







































Abstract: Lyme borreliosis is the most prevalent vector-borne disease in the United States caused by the transmission 
of bacteria Borrelia burgdorferi harbored by the Ixodus scapularis ticks (Sharma, Brown, Matluck, Hu, & Lewis, 2015). 
Antibiotics currently used to treat Lyme disease include oral doxycycline, amoxicillin, and ce!riaxone. Although 
the current treatment is e"ective in most cases, there is need for the development of new antibiotics against Lyme 
disease, as the treatment does not work in 10-20% of the population for unknown reasons (X. Wu et al., 2018). Use 
of antibiotics in the treatment of various diseases such as Lyme disease is essential; however, the downside is the 
development of resistance and possibly deleterious e"ects on the human gut microbiota composition. Like other 
organs in the body, gut microbiota play an essential role in the health and disease state of the body (Ianiro, Tilg, & 
Gasbarrini, 2016). #Of importance in the microbiome is the genus Bacteroides, which accounts for roughly one-third 
of gut microbiome composition (H. M. Wexler, 2007). $e purpose of this study is to investigate how antibiotics 
currently used for the treatment of Lyme disease in%uences the Bacteroides cultures in vitro and compare it with a 
new antibiotic (antibiotic X) identi&ed in the laboratory to be e"ective against B. burgdorferi. Using microdilution 
broth assay, minimum inhibitory concentration (MIC) was tested against nine di"erent strains of Bacteroides. 
Results showed that antibiotic X has a higher MIC against Bacteroides when compared to amoxicillin, ce!riaxone, 
and doxycycline, making it a promising new drug for further investigation and in vivo studies.

Aisthesis      Volume 11,  202040

Assessing Lyme Disease Relevant Antibiotics through Gut 
Bacteroides Panels

by Sohum Sheth 

I. Introduction
 Lyme borreliosis is the most prevalent vector-
borne disease in the United States, where it is 
transmitted by Ixodes scapularis ticks carrying the 
bacteria Borrelia burgdorferi, the causative agent 
of Lyme disease (Sharma, Brown, Matluck, Hu, & 
Lewis, 2015). B. burgdorferi belongs to the phylum 
Spirochaetes, which is characterized by highly 
motile, spiral-shaped bacteria. Since a standardized 
case de&nition of Lyme disease was issued in 1991, 
the geographic range of ticks carrying B. burgdorferi 
has expanded past the mid-Atlantic and New 
England regions to encompass the entire East coast, 
majority of the West coast, and much of the North-
central portions of the United States (X. Wu et al., 
2018). Similar spirochetes, such as Borrelia duttonii 
and Borrelia garinii, cause Lyme disease in Europe 
and Asia (X. Wu et al., 2018). While approximately 
30,000 cases of Lyme disease are reported every 
year in the U.S., the true incidence might range 
from 300,000 to 1,000,000 cases per year (Stricker & 
Johnson, 2014). 
 $e most common sign of Lyme disease is 
erythema migrans, which is an expanding rash 

occurring in roughly 80% of patients (Cairns, 2020; 
Shapiro, 2014). When detected at an early, localized 
stage of Lyme disease, the typical antibiotic treatment 
of oral doxycycline (100 mg twice daily for 21 days) 
cures the disease in most patients (Cairns, 2020). 
Other common antibiotics used include amoxicillin 
and ce!riaxone (Shapiro, 2014). However, when the 
pathogen delocalizes and enters the bloodstream, 
patients can experience Bell’s Palsy (facial paralysis), 
arthritis, nerve pain, vision loss and severe ear pain 
(Cairns, 2020). While most patients are cured and no 
longer experience symptoms 2 to 4 weeks following 
antibiotic treatment, some patients still experience 
symptoms, particularly arthritis, in which a second 
course of antibiotics is recommended (Weitzner et 
al., 2015). Roughly 10 to 20% of Lyme disease patients 
continue to experience varying levels of muscle and 
joint pain a!er treatments, a condition referred to 
as post-treatment Lyme disease syndrome (PTLDS) 
(Weitzner et al., 2015). 
 Very little is currently known about PTLDS, and 
e"orts to isolate Borrelia from patients with PTLDS 
in the past have had no success (Klempner et al., 
2001). It is not known what factors may predispose 



