This document summarizes a study investigating horizontal gene transfer (HGT) of antibiotic resistance genes between gut bacteria under conditions mimicking the human gut. Through conjugation experiments, E. coli gained chloramphenicol resistance after co-culture with B. uniformis or E. fergusonii. A transformation experiment found that A. baylyi gained chloramphenicol resistance after exposure to Bacteroidetes DNA. The results suggest HGT of antibiotic resistance genes can readily occur between gut bacteria, and that chloramphenicol resistance genes may spread most easily. Further experiments are needed to confirm the transferred genes and understand factors influencing HGT frequency in the gut.

1. Horizontal Gene Transfer of Antibiotic Resistance Genes in the Human Gut
Sarah Maciorowski
Abstract
Horizontal gene transfer (HGT) of antibiotic resistance genes is of increasing interest
because of its impact on the evolution of bacteria. There are millions of genes and hundreds of
bacterial species in the human gut, so HGT likely occurs frequently in the gut microbiota. To test
HGT through conjugation, specific obligate and facultative anaerobes were co-cultured with E.
coli in anaerobic conditions then selected on antibiotic plates. To test HGT by transformation,
bacterial strains commonly found in the human gut microbiota were studied by pooling genomic
DNA and culturing recipient A. baylyi strains with the pooled DNA. After co-culturing antibiotic
sensitive E. coli with antibiotic resistant B. uniformis, E. coli gained the ability to grow in the
presence of the antibiotic Chloramphenicol. E. coli also grew in the presence of
Chloramphenicol when co-cultured with E. fergusonii. A. baylyi was similar in its growth in the
presence of Chloramphenicol when transformed with Bacteroidetes DNA. By studying and
experimenting with gut microbiota conditions, we can further understand the driving factors in
HGT of antibiotic resistance genes.
Introduction
In the human body, the ratio of bacterial cells to human cells and bacterial genes to
human genes is 10:1 and 100:1, respectively (Gill et al., 2006). The majority of the microbes that
are found in the human body are located in the gut (Liu et al., 2012). There are approximately
2.5 million bacterial genes making it a prime location to study horizontal gene transfer (HGT).
HGT is the exchange of genes between bacteria, independent of reproduction (Syvanen,
1994). The main mechanisms of HGT include transformation, transduction, and conjugation.
Transformation occurs when the bacterium intakes exogenous DNA from its surroundings
(Kaznowski and Wlodarczak, 1991). Transduction occurs when a virus injects DNA into the cell,
while conjugation is the exchange of genes between donor and recipient bacteria through direct
contact (Lederberg and Tatum, 1946). One particularly interesting class of genes that can be
exchanged through HGT is antibiotic resistance genes. While antibiotics target a certain area of
the cell to kill the bacteria or prevent it from replicating, antibiotic resistance genes encode
proteins allowing the bacteria to grow in the presence of an antibiotic (Bush, 1988).
Antibiotic resistance genes have become of increasing interest in the health field because
of their ability to alter the genome sequence of a bacterium and complicate treatment for
bacterial infections. Any bacteria that can collect resistance genes can become resistant to one or
more antibiotics. Bacteria acquire antibiotic resistance genes either through mutations or from
another bacterium through vertical or horizontal gene transfer. Vertical transfer happens within a
generation as the resistant bacteria reproduce, while horizontal transfer occurs across the
Bacteria phylogeny (Freeman, 1951). Over time, bacteria can collect multiple resistance genes
and become multidrug-resistant—resistant to many different antibiotic classes (Amábile-Cuevas,

