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First published online: November 12, 2015 ISSN 2062-8633 © 2015 The Author(s)
European Journal of Microbiology and Immunology 5 (2015) 4, pp. 268–280
DOI: 10.1556/1886.2015.00031
Original article
* Corresponding author: Eva Sapi; Department of Biology and Environmental Science, University of New Haven,
1211 Campbell Avenue, Charger Plaza LL16, West Haven, CT, USA; E-mail: esapi@newhaven.edu
EFFECