrointestinal Tract

In the nineteenth century, it was discovered that there exists communication

between the nervous system within the brain and that within the gut. The enteric nervous

system, or the nervous system within the GI tract, is considered a branch of the

autonomic nervous system and is responsible for maintaining gastrointestinal

homeostasis. It has also been linked to affecting higher cognitive functions including

affect, motivation, and decision-making. Based on its complexity regarding its neuronal

pathways and neurotransmitter signaling molecules used, it has even been referred to as

“the second brain” (Mayer, 2011; Gershon, 1999).

While serotonin is largely thought to be involved in regulating mood and behavior

within the brain, most of this molecule within the human body is synthesized from cells

in the intestinal mucosa. Serotonin, abbreviated as 5-HT, has many functions within the

gut including propulsive and segmentation motility, vasodilation, and epithelial cell

secretion. 5-HT can also affect satiation, pain, nausea, and vomiting. Investigative studies

have demonstrated that 5-HT levels are significantly altered in GI motility disorders,

including coeliac disease and inflammatory bowel disease (IBD). Interestingly, 5-HT

tissue content is elevated in individuals with coeliac disease and lowered in those with

IBD. Several drugs have been created which target 5-HT signaling as GI motility disorder

treatment (Mawe & Hoffman, 2013; Spohn et al., 2016).

Gut 5-HT synthesis and storage begins in enterochromaffin (EC) cells, which are

specialized enteroendocrine cells located in the intestinal mucosa. As the nerves within

the gut do not directly encounter the lumen, EC cells act as the transducers that are

5
capable of transmitting chemical and mechanical stimuli (Mawe & Hoffman, 2013). The

initial and rate-limiting step of 5-HT synthesis within EC cells involves the enzyme

tryptophan hydroxylase and the essential amino acid, tryptophan (Trp) (Figure 2). While

the diet is the principal source of Trp, the microbiota is another potential source of this

amino acid. After Trp has been converted to L-5-hydroxytryptophan (5-HTP), aromatic

L-amino acid decarboxylase quickly acts on the molecule to form 5-HT. Low levels of 5-

HTP are typically found during tissue analysis because of how rapidly it is converted to

serotonin. 5-HT can be further metabolized by monoamine oxidase and aldehyde

dehydrogenase to form 5-hydroxindole acetic acid, or 5-HIAA. Metabolism of 5-HT can

be measured by the amount of 5-HIAA found in intestinal tissue (Hoglund et al., 2019).

6
Figure 2:

Biosynthetic pathway of serotonin (5-HT). The initial and rate-limiting step of serotonin

synthesis involves tryptophan hydroxylase and L-tryptophan. (Höglund et al., 2019).

Upon mechanical or chemical stimulation, 5-HT is released from the basal surface

of the EC cell into the interstitial space of the lamina propria where it is available to bind

to nerve fiber receptors such as those within the myenteric plexus (Figure 3). It is

suggested that approximately 2% of the neurons within the myenteric plexus are

serotonergic and will also synapse to other serotonergic neurons along the GI tract wall

(Costa et al., 1996). There are currently 7 identified serotonin receptors within the gut,

with the 5-HT3 and 5-HT4 receptors being the most extensively studied given their

involvement in constipation and diarrhea. Once released, 5-HT can be transported into

gut epithelial cells via SERT (serotonin-selective reuptake inhibitor) where it is

7
enzymatically degraded or brought into the bloodstream into platelet cells for further use

(Wade et al., 1996; Mawe & Hoffman, 2013).

Figure 3:

A schematic detailing serotonin (5-HT) signaling within the gut. Upon mechanical

stimulation, an enterochromaffin cell (EC) will release 5-HT into the interstitial space of

the lamina propria where it can bind to serotonergic sensory fibers within the myenteric

plexus. During the recovery phase, a serotonin transporter (SERT) can transport 5-HT

back into gut epithelium for breakdown, or 5-HT is sent to the bloodstream for platelet

storage (Mawe & Hoffman, 2013).

5-HT3 receptors, or 5-HT3Rs, are found expressed on neurons extending into the

gut mucosa, smooth muscle cells, and enterocytes. When activated by 5-HT released

from EC cells, they have been demonstrated to have prokinetic effects on propulsive

motility and on GI secretion (Mawe & Hoffman, 2013). 5-HT3R agonists were first used

8
therapeutically in 1986 as an anti-nausea medication for patients receiving chemotherapy,

however, it had a common adverse side effect of inducing constipation (Costall et al.,

1986; Miner & Sanger, 1986). Given this, when administered to patients with IBS-D

(irritable bowel syndrome, predominant diarrhea stool pattern), it has been found to

decrease symptoms of diarrhea by decreasing propulsive motility (Talley et al., 1990).

Conversely, 5-HT4 receptors, or 5-HT4Rs, have been identified to accelerate

propulsive motility. 5-HT4Rs are G-protein coupled receptors that are expressed on a

variety on gut epithelial cells, primarily within the colonic mucosa. 5-HT4R agonistic

activation has been found to excite peristaltic neurons, encouraging prokinetic effects. 5-

HT4R agonists can be used to alleviate pain and constipation in those with IBS-C

(predominant constipation stool pattern) (Evans et al., 2007). Moreover, transgenic mice

lacking 5-HT4Rs have slower colonic motility times (Mawe & Hoffman, 2013; Hoffman

et al., 2012). Interestingly, tryptamine, a tryptophan-derived indole, also has a high

affinity for 5-HT4Rs (Van Oekelen et al., 2002; Bhattarai et al., 2002). As tryptamine

(along with tryptophan itself) may be produced by microbiota, it is thought that gut

bacteria products may influence GI motility and other 5-HTR mediated functions

(Bhattarai et al., 2018).

III. The Gut Microbiome

Gut Bacterial Biogeography

It has become increasingly clear that there is bidirectional communication

between the microbes that exist in the lumen of the GI tract and the nervous system.

9
Research within the last 20 years has revealed GI bacterial metabolites have significant

effects on the brain, spinal cord, and enteric nervous system (Cryan et al., 2019).

The human gastrointestinal system provides complex microhabitats that are

conducive to harboring an estimated 1013 bacterial cells, which is approximately equal to

the number of cells that comprise the human body. From the start of the GI tract at the

oral cavity to the end at the anus, the chemical and environmental conditions vary

immensely. These differences impact the GI microbes in terms of types of bacterial

species present as well as density. Where bacteria colonize within the tract as well as the

ingested substance passing through also affects the bacteria’s metabolic functional role.

Genomic sequence analysis has revealed that the gut microbiota varies greatly between

hosts and that there are many factors that contribute to microbiome diversity. The host

genome and immune system have large influence on gut microbiota composition

(Hillman et al., 2017). Additionally, diseases such as inflammatory bowel disease, cancer,

diabetes mellitus, and cardiovascular pathology have significant impact on microbiota-

host interactions (Cho & Blaser, 2012; Lynch & Pederson, 2016).

At the start of the GI tract, the oral cavity, there exists populations of transient

bacteria as well as commensal populations which form biofilms within the mouth.

According to the Human Oral Microbiome Database (HOMD), the most prevalent oral

bacterial genus is Streptococcus, followed by Neisseria, Granulicatella, and Veillonella

(Dewhirst et al., 2010). Some of the bacterial species within the oral cavity can be

metabolically complementary. For example, Porphyromonas gingivalis has been found to

exist symbiotically with other microbial species (Mark Welch et al., 2016). Conversely,

other species such as Streptococcus oralis and Streptococcus gordonii require the same

10
nutritional niche and are therefore highly antagonistic towards each other (Levy &

Borenstein; 2013).