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2006). If bacteria in the gut transfer antibiotic resistance genes to pathogens, it becomes much
more difficult to treat bacterial infections. Over 70% of the bacteria that cause infections are
resistant to at least one commonly used drug (Stone, 2010). Antibiotic resistant pathogens cause
patients to undergo more difficult treatments and purchase costly antibiotics. Antibiotic resistant
bacterial infections cost the United States healthcare industry an estimated $28 to $45 billion per
year in extra care (Stone, 2010). The inability to treat infectious pathogens in patients has caused
the death rate from bacterial infections to increase by 675% over the past 20 years (NIAID,
2012). Treating infectious diseases has become more difficult with the increase of antibiotic
resistant genes in bacteria.
Two recent studies examined HGT in the human microbiome (Smillie, 2011; Liu, 2012).
Smillie et al. focused on the HGT between human, non-human, and environmental sites, while
Liu et al. focused on HGT between different sites in the human body. Although these two studies
concentrated on HGT, HGT among bacteria at the same body habitat has not been closely
studied. The experimental or computational conditions in these studies tend to create ideal
conditions for detection of HGT, so the driving factors of transfer are still unknown.
In order for HGT to occur, bacteria must incubate in their intended growing conditions.
Growth conditions include temperature, moisture, pH, etc. When it comes to oxygen, some
bacteria thrive in its presence, while others find it a toxic substance. Obligate aerobes require
oxygen for growth, while obligate anaerobes have to grow in the absence of oxygen. Facultative
anaerobes are organisms that can switch between aerobic and anaerobic. These organisms grow
by fermentation or respiration depending on the oxygen condition they find themselves. Similar
to facultative anaerobes, aerotolerant anaerobes can grow with or without oxygen (Fox, 2011).
Since the conditions in the human gut are anaerobic, we chose bacteria strains that could live
anaerobically to mimic the gut.
We aimed to test and understand the factors of HGT without forcing ideal conditions for
conjugation or transformation. By using genetically un-manipulated bacteria and standard culture
conditions, we were able to replicate the kind of conditions for horizontal gene transfer seen in
the gut and test if antibiotic-resistance arose. If the originally antibiotic-sensitive recipient
bacterium was resistant to an antibiotic, then HGT occurred and an antibiotic resistance gene was
passed along. We used E. coli and A. baylyi as antibiotic-sensitive bacteria, hypothesizing HGT
would occur (Rouxel et al., 1991). After examining both conjugation and transformation,
Chloramphenicol resistance seems to be the most readily transferred type of resistance gene.
Materials and Methods
Bacteria and Antibiotics
Each donor bacterial strain was originally isolated from the human gut (Table 1). Strains
were grown statically in Mega Media (Washington University, St. Louis) at 37° C for 24 hours.
The recipient strains came from BioBricks and ATCC and were frozen in 15% glycerol (Table
2). Each antibiotic came from Sigma in St. Louis. Penicillin and Tetracycline were stored at
room temperature, while Chloramphenicol and Trimethoprim were stored at 4°C. Stock

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solutions of each antibiotic were made at the following concentrations: 100 mg/mL Penicillin, 5
mg/mL Tetracycline, 50 mg/mL Chloramphenicol, and 50 mg/mL Trimethoprim.
Validating Donor Bacteria through Sequencing
The identities of the bacterial cultures used were confirmed through the sequence of the
culture’s 16S rRNA gene. For each strain used, colony PCR was performed using commercially
available PCR mix (Thermo Scientific, ReddyMix) and the following primers: forward primer
(Bac_27F: 10um; 5’-AGAGTTTGATCATGGCTCAG-3’) and reverse primer (Bac_1391R:
10um; 5’-GACGGGCGGTGTGTGCA-3’). PCR products were confirmed on an agarose gel
before purification with QIAquick PCR Purification Kit (QIAGEN). Sanger sequencing was
performed by GeneWiz, Inc. Sequences from GeneWiz were analyzed using the Ribosomal
Database Project classifier (http://rdp.cme.msu.edu/classifier/classifier.jsp).
Validating Recipient Bacteria through Fluorescence
To verify that our stock of E. coli was CFP-positive, we diluted the culture to get
individual colonies on Luria-Bertani (LB) plates (US Biological). Single colonies were picked
and added to the wells of a 96-well plate, each containing 200 uL of LB Broth (US Biological).
Fluorescence was read every 15 minutes for 24 hours on a fluorescence reader kept at 37°C, and
recorded using the Gen5 Secure software package (BioTek Instruments, 1.11.5).
Plates Containing Antibiotics
Standard LB agar (LB Agar Lennox, US Biological) plates were used to grow CFP-
positive E. coli with or without antibiotics at one of the following concentrations: 128 ug/mL
Penicillin, 8 ug/mL Tetracycline, 8 ug/mL Chloramphenicol, or 8 ug/mL Trimethoprim (Sigma,
St. Louis). Standard LB agar plates were used to grow A. baylyi with or without antibiotics at one
of the following concentrations: 128 ug/mL Penicillin, 8 ug/mL Tetracycline, 12 ug/mL
Chloramphenicol, or 50 ug/mL Trimethoprim.
Plates for Coy Chamber
To prepare plates for the obligate-anaerobe bacteria, components for part 1 (Table 3)
were sterilized by autoclave, while the components for part 2 were combined and filter-sterilized
(FS). FS-part 2 was added to autoclaved part 1, and the solution was mixed until combined.
Plates were then poured and left to solidify. Plates were covered in aluminum foil due to the light
sensitivity of Vitamin K, Histidine-Hematin, and Resazurin. Once plates were solid, they were
moved into the anaerobic chamber (Coy Laboratory Products) to become anaerobic.
Conditions for Conjugation of Obligate Anaerobic Donor
In an anaerobic chamber, a 1:1 volume of donor and recipient bacterial strains were co-
cultured together to create a 650 uL culture. The co-culture was grown at 37 °C in the anaerobic