Assessing Lyme Disease Relevant Antibiotics through Gut Bacteroides Panels

Aisthesis      Volume 11,  202041

one to PTLDS and why only some patients develop 
it (X. Wu et al., 2018). Some animal models of Lyme 
disease do suggest that residual bacteria or bacterial 
products remain a!er antibiotic treatment, but 
whether this has any e"ect on PTLDS is currently 
unknown (Jutras et al., 2019; X. Wu et al., 2018). 
Despite the lack of knowledge concerning PTLDS, 
more e"ective management of B. burgdorferi would 
decrease the ability of bacterial recalcitrance and 
in%ammation-inducing bacterial products. $e 
pressing need for a more e'cacious antibiotic 
regimens to kill growing and non-growing forms of 
B. burgdorferi is clear. 
 Antibiotics, however, are no longer considered 
solely bene&cial to humans, as repeated use of 
these drugs are linked to numerous disorders 
associated with microbiota imbalance (Ianiro, 
Tilg, & Gasbarrini, 2016). Most antibiotics on the 
market have a wide array of action, impacting not 
only harmful bacteria, but also healthy bacteria. 
Furthermore, repeated abuse of antibiotics has been 
shown to have a direct link to the development of 
antibacterial resistance (Jernberg, Lofmark, Edlund, 
& Jansson, 2010). $e primary negative e"ect of 
current antibiotics is decreased diversity of gut 
microbiota, which are of clinical importance due 
to the wide range of disorders associated with their 
imbalance (Lange, Buerger, Stallmach, & Bruns, 
2016). Microbial life prospers in the anaerobic 
environment of the human gastrointestinal 
tract, representing “one of the densest microbial 
communities known to nature” (A. G. Wexler & 
Goodman, 2017). Bacteria in the gut play a major 
role in critical bodily functions, from immunity to 
digestion to protection against disease (Jernberg et 
al., 2010; Lange et al., 2016; G. D. Wu et al., 2011). 
Modulation of gut microbiome has been linked to 
obesity, in%ammatory bowel disease (Crohn’s disease 
and ulcerative colitis), type 2 diabetes, and numerous 
other metabolic disorders (Hills et al., 2019; Lange 
et al., 2016; Rinninella et al., 2019). Recent studies 
have shown that early life appears to be a critical 
period for maturation of metabolic function (Ianiro 
et al., 2016). $erefore, use of antibiotics that may 
cause unnatural %uctuations of the gut microbiota 
in children may be particularly worrisome. $e 
goal of e"ective antibiotics against Lyme disease in 
any population should be to kill the pathogenic B. 

burgdorferi while simultaneously leaving minimal 
impact on healthy gut microbes.
 Although the human gut microbiome consists of 
thousands of bacteria, the most predominant genus 
found in lower intestinal tract isolates from the United 
States and Canada—accounting for roughly one-
third of gut microbiome composition—is Bacteroides 
(King et al., 2019; Nishijima et al., 2016; Sears, 2005). 
$e genus Bacteroides is the principal component of 
the phylum Bacteroidetes, which constitutes roughly 
half of the relative abundance of bacteria in the gut 
(Garcia-Bayona & Comstock, 2019). Laitinen and 
Mokkala (2019) found that the median relative 
abundance of Bacteroides in the gut is 31.16%, with 
some individuals having a microbiome composition 
of up to 40.83% Bacteroides. To put into perspective, 
as many as 1010–1011 cells of Bacteroides are found 
in one gram of human feces (Hong, Wu, & Liu, 2008). 
Numerous species of Bacteroides are present in the 
intestines, with the most common being B. fragilis 
(A. G. Wexler & Goodman, 2017). As mutualists and 
commensals, Bacteroides generally have a bene&cial 
and complex relationship with its host. Bacteroides 
can break down food to produce valuable energy, 
digest complex sugars, and help in other metabolic 
activities of the colon (Karlsson, Ussery, Nielsen, & 
Nookaew, 2011; H. M. Wexler, 2007). Studies have 
shown that people with healthier diets and less-obese 
individuals have a higher composition of Bacteroides 
in their microbiome (Gorvitovskaia, Holmes, & 
Huse, 2016). 
 Despite their many bene&ts, imbalances in 
Bacteroides levels are responsible for infections 
such as intraabdominal sepsis, appendicitis, and 
gynecological infections that cause signi&cant 
morbidity and mortality (Johnson, Heaver, Walters, & 
Ley, 2017). In fact, most anaerobic human infections 
have an abnormal prevalence of B. fragilis. B. fragilis 
related disorders have an associated mortality rate of 
19%, increasing to 60% when le! untreated (H. M. 
Wexler, 2007). 
 Given the importance of microbiome balance 
to the health of individuals and the prevalence 
of Bacteroides in the gut microbiome, antibiotic 
dosages that strongly impact Bacteroides should be 
closely monitored. Studies have attempted to discern 
the range of Bacteroides species present in the gut 
but have yielded varied results due to individual 



