The Impact of Oral Antibiotics on Lactobacillus Populations: A Potential Risk Factor for

Depression

Mihret Gebru

Dorothy Perkins

Central Magnet School

Dr. Mary Farone

Dr. Melanie Thomas

Mr. Gene Cowart

Mrs. Eve Harrison

May 2019
To my mom, who regardless of her own struggles, constantly helped me through mine.
To my dad, who always made a way to ensure that I got everything I needed.

ii
 ACKNOWLEDGEMENTS
I would like to extend my deepest gratitude to my mentor, Dr. Mary Farone, who

provided all the resources and gave such a huge amount of her time in helping me

navigate through this investigation. This thesis could not have been completed without

her guidance. Also, I would like to extend greatest appreciation to my Field of Study

advisor, Mrs. Eve Harrison, as well as my additional thesis advisors, Mr. Gene Cowart

and Dr. Melanie Thomas, who have each helped me greatly with providing countless

tools and tips in making this thesis easier to accomplish. Finally, I would love to thank

my thesis partner, Dorothy Perkins, who got me through the stresses and triumphs of this

thesis.

iii
 ABSTRACT
The gut-brain axis is a recent discovery demonstrating a strong connection
between the gastrointestinal tract and the brain in which one can influence the other and
vice versa. Many studies have been conducted in better understanding the complexity of
this bond in that a lack of Lactobacillus can increase the risk of developing depression.
This study aims on delving further on the function of the gut-brain axis, specifically
Lactobacillus and its relation to depression, but in relation to antibiotics. The growing use
of antibiotics constantly begs the question of its impact on the bacterial composition of
the gastrointestinal tract. Bacterial composition has shown a link to the development of
certain mental health issues, so antibiotics could possibly play a role. The experimental
procedure of this study focused on Colistin and Erythromycin with the hypothesis being
that use of those antibiotics would result in a decrease of Lactobacillus and increased
depression susceptibility. All the bacteria were put in a mixed culture to mimic the
environment of the gastrointestinal tract and to help created competition to see the
different growth of the bacteria.

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 TABLE OF CONTENTS

LIST OF TABLES AND FIGURES................................................................................. vii

INTRODUCTION .............................................................................................................. 1
Research Question ........................................................................................................... 1
Research Purpose ............................................................................................................ 1
Background Information ................................................................................................. 2
Possible Treatments or Solutions .................................................................................... 4
Hypothesis ....................................................................................................................... 4
REVIEW OF LITERATURE ............................................................................................. 5
Gut Bacteria..................................................................................................................... 5
The Development of Depression ..................................................................................... 6
The Gut-Brain Axis ......................................................................................................... 7
Dietary Intake Affects the Composition of Gut Bacteria ................................................ 9
The Growing Use of Antibiotics and Its Effects on Bacteria Composition .................. 10
Methodology ................................................................................................................. 11
METHODOLOGY ........................................................................................................... 13
Apparatus/Materials ...................................................................................................... 13
Procedures for Phase One ............................................................................................. 15
Procedure for Phase Two .............................................................................................. 16
RESULTS ......................................................................................................................... 22
Trial One ....................................................................................................................... 23
Phase One .................................................................................................................. 23
Phase Two.................................................................................................................. 24
Trial Two ....................................................................................................................... 29
Phase One .................................................................................................................. 29
Phase Two.................................................................................................................. 31
Analysis of Data ............................................................................................................ 36
DISCUSSION ................................................................................................................... 40
CONCLUSIONS............................................................................................................... 42

v
References ......................................................................................................................... 43
Appendices ........................................................................................................................ 46
Appendix 1: Trial One Raw Colony Counts ................................................................. 47
Tube 1 ........................................................................................................................ 47
Tube 2 ........................................................................................................................ 47
Tube 3 ........................................................................................................................ 48
Appendix 2: Trial Two Raw Colony Counts ................................................................ 49
Tube 1 ........................................................................................................................ 49
Tube 2 ........................................................................................................................ 49
Tube 3 ........................................................................................................................ 50

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 LIST OF TABLES AND FIGURES
Table 1. The diameters of the inhibition zones of the bacteria of Various Oral Antibiotics

........................................................................................................................................... 23

Table 2. The Diameter Measurements of the Inhibition Zones for the Lactobacillus Strain

in Trial One and the Lactobacillus Strain for Trial Two .................................................. 30

Table 3. The Descriptive Statistics of L. rhamnosus for Trial One .................................. 37

Table 4. Paired Samples T-Test for Trial One L. rhamnosus ........................................... 37

Table 5. Descriptive Statistics for Trial Two L. rhamnosus Colony Counts .................... 38

Table 6. Paired Samples T-Test for L. rhamnosus............................................................ 39

Figure 1. The Dilution Method Used for E. faecalis and E. coli in Phase Two................ 18

Figure 2. The dilution method used for the FTM tubes of the mixed bacterial culture. ... 20

Figure 3.Trial One L. rhamnosus Colony Counts ............................................................. 24

Figure 4. Trial One E. faecalis Colony Counts ................................................................. 25

Figure 5. Trial One E. coli Colony Counts ....................................................................... 26

Figure 6. Trial One C. sporogenes Colony Counts ........................................................... 27

Figure 7. Unaltered Average Colony Counts .................................................................... 28

Figure 8. Altered Average Colony Counts ....................................................................... 29

Figure 9. Trial Two L. rhamnosus Colony Counts ........................................................... 31

Figure 10. Trial Two E. faecalis Colony Counts .............................................................. 32

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Figure 11. Trial Two E. coli Colony Counts .................................................................... 33

Figure 12. Trial Two C. sporogenes Colony Counts ........................................................ 34

Figure 13. Unaltered Average Colony Counts .................................................................. 35

Figure 14. Altered Average Colony Counts ..................................................................... 36

ii
 INTRODUCTION

Research Question

How do antibiotics affect the likelihood of developing depression through the

disruption of the microbiome?