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chamber for 48 hours or 5 days. 100 uL of co-culture was spread on a LB agar plate containing
one of the antibiotics. The bacteria were incubated at 37 °C for 24 hours.
Conditions for Conjugation of Facultative Anaerobic Donor
A 1:1 ratio of donor and recipient strains of bacteria were co-cultured together to create a
650 uL culture. The co-culture was grown at 37°C for 5 days. 100 uL of co-culture was spread
on a LB agar plate containing one of the antibiotics. The bacteria were incubated at 37 °C for 24
hours. To quantify fluorescent colonies, CFP was read at 480 nm using DPController and
DPManager programs (Olympus Optical Co. 1.2.1.108).
Conditions for Transformation of Pooled DNA
To test the transformation between bacterial DNA and A. baylyi, four pools of genomic
DNA were collected (Table 4). 50 uL of A. baylyi was placed on standard BHI plates in a single
drop. 5 uL of pooled DNA from different bacterial phyla was added to the drop. Once all the
liquid was absorbed into the agar, plates were incubated for 24 hours at 37°C. 750 uL of LB
broth was added to the plates, and cells were scraped. To assay for the potentially transformed
colonies, 100 uL of the suspended cells were spread on LB plates inoculated with antibiotics.
Plates were incubated at 37°C for 24 hours.
Validating Resistance
To validate the presence of a resistance gene in the colony, half of the colony was added
to LB broth with the antibiotic concentration that corresponded to the plate on which the colonies
grew. Colonies grew overnight in the shaker at 37°C.
Results
Colony Growth between B. uniformis and E. coli
We used anaerobic conditions to test the horizontal gene transfer between B. uniformis
and E. coli because B. uniformis is an obligate anaerobe. After 48 hours, E. coli co-cultured with
B. uniformis grew on standard LB plates but did not grow in the presence of any antibiotic
(Figure 1). After 5 days, E. coli co-cultured with B. uniformis was able to grow in the presence of
Chloramphenicol.
Colony Growth between E. fergusonii and E. coli
We tested the horizontal gene transfer between E. fergusonii and E. coli through
conjugation by using conditions for facultative anaerobes. After co-culturing E. coli with E.
fergusonii for five days, we saw growth on Penicillin, Tetracycline, and Chloramphenicol plates
(Figure 2). Because E. fergusonii and E. coli grow in the presence of oxygen, we looked for the
fluorescent property of CFP to determine if E. coli grew in the presence of any antibiotics. To
identify CFP-positive colonies, we first looked at the colonies through white light and then at