After swallowing, food is transported down through the esophagus into the

stomach. Given its acidic pH and comparatively shorter transit time to the lower GI, the

upper GI tract generally harbors a smaller and less diverse microbial population (Hillman

et al., 2017). Similar to the oral cavity, Streptococcus is also the most prevalent bacterial

genus in the esophagus (Benitez et a.l., 2015). The stomach contains the smallest

microbial population within the GI tract, with 101 to 103 CFU g-1. As the pH of gastric

juice is so low (pH <4), it is thought that these stomach conditions have evolved as a

method of protection against harmful pathogens as it inhibits most microbial growth.

There are two apparent categories of stomach bacterial diversity in humans: those with

Helicobacter pylori, and those without. Individuals with H. pylori have higher

populations of Proteobacteria and overall lower alpha diversity compared to individuals

without (Bik et al., 2006).

As digested chyme is transported from the stomach into the intestines, the

microbiota present becomes more abundant and diverse. Within the small intestine, the

most prevalent genera of bacterial species are Clostridium, Streptococcus, and

Bacteroides. The colon is the most microbially rich area, accounting for approximately

70% of all human bacteria. The colon supports mainly anaerobic bacteria capable of

catabolizing undigested food product, with its dominating phyla being Prevotellaceae,

Lachnospiraceae, and Rickenellaceae (Hillman et al., 2017; Thursby & Juge, 2017).

Lower GI diversity at the phylum level is highly stable, however, there is

microbial variability spatially and depending on several factors, including time and type

11
of food intake. While the lower GI tract is largely anaerobic, there are areas of the

mucosa with increased oxygen concentrations allowing for aerobic bacterial growth. The

gastrointestinal oxygen gradient benefits and provides microenvironments where

microbes, especially facultative anaerobes, can thrive. Most of the gut microbiota exists

in the lumen, where it can interact with and utilize food substrate. It has become evident

that host cells and gut microbiota have a mutualistic relationship, where bacterially

produced metabolites can be used by the host and vice versa. Food that cannot be

digested by the host in the upper GI tract is fermented by bacteria in the lower gut.

Moreover, a diverse gut microbiota has been linked to enhanced energy reabsorption and

immune system strengthening (Sommer & Backhed, 2016; Hillman et al., 2017).

Notably, it has been difficult to characterize microbial diversity in host upper

GI/small intestine as it requires serial biopsies in healthy individuals. Colonic microbial

diversity is typically assessed using fecal analysis, however, even this method is not

entirely accurate. Fecal samples do not represent the spatial diversity across the colon and

instead contain a mixture of commensal and transient flora (Hillman et al., 2017). For

example, research has shown that in mice, fecal samples show Bacteroidetes to be most

abundant, while Firmicutes are most prevalent in colonic mucosal samples (Thursby &

Juge, 2017).

Of note, while the human microbiome is comprised largely of bacteria, it is also

influenced by archaea, fungi, and viruses which also crosstalk with the host. Archaea,

fungi, and viruses comprise <0.1% of the microbiome and are thus called the “rare

biosphere” (Sogin et al., 2006). Fungi comprises approximately 0.03% of the fecal

microbiome, with its most abundant genus being Candida. Although fungi can colonize

12
the human gut, fungi populations are considerably more unstable than bacterial

populations and can fluctuate over a course of a few months (Scanlanet al., 2006;

Hillman et al., 2017). Archaea species and viruses are the smallest components of the

microbiota, with Methanobrevibacter and bacteria-infecting phage viruses being most

abundant, respectively (Dridi et al., 2011; Reyes et al., 2012).

13
Figure 4:

Microbial composition varies widely across the GI tract. There is limited microbiota

within the upper GI due to an acidic environment and short transit times. The colon

harbors approximately 70% of all bacteria in the human body and is very microbially

diverse. There are several factors that can influence the gut microbiota across an

individual’s lifetime including genetics, location, antibiotic use, diet, and other lifestyle

choices (Clarke et al., 2019).

Factors Affecting Gut Biodiversity

The human gut microbiome has been understood to be influenced by several

factors and across an individual’s lifetime. In utero, an infant’s gut is considered largely

to be sterile. Infants born via vaginal delivery are exposed to microbiota present in the

birth canal, which allows for maternal bacteria to populate the newborn’s gut.
14
Conversely, infants born via Cesarean delivery have decreased exposure to maternal

bacteria and therefore have a diminished gut microbial population. Studies have

demonstrated how infants born via “C-section” only have 41% similar fecal microbiota to

their mother’s, compared to 72% in infants born vaginally (Thursby & Juge, 2017).

Infants born via C-section have been found to be more susceptible to asthma, allergies,

type I diabetes, and obesity. Moreover, how an infant is fed can also influence its gut

microbial composition later in life. Infants who were formula-fed were more likely to

have impaired immune development as well as impaired metabolism compared to

breastfed counterparts (Bokulich et al., 2016). An individual’s microbiome is thought to

reach “adult-like complexity” by age 3.

There are many additional factors that have been identified to affect gut microbial

composition. Antibiotic use, especially at a young age, can deplete host microbiota and

impact species diversity. Furthermore, research conducted using mice as an animal model

has shown that when gut microbiota is significantly altered by antibiotic use, bile acid

and serotonin metabolism are decreased, resulting in slower GI transit times (Ge et al.,

2017). Microbiota depletion by antibiotics can make host organisms more susceptible to

pathogenic infections due to changes in nutrient availability that are usually maintained

by GI bacteria (Thursby & Juge, 2017). Diet and nutrition can also affect microbiota, for

example, diets high in fiber have been linked to increased gut microbial diversity while

Westernized diets may impart microbial instability. Aging may slowly decrease gut

microbial diversity, whereas frequent exercise has been linked to preventing this. There

15
are several other possible influences on the gut microbiome, including but not limited to

host genetics, geographical location, and stress. (Clarke et al., 2019).

Several issues arise within the human body when there is not sufficient microbial

diversity within the GI tract. Treatment for this phenomenon, termed dysbiosis, include

fecal microbiota transplant (FMT), which involves transplanting fecal microbes from a

healthy donor into a recipient whose GI microbiota has been altered. As genetic analysis

has revealed human colonic flora and fecal flora are nearly identical, FMT is a successful

procedure to help restore GI microbial diversity (DeFillipp et al., 2019; Hillman et al.,

2017).

IV. GI Bacterial Metabolites and the Host

The human GI tract is estimated to harbor approximately 1013 bacterial cells, and

there has been mounting evidence to support that the host-gut bacteria relationship is

highly mutualistic. Due to the large variety of genetic makeup and therefore metabolic

capabilities, GI bacteria provide many different benefits to the host. GI bacteria play a

major role in the fermentation of carbohydrates to produce short-chain fatty acids

(SCFAs) including propionate, butyrate, and acetate. These SCFAs absorbed by GI

epithelial cells and are involved in regulation of epithelial gene expression, proliferation,

and apoptosis (Correa-Oliveira et al., 2016). Moreover, SCFAs, along with other bacterial

metabolites, are involved in the host immune system by generating mucins that maintain

the integrity of the GI mucosal barrier. SCFAs also stimulate the synthesis of interleukin

18, which protects GI epithelial cells by stimulating an immune inflammatory response

(Thursby & Juge, 2017; Bickel, 1993).

16
 GI bacteria can also synthesize vitamins that host organisms are incapable of

producing and are heavily involved in host cellular processes. For instance, gut

microbiota produce folate, which is essential for host DNA synthesis and repair (Pompei

et al., 2007). They are also involved in co-metabolism of bile acids with the host.

Alterations in this pathway have been associated with the development of type II diabetes

mellitus and obesity (Palau-Rodriguez et al., 2015). Additionally, gut bacteria can provide

a protective response against pathogenic bacteria. The presence of commensal bacteria

will limit the nutrient resources and colonization sites available for unfamiliar bacterial

species, as well as synthesize antimicrobial compounds to limit the expansion of

pathogens (Thursby & Juge, 2017).