Table 1: MIC values of ten antimicrobial agents 
against B. fragilis. MIC values represent the minimum 
concentration (in µg/mL) necessary to kill 50% (MIC50) 
and 90% (MIC90) of bacteria. 198 samples of B. fragilis 
were tested. Table adapted from Fernandez-Canigia et 
al. (2012).

Figure 1: Gel electropho-
resis results depicting the 
PCR products used for 
16S rRNA gene sequenc-
ing Lane 1 represents the 
NEB 1kb ladder of known 
lengths, with desired re-
gion of DNA from di"er-
ent Bacteroides samples 
in lanes 2-6.

Assessing Lyme Disease Relevant Antibiotics through Gut Bacteroides Panels

Aisthesis      Volume 11,  202042

di"erences in geography, diet, lifestyle, and other 
factors that contribute to the composition of the gut 
microbiome (Aldridge & Johnson, 1997; Snydman et 
al., 2011; Yim et al., 2015). 
 Nevertheless, Table 1 attempts to summarize 
known minimum-inhibitory concentration (MIC) 
values of ten antimicrobial agents against B. fragilis 
(Fernandez-Canigia et al., 2012). Clinical isolates 
in this study were recovered from several body 
sites, with 58%  recovered from the human gut. 
MIC values refer to the minimum concentration of 
drug (in µg/mL) needed to kill a certain percentage 
of bacteria. Limited information is known about 
Bacteroides MIC values of speci&c species (i.e. B. 
vulgatus, B. nordii, etc.) with respect to clinically 
relevant antibiotics for Lyme disease.
 A promising antibiotic—herea!er referred to as 
antibiotic X—has been identi&ed by researchers at 
the Antimicrobial Discovery Center at Northeastern 
University. Antibiotic X has been found to be active 
against B. burgdorferi and shows signs of selectivity 

against spirochetes. Antibiotic X was kindly 
provided for this study by researchers led by Dr. Kim 
Lewis at the Antimicrobial Discovery Center. $e 
purpose of this study is to investigate how antibiotic 
X in%uences Bacteroides, the predominant genus of 
bacteria in humans, as compared to other clinically 
relevant antibiotic treatments for Lyme disease. 

II. Results
 16S sequencing analysis showed that majority of 
the tested samples were not contaminated. Various 
Bacteroides isolates provided by the Antimicrobial 
Discovery Center were taken from lab freezer 
stock for use in the present study. However, freezer 
stocks are prone to contamination, so the identity 
of the isolates must &rst be genetically con&rmed. 
Once bacterial colonies were cultured, the colony 
used in MIC analysis for each strain of Bacteroides 
was assessed to ensure that contaminated samples 
of bacteria were not used in this study. 16S rRNA 
gene sequencing was done to con&rm the species 
of bacteria. 16S rRNA gene sequencing is valuable 
because the 16S rRNA gene region is highly conserved 
among bacteria, allowing for taxonomic resolution at 
the species and strain level. 27F and 1492R universal 
primers encompass nearly the entire 16S rRNA gene, 
which is about 1,500 base pairs long. $ese universal 
primers can amplify any gene in any organism. Gel 
electrophoresis results (Figure 1) show that PCR 
yielded the desired product length and was used for 
sequencing.  All PCR product in lanes 2-6 are located 
around the 1,500 bp stain mark of the DNA ladder 
in lane 1, con&rming gDNA isolation and PCR were 
successfully conducted. 16S sequencing analysis was 
conducted on four separate occasions. As shown 
in Table 2, of the 24 samples of Bacteroides tested, 
seven were shown to be contaminated. Multiple 
samples of B. stercoris and B. dorei were shown to 



Table 2: 16S rRNA gene sequence analysis was performed 
on di"erent freezer stocks of Bacteroides. At 7 (of 24) occa-
sions, the sequencing results were di"erent than expected.