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480 nm to determine if the colonies contained CFP (Figure 3). After looking for CFP, E. coli
only grew in the presence of Chloramphenicol when co-cultured with E. fergusonii (Figure 4).
Colony Growth between A. baylyi and DNA
We tested transformation of HGT by using A. baylyi combined with genomic DNA pools
from one of the following four phyla: Firmicutes, Actinobacteria, Proteobacteria, and
Bacteroidetes (Table 4). After incubation, A. baylyi was present on each of the standard control
plates but was unable to grow in the presence of most antibiotics. However, A. baylyi combined
with Bacteroidetes DNA was able to grow in the presence of Chloramphenicol (Figure 5).
Discussion
Our results highly suggest horizontal gene transfer between donor and recipient strains of
bacteria without forcing ideal conditions for such gene transfer. The growth of E. coli co-
cultured with B. uniformis in the presence of Chloramphenicol strongly implies that horizontal
gene transfer of antibiotic resistance genes occurred between bacteria that are members of the
human gut microbiota, which supports the HGT mechanism of conjugation. E. coli also grew in
the presence of Chloramphenicol after being co-cultured with a facultative anaerobe. Because
antibiotic-sensitive E. coli grew in the presence of Chloramphenicol after co-culturing with E.
fergusonii, HGT between the donor and recipient bacteria strain is strongly suggested. Through
the HGT mechanism of transformation, we were able to test transfers between A. baylyi and
bacterial DNA from different phyla. A. baylyi combined with Bacteroidetes DNA growing in the
presence of Chloramphenicol suggests that HGT occurred in the transformation experiment.
After testing HGT transformation and conjugation, it seems that Chloramphenicol resistance
genes are most readily transfer from donor and recipient bacterial strain.
Although we expected more antibiotic resistance genes to transfer from donor to recipient
strain, there were multiple variables that could have played a part in the abundance of
unexpected colony growth. Co-culturing B. uniformis and E. coli together in the Coy Chamber
could have contaminated B. uniformis. The sterile technique used in the Coy Chamber is UV
light, which might have not been strong enough to prevent contamination in pure B. uniformis
cultures, which allowed it to grow in aerobic conditions. The growth of E. coli co-cultured with
Chloramphenicol in the presence of Chloramphenicol could have been due to adaptation of E.
coli to Chloramphenicol. Also, the lack of growth on antibiotic plates in the conjugation and
transformation experiments could be due to the fact that horizontal gene transfer did not occur,
and the two strains did not exchange DNA.
In order to further confirm the presence of an antibiotic resistance gene in the recipient
strain of bacteria, PCR amplification of the putative antibiotic resistance gene will be performed.
Antibiotic resistant colonies will be grown up in an antibiotic liquid culture to select for
antibiotic resistance genes again. The antibiotic resistance gene that was passed from B.
uniformis or E. fergusonii and is now present in the E. coli will be amplified through PCR. The
resistance gene that was transferred from the Bacteroidetes phyla to A. baylyi would also be

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tested with PCR and sequenced. If the gene is confirmed through sequencing, then the horizontal
gene transfer mechanism did indeed occur.
After testing two main methods of horizontal gene transfer, Chloramphenicol resistance
genes most readily transfer from donor to recipient strains of bacteria in the conditions studied.
Chloramphenicol and possibly Amphenicol antibiotics could quickly become ineffective to treat
certain infections since Chloramphenicol resistance genes most readily transferred out of the four
antibiotics we tested. Since Chloramphenicol resistance genes readily transfer, it is likely that
other Amphenicol antibiotics transfer just as easily creating bacteria that are multi-drug resistant.
Multi-drug resistant bacteria will make treating infectious diseases increasingly difficult
especially because antibiotic resistance from HGT events can occur in as little as five days. It is
possible that HGT can occur even more frequently in the human gut because of the 2.5 million
bacterial genes and high bacterial density of the gut. Now that we have demonstrated that
horizontal gene transfer can occur under more realistic conditions between donor and recipient
bacterial strains, more antibiotics along with various donor strains can be tested to increase our
knowledge about HGT of antibiotic resistance genes in the human gut.
Resources
1. Amábile-Cuevas, Carlos et al. Antimicrobial Resistance in Bacteria. Horizon Bioscience
(2006).
2. Bush. Beta-lactamase inhibitors from laboratory to clinic. Clinical Microbiology Reviews
1, 109-123 (1988).
3. Fox, Alvin. Microbiology and Immunology On-line. University of South Carolina
Available online at: http://pathmicro.med.sc.edu/fox/nutrition.htm (2011).
4. Freeman, Victor J. Studies on the Virulence of Bacteriophage-infected strains of
Corynebacterium Diphtheriae. Journal of Bacteriology 61, 675-688 (1951).
5. Gill, Steven R. et al. Metagenomic Analysis of the Human Distal Gut Microbiome.
Science 312, 1355-1359 (2006).
6. Kaznowski, A and Wlodarczak K. Susceptibilities of motile Aeromonas sp. to
antimicrobial agents. Zentralbl Bakteriol 275, 85-93 (1991).
7. Lederberg, Joshua and Tatum, Edward. Gene recombination in Escherichia-
coli Nature 158, 558 (1946).
8. Liu, Li. et al. The human microbiome: A hot spot of microbial horizontal gene transfer.
Elsevier 100, 265-270 (2012).
9. NIAID. Antimicrobial (Drug) Resistance: Quick Facts. National Institutes of Health
Available online at: http://www.niaid.nih.gov/topics/antimicrobialResistance/
Understanding/ Pages/quickFacts.aspx (2012).
10. Rouxel, T. et al. Evidence for horizontal gene transfer in Escherichia coli speciation.
Elsevier 222, 851-856 (1991).
11. Smillie, Chris S. et al. Ecology drives a global network of gene exchange connecting the
human microbiome Nature 480, 241-244 (2011).