Research Purpose

Mental health has become a prevalent topic in today’s society, especially the

various factors that influence an individual's mental stability. Recently, studies have

found that there is a strong connection between the brain and gastrointestinal tract in that

shifts within one organ can influence the other, demonstrating that there is a

physiological aspect in determining a person’s mental health. Through this study, the goal

is to contribute to the conversation of this gut-brain axis and to identify how the

disruption of the gut bacteria, specifically Lactobacillus, due the use of antibiotics can

affect the brain. Demonstrating this correlation between the brain and gut can clearly

convey the intersection of psychology and medicine, two fields that are often seen to be

completely independent of each other. In addition, showing that antibiotics have a

detrimental effect on an individual's physical health but also mental health may cause

patients to be more wary and conscious of their usage of antibiotics, especially in an age

where the overuse and misuse of antibiotics has become a widespread issue.

Furthermore, the study may be a way to explore new methods of the assessment of

mental health as well as the development of a more microbial treatment for depression

and possibly other mental health issues.

Background Information

In recent studies, researchers have identified a direct connection between the brain

and the gastrointestinal (GI) tract in the human body. According to the Cleveland Clinic

(2016), this connection, known as the gut-brain axis, occurs due to the enteric nervous

system, an extension of the central nervous system that is located along the digestive

tract. Like the central nervous system, the enteric nervous system utilizes a network of

nerves, neurons, and neurotransmitters. As a result, it acts in a similar way and

communicates with the brain and spinal cord, which explains the reason the gut is often

referred to as the “second brain.” An instance exemplifying this connection is the

common “butterflies in the stomach” feeling when encountering something that causes an

individual to be nervous or excited (Cleveland, 2016). This feeling is prompted by the

emotions that are occurring within the brain that are inducing changes within the

digestive tract as a result of the communication between the two nervous systems.

Reversely, the digestive tract can also influence changes within the brain.

Delving further into the complexity of the gut-brain axis, research has looked into

identifying the neural aspects of prominent bacteria in the GI tract in finding that some of

the bacteria release specific neurotransmitters that possess functions similar to the ones

located in the brain. For instance, Bifidobacterium releases Gamma-aminobutyric acid

(GABA), which is a neurotransmitter involved in controlling the firing of neurons but

also helps in the body’s response to stress (Kohn, 2015). Bacillus releases dopamine, a

neurotransmitter that is deemed as the “feel good” hormone and is responsible for

reward-motivated behavior (Kohn, 2015). With this information, studies are being

conducted to understand the effects of the bacteria, especially Lactobacillus, on mental

2
stability. This rod-like bacterium that usually inhabits areas of the digestive tract is shown

to release GABA as well as acetylcholine, which both are shown to play influential roles

in reducing the effects anxiety, stress, but especially depression (Miller, 2016). For

instance, one study, conducted by researchers of Pennsylvania State University, found

that when mice were given Lactobacillus, their depressive-like symptoms were reduced

(Miller, 2016). Similar studies that have been conducted by other institutions have

confirmed the same results. Lactobacillus acts like an antidepressant, and many other

studies demonstrate this strong connection between the brain and gut but also as a

possible means to ease mental health issues.

Along with expanding the knowledge in which strains of bacteria affect certain

parts of the brain, an area of research is looking further into understanding different

factors that affect the bacterial equilibrium. Nutrition has been a popular topic, such as

yogurt that often contains Lactobacillus and other probiotics, as a method to improve gut

health and overall mood as shown by a study conducted by University College Cork’s

John Cray (Sanders, 2016). Cray found that the participants who ate yogurt infused with

probiotics like Lactobacillus had more blunted emotional responses compared to those

who did not (Sanders, 2016). However, while dietary consumption is continually being

researched, chemical consumption like antibiotics, which are as widely consumed as

food, has not been as extensively investigated on its effects in relation to the gut-brain

axis. The recent findings relating fluoroquinolones to detrimental effects on mental health

has been the only significant research relating to antibiotics and mental health (Pasternak,

2018). Even then, fluroquinolones are only related in regard to neurological damage that

can cause issues with movement rather than a focus on the development of mental health
3
issues. Antibiotics work to rid the body of harmful bacteria, but also, unintentionally, rid

the body of beneficial bacteria as well. Lactobacillus has been shown to be linked to

decreasing the risk of depression, and the lack of presence of the bacteria could possibly

lead to increased depressive symptoms.

Possible Treatments or Solutions

If antibiotics are shown to be weakening the gut through the disruption of

Lactobacillus, additional screening could be implemented in prescribing drugs to lessen

the overall risk. In addition, with this understanding of the relationship between

Lactobacillus and depression, a different branch of treatments for mental health issues

could be developed. By using a more biopsychological approach with the treatment for

mental health, a form of an immunological therapy could be used in which patients could

help ease their anxiety by restoring their bacterial equilibrium of Lactobacillus and other

depression-reducing bacteria in their gut.

Hypothesis

If Lactobacillus is treated with Erythromycin or Colistin, its population will

decrease in the mixed culture, possibly indicating an increase risk of depression.

4
 REVIEW OF LITERATURE

Gut Bacteria

Gut bacteria refer to the bacteria that is located along the lining of the

gastrointestinal tract. These microbes play a huge role in sustaining homeostasis within

the body as well as regulating metabolism. However, looking more closely, this region of

the GI tract, known as the gut microbiota, has its own role and function. Much research

has been conducted on gut bacteria in illustrating the unique properties of each species to

a point in which it appears that researchers have developed a solid foundation of

information regarding the gut bacteria. In recent years, however, more studies have

surfaced to dismiss this conclusion, demonstrating the functions of gut bacteria extending

beyond the physiological activities of homeostasis or metabolism and directly impacting

psychological activities as well. In “How Gut Microbes Influence Physiology,” Viviane

Callier, PhD discusses a study conducted by Sean Brady and Louis Cohen from

Rockefeller University in which they observed the ligands, molecules that are used to

bind to other molecules, of G-protein coupled receptors (GPCR), which are known to

play a role in the binding of different neurotransmitters in the brain. The researchers

found that these ligands were produced by a family of genes that also code for Nacyl

molecules in the gut bacterial genome. With a shown similarity between the genes used

to create GPCR used in the brain and Nacyl molecules in gut bacteria, the vital signaling

molecules of bacteria and signaling molecules of human cells are shown to have some

shared characteristics (Callier, 2018). This similar expression could demonstrate an active

role of gut bacteria in the changes of the overall human physiology.