Table 3: MIC values of the di"erent antibiotics against 
the lab isolates of Bacteroides strains cultivated under 
anaerobic conditions. ND = Not determined

Assessing Lyme Disease Relevant Antibiotics through Gut Bacteroides Panels

Aisthesis      Volume 11,  202043

be of other species, suggesting that the freezer stock 
of these strains may be contaminated. Common 
contaminants were Streptococcus parasanguinis and 
Aneurinibacillus aneurinilyticus. Once correct strains 
of Bacteroides were con&rmed via 16S rRNA gene 
sequencing, 96-well MIC assays could be conducted 
on the correctly identi&ed strains to determine drug 
susceptibilities to the di"erent antibiotics relevant to 
Lyme disease.  

 Antibiotic X has comparatively higher MIC 
for Bacteroides. Compared to clinically relevant 
antibiotics for Lyme disease, higher concentrations 
of antibiotic X were required to kill the di"erent 
Bacteroides species tested, suggesting that antibiotic 
X has minimal e"ect on the human microbiome. 
MIC was performed using microdilution broth assay 
on laboratory isolates of Bacteroides to determine 
the drug concentrations of antibiotic X, ce!riaxone, 
amoxicillin, and doxycycline. In a few instances, 
the MIC values of ce!riaxone and amoxicillin were 
high, suggesting that the strains used in this study 
may have developed resistance to them. Importantly, 
in four of the seven species tested, antibiotic X had 
a higher MIC value than all other antibiotics tested, 

and overall potency against all the strains were low. 
Of special signi&cance is B. fragilis, whose imbalance 
is the cause of a variety of metabolic disorders. 
Antibiotic X and amoxicillin had the highest MIC 
values (32 µg/mL) of the four drugs against B. fragilis. 
 Mean MIC values against Bacteroides. Of the 
four drugs tested, doxycycline had the lowest overall 
MIC values, showing that even small concentrations 
of doxycycline have a large impact on Bacteroides. 
When the four drugs were ranked on their mean MIC 
values for Bacteroides, doxycycline had the smallest 
mean MIC (0.72 µg/mL), followed by amoxicillin 
(50.43 µg/mL), ce!riaxone (53.67 µg/mL), and 
then antibiotic X (107.43 µg/mL). Amoxicillin 
and ce!riaxone had comparable mean MIC values 
against Bacteroides in this study, both of which were 
still less than half the mean MIC for antibiotic X. 
$e mean MIC for antibiotic X was more than 149 
times higher than the mean MIC for doxycycline. 
Antibiotic X had a higher MIC than doxycycline in 
each of the species tested in this study. $e low MIC 
values for doxycycline suggest that small dosages of 
doxycycline have the potential to dramatically shi! 
the nature of the Bacteroides in the gut microbiome. 
Antibiotic X had a higher MIC value than the other 
clinically relevant treatments for Lyme disease. $ese 
results suggest that antibiotic X—when compared 
to doxycycline, amoxicillin, and ce!riaxone—has 
the least e"ect on Bacteroides in the human gut 
microbiome.