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12. Stone, Patricia W. Economic burden of healthcare-associated infections: an American
perspective. Expert Rev Pharmacoecon Outcomes Res.9, 417-422 (2010).
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Acknowledgments
The author is grateful for the opportunity that Dr. Gautam Dantas provided. The utmost
thanks goes out to her mentor Molly Gibson and tutor Vitas Wagner as well as everyone
involved in the Young Scientist Program at Washington University.
Table 1. Donor Strains Used.
Table 2. Recipient Strains and Antibiotics Used.
Donor Strains
Strain Phyla Anticipated Growth Conditions
Escherichia fergusonii Proteobacteria Facultative Anaerobe
Bacteroides uniformis Bacteroidetes Obligate Anaerobe
Recipient Strains Antibiotics
Strain Phyla Antibiotic Antibiotic Class
Escherichia coli (MG1655) Proteobacteria Penicillin (PE) Beta lactam
Acinetobacter baylyi (ADP1) Proteobacteria Tetracycline (TE) Tetracycline
Chloramphenicol (CH) Amphenicol
Trimethoprim (TR) Pyrimidine derivative

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Table 3. Components for Anaerobic Plates.
Components for Part 1 Amount Components for Part 2 Amount
Tryptone Peptone (US Biological, MA) 5 g D-(+)-Glucose (Sigma) 1 g
Yeast Extract (US Biological, MA) 2.5 g Vitamin K Solution (Sigma) 500 uL
L-Cysteine HCl (Sigma) 0.25 g Distilled H2O 10 uL
1 M Potassium Phosphate Buffer, pH 7.2
(1M KH2PO4 and 1M K2HPO4)
50 mL Resazurin (Sigma) 2 mL
TYG Salts Solution (MgSO4·7H2O,
NaHCO3, NaCl, Distilled H2O)
20 mL Histidine-Hematin (0.2 M histidine,
pH 8.0 and hematin (Sigma))
500 uL
0.8% CaCl2 500 uL
FeSO4 ·7H2O (0.4 mg/ml) 500 uL
Agar (US Biological, MA) 7 g
Distilled H2O 416 mL
Total: 487 mL Total: 13 mL
Figure 2. Quantified Colony Growth between B. uniformis and E. coli. A combination of B. uniformis, E. coli,
and Penicillin, Tetracycline, Trimethoprim, or Chloramphenicol were co-cultured and plated on LB and LB with the
corresponding antibiotic. The quantity of colonies is shown for all combinations of B. uniformis, E. coli, and an
antibiotic. *The colonies that grew in the presence of an antibiotic were validated to have resistance genes.
*
Figure 1. Colony Growth between E. fergusonii and E. coli. A combination of E. fergusonii, E. coli, and
Penicillin, Tetracycline, Trimethoprim, or Chloramphenicol were co-cultured and plated on LB and LB with the
corresponding antibiotic. The quantity of colonies is shown for all combinations of E. fergusonii, E. coli, and an
antibiotic. *The colonies that grew in the presence of an antibiotic were validated to have resistance genes.
*

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Figure 3. CFP Fluorescent Colonies. To measure E. coli growth between E. coli and E.
fergusonii, the CFP of the colonies was read. A1, B1, and C1 are colonies that can be seen in white
light (1a). Colonies A2 and B2 can be seen at 480 nm, while colony C1 is nonexistent (1b).
1a
1b
Figure 5. Quantified Colony Growth between A. baylyi and DNA. A.
baylyi combined with bacteria DNA from Firmicutes, Actinobacteria,
Proteobacteria, or Bacteroidetes phyla were spread on antibiotic plates.
*The colonies that grew in the presence of an antibiotic were validated to
have resistance genes.
*
Figure 4. Quantified E. coli Growth between E. fergusonii and E. coli. All colonies that grew from
co-cultures containing combinations of E. fergusonii, E. coli, and an antibiotic were analyzed for E. coli.
After looking at the CFP fluorescent colonies, we quantified E. coli. *The colonies that grew in the
presence of an antibiotic were validated to have resistance genes.
*

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Table 4. Pooled Bacteria DNA.