5
 Furthermore, not all microbes in the human body have been identified. An

entirely new array of bacteria has been discovered as the result of different conditions,

such as suffering from an autoimmune system disease or going through a pregnancy. In

“Hidden Microbial Treasures,” Meenakshi Prabhune, Ph.D. delves further into the

discussion with the findings of Mark Kowarsky from Stanford University. Kowarsky and

his team utilized a method of detecting organ rejection in patients by identifying

circulating cell-free (cf) DNA, which is simply free-floating fragmented DNA, and

determining whether it was the patient’s DNA, the organ donor’s DNA, or microbial and

viral, nonhuman DNA using previously analyzed blood samples. To their surprise, nearly

“99% of the non-human DNA fragments circulating in our bloodstream did not match

any of those in the existing database...” (Prabhune, 2019). This finding is quite interesting

in that despite the extensive work that has been be done to identify microbes, there is still

much to be discovered just on the bloodstream itself. In addition, the later findings by

Kowarsky echoes what was stated in Brady and Cohen's study to establish the mass

diversity of the human microbiome (Callier, 2018). Due to this information, the

knowledge of gut bacteria is constantly growing and its influence impacts on human

physiology is greater than expected. The field of gut bacteria research has endless

discoveries of different roles of the gut microbiota and how its effects on the human body

as a whole.

The Development of Depression

Mental health has become a greater topic in today’s society, but the roots of its

development has not been clearly identified. However, depression is one mental health

condition that has been thoroughly studied with possible causes. Major depressive disorder
6
(MDD), or clinical depression, as defined by Mayo Clinic (2018) “is a mood-related

disorder that results in a prolonged feeling of sadness and emptiness, often characterized

by a loss of interest in pleasures, lack of energy, and can also exhibit anxiety or agitation.”

No simple root cause of depression can be solidified, but rather, there is a combination of

factors that lead to its development. For instance, social factors, such as family conflicts or

peer pressure, could be a risk factor in the progression of depression.

Also, in conjunction with social factors physiological and genetics roots have been

identified in playing role in the development of depression as well. In “The Development

and Course of Major Depressive Disorder,” Rashmi Nemade, Ph.D. emphasizes the

hereditary link of major depressive disorder in that those with a parent or sibling with

depression are two to four times more likely to develop it and that “40% of those with

MDD have a genetic link to the disorder.” Still some more research is being conducted in

understanding the development of other mental health issues with many indicating that

physiology and genetics could be a huge contributing factor.

The Gut-Brain Axis

In recent years, researchers have identified a connection between the brain and the

gastrointestinal tract, which has been deemed as the gut-brain axis. Obviously, a

connection between the brain and gut is not ordinary as the brain controls all the

functions of the organs in the body. However, the more fascinating point is just how

closely related the gut and brain are as well as the direct link between the two systems.

The brain secretes neurotransmitters, such as serotonin and dopamine, which then release

hormones affecting a person’s mental state whether that be their emotions or sense of

7
satiety in regards to hunger. However, the gut performs similarly in that it also secretes

neurotransmitters. These neurotransmitters come from different gut bacteria.

Due to this similarity, the brain and gut can communicate directly to each other.

This method of communication is established by the vagus channel, in which the

neurotransmitters from the brain can reach the gut and conversely, neurotransmitters from

the gut can reach the brain. As a result, the nervous system and immune system are

interconnected, which has been demonstrated through various instances. For example,

issues with mental health, which may be rooted by imbalances of the brain, could also

affect physical health. According to “When Gut Bacteria Change Brain Function,” Kohn

writes how studies have shown that people with autism were found to have a lack of

Bacteroides fragilis, which is a bacterium in the gut that is contributing factor to reducing

anxiety. Decreased levels of Bacteroides fragilis may result in an increased likelihood of

developing anxiety (Kohn, 2015). Furthermore, daily struggles that deteriorate an

individual’s mental health, such as stress and sleep deprivation, can also affect the

physical well-being as the body attempts to function at top capacity despite not having

enough energy.

Additionally, physical illnesses can induce mental health issues as a result of

stress that is put on the body to combat the illness but also due to the deeply connected

gut-brain axis. In “A Gut Feeling About Irritable Bowel Syndrome,” Lizzie Harrett

discusses a study by Emeran Mayer of the University of California, Los Angeles

demonstrates a correlational relationship between gut health issues and depression and

anxiety in that participants who suffered from Irritable Bowel Syndrome were more

8
likely to exhibit symptoms of anxiety and depression. This finding clearly exemplifies the

gut-brain axis. Similarly, in “Microbes and the mind: The bacteria in our guts may help

decide who gets anxiety and depression,” Laura Sanders mentions a study conducted by

psychiatrist Ted Dinan on the people of Walkerton, Canada who were getting ill from

their water that was overrun by Escherichia coli and Campylobacter because of a flood.

While most recovered, Dinan and his team found that the rates of depression actually

increased possibly due the bacterial strains in the water that now has inhabited their

microbiota (Sanders, 2016). Both studies establish the direct influence that bacteria can

have on mental health and the development of mental health issues. The results of these

studies can be supported by the gut -brain axis in the communication between the two

body systems that result in changes of mental and physical health.

Dietary Intake Affects the Composition of Gut Bacteria

While the health of the brain plays a huge role, the effects of the gut-brain axis are

heavily dependent on the composition of the gut bacteria, which are partially controlled

by dietary intake. However, there are also methods to improve the health of the

microbiota. Probiotics, such as those found in yogurt, have been shown to boost the

number of beneficial bacteria that is found in the microbiota. For instance, Selhub’s

“Fermented Foods, Microbiota, and Mental Health,” emphasizes ways to alter the gut

bacteria to mental state such as consuming probiotics. A lack of those vital gut bacteria

can be detrimental. In “Depression-fighting Bacteria,” Jesse Jenkins argues that the lack

of Lactobacillus is not only harmful in resulting in an unbalanced microbiome but also

causing an increase of the metabolite kyurriene, which can lead to increased depressive

symptoms. Furthermore, simply a high fat diet can lead to altered microbiome in that it
9
can cause the brain to be have reduced ability to rewire connections, resulting in anxiety-

like behavior (Jenkins, 2018).

The Growing Use of Antibiotics and Its Effects on Bacteria Composition

Along with food, antibiotics are just as frequently taken by people and have been

used constantly as a method to cure or alleviate the side effects of a disease. They work to

ride the body of harmful bacteria that may be found in the body, but often, rid the body of

beneficial bacteria. A current issue in today’s society is antibiotic resistance which has

sprouted from two different scenarios. One is the misuse of antibiotics, which extends

behind not using the correct medication but rather using it when it is not needed.