III. Discussion
 $e current study was aimed at identifying 
the e"ect of antibiotic X on Bacteroides species 
of the human gut microbiota as compared with 
clinically relevant antibiotics for Lyme disease. 
Results were obtained using laboratory isolates of 
a Bacteroides panel of seven distinct species of the 
genus. Importantly, the species most abundant in the 
human gut microbiome, B. fragilis, was present in 
the analysis. 
 MIC assays performed using microbroth 
dilution in a 96-well plate format showed that 
antibiotic X had minimal e"ect on the Bacteroides 
human microbiome when compared to doxycycline, 
amoxicillin, and ce!riaxone. MIC values for 
antibiotic X were signi&cantly higher than the 
clinically relevant treatments for Lyme disease tested 
in this study, suggesting higher doses of antibiotic X 



Assessing Lyme Disease Relevant Antibiotics through Gut Bacteroides Panels

Aisthesis      Volume 11,  202044

are needed to modulate the Bacteroides diversity in 
the microbiome. To the best of our knowledge, there 
are only a few recent studies assessing the MIC values 
of amoxicillin, ce!riaxone, and doxycycline against 
Bacteroides. However, one study from 1997 describes 
the MIC of ce!riaxone against unseparated isolates of 
24 species of Bacteroides, which were predominantly 
B. fragilis (Aldridge & Johnson, 1997). Interestingly, 
the MIC values found in our study were di"erent 
from the results from Aldridge and Johnsen (1997). 
Aldridge and Johnsen showed that ce!riaxone had 
an MIC of 128 µg/mL against the 24 species of 
Bacteroides. $is study showed that ce!riaxone had 
an MIC of 1 µg/mL against B. fragilis and an average 
MIC of 53.67 µg/mL against all Bacteroides. $is 
discrepancy may be explained by the fact that our 
study looked at Bacteroides on a species level rather 
than testing all Bacteroides together. 
 In our study, 16S rRNA gene sequencing was 
performed with every experiment. 7 of the 24 
sequencing results yielded unexpected results. 
$e most common contaminants found a!er 
16S sequencing were Streptococcus parasanguinis 
and Aneurinibacillus aneurinilyticus. $ese 
contaminations may arise during lab isolation of 
the strains from the source or due to non-sterile 
conditions that may have been present in the 
anaerobic chamber during the culturing of bacteria. 
MIC results for ce!riaxone against B. eggerthii 
were not determined due to a tailing-o" e"ect in 
the MIC plates. $e exact reason for this tailing o" 
e"ect is unknown, but possibilities include incorrect 
pipetting of the drug into the 96-well plate or 
contamination of the 96-well plate. $is tailing-o" 
e"ect did not render a readable MIC value by visual 
search or plate reader.  
 Given the array of impacts antibiotics may have 
on the human gut microbiome, performing a high-
throughput Bacteroides panel is crucial in the drug 
development pipeline, as the genus plays important 
roles in human health. $e in vitro experiments 
performed in this study with individual assessment 
of di"erent Bacteroides species allows for in-depth 
analysis of how drugs may impact the most prevalent 
Bacteroides found in the microbiota. However, this 
method does not consider the in vivo interaction of 
Bacteroides, both with the host and other microbes 
present in the body. Additionally, in vitro analysis of 

individual Bacteroides species does not mimic the 
true diversity and relative prevalence of Bacteroides 
in the proportions present in the human gut. 
 Considering that the typical dose of oral 
doxycycline prescribed to Lyme disease patients is 
100 mg twice a day for ten to 21 days, the results 
found in this study suggest that doxycycline may 
have signi&cant adverse e"ects on the Bacteroides 
population in the human gut microbiome. $is 
arti&cial alteration of gut microbiota composition 
may have adverse health and lifestyle e"ects, in both 
the short and long term. B. fragilis, whose imbalance 
accounts for a majority of the adverse health impacts 
associated with gut microbiota, had an MIC of 0.25 
µg/mL, compared to 32 µg/mL for antibiotic X. 
Of the four antibiotics tested, doxycycline had the 
greatest e'cacy in killing the Bacteroides strains 
while antibiotic X had the least impact.
 Although amoxicillin showed higher MIC 
values than doxycycline in this study, it suggests that 
many of the strains used in this study developed 
resistance to amoxicillin. Numerous studies have 
shown that it is not uncommon for Bacteroides to 
develop resistance towards amoxicillin. Nagy et al. 
(2011) showed that 10.4% of Bacteroides tested in 
his study developed resistance to amoxicillin. $e 
typical dosage for amoxicillin in Lyme disease is 500 
mg three times per day, compared to the 100 mg 
dosage of doxycycline twice a day (Wormser et al., 
2000). Drug-induced rashes have also been common 
side e"ects of amoxicillin in the treatment of Lyme 
disease. As a result, treatment of Lyme disease with 
amoxicillin poses additional risk. In this study, we 
observe that antibiotic X had minimum impact 
on the Bacteroides strains tested when compared 
to clinically relevant antibiotics. Preliminary 
investigations have shown that antibiotic X is 
e"ective in selectively killing B. burgdorferi, the 
causative agent of Lyme disease. Taken together, 
these results suggest that antibiotic X may prove to 
be a more e"ective drug in the treatment of Lyme 
disease, as the antibiotic suppresses Lyme bacteria 
while preserving gut Bacteroides. Additional studies 
are required to investigate the exact in vivo e"ect of 
the di"erent drugs on microbial diversity for a better 
understanding and approach towards the treatment 
of Lyme disease. 



Table 4: Strains 
of Bacteroides ex-
pected based on 
freezer stock.

Table 5: Directions to prepare components for BHIymch 
media

Table 6: Schematic for &nal drug concentrations (in µg/
mL) of amoxicillin, ce!riaxone, and doxycycline used for 
the MIC assay. Triplicates were used for each drug.

Assessing Lyme Disease Relevant Antibiotics through Gut Bacteroides Panels

Aisthesis      Volume 11,  202045

IV. Materials and Methods
 Bacteria strains used in this study. Bacteroides 
strains listed in Table 4 are from lab isolates and 
were used in all the in vitro studies. Seven di"erent 
species of Bacteroides were used for analysis and kept 
at -80°C. 

 Media preparation. To ensure optimal Bacteroides 
colony formation, careful attention was placed in 
preparing the nutrient-rich growth medium for 
strains to proliferate. Both solid BHIymch media 
and liquid BHIymch media were made. Directions 
for making each component of BHIymch media 
(both liquid broth and solid agar) are shown in Table 
5.

 Once autoclaved, the BHI-y media was cooled in 
a water bath. MOPS, cysteine, and hemin were added 
to their &nal concentration of 1X. To prepare the 
BHI-ymch plates, the media with agar was poured 
into petri dishes. All media and plates were stored in 
an anaerobic chamber.
 Culturing conditions and microdilution broth 
assay for MIC determination. Bacteroides strains 
were removed from a -80°C freezer and taken to 
an anaerobic chamber. BHIymch agar plates were 
streaked with Bacteroides using a sterilized wire loop. 

Plates were incubated in an anaerobic chamber for 
24-48 hours for colonies to appear. Single colonies 
were then used to transfer to the 3 mL BHI-ymch 
media and grown for 12-16 hours to allow cultures 
to proliferate in the liquid media. 
 Stock solutions of 50 times concentrated (i.e. 6.4 
mg/ml) ce!riaxone, doxycycline and amoxicillin 
were prepared and serially diluted 2-fold. In a 96-well 
plate, 2 µL of the 50 times concentrated antibiotic 
solution was pipetted according to the scheme below 
(Table 6) in aerobic conditions. $is gives a &nal 
concentration range of 128 µg/ml to 0.01 µg/ml to be 
tested. 

 For MIC plates of antibiotic X, a similar procedure 
was performed with the &nal concentration range of 
512 µg/mL to 0.002 µg/ml. A total of 4 MIC plates, 
each containing triplicates of the drug dilutions and 
drug controls per Bacteroides strain were prepared 
and placed in the anaerobic chamber. 100µL of the 
1 in 100 dilution of the stationary phase cultures of 
the Bacteroides strains growing in the liquid BHI-
YMCH media was then pipetted to each well using 
a multichannel pipette. $e plates were incubated 
in the anaerobic chamber for 12-16 hours, and the 
MIC was determined as the lowest concentration 
of compound that inhibits growth of the bacteria as 
detected by the unaided eye. 
 Genomic DNA (gDNA) isolation. To verify 
species, a cell lysis-based method of DNA extraction 
consisting of a lysis solution of 500 mM EDTA, 1M 
Tris-HCl, and Triton X-100 was initially attempted. 
Polymerase chain reaction (PCR) was performed 
directly using the lysis solution. However, the PCR 
reaction did not yield any products. $erefore, gDNA 



Assessing Lyme Disease Relevant Antibiotics through Gut Bacteroides Panels

Aisthesis      Volume 11,  2020

isolation was performed. gDNA was isolated from 
the Bacteroides strains used for MIC assay in order to 
perform 16S rRNA gene sequencing to con&rm the 
species. Qiagen kits were used for gDNA isolation, 
and protocol from the Qiagen DNeasy Blood & 
Tissue handbook for gram-negative bacteria was 
followed. 
 1 µL of isolated gDNA was measured using 
a NanoDrop spectrophotometer to assess the 
concentration of the gDNA isolated in ng/µL. 
Using the DNA concentrations, 50 µL of 20 ng/
µL gDNA stock solution was prepared in water. 
PCR using Q-load M0271S NEB was performed 
where each PCR tube with 25 µL reaction 
contained 0.2 µM  of universal 27F forward 
primer (5’ AGAGTTTGATCMTGGCTCAG 
3’), 0.2 µM of universal 1492 reverse primer (5’ 
TACGGYTACCTTGTTACGACTT 3’), 12.5 µL of 
2X master mix, 6.5 µL of water, and 100 ng template 
gDNA. 35 cycles of PCR cycles were performed 
as follows: denaturation at 95°C for 30 seconds, 
annealing at 60°C for 30 seconds, and extension 
at 68°C for 70 seconds. Gel electrophoresis was 
conducted in 1% agarose gel to con&rm PCR 
product was of desired length. Ethidium bromide 
was used to stain DNA in the gel and NEB 1kb 
ladder was used as marker. 20 µL PCR products were 
sent for sequencing (Macrogen), and the results were 
analyzed using NCBI nBLAST program. 

V. References
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comparison of susceptibility results of the 
Bacteroides fragilis group and other anaerobes 
by traditional MIC results and statistical 
methods. J Antimicrob Chemother, 39(3), 319-
324. doi:10.1093/jac/39.3.319

Cairns, V. (2020). Lyme disease: implications for 
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Fernandez-Canigia, L., Litterio, M., Legaria, M. 
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antibiotic susceptibility of the Bacteroides fragilis 
group: emerging resistance to carbapenems in 
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Garcia-Bayona, L., & Comstock, L. E. (2019). 
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Gorvitovskaia, A., Holmes, S. P., & Huse, S. M. 
(2016). Interpreting Prevotella and Bacteroides 
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4, 15. doi:10.1186/s40168-016-0160-7

Hills, R. D., Jr., Pontefract, B. A., Mishcon, H. R., 
Black, C. A., Sutton, S. C., & $eberge, C. R. 
(2019). Gut Microbiome: Profound Implications 
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Hong, P. Y., Wu, J. H., & Liu, W. T. (2008). 
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Microbiol, 74(9), 2882-2893. doi:10.1128/
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Ianiro, G., Tilg, H., & Gasbarrini, A. (2016). 
Antibiotics as deep modulators of gut microbiota: 
between good and evil. Gut, 65(11), 1906-1915. 
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Jernberg, C., Lofmark, S., Edlund, C., & Jansson, J. 
K. (2010). Long-term impacts of antibiotic 
exposure on the human intestinal microbiota. 
Microbiology, 156(Pt 11), 3216-3223. doi:10.1099/
mic.0.040618-0

Johnson, E. L., Heaver, S. L., Walters, W. A., & Ley, 
R. E. (2017). Microbiome and metabolic disease: 
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016-1492-2

Jutras, B. L., Lochhead, R. B., Kloos, Z. A., Biboy, J., 
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doi:10.1073/pnas.1904170116

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Aisthesis      Volume 11,  2020

Karlsson, F. H., Ussery, D. W., Nielsen, J., & Nookaew, 
I. (2011). A closer look at bacteroides: 
phylogenetic relationship and genomic 
implications of a life in the human gut. Microb 
Ecol, 61(3), 473-485. doi:10.1007/s00248-010-
9796-1

King, C. H., Desai, H., Sylvetsky, A. C., LoTempio, 
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