Given the large variety of metabolites GI microbiota can produce, it has been

thought that bacterial products may have a significant impact on serotonin signaling

within the GI tract as well as GI motility. The prokinetic effects of gut bacteria are

discussed heavily in this study and are described in the next section.

V. Microbial Effects on Serotonin Signaling and Gastrointestinal Motility

Tryptophan Modulating Bacteria

Bacteria within the GI tract are considered to have a significant effect on gut

motility, and some of these actions involve influencing serotonin signaling in the

epithelial layer of the intestines. For instance, several bacterial species have been

experimentally determined to modulate the tryptophan (Trp)/5-HT pathway. As described

above in section II, the amino acid tryptophan is the precursory molecule for 5-HT. In the

gut Trp is first acted on by tryptophan decarboxylase, converting the molecule into L-5-

17
hydroxytryptamine (5-HTP). Aromatic L-amino acid decarboxylase then binds to 5-HTP

and quickly forms 5-HT (Hoglund et al., 2019). A recent study conducted by Lallemand

Health Solutions revealed that there are many probiotic bacterial strains that are able to

up-/down-regulate tryptophan concentrations in vitro (Figure 5). One species that has

been demonstrated to increase tryptophan concentrations that our lab previously focused

on is Bacillus subtilis.

18
Figure 5:

In vitro tryptophan consumptions from various probiotic bacterial strains. Bacillus

subtilis has been found to increase tryptophan concentrations in vitro. Courtesy of

Lallemand Health Solutions.

19
 Bacillus subtilis is gram-positive, spore-forming, facultative anaerobe that can

inhabit the GI tract. Certain B. subtilis strains, when included in probiotic formulations,

have been found to exhibit benefits on GI and brain system health, however, the exact

mechanisms are not yet understood (Ilinskaya et al., 2017). B. subtilis synthesizes Trp

through its trp operon. When the cell detects low levels of Trp, an RNA polymerase

enzyme will bind to the promoter of the operon to initiate transcription. The genes within

the operon each synthesize an enzyme that is essential for the synthesis of Trp, including

the enzyme tryptophan synthase, which synthesizes tryptophan from indole (Carlton,

1967).

Since EC cells use Trp as a precursor for the biosynthesis of 5-HT, it has been

hypothesized that Trp-synthesizing bacteria such as B. subtilis could be utilized to

increase 5-HT levels within the gut. 5-HT is an essential molecule in gut signaling,

therefore, greater circulating levels of its precursor might ultimately lead to enhanced 5-

HT-mediated gut functions including motility. Recent research conducted by the Mawe

lab has demonstrated that orally administering Trp-synthesizing B. subtilis spores (strain

R0179; a gift from Lallemand Health Solutions) leads to higher Trp and 5-HT signaling

levels, as measured by HPLC (Figure 6), as well as faster gut motility and increased fecal

water content in mice, compared to mice treated with vehicle control (Legan et al., 2023).

20
Figure 6:

Recent research conducted by the Mawe lab has demonstrated that administering B.

subtilis R0179 spores to mice increases Trp and 5-HT colonic signaling levels, measured

via HPLC (Legan et al., 2023).

21
Figure 7:

Recent research conducted by the Mawe lab has demonstrated that administering B.

subtilis R0179 spores to mice increases colonic motility when compared to a vehicle

control (Legan et al., 2023).

Tryptamine Modulating Bacteria

Another bacterially derived molecule that has been associated with 5-HT

signaling in the GI tract is tryptamine. Tryptamine is a biogenic amine that serves as a

precursory molecule for several compounds referred to as “tryptamines”. Tryptamines

include many biologically significant compounds including 5-HT and melatonin (Figure

8), as well as psychedelic drugs (Kema et al., 2000; Itoh et al., 1999; Jacob & Presti,

2005). In mammals, tryptamine is synthesized first by the decarboxylation of Trp,

catalyzed by the enzyme aromatic L-amino acid decarboxylase. Tryptamine can be

22
further metabolized into 3-indolacetaldehyde enzymatically through oxidative

deamination by monoamine oxidase (Figure 9) (Dragulska & Kanska, 2014).

Figure 8:

Several products including the tryptamine subunit, highlighted in yellow (Simonetti et al.,

2021).

Figure 9:

The enzymatic of from L-Trp to 3-IAL. In mammals, L-Trp is converted by L-amino acid

decarboxylase to tryptamine. Tryptamine can then undergo oxidative deamination to

produce 3-IAL (Dragulska & Kanska, 2014).

23
 The tryptamine molecular structure is remarkably like that of 5-HT, although it is

significantly less present in mammalian tissue. Tryptamine’s possible involvement within

the enteric nervous system was first introduced in 1985, when it was discovered that

tryptamine triggered the release of physiologically active 5-HT within the myenteric

plexus of guinea pigs (Takaki et al., 1985). More recently, it was revealed that tryptamine

can act as a ligand for multiple 5-HT receptors, with its highest affinity being towards 5-

HT4 receptors (5-HT4Rs). 5-HT4Rs are G-protein coupled receptors that while ubiquitous

across the entire GI tract, are highly expressed in mouse colon. When bound to 5-HT, 5-

HT4Rs have been found to induce prokinetic effects and colonic mucus secretion. It has

been discovered that tryptamine-activated 5-HT4Rs can also establish these effects

(Bhattarai et al., 2018; Mawe & Hoffman, 2013).

Tryptamine has been found to be present at high concentrations in both human

and rodent feces (Brooks et al., 1984). Moreover, GF mice have significantly lower fecal

tryptamine content compared to mice colonized with human GI flora, suggesting that gut

microbiota play an important role in tryptophan/tryptamine metabolism in the GI tract

(Marcobal et al., 2013). While the majority of gut commensal bacteria is not inherently

capable of producing tryptamine from tryptophan, it has been estimated that

approximately 10% of human GI tracts contain at least one bacterial strain capable of

synthesizing tryptophan decarboxylase. Two reported commensal tryptamine-producing

bacterial species include the Firmicutes Clostridium sporogenes and Ruminococcus

gnavus (Williams et al., 2014).

24
 Recently, a study conducted by Kashyap and colleagues assessed how in vivo

tryptamine production may affect gastrointestinal transit by using an engineered microbe

capable of synthesizing tryptamine decarboxylase. To accomplish this, they isolated the

gene encoding tryptamine decarboxylase from R. gnavus, codon optimized it, and

integrated it into the host chromosome of Bacteroides thetaiotmicron under a

constitutively active phage-derived promoter (B. thetaiotomicron Trp D+) (Bhattarai et

al., 2018). B. thetaiotomicron is a gram-negative, non-spore forming, obligate anaerobic

bacterial species. It is a bile-resistant, commensal bacteria easily able to colonize the GI

tract. Moreover it is genetically tractable, rendering it an ideal microbe to study for

tryptamine production in the gut (Wexler, 2007; Bechon et al., 2022).

When GF mice were colonized with the manipulated B. thetaiotomicron Trp D+,

they were discovered to have a higher water content as well as faster GI transit times by

stimulating colonic mucus release. Interestingly, B. thetaiotomicron Trp D+ colonization

did not elicit any changes in feces or serum 5-HT concentrations (Bhattarai et al., 2018;

Bhattari et al., 2020). However, the prokinetic effects of B. thetaiotomicron Trp D+ on

conventional mice with normal gut microbiota are currently unknown as well as its

effects in probiotic formulations. This investigation sought to further understand how

administering B. thetaiotomicron Trp D+ in combination to B. subtilis in mice may affect

GI motility by modulating serotonin signaling.

VI. Specific Aims

Previous work conducted by the Mawe lab has demonstrated that administering

Trp producing bacteria, Bacillus subtilis strain R0179, to conventional mice has led to

25
decreased colonic transit times and higher fecal water content by encouraging the

conversion of 5-HT from Trp as well as higher colonic tissue concentrations of 5-HT and

5-HIAA (Legan et al., 2023). Moreover, recent work conducted by Kashyap and

colleagues have demonstrated that administering an engineered Trp decarboxylating

bacteria, B. thetaiotomicron Trp D+, to germ-free mice has led to decreased whole GI

transit times by enhancing epithelial secretion and activating 5-HT4Rs (Bhattarai et al.,

2018).

The primary aim of the present investigation is to assess whether the conversion

of Trp to tryptamine via B. thetaiotomicron Trp D+ may contribute to the prokinetic

effects of B. subtilis in conventional mice. We hypothesized that administering the two

bacterial strains to conventional mice would lead to lower GI motility times and higher

fecal water content, consistent with what was described in the above studies. To assess

this, conventional mice were either pre-colonized with B. thetaiotomicron Trp D+, B.

thetaiotomicron wild-type, or administered a vehicle control. They were then provided a

seven-day treatment of B. subtilis R0179 spores or a vehicle control. Whole gut transit

times, colonic motility times, and fecal water content were measured pre- and post-

treatment and used to assess changes in gut function as a response to bacterial treatment.

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32
CHAPTER II: ASSESSING PROKINETIC EFFECTS OF
TRYPTOPHAN AND TRYPTAMINE PRODUCING BACTERIA IN
MICE

I. Introduction

The gut-brain-microbiome axis is the concept where there is extensive

communication between the bacteria within the gastrointestinal tract and the nervous

system. Research within the last 20 years has revealed GI bacterial metabolites have

significant effects on the brain, spinal cord, and enteric nervous system (Cryan et al.,

2019). This interaction has proposed the idea that microbiota within the gastrointestinal

(GI) system affect GI function by influencing serotonin signaling in the epithelial layer of

the intestines.

Serotonin, abbreviated as 5-HT, has many roles within the GI tract including

propulsive and segmentation motility, vasodilation, and epithelial cell secretion. Within

the gut, 5-HT is synthesized and released by enterochromaffin (EC) cells that are found in

the intestinal mucosa (Mawe & Hoffman, 2013). Once released, 5-HT can bind to one of

many gut epithelial receptors including 5-HT4Rs. 5-HT4Rs are heavily expressed within

mouse colonic mucosa, and when activated, can accelerate propulsive motility (Hoffman,

2012). Because of this ability, several drugs have been created that can target this

receptor as a possible treatment for individuals with motility disorders. For instance, 5-

HT4R agonistic activation has been found to promote peristaltic effects and reduce

constipation in individuals with constipation predominant IBS (Evans et al., 2007).

Research investigating the gut-brain-microbiome axis has identified several

microbially synthesized metabolites that may influence serotonin signaling in the gut and

thus GI motility. Several bacterial species can produce tryptophan (Trp), an essential
33
amino acid which can be enzymatically converted into 5-HT. For example, Bacillus

subtilis strain R0179 encodes the enzyme Trp synthase, which synthesizes Trp from

indole (Carlton, 1967). A recent study conducted by the Mawe lab has demonstrated that

orally gavaging a short daily course of B. subtilis R0179 spores to mice resulted in

increased colonic motility times as well as higher colonic tissue concentrations of 5-HT

and its breakdown product, 5-HIAA (Legan et al., 2023).

Another bacterial metabolite that has been associated with enhanced GI motility

function is tryptamine. Tryptamine, an indolamine derived from the decarboxylation of

Trp, has been linked to increased GI transit times by prompting intestinal epithelial

mucus secretion. Moreover, tryptamine has been discovered to function as a 5-HT4R

agonist. Kashyap and colleagues have recently found that when germ free (GF) mice

were colonized with a genetically engineered bacteria, B. thetaiotomicron Trp D+, that

synthesizes tryptamine by encoding Trp decarboxylase, they demonstrated faster whole

GI transit times as well as higher fecal water content (Bhattarai et al., 2018).

The current study was conducted to assess the collaborative effects of B. subtilis

R0179 and B. thetaiotomicron Trp D+ on gastrointestinal motility in conventional mice.

Given that tryptophan and tryptamine both individually elicit faster GI transit times via 5-

HT signaling modulation, it was hypothesized that administering two bacterial strains

capable of synthesizing one or the other metabolite would elicit similar prokinetic effects.

Conventional mice were either pre-colonized with B. thetaiotomicron Trp D+, B.

thetaiotomicron WT, or administered a vehicle control. They were then provided a seven-

day treatment of B. subtilis R0179 spores or a vehicle control. The primary outcome

measures were changes in whole GI motility, colonic motility, and fecal water content.

34
 II. Materials and Methods

Animals

For the in vivo GI motility assays, C57/BL6 mice approximately 8 weeks of aged

were utilized. Mice were chosen as opposed to other animal models due to the

physiological similarity between mice and human GI systems. The mice were purchased

from Jackson Laboratories in Bar Harbor, ME. All experimental protocols used for this

study were approved by the University of Vermont Institutional Animal Care and Use

Committee.

Bacteria

Bacterial Strains

Freeze-dried spores for the Bacillus subtilis strain R0179 were obtained from

Lallemand Health Solutions. Bacteroides thetaiotomicron WT and Bacteroides

thetaiotomicron Trp D+ were gifted from Purna Kashyap at the Mayo Clinic as glycerol

cryovials. B. thetaiotomicron Trp D+ is a genetically manipulated strain capable of

synthesizing tryptophan decarboxylase. The Trp decarboxylase gene was obtained from

Ruminococcus gnavis, codon optimized, and cloned into B. thetaiotomicron under a

phage promoter (Bhattarai, 2018).

Bacterial Preparation

As B. subtilis R0179 was directly administered to mice as spores, they did not

require any culturing. Freeze-dried spores were kept at 4°C. Each day of mouse

treatment, spores were suspended in a phosphate buffered saline solution at a

35
concentration of 109 CFU/mL. Mice receiving this treatment were administered 100µl via

oral gavage daily.

B. thetaiotomicron WT and B. thetaiotomicron Trp D+ were received as glycerol

cryovials. As B. thetaiotomicron is an anaerobic bacterial species, all culturing was

completed within an anaerobic chamber. 1 mL of the B. thetaiotomicron WT and B.

thetaiotomicron Trp D+ cryovials were combined with 6 mL of Brain Heart Infusion

(BHI) broth supplemented with hemin, vitamin K, cysteine, and yeast extract. BHI is a

nutrient-rich medium used to culture fastidious bacteria species such as B.

thetaiotomicron. Both strains of B. thetaiotomicron were cultured for three days before

gavage to ensure sufficient bacterial growth. B. thetaiotomicron culture purity was

confirmed by performing a boiling prep to extract plasmid DNA and by completing

qPCR using B. thetaiotomicron and Eubacterial specific primers.

Mice receiving B. thetaiotomicron were orally gavaged 300µl of the bacteria

suspended in BHI once a week to establish colonization. To ensure that mice treated with

B. thetaiotomicron were in fact colonized, QIAamp PowerFecal DNA Kit was used to

extract DNA from mouse stool after one week following B. thetaiotomicron

administration. qPCR using B. thetaiotomicron specific primers confirmed the presence

of the bacteria after 1 week.

Motility Assays

Whole GI transit: A solution was made consisting of 6% unabsorbable carmine red dye,

0.5% methylcellulose, and water. This solution (300 μl) was orally gavaged and the mice

36
were placed in individual cages without bedding. The time required for excretion of an

entirely red pellet was recorded as the time for whole GI transit.

Colonic motility: Mice were placed into a clean cage and allowed to acclimate for 1 hour

prior to performing colonic motility. Mice were then lightly anesthetized with 3%

isoflurane. A bead (size 10) was inserted 2cm into the rectum of a mouse using a gavage

needle. The mouse was then placed individually in a clear cage until the bead was

expelled. The time required for the bead to be expelled was recorded as fecal bead

expulsion time.

Fecal water content: Mice were placed into a cage and fresh fecal pellets were collected

for one hour. After an hour, the fecal pellets were weighed and placed in a 50°C for 24

hours to dry. The dry fecal pellets were weighed. The dry weights were subtracted from

the weight wets to calculate fecal water content.

Contamination assessment: Fecal pellets were collected from experimental and control

mice on days 0, 7, and 14 of treatment. A QIAamp PowerFecal DNA Kit was used to

extract DNA from mouse stool. Quantitative PCR using B. thetaiotomicron specific and

Eubacterial primers was performed on the fecal DNA using SYBR Green Master Mix and

confirmed minimal bacterial contamination between control groups.

Experiment 1: B. thetaiotomicron Trp D+ only

This experiment was completed to assess any prokinetic effects of B.

thetaiotomicron Trp D+ and to ensure the BHI medium was not eliciting any prokinetic

effects on its own. On day 0 and on day 7, mice were orally administered B.

thetaiotomicron Trp D+, BHI, or PBS. The motility assays described above were

37
performed on day 0 (pre-treatment baseline), day 7, and day 14 (Figure 10). Mice were

weighed on days 0, 7, and 14 to confirm no significant weight gain or weight loss was

occurring. Mice were euthanized on day 15 following the final motility assay.

Figure 10:

Timeline for experiment 1. Mice received B. thetaiotomicron Trp D+, BHI, or PBS on day

0 and day 7. Motility assays were performed on day 0 (pre-treatment baseline), day 7,

and day 14. Image created using BioRender.com.

Experiment 2: B. thetaiotomicron Trp D+ and B. subtilis R0179

This experiment was completed to compare the effects of administering both B.

thetaiotomicron Trp D+ and B. subtilis R0179 versus B. subtilis R0179 alone. Mice were

organized into three treatment groups: B. thetaiotomicron Trp D+ and B. subtilis R0179,

B. subtilis R0179 only, and PBS. Mice receiving the B. thetaiotomicron Trp D+ and B.

subtilis R0179 received B. thetaiotomicron Trp D+ on day 0 and day 7 via oral gavage to

establish colonization in the gut, while the other mice received PBS of the same volume.
38
All mice receiving B. subtilis R0179 were administered it via daily gavage days 7

through 14, while the PBS group received a PBS gavage instead. The motility assays

were performed on day 0 (pre-treatment baseline), day 7, and day 14 (Figure 11). Mice

were weighed on days 0, 7, and 14 to confirm no significant weight gain or weight loss

was occurring. Mice were euthanized on day 15 following the final motility assay.

Figure 11:

Timeline for Experiment 2. Mice received B. thetaiotomicron Trp D+ or PBS on day 0 and

day 7. Mice then received B. subtilis R0179 or PBS daily days 7-14. Treatment groups

include B. thetaiotomicron Trp D+ along with B. subtilis R0179, B. subtilis R017 alone,

or PBS. Motility assays were performed on day 0 (pre-treatment baseline), day 7, and day

14. Image created using BioRender.com.

39
Experiment 3: B. thetaiotomicron WT and B. subtilis R0179

This experiment was completed to compare the effects of administering both B.

thetaiotomicron WT and B. subtilis R0179 versus B. subtilis R0179 alone, as well as to

determine any differences between Trp decarboxylating B. thetaiotomicron and the wild-

type. Mice were organized into three treatment groups: B. thetaiotomicron WT and B.

subtilis R0179, B. subtilis R0179 only, and PBS. Mice receiving B. thetaiotomicron WT

and B. subtilis R0179 received B. thetaiotomicron WT on day 0 and day 7 via oral gavage

to establish colonization in the gut, while the other mice received PBS of the same

volume. All mice receiving B. subtilis R0179 were administered it via daily gavage days

7 through 14, while the PBS group received a PBS gavage instead. The motility assays

were performed on day 0 (pre-treatment baseline), day 7, and day 14 (Figure 12). Mice

were weighed on days 0, 7, and 14 to confirm no significant weight gain or weight loss

was occurring. Mice were euthanized on day 15 following the final motility assay.

40
Figure 12:

Timeline for Experiment 2. Mice received B. thetaiotomicron WT or PBS on day 0 and

day 7. Mice then received B. subtilis R0179 or PBS daily days 7-14. Treatment groups

include B. thetaiotomicron WT+ along with B. subtilis R0179, B. subtilis R017 alone, or

PBS. Motility assays were performed on day 0 (pre-treatment baseline), day 7, and day

14. Image created using BioRender.com.

Statistical Analysis

The results of the motility assays were assessed using paired t-tests via GraphPad

Prism Version 9 software. Paired t-tests were used because each mouse’s whole GI transit

and colonic motility after being administered bacterial treatment can be compared to their

own baseline. Unpaired t-test analysis was used to compare percent change in colonic

motility between bacterial treatment groups. A p-value of < 0.05 is considered statistically

significant.

41
 III. Results

B. thetaiotomicron Trp D+ alone does not enhance GI motility in conventional mice

Whole GI transit times were obtained for a control group of conventional mice

treated with PBS, a control group of mice treated with BHI medium, and an experimental

group treated with live B. thetaiotomicron Trp D+ cells (Figure 13). Mice were

administered these treatments once on day 0 and once on day 7 to ensure bacterial

colonization, and motility assays were performed on days 7 and 14. As mice were orally

gavaged B. thetaiotmicron suspended in BHI throughout this entire study, BHI was also

separately gavaged to serve as an additional control and ensure medium alone did not

elicit an effect on motility. There was no statistical significance between day 7 and day 14

transit times for the B. thetaiotomicron Trp D+ or BHI treatment groups. Interestingly,

there was significance for the PBS control group whole GI transit times however, this is

inconsistent with prior whole GI data obtained by the Mawe lab and is likely erroneous.

Colonic motility times were also obtained on days 7 and 14 (Figure 14). There were no

significant changes in colonic transit times between the three treatment groups.

Moreover, fecal water content between for mice treated with B. thetaiotomicron

Trp D+, BHI, or PBS were also assessed on days 7 and 14 (Figure 15). Mice treated with

B. thetaiotomicron Trp D+ had significantly drier stools compared to their baseline with

lower fecal water content. Lower fecal water content suggests stool being inside the colon

for a longer period of time, allowing for increased water absorption. Mice treated with

BHI or the PBS did not experience similar changes in fecal water content.

42
Mice colonized with B. thetaiotomicron Trp D+ combined with daily B. subtilis R0179

treatment do not have enhanced GI transit.

Whole GI transit times were obtained for a control group of mice treated with

PBS, an experimental group treated with one week B. subtilis R0179 spores alone, and an

experimental group pre-colonized with live B. thetaiotomicron Trp D+ cells in addition to

one week of daily B. subtilis R0179 spores (Figure 16). Mice given B. thetaiotomicron

Trp D+ cells were given so on days 0 and 7, while mice given B. subtilis R0179 spores

were given so daily on days 7-14. Mice receiving both B. thetaiotomicron Trp D+ and B.

subtilis R0179 had significantly increased whole GI transit times on day 14 compared to

day 7. There was no statistical significance between day 7 or day 14 transit times for the

B. subtilis R0179 or PBS vehicle control groups. Previous GI transit studies in the Mawe

laboratory with R0179 spores have indicated a decrease in transit times, however, colonic

motility times has been typically shown to have more significant results than whole GI

transit.

Colonic transit times were also recorded for the same groups of mice on days 7

and 14 (Figure 17a). Like what was described in Legan et al., 2023, mice administered B.

subtilis R0179 showed statistically faster colonic transit times compared to their baseline.

Mice administered both B. thetaiotomicron Trp D+ and B. subtilis R0179 also had

significantly faster colonic motility. However, comparing percent change from baseline

for the combined bacterial treatment group and the group receiving B. subtilis R0179

alone indicates no significant difference between the two groups, indicating that one

treatment group did not have significantly faster colonic transit times than the other

43
(Figure 17b). The PBS vehicle control group did not exhibit any statistically significant

changes in colonic motility.

Fecal water content was also compared on day 7 and day 14 for the three

treatment groups (Figure 18). Like the mice treated with B. thetaiotomicron Trp D+ alone,

mice treated with both B. thetaiotomicron Trp D+ and B. subtilis R0179 had significantly

lower fecal water content indicating drier stools. Mice treated with B. subtilis R0179

alone or PBS vehicle control did not demonstrate any statistically significant changes in

fecal water content. Taken together, combined B. thetaiotomicron Trp D+ and B. subtilis

R0179 did not elicit any prokinetic effects in conventional mice as hypothesized.

Mice colonized with B. thetaiotomicron WT combined with daily B. subtilis R0179

treatment do not have enhanced GI transit

To assess whether any changes in motility were due to the tryptamine-producing

property of B. thetaiotomicron Trp D+, the previous experiment was re-produced using B.

thetaiotomicron WT bacteria. Mice were administered B. thetaiotomicron WT on days 0

and 7 as well as daily B. subtilis R0179 on days 7-14, daily B. subtilis R0179 alone on

days 7-14, or a PBS vehicle control. Whole GI transit times were obtained on days 7 and

14 (Figure 19). There were no statistically significant differences in day 7 vs day 14 GI

transit times in all three treatment groups.

Colonic transit times were also obtained on day 7 and on day 14 for the three

treatment groups (Figure 20). As described in Legan et al., 2023, mice treated with a one

week daily treatment of B. subtilis R0179 had significantly faster colonic motility times

44
compared to their baseline. However, mice treated with B. thetaiotomicron WT and B.

subtilis R0179 did not have any statistically significant changes in colonic motility.

Lastly, fecal water content for the B. thetaiotomicron WT combined with B.

subtilis R0179, B. subtilis R0179 alone, and PBS vehicle treatment groups were assessed

on days 7 and 14 (Figure 21). There was no statistically significant change in fecal water

content in all three treatment groups.

45
Figure 13:

Treatment with B. thetaiotomicron Trp D+ (n=7) or BHI medium (n=7) did not affect

whole GI motility. Paired t-tests indicate that transit times for B. thetaiotomicron Trp D+

and BHI are not statistically significant (B. thetaiotomicron Trp D+ p=0.4408, BHI

p=0.2245). The PBS control group (n=7) was significant (p=0.0059), however, this is

inconsistent with previous data obtained by the Mawe lab.

46
Figure 14:

Treatment with B. thetaiotomicron Trp D+ (n=7), BHI (n=7), or PBS (n=7) did not affect

colonic motility. Paired t-tests indicate no statistical significance in colonic transit times

(B. thetaiotomicron Trp D+ p=0.4926, BHI p=0.8747, PBS p=0.5339).

47
Figure 15:

Treatment with B. thetaiotomicron Trp D+ (n=7) revealed significantly lower fecal water

content via paired t-test analysis (p=0.0248). Mice treated with BHI (n=7) or PBS (n=8)

did not have any statistically significant differences in fecal water content (BHI

p=0.3782, PBS=0.1021).

48
Figure 16:

Mice treated with for B. thetaiotomicron Trp D+ and B. subtilis R0179 combined (n=8)

showed significantly slower whole GI transit times via paired t-test analysis (p=0.0389).

GI transit times for B. subtilis R0179 alone (n=7) and PBS (n=6) groups were not

statistically significant (B. subtilis R0179 p=0.1014, PBS p=0.3213).

49
Figure 17a:

Mice receiving B. thetaiotomicron Trp D+ and B. subtilis R0179 treatment (n=8) showed

accelerated colonic motility via paired t-test (p=0.0109). B. subtilis R0179 treatment

alone (n=7) also had statistically decreased colonic transit times (p=0.0182). There was

no change in colonic motility in the group receiving PBS (n=6) (p=0.8935).

50
Figure 17b:

There is no significant difference in colonic motility percent change from baseline for B.

thetaiotomicron Trp D+ and B. subtilis R0179 combined treatment group (n=8) and B.

subtilis R0179 alone group (n=7). Both treatment groups demonstrated accelerated

colonic transit times, however, there was no significant difference when comparing

percent change via unpaired t-test analysis (p=0.4511).

51
Figure 18:

Mice treated with B. thetaiotomicron Trp D+ and B. subtilis R0179 combined (n=8) had

significantly lower fecal water content via paired t-test analysis R0179 (p=0.0015). B.

subtilis R0179 alone and PBS treatment groups did not have any statistically significant

changes in fecal water content (B. subtilis R0179 p=0.7113, PBS p=0.1308).

52
Figure 19:

There were no significant changes in whole GI motility for mice treated with B.

thetaiotomicron WT and B. subtilis R0179 combined (n=7), B. subtilis R0179 alone

(n=8), or PBS vehicle control (n=8). Paired t-tests indicate that there are no statistically

significant changes in whole GI transit time in all three treatment groups (B.

thetaiotomicron WT and B. subtilis R0179 p=0.1757, B. subtilis R0179 alone p=0.2576,

PBS p=0.6835).

53
Figure 20:

Mice treated with one week of daily B. subtilis R0179 (n=8) showed accelerated colonic

motility via paired t-test (p=0.0281). However, mice treated with both for B.

thetaiotomicron WT and B. subtilis R0179 (n=6) as well as the PBS vehicle control group

(n=4) did not have any significant changes in colonic motility (B. thetaiotomicron WT

and B. subtilis R0179 p=0.3465, PBS p=0.2422).

54
Figure 21:

There was no significant change in fecal water content for mice treated with B.

thetaiotomicron WT and B. subtilis R0179 combined (n=6), B. subtilis R0179 alone

(n=8), or PBS (n=7). Paired Paired t-test analysis revealed no statistically significant

difference in any treatment group compared to their baseline (B. thetaiotomicron WT and

B. subtilis R0179 p=0.9693, B. subtilis R0179 p=0.0532).

55
 IV. Discussion

Several bacterial species, including B. subtilis R0179, have been discovered to

produce Trp, the precursory molecule for 5-HT, in vivo by encoding the gene Trp

synthase. Recent research conducted by the Mawe lab has discovered that administering

B. subtilis R0179 spores to mice elicited prokinetic effects by increasing colonic motility

(Legan et al., 2023). Moreover, Bacteroides thetaiotomicron Trp D+ is a commensal gut

bacterial species that has been genetically manipulated to synthesize Trp decarboxylase,

allowing it to convert Trp to tryptamine. Tryptamine has been discovered to promote

propulsive motility in mice by enhancing epithelial cell secretion and by activating 5-

HT4Rs (Bhattarai et al., 2018). In the current investigation, we aimed to determine

whether administering both B. subtilis R0179 and B. thetaiotomicron Trp D+ leads to the

conversion of Trp to tryptamine and therefore enhanced gastrointestinal motility.

Mice were given one of three separate bacterial treatments: B. thetaiotomicron

Trp D+ only, B. thetaiotomicron Trp D+ and B. subtilis R0179, and B. thetaiotomicron WT

and B. subtilis R0179. Whole GI transit time, colonic transit time, and fecal water content

was measured to assess any changes in gut motility before and after treatment.

Taken together, it does not appear that orally administering B. thetaiotomicron Trp

D+ elicits any prokinetic effects on conventional mice. In fact, mice treated with this

bacteria showed overall a lower fecal water content. A lower fecal water content may

indicate increased water absorption due to prolonged time in the colon. Stools with lower

water content is considered a hallmark characteristic of constipation. Furthermore, mice

treated with both B. thetaiotomicron Trp D+ and B. subtilis R0179 also had stools with an

overall lower fecal water content as well as increeased whole GI transit time.

56
 Interestingly, during tissue dissection following euthanasia, it was noted that

several mice treated with B. thetaiotomicron either Trp D+ or WT bacteria had bright-red

appearing ceca and fecal pellets (Figure 22). As described in the methods section of this

paper, carmine red is orally administered to mice during the whole GI transit assay. While

the red dye is typically expulsed from mice soon after, this dye was present at higher

levels in mice receiving B. thetaiotomicron over 24 hours after gavage. This could

suggest slower gastrointestinal motility in these mice. Of note, no mice receiving B.

subtilis R0179 or PBS had similarly red ceca.

Figure 22:

Several mice treated with B. thetaiotomicron had bright red ceca 24 hours following

carmine red for the whole GI motility assay (right). Mice treated with B. subtilis R0179

or PBS did not have similar appearing ceca (left).

57
 Despite having slower whole GI transit times and a lower fecal water content,

mice administered both B. thetaiotomicron Trp D+ and B. subtilis R0179 did have

significantly accelerated colonic transit times. One possible explanation for this

phenomenon could be explained by the procedure for the colonic motility assay itself. As

previously described, this experiment measures colonic transit times by rectally inserting

a small bead into a mouse and recording the time required for expulsion. If a mouse is

constipated, which is reflected via whole GI transit and lower fecal water content,

perhaps rectal/colonic stimulation would promote movement and therefore bead

expulsion, which would explain apparently accelerated transit times. However, this

cannot be definitively concluded. Moreover, it has been discovered that mice treated with

B. thetaiotomicron WT and B. subtilis R0179 do not exhibit any differences in whole GI

transit time, colonic motility, or fecal water content.

To further understand the combinative effect of B. thetaiotomicron Trp D+ and B.

subtilis R0179 on conventional mouse GI motility, one could look at various metabolite

levels within GI treatment. At the end of each motility assay on day 15 following

euthanasia, several tissue samples were collected including serum, duodenum, ileum,

proximal colon, distal colon, and cecum for further processing. These samples can be

processed in perchloric acid and analyzed using high performance liquid chromatography

(HPLC). Tissue Trp/tryptamine levels can be assessed to determine the amount of

metabolite that is produced by each bacteria, and how bacterial interaction may influence

the amount of metabolite produced. This can also provide insight as to where the most

Trp/tryptamine synthesis and metabolism is occurring within the GI tract. Moreover,

levels of 5-HT and its breakdown product, 5-HIIAA, can also be assessed to learn how

58
the conversion of Trp to tryptamine may influence 5-HT signaling. HPLC metabolite

analysis may help explain how B. thetaiotomicron Trp D+ and B. subtilis R0179

administration is directly affecting GI motility.

While it has been considered B. thetaiotomicron Trp D+ synthesizing tryptamine

may cause slower GI motility, this may be unlikely given the current understanding of

tryptamine’s role in activating 5-HT4Rs and therefore prokinetic effects (Bhattarai et al.,

2018). It is more likely that B. thetaiotomicron treatment itself may have constipating

effects.

On further investigation, there have been numerous reports linking B.

thetaiotomicron with constipation. A recent study has discovered that patients suffering

from constipation have a more abundant colonic mucosal Bacteroidetes population than

individuals without (Ge et al., 2017). More specifically, a study analyzing constipation in

patients receiving peritoneal analysis found an increased B. thetaiotomicron presence in

stool samples compared to the control (Peng et al., 2023). One possible explanation for

this phenomenon is that B. thetaiotomicron have a cell-envelope-associated multi-protein

system referred to as the “starch utilization system”. This system may produce excessive

organic acids in the colon, which may result in abdominal distension and constipation

(Zhao & Yu, 2016; Lammerts van Bueren et al., 2015).

Conversely, it has also been identified that B. thetaiotomicron improves colonic

ENS innervation and increases propulsive contraction frequency in germ-free mice

(Aktar et al., 2020). It is clear there are several avenues of future research to further

understand B. thetaiotomicron and its potential role in constipation/colonic motility.

59
 Taken together, this study demonstrates that a genetically manipulated B.

thetaiotomicron capable of converting tryptophan to tryptamine via tryptophan

decarboxylation may not be an appropriate adjunct to probiotic formulations. While B.

subtilis R0179 has been identified to promote colonic motility through tryptophan

production, it does not appear that combining it with B. thetaiotomicron Trp D+ enhances

its prokinetic effects.

V. Works Cited

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(2020). Human resident gut microbe Bacteroides thetaiotaomicron regulates
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1757. doi:10.1080/19490976.2020.1766936

Bhattarai, Y., Williams, B. B., Battaglioli, E. J., Whitaker, W. R., Till, L., Grover, M., . . .
Kashyap, P. C. (2018). Gut Microbiota-Produced Tryptamine Activates an
Epithelial G-Protein-Coupled Receptor to Increase Colonic Secretion. Cell Host
Microbe, 23(6), 775-785 e775. doi:10.1016/j.chom.2018.05.004

Carlton B. C. (1967). Transformation mapping of the genes controlling tryptophan

biosynthesis in Bacillus subtilis. Journal of bacteriology, 94(3), 660–665.

Cryan, J. F., O'Riordan, K. J., Cowan, C. S. M., Sandhu, K. V., Bastiaanssen, T. F. S.,
Boehme, M., . . . Dinan, T. G. (2019). The Microbiota-Gut-Brain Axis. Physiol
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Evans, B. W., Clark, W. K., Moore, D. J., & Whorwell, P. J. (2007). Tegaserod for the
treatment of irritable bowel syndrome and chronic constipation. Cochrane
Database Syst Rev(4), CD003960. doi:10.1002/14651858.CD003960.pub3

Ge, X., Zhao, W., Ding, C., Tian, H., Xu, L., Wang, H., . . . Li, N. (2017). Potential role
of fecal microbiota from patients with slow transit constipation in the regulation
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Hoffman, J. M., Tyler, K., MacEachern, S. J., Balemba, O. B., Johnson, A. C., Brooks, E.
M., . . . Mawe, G. M. (2012). Activation of colonic mucosal 5-HT(4) receptors
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Lammerts van Bueren, A., Saraf, A., Martens, E. C., & Dijkhuizen, L. (2015).
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Legan, T. B., Lavoie, B., Norberg, E., Ley, I. C., Tack, S., Tompkins, T. A., . . . Mawe,
G. M. (2023). Tryptophan-synthesizing bacteria enhance colonic motility.
Neurogastroenterol Motil, e14629. doi:10.1111/nmo.14629

Mawe, G. M., & Hoffman, J. M. (2013). Serotonin signalling in the gut--functions,

dysfunctions and therapeutic targets. Nat Rev Gastroenterol Hepatol, 10(8), 473-
486. doi:10.1038/nrgastro.2013.105

Peng, Y., Zeng, Y., Zheng, T., Xie, X., Wu, J., Fu, L., . . . Wang, L. (2023). Effects of
Tiaopi Xiezhuo decoction on constipation and gut dysbiosis in patients with
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Zhao, Y., & Yu, Y. B. (2016). Intestinal microbiota and chronic constipation.
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61
CHAPTER III: FINAL CONCLUSIONS AND FUTURE DIRECTIONS

I. Summary and Conclusions

The creation of various probiotic formulation is a mounting field of research with

many possible avenues. As the gut-brain-microbiome axis is becoming increasingly

understood, recent studies have postulated that bacterial metabolites may have beneficial

effects on the enteric nervous system and GI motility (Cryan et al., 2019). One specific

area that may be targeted microbially is the serotonin signaling system within the GI

tract.

Serotonin, abbreviated as 5-HT, is a major signaling molecule in the GI tract and

is involved heavily in intestinal motility. Several bacterial metabolites that may influence

enteric 5-HT signaling have been identified, including the essential amino acid

tryptophan. It has been discovered that orally administering a short daily course of Trp-

synthesizing bacteria, Bacillius subtilis strain R0179, to conventional mice results in

increased colonic motility (Legan et al., 2023). Another bacterial metabolite thought to

modulate 5-HT signaling in the gut is tryptamine, a biogenic amine that can be produced

by the decarboxylation of Trp. When bound to 5-HT4Rs, tryptamine can mimic the effects

of 5-HT by inducing propulsive motility and colonic mucus secretion. A recent study has

identified a genetically manipulated strain of Bacteroides thetaiotomicron capable of

producing Trp decarboxylase, termed B. thetaiotomicron Trp D+. Germ-free mice

colonized with B. thetaiotomicron Trp D+ have accelerated GI transit times (Bhattarai et

al., 2018).

The aim of this study was to determine whether providing both B. subtilis R0179

and B. thetaiotomicron Trp D+ to conventional mice enhances the prokinetic effects of B.

62
subtilis R0179. In this investigation, mice were either pre-colonized with B.

thetaiotomicron Trp D+, B. thetaiotomicron WT, or administered a vehicle control. They

were then provided a seven-day treatment of B. subtilis R0179 spores or a vehicle

control. Whole gut transit times, colonic motility times, and fecal water content were

measured pre- and post-treatment and used to assess changes in gut function.

As described in Legan et al., 2023, mice administered B. subtilis R0179 alone did

have significantly faster colonic transit times compared to their baseline and to the PBS

vehicle control. However, mice administered B. thetaiotomicron Trp D+ bacteria did not

experience similarly enhanced motility. Mice treated with B. thetaiotomicron Trp D+ and

B. subtilis R0179 had increased whole GI transit times and lower fecal water content,

consistent with constipation. These mice did show improved colonic transit time,

however, it is possible that rectal stimulation from the colonic motility assay encouraged

bead expulsion from constipated mice instead of true accelerated colonic motility. Taken

together, it can be concluded that B. thetaiotomicron Trp D+ may not be an appropriate

adjunct to probiotic formulations including B. subtilis R0179 to modulate 5-HT signaling.

Further investigation is required to fully understand B. thetaiotomicron Trp D+’s role in

gastrointestinal motility.

II. Future Directions

Tissue 5-HT/5-HIAA Analysis

Following mouse euthanasia on day 15 of bacterial treatment, several tissue

samples were collected including serum, duodenum, ileum, proximal colon, distal colon,

and cecum. These tissues can be analyzed via high-performance liquid chromatography

63
(HPLC) to measure 5-HT, 5-HIAA (metabolic product of 5-HT), Trp, and tryptamine

content. Doing so may help us understand the conversion of Trp to tryptamine as well as

5-HT synthesis and breakdown in mice treated with B. thetaiotomicron Trp D+.

Uncovering these metabolite levels may provide insight on B. thetaiotomicron Trp D+’s

direct impacts on enteric 5-HT signaling.

Alternative Methods for Mouse Tryptamine Administration

It is possible that the bacterium B. thetaiotomicron may be directly impacting

gastrointestinal motility itself instead of the actions of tryptamine. To directly understand

tryptamine and its prokinetic effects in combination with B. subtilis R0179, different

routes of mouse administration may be considered. For example, mice can be provided

tryptamine directly in drinking water or via oral gavage along with B. subtilis R0179

treatment. Another potential avenue would include genetically manipulating B. subtilis

R0179 or another bacterial strain commonly used in probiotic formulations to synthesize

Trp decarboxylase. Certain strains of Latilactobacllus curvatus, including L. curvatus

LFC-1 and L. curvatus WSV-1, have also been identified to produce Trp decarboxylase

and can be administered to mice (Bover-Cid et al., 2001; Latorre-Moratalla et al., 2010).

These different avenues of tryptamine administration in conventional mice may all elicit

interesting effects on GI motility.

Tryptophan Decarboxylase Inhibition

To further assess how the conversion of Trp to tryptamine may impact GI motility,

mice can be administered a Trp decarboxylase inhibitor along with bacterial treatment.

64
Alpha-monofluoromethyldopa is a known amino acid decarboxylase inhibitor. In future

experiments, this drug can be orally given to mice along with B. subtilis R0179 alone and

with B. thetaiotomicron Trp D+. Motility assays including whole GI transit time, colonic

transit time, and fecal water content can be repeated before and after treatment to

understand the effects of Trp/tryptamine metabolism on gut motility.

Probiotic Effects on Host Gut Microbial Diversity

Preliminary data collected by the Mawe lab has suggested that B. subtilis R0179

administration may influence host gut microbial diversity. As B. subtilis R0179 is

expected to be transient when used as a probiotic additive and pass through the GI tract

quickly, it is important to understand its effect on the host gut microbiome. To assess this,

fecal samples were collected from mice before and after seven-day daily treatment with

B. subtilis R0179 or with PBS. Fecal DNA from these samples was extracted using a

QIAamp Fecal Pro DNA kit and were sent to Mark Band at the University of Illinois for

16S sequencing. 16S data analysis was kindly completed by Theresa Montgomery, a

post-doctoral fellow at the University of Vermont, using QIIME2 and R analysis

platforms.

16S analysis revealed that alpha diversity, or how species richness within each

group differs, is significantly reduced by experimental treatment with B. subtilis R0179

(Figure 23). B. subtilis R0179 treatment did not impact beta diversity, or how species

richness between each group differs (Figure 24).

Opposed to B. subtilis R0179, B. thetaiotomicron is a commensal gut bacterial

species capable of host gut colonization (Wexler, 2007; Bechon et al., 2022). Similar 16S

65
analysis can be completed before and after treatment with B. thetaiotomicron Trp D+, B.

thetaiotomicron WT, and treatment in combination with B. subtilis R0179 to understand

any treatment effects on the host gut microbiome.

Figure 23:

The three major metrics for species diversity suggest that one week daily experimental

treatment of B. subtilis R0179 significantly reduced alpha diversity in the microbiome.

Image courtesy of Theresa Montgomery, University of Vermont.

66
Figure 24:

The three major metrics for species diversity suggest that one week daily experimental

treatment of B. subtilis R0179 does not significantly affect beta diversity in the

microbiome. Image courtesy of Theresa Montgomery, University of Vermont.

III. Concluding Remarks

While B. thetaiotomicron Trp D+ may not be the most effective bacteria when

combined with B. subtilis R0179 to enhance gastrointestinal motility, this study has

provided valuable insight on how to improve probiotic formulations and how substantial

the gut-brain-microbiome axis truly is. Overall, this experience has taught me so much on

both the enteric nervous system and bacteria. I have learnt about how to design and

perform a scientific experiment, troubleshoot when needed, perform bacterial culturing,

complete animal care and management, become a better scientific writer, and much more.

I am so grateful for the opportunities that this project and the Mawe lab have provided

me.

67
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