Antibiotics were created to combat illness that is a result of a bacterial attack in which it

can specifically target those foreign bodies. They cannot be used to treat viral illnesses

such as the flu in which viral DNA has hijacked and ingrained in the DNA of a human

cell. As mentioned by Elana Pearl Ben-Joseph, MD, in “The Danger of Antibiotic

Overuse,” the overuse of antibiotics has the ability to treat pneumococcal infections that

result in pneumonia, skin infections, meningitis, tuberculosis, and ear infections more

difficult.

On the opposite side of the spectrum is the misuse of antibiotics in not completing

the proper, prescribed of antibiotics in that once the symptoms are resolved there is no

need to take continue the course of the medication. However, this logic flawed in that

often times bacteria still continue to thrive and reproduce due to failure to the completion

of prescribed antibiotics. The bacteria can learn how to become resistant to the

antibiotics, so then when it is exposed to it again, they can survive. Eventually, the

10
bacteria can develop into a superbug and become multi-resistant to various classes of

antibiotics.

Antibiotics have shown to be capable of wiping out both good and bad bacteria in

other order to combat illnesses as a result of a foreign body residing in the body. As

mentioned, any intake whether that be dietary or medicinal can be an alteration to the gut

bacteria equilibrium. The impact of this action could possibly be detrimental to the vital

bacteria composition and equilibrium in the microbiome, which plays a major role in

maintaining homeostasis and regulating metabolism to allow the body to function

properly. However, furthermore, this gut bacterium has been linked to a connection to the

brain, inducing psychological effects such as the development of depression. So, the

analysis of the series of effects ranging from the consumption of antibiotics to the

changes of mental health to clear evaluate the possible side effects that can occur from

the antibiotic use, specifically its misuse or overuse.

Methodology

Much of the studies involving studying the gut-brain axis involve the use of

human subjects to analyze their behavior. For instance, one study was conducted with

twelve women who ate yogurt with probiotics and twelve women who ate yogurt with

bacteria, and the researchers analyzed their behavior and how it related to the gut-brain

axis (Sanders, 2016). Another method, however, is also analyzing the growth of bacteria

outside the human body and analyze the increase and decrease of its population in

responses to stimuli. The main growth medium that is used is an agar plate where the

bacteria can expand during its incubation. In order to control the population of the

11
bacteria, a spectrophotometer is also used. A spectrophotometer is a device in which it

passes light through the bacteria in order to determine a value of optical density, which is

the amount of light the bacteria can refract (Godbey, 2014). This optical density can be

helpful in understanding the concentration of bacteria in a cuvette but also ensure that the

bacteria that is grown on the agar plate can accurately be evaluated (Godbey, 2014).

In addition, within research of antibiotic resistance, inhibition zones are often

evaluated to understand the extent in which an antibiotic hinders the growth of the

bacteria. The zone of inhibition is a ring surrounding an antibiotic where there is no

bacterial growth (“Observing microbes,” n.d). The width of this area can help to

determine if the bacteria was resistant to the antibiotic in that it had a smaller inhibition

zone. A larger inhibition zone indicates that the bacteria is not resistant to the antibiotic

(“Observing microbes,” n.d). Measurements of inhibition can provide concrete evidence

of the behavior of gut bacteria. Along with inhibition zones and agar plates, the need for

oxygen of the bacteria is important to consider. The use of certain broths may be used to

help in determining the presence of oxygen in the experiment.

12
 METHODOLOGY

Apparatus/Materials

The investigation was divided into two phases. In order to understand the

connection between the effects of antibiotics on gut bacteria composition and possibly

mental health, the impact of antibiotics on bacteria individually must first be observed.

This analysis was done in Phase One in which an antibiotic sensitivity test was conducted

on each of the bacteria to better understand how their growth was affected by the

antibiotics. Using the results from Phase One, Phase Two involved developing a mixture

of the different types of bacteria and how their individual growth was affected by the

presence of other bacteria but also the antibiotics. The antibiotics used in Phase Two

were ones that caused the most infringement on the growth of Lactobacillus as shown by

the results of the antibiotics sensitivity tests in Phase One (shown in Table 1). The

effects of the chosen antibiotics, Erythromycin and Colistin, along with a control group

were each compared in a mixed culture that loosely resembles the environment of the

microbiota. This artificial gut microbiota was developed to observe whether there was an

increase or decrease of the other bacteria in response to a decreased Lactobacillus

population.

In regards to materials, various species of bacteria were used to carry out the

purpose of the study. First off, Lactobacillus (L. rhamnosus) was used due to the growing

research with its connection to decreasing the symptoms of depression as well as being

the primary focus of the study. In addition, common intestinal bacteria, specifically

Enterococcus faecalis (E. faecalis), Escherichia coli (E. coli), Clostridium sporogenes (C.

13
sporogenes), were also cultured and analyzed. The wide range of bacteria was utilized in

order to provide an adequate example of the bacterial diversity exhibited in the

microbiota. In addition, for Phase Two, the use of various types of bacteria was vital to

observe the competition between Lactobacillus and the other prominent gut bacteria.

Furthermore, different types of agar plates were used based on which would

provide the most ideal environment for the different bacteria to grow. In Phase Two,

MRS agar plates were used for Lactobacillus, BHI agar plates for C. sporogenes,

MacConkey agar plates for E. coli, and KF strep agar plates for E. faecalis. For

Lactobacillus and C. sporogenes, both are anaerobic bacteria, so GasPaks were used to

eliminate oxygen surrounding those plates. To test antibiotic sensitivity, the antibiotics

that were chosen is as follows: Amoxicillin-Clavulanic Acid (AMC), Colistin (CC2),

Clindamycin (CL10), Ciprofloxacin (CIP5), Erythromycin (E15), Ampicillin (AM10),

Minocycline (MI30), Sulfamethoxazole-trimethoprim (SXT), and Cefaclor (CEC30).

These antibiotics are some of the most common oral antibiotics used and therefore, are

considered to frequently interact with the microbiota and alter the gut bacteria

composition. To measure the bacterial content prior to plating, a spectrophotometer was

utilized.

For Phase One, the complete list of materials used were as follows: three brain

heart infusion agar plates for Lactobacillus, nine non-selective agar plates (three for C.

sporogenes, for E. faecalis, and for E. coli), twelve swabs, two GasPaks, GasPak

generators, four one mL pipettes, a pipettor, a spectrophotometer, cuvettes for

spectrophotometer, and four tubes each containing the bacterial cultures. For Phase Two,

14
the complete list of materials includes four tubes of the bacterial cultures, cuvettes for

spectrophotometer, 0.1 mL pipettes and pipettors, 0.9 mL TSB dilution blanks in snap-

cap tubes, sterile snap-cap tubes, additional TSB for dilution, tubes of sterile distilled

water, nine screw-cap tubes of 8.5 mL sterile FTM (three for the control and three for

each antibiotic treatment), eighteen MacConkey agar plates (six for the control, six for

each antibiotic treatment) , eighteen KF-Strep agar plates, eighteen BHI agar plates, 18

MRS agar plates, GasPaks, and GasPak generators.

Procedures for Phase One

In Phase One, the primary focus was on the individual species of bacteria and

analyzing the growth of bacteria outside the human body. An additional aim was to

analyze the increase and decrease of the bacterial populations in response to stimuli,

specifically common oral antibiotics. The main growth medium that was used is an agar

plate, which is a nutrient-based Petri dish where the bacteria can expand during its

incubation. For the Lactobacillus, brain heart infusion agar plates were used as it was

deemed to have more nutritional value for the bacteria and better suited to help with its

growth. In order to control the population of the bacteria on each plate, a

spectrophotometer was also used. With each species, one microliter of the bacteria was

placed in a cuvette from a prepared sample tube to measure the concentration of bacteria.

The ideal concentration ranges between .4 and .6.

Once the concentration was achieved, the bacteria was extracted with a swab and

streaked vertically, horizontally, and then diagonally in order to spread out the

concentration of the bacteria and isolate the colonies on three agar plates for each type of

15
bacteria. After the plates were streaked, three different oral antibiotics were placed on

each plate.

The plates were incubated at 36 ℃ for 24 hours. The sensitivity of the bacteria to

the antibiotics was recorded through the measurement of the diameter of the inhibition

zones surrounding the antibiotic disk. A larger inhibition zone diameter indicates an

inhibition of bacterial growth in response to the antibiotics. In other, having a larger zone

of inhibition demonstrates that the antibiotic was effective in keeping the bacteria from

growing. A smaller or even a lack of an inhibition zone indicates the bacteria’s resistance

to the antibiotics.

The information of the antibiotic sensitivity test determined which antibiotics were

to be used in Phase Two (refer to Table 1 in Results). According to the data, the antibiotics

that produced these results were Colistin (CC2) and Erythromycin (E15).

Procedure for Phase Two

For Phase Two of the investigation, the focus was to analyze how the growth of

Lactobacillus as well as the other intestinal bacteria was affected by the antibiotics in a

setting that offered a resemblance to the environment created in the gastrointestinal tract.

In order to do so, Lactobacillus, E. faecalis, E. coli, and C. sporogenes had to be mixed

together and exposed to the antibiotics separately, which was done through a plate

culture.

Prior to the experiment, the Lactobacillus was inoculated, or implanted, into MRS

broth for 48 hours at 37 ℃. E. coli and E. faecalis were inoculated into tryptic soy broth

(TSB) for 24 hours prior to the experiment. C. sporogenes was also inoculated into Fluid

16
Thioglycolate medium (FTM) Brewer’s medium at 37 ℃ for 24 hours prior to the

experiment. In addition, 8.5 mL of sterile FTM broth tubes were prepared, in which three

were assigned to the control, the Colistin antibiotic treatment, and the Erythromycin

antibiotic treatment.

For the experiment, a spectrophotometer was first used. A spectrophotometer

blank cuvette with 1 mL of TSB was run through the machine to obtain an optical density

of 600 nm to 0 nm. Once this value was obtained, .9 mL of the sterile TSB was added to

four cuvettes. Then, .1 mL of the bacterial cultures were added to one cuvette. Once each

bacterium was put in a cuvette, the optical density was observed again and adjusted to

obtain the ideal values for each type of bacteria. For E. coli and E. faecalis, 0.08-0.12 is

the range needed for 1-1.5x 10^8 cells/mL. C.sporogenes required 0.08-0.12 for

concentration of approximately 2.6 x10^6 cells/mL. For Lactobacillus, the optical density

needed is .38-.42 for a concentration of approximately 1.5x10^8 cells/mL. Dilutions, or

the gradual changes to the bacterial concentrations, used to obtain the ideal optical

density of each type of bacteria were recorded in order to keep them sterile.

Once the optical densities have been adjusted in the cuvettes, the bacteria

underwent serial dilutions. The information from the previous dilutions regarding the

appropriate concentration of each bacterium needed was used to determine how many

serial dilutions would be conducted for each type of bacteria. These calculations were

used to reproduce similar results of the previous dilutions with the use of sterile snap-cap

tubes and sterile TSB. Through the use of .9 mL TSB blanks in snap tubes, E. faecalis

and E. coli were diluted as follows:

17
 Figure 1. The Dilution Method Used for E. faecalis and E. coli in Phase Two

Figure 1, developed by the author’s mentor, Dr. Mary Farone, illustrates how .1 ml is

transferred from the first (sample) tube into a .9 ml tube. Then, .1 ml of the second tube is

transferred into another .9 ml tube.

First, .1 mL of the bacteria from the cuvette was added to the .9 mL TSB, creating

a 1: 10 dilution and diluting the bacteria 1x10^7 cells/mL. Then, with a new pipette, .1

mL of the TSB and bacteria mixture was added to another .9 mL TSB, creating a 1:10

and diluting the bacteria to approximately 1x10^6 cells/mL. From there, .1 mL was taken

from the second serial dilution (the third tube) and added to the nine FTM tubes. At the

end of the serial dilution, a 10 mL volume mixture was created and ended with a 1 x 10^4

cells/mL as the final concentration amount. This process was individually done for E.

faecalis and E. coli.

18
 With Lactobacillus, it only needed to be diluted once, so .1 mL of the bacteria

(adjusted to the correct optical density) was added to a snap tube of .9 mL of TSB,

creating a 1:10 dilution and diluting the bacteria to approximately 1.5 to 10^7 cells/mL.

Unlike the other bacteria, .2 mL of the diluted Lactobacillus (instead of .1 mL)

was added to the FTM tubes to get a final concentration of approximately 3 x 10^5

cells/mL. C.sporogenes did not need to be diluted, so .1 mL of the bacterial culture was

directly added to the FTM tubes.

After the concentrations were set in the FTM broth tubes, an appropriate

concentration of Colistin was added to 3 tubes, an appropriate concentration of

Erythromycin was added to 3 tubes, and an appropriate concentration of sterile water was

added to the last 3 tubes. The sterile water served as the control group. The FTM tubes

were pipetted up and down once by inserting the pipette at the bottom to pull up 1.2 mL

of the mixture and then pushing 1.0 mL of the volume to avoid adding oxygen to the

broth. The last .2 mL of the volume was gently added to the top of the broth. The snap

tubes, with screw tops to keep out the oxygen, were all incubated for two hours at 37 ℃.

While the tubes were being incubated, two .9 mL dilution tubes were arranged for each of

the nine FTM tubes, resulting in a total of eighteen dilution tubes. Also, various agar

plates were gathered and labeled with the type of agar plate, the treatment group, and the

appropriate dilutions. Once the 2 hour incubation period was finished, the tubes were

removed, and each tube was diluted as shown below.

19
 Figure 2. The Dilution Method Used for the FTM Tubes of the Mixed Bacterial Culture.

Figure 2, developed by the author’s mentor, Dr. Mary Farone, illustrates how .1

ml is transferred from the first (sample) tube into a .9 ml tube of TSB. Then, .1 ml of the

second tube is transferred into another .9 ml tube of TSB to achieve a 1:100 dilution, or 1

x 10 ^-2 concentration of bacteria.

After, .1 mL were taken from the appropriate tubes placed on the center of the

corresponding plates. Two plates were assigned for each tube used. So, for the

MacConkey plates, 0.1 mL of the FTM mix from the 1:10 and 1:100 dilution was placed.

For the KF strep plates, 0.1 mL of the FTM mix from 1:10 and 1:100 dilution was placed.

For the BHI agar plates, 0.1 mL was placed from the 1:10 dilution and 1:100 dilution. For

the MRS agar plate, .1 mL from the 1:10 dilution and 1:100 dilution was plated for the

control group, and .1 mL from the original FTM mix and the 1:10 dilution for the

antibiotic treatment groups.

20
 Then, the LAB and BHI plates were placed in GasPaks at 37 ℃, and the

MacConkey and KF Strep plates were placed at 37 ℃. After 48 hours of incubation, the

number of colonies was observed and counted. Some were too numerous to count, so

those were not included. For some plates with more clustered colonies, a machine was

used to be able to magnify the colonies and electronically keep hold of the counts of

colonies seen on the plates as each colony is tapped. Using this information, the number

of bacteria was calculated using the following formula:

Concentration of bacteria = colony number (CFU, colony forming units

1.1 mL x dilution of plate with countable colonies

21
 RESULTS

The investigation was divided into two phases and was repeated for an additional

trial. Phase One involved the antibiotic sensitivity tests of the Lactobacillus, E. faecalis,

C. sporogenes, and E. coli to observe the effects of the oral antibiotics on the growth of

the bacteria and to determine which was the most impactful on Lactobacillus but not as

effective on the other bacteria. Phase Two involved the mimicking of the gut

environment by creating a mix culture to allow for competition. In addition, Phase Two

allowed for observation of how the types of bacteria were affected by the Erythromycin

and Colistin, and how the decline of one type of bacteria could result in the increased

growth of another bacteria.

22
 Trial One

Phase One

Type of Antibiotic (Abbreviated)

Type of AMC CC2 CL10 CIP5 EI5 AM10 MI30 SXT CEC30

Bacteria

E. faecalis None None None 1.5 cm 1.4 cm 3.6 cm 1.3 cm None

E. coli None None 1.2 cm 4.0 cm None 2.2 cm 2.1 cm 2.8 cm 2.0 cm

C.sporogenes 5.3 cm 2.3 cm None 3.6 cm 1.9 cm 6.1 cm 3.4 cm none 1.7 cm

L.rhamnosus 3.0 cm 2.6 cm None 1.7 cm 3.0 cm 2.7 cm 3.4 cm none .8 cm

Table 1. The Diameters of the Inhibition Zones of the Bacteria of Various Oral Antibiotics

Table 23 represents the diameter measurements of the inhibition zones of the all the

bacteria that was tested in Phase One. Based on a greater inhibition zone for

Lactobacillus, Colistin and Erythromycin were chosen to be the variables to be tested in

Phase Two. In order to understand the competition between the types of intestinal

bacteria in a mixed culture with the decrease of Lactobacillus, the antibiotics that were

chosen were ones in which Lactobacillus had the largest inhibition zone while also not

being as detrimental to other bacteria (which is why MI30 was not used).

23
Phase Two

Figure 3.Trial One L. rhamnosus Colony Counts

The data from Figure 3 was taken from the MRS agar plates and represents the

number of colonies recorded for Lactobacillus in the control, Erythromycin, and Colistin

group across each tube in Phase Two. The Colistin group, with tube 1 having more than 2

times the colony count of the other tubes, shows to be an outlier compared to the data for

the control group and the Erythromycin group.

24
Figure 4. Trial One E. faecalis Colony Counts

The data from Figure 4 was taken from the KF Strep agar plates and represents

the number of colonies recorded for E. faecalis in the control, Erythromycin, and Colistin

group across each tube. Generally, the colony counts for E. faecalis decreased due to the

use of antibiotics, and Colistin completely eradicated the E. coli population in all tubes.

25
Figure 5. Trial One E. coli Colony Counts

The data from Figure 5 was taken from the MacConkey agar plates represents the

number of colonies recorded for E. coli in the control, Erythromycin, and Colistin group

across each tube. Generally, the colony counts for E. coli decreased due to the use of

antibiotics, and Colistin completely eradicated the E. coli population in all tubes.

26
Figure 6. Trial One C. sporogenes Colony Counts

The data from Figure 6 was taken from the BHI agar plates and represents the

number of colonies recorded for C. sporogenes in the control, Erythromycin, and Colistin

group across each tube. There was much variability with the colonies between each group

in which the control had an outlier in tube 1 and also an increase in bacterial growth in

tube two within the Erythromycin group.

27
Figure 7. Unaltered Average Colony Counts

The data in Figure 7 represents the average colony counts for the control,

Erythromycin, and Colistin group for each type of bacteria across the three tubes that

were used for each type.

28
Figure 8. Altered Average Colony Counts

Similar to Figure 7, the data in Figure 8 represents the average colony counts for

the control, Erythromycin, and Colistin group for each type of bacteria. However, this set

of data excludes outliers. For instance, the value of the average number of L. rhamnosus

colonies became less than the value of the average number of L. rhamnosus colonies in

the control.

Trial Two

Phase One

With Trial One resulting in a null hypothesis and the possibility of error to have

been a confounding variable, an additional trial was conducted in attempt to reduce the

effects of error as well as provide validity. The experimental setup and methodology in

Trial One and Trial Two were the same. However, the strain of Lactobacillus that was

used in Trial One was not used in Trial Two, but rather a strain known as ATCC 53101

29
 that was found to be more responsive to the antibiotics was used. In Trial Two, Phase

One only consisted of conducting an antibiotic sensitivity test for the new strain of

Lactobacillus.

Type of Antibiotic (Abbreviated)

AMC CC2 CL10 CIP5 E15 AM10 MI30 SXT CEC30

Trial 1 3.0 2.6 None 1.7 3.0 2.7 3.4 None .8 cm

Lactobacillus cm cm cm cm cm cm

Trial 2 4.3 3.9 N/A 2.9 5.3 3.4 4.4 .9 N/A

Lactobacillus cm cm cm cm cm cm cm

Table 2. The Diameter Measurements of the Inhibition Zones for the Lactobacillus Strain in Trial One and the
Lactobacillus Strain for Trial Two

Table 2 compares the diameter measurements of the inhibition zones for the

Lactobacillus strain in Trial One and the Lactobacillus strain for Trial Two that were

done through the antibiotic sensitivity test done during Phase One for both trials. The

measurements show a larger inhibition zones for the Trial Two Lactobacillus, indicating

a stronger response to antibiotics. CEC30 and CL10 were unavailable for the testing for

Phase One of Trial Two and therefore have been denoted as N/A in Table 2.

30
Phase Two

Figure 9. Trial Two L. rhamnosus Colony Counts

The data from Figure 9 was taken from the MRS agar plates and represents the

number of colonies recorded for Lactobacillus in the control, Erythromycin, and Colistin

group across each tube in Phase Two. The Colistin group was shown to have a slight

decrease in the number of Lactobacillus colonies counted. However, the Erythromycin

group had a more significant decrease over the tubes.

31
Figure 10. Trial Two E. faecalis Colony Counts

The data from Figure 10 was taken from the KF Strep agar plates and represents

the number of colonies recorded for E. faecalis in the control, Erythromycin, and Colistin

group across each tube. There was much variability with the colonies between each group

in which the control in which an increase in bacterial growth in tube two within the

Erythromycin group, but also, E. faecalis was completely eradicated in tube one of the

Colistin group.

32
Figure 11. Trial Two E. coli Colony Counts

The data from Figure 11 was taken from the MacConkey agar plates represents

the number of colonies recorded for E. coli in the control, Erythromycin, and Colistin

group across each tube in Phase Two. The treatment with Colistin was a bit of an outlier

in that the number of colonies reported for E. coli surpassed the number of colonies found

in the control group and the Erythromycin group. However, with the Erythromycin

group, the E. coli were greatly reduced with the population nearly being eradicated in

tube three.

33
Figure 12. Trial Two C. sporogenes Colony Counts

The data from Figure 12 was taken from the BHI agar plates and represents the

number of colonies recorded for C. sporogenes in the control, Erythromycin, and Colistin

group across each tube. There was much variability with the data with the Colistin having

the greatest number of C. sporogenes colonies recorded with the exception of tube two

despite the bacteria being treated with an antibiotic.

34
Figure 13. Unaltered Average Colony Counts

The data in Figure 13 represents the average colony counts for the control,

Erythromycin, and Colistin group for each type of bacteria.

35
Figure 14. Altered Average Colony Counts

Similar to Figure 13, the data in Figure 14 represents the average colony counts

for the control, Erythromycin, and Colistin group for each type of bacteria. However, this

set of data excludes outliers.

Analysis of Data

Since the main focus of the investigation is the effects of Erythromycin and

Colistin on the growth of Lactobacillus, only the results regarding Lactobacillus was

analyzed through descriptive statistics to understand the variation of the values and

evaluated for statistical significance through the use of a t-test.

36
Descriptive Statistics- Trial One L. rhamnosus Colony Counts

Control Erythromycin Colistin

Valid 3 3 3
Missing 0 0 0
Mean 166.0 168.0 218.3
Std. Deviation 51.39 17.78 186.0
Minimum 121.0 148.0 86.00
Maximum 222.0 182.0 431.0

Table 3. The Descriptive Statistics of L. rhamnosus for Trial One

The standard deviation of the colony counts varied the most in the Colistin group

compared to the others which is contributed the great range number of colonies found in

that group. The average colony counts, however, did not vary much between the control,

Erythromycin, and Colistin group, and the data overall did not exhibit much variation

other than the maximum and standard deviation for Colistin group.

Paired Samples T-Test- Trial One L. rhamnosus

t df p

Control - Erythromycin -0.063 2 0.956

Control - Colistin -0.667 2 0.574

Note. Student's t-test. A paired sample t-test of treatment groups

for L. rhamnosus Trial One.

Table 4. Paired Samples T-Test for Trial One L. rhamnosus

In order to exemplify statistical significance, the p-values for Erythromycin and

Colistin must be less than .05, which indicates that the hypothesis is valid. A p-value

37
greater than .05 results in a null hypothesis. Based on the p-values represented in Table 4,

the data of Trial One results in a null hypothesis.

Descriptive Statistics- Trial Two L. rhamnosus Colony Counts

Control Erythromycin Colistin
Valid 3 3 3
Missing 0 0 0
Mean 120.7 37.00 92.00
Std. Deviation 31.50 6.245 18.19
Minimum 101.0 30.00 71.00
Maximum 157.0 42.00 103.0

Table 5. Descriptive Statistics for Trial Two L. rhamnosus Colony Counts

The descriptive statistics presented in Table 5 demonstrates a greater variation of

the values for Erythromycin group compared to the control group and Colistin. While the

Colistin group had smaller mean value and maximum in comparison to the control group,

the Erythromycin group mean value of 37.00 and maximum value of 42.00 were

significantly lower, only equating to approximately one fourth of the control group mean

value of 101.0 and maximum value of 157.0. The decreased values in the Erythromycin

values indicates that the antibiotic may have greatly inhibited the growth of the

Lactobacillus.

Paired Samples T-Test- Trial Two L. rhamnosus

t df p

Control - Erythromycin 4.726 2 0.042

Control - Colistin 1.837 2 0.208

38
Note. Student's t-test. A paired t-test for L. rhamnosus Trial One.

Table 6. Paired Samples T-Test for L. rhamnosus

Similarly, to Trial One, the statistical significance based on whether the p-values

for Erythromycin and Colistin were less than .05, indicating the results were most likely

not due by chance and that the hypothesis is valid. Based on the p-values represented in

Table 6, the p-value of Colistin was over .05, but the p-value for Erythromycin was less

than .05. So, the hypothesis was not proven to valid for Colistin and was most likely due

to chance. However, for Erythromycin, the hypothesis was proven to valid.

39
 DISCUSSION
The overall goal of the investigation was to determine the effects of antibiotics,

specifically Erythromycin and Colistin, on Lactobacillus and how it affects its growth.

Through the gut-brain axis, a connection has been made with the presence of

Lactobacillus to reduce inflammation, which in turn reduces a risk of developing

depression. In addition, the bacterial composition of the gastrointestinal tract has been

discussed greatly in how changes in the gut could also affect the brain, so studying

antibiotics, which often alter bacterial composition, was the focus of the study. After

narrowing down Erythromycin and Colistin as having the most detrimental impact on the

bacteria, the hypothesis developed in that the use of Erythromycin and Colistin would

result in a decrease of Lactobacillus in the mix culture, which could correlate to a greater

depression susceptibility. While Colistin was not proven to be valid, Trial Two proved

that Lactobacillus was greatly reduced after being treated with Erythromycin, presenting

the possibility there could be some unknown effects of the use of Erythromycin,

specifically if used inappropriately.

Due to the complex nature of the topic of this study, there were several limitations

to the methodology as well as well as the validity of the results in its application to the

field of psychology and medicine. First, the methodology does not directly analyze

mental health but is rather connected to the topic of mental health based on the results of

the investigation and how that data relates to previous gut-brain axis research. In

addition, human subjects could not be used to truly understand the full effects of the

40
antibiotics on the bacterial composition in the gastrointestinal tract, which again explains

the reason for using an indirect methodology. With any study, time constraints are

always a limitation, and it was played a role in this study as well. Repetition of the

experiment would have been valuable for validity and accuracy, but short amount of time

only allowed for two trials. Since human subjects were not used, the environment of the

gastrointestinal tract had to be reproduced in the lab, which was not necessarily an

accurate representation because there are thousands of bacteria that still not been

discovered in the gut and other microbes and enzymes could affects the growth of the

bacteria as well. Also, the bacterial composition of the gut also varies from person to

person. Lastly, human error can occur and alter the results of the investigation, and it was

exhibited in this investigation with the possibility that self-poured agar plates may have

offered an excess or a lack of sufficient nutrients for the bacteria or ensuring that the snap

cap tubes containing the bacteria were flipped once to ensure the bacteria do not settle at

the bottom.

41
 CONCLUSIONS

Through this study, the focus was to identify the effects of antibiotics on

Lactobacillus and possibly being a risk factor for depression. However, there are many

topics engulfed in this investigation, such as the ongoing antibiotic resistance crisis.

While there have been several physical health conditions that can be caused by the

overuse or misuse of antibiotics, there is not much research regarding the mental effects.

By exploring the issues that can arise in mental health as a result of the inappropriate use

of antibiotics could possibly be one way to resolve the issue of antibiotics in today’s

society.

In addition, some believe mental health is only associated with psychology and

physical health with medicine. Psychology has always been seen as a “lesser science.”

However, the gut-brain axis proves that medicine and psychology are dependent on each

other, offering a new outlook on the two fields. Also, new and possibly more effective,

microbial treatment for mental health conditions could be developed with the indication

that gastrointestinal tract plays a major role in mental health. Future research in

understanding the gut-brain axis is endless from comparing the bacterial composition of

the gastrointestinal tract of people who have a mental health or observing how other

gastrointestinal problems can influence the risk of developing depression or stress. While

the results of this investigation do not necessarily equate to a causational relationship

with antibiotics and depression, it definitely offers more questions and opportunities to

delve further in understanding the complexity of the gut-brain axis.

42
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45
Appendices

46
Appendix 1: Trial One Raw Colony Counts

Tube 1

MRS agar KF strep agar MacConkey agar BHI agar

(Lactobacillus) (E. faecalis) (E. coli) (Clostridium)

Control 222 203 97 80

Erythromycin Treatment 174 67.2 23 21

Colistin Treatment 431 0 0 26

Tube 2

MRS agar KF strep agar MacConkey agar BHI agar

(Lactobacillus) (E. faecalis) (E. coli) (Clostridium)

Control 121 117 87 14

Erythromycin Treatment 182 36.9 38 36

Colistin Treatment 86 0 0 16

47
Tube 3

MRS agar KF strep agar MacConkey agar BHI agar

(Lactobacillus) (E. faecalis) (E. coli) (Clostridium)

Control 155 127 41 33

Erythromycin Treatment 148 57.1 21 20

Colistin 138 0 0 24

Treatment

48
Appendix 2: Trial Two Raw Colony Counts

Tube 1

MRS agar KF strep agar MacConkey agar BHI agar

(Lactobacillus) (E. faecalis) (E. coli) (Clostridium)

Control 104 264 175 137

Erythromycin Treatment 30 223 53 204

Colistin Treatment 103 0 485 312

Tube 2

MRS agar KF strep agar MacConkey agar BHI agar

(Lactobacillus) (E. faecalis) (E. coli) (Clostridium)

Control 101 313 285 143

Erythromycin Treatment 42 316 18 268

Colistin Treatment 71 341 381 312

49
Tube 3

MRS agar KF strep agar MacConkey agar BHI agar

(Lactobacillus) (E. faecalis) (E. coli) (Clostridium)

Control 157 337 267 337

Erythromycin Treatment 39 261 6 261

Colistin 102 267 449 267

Treatment

50
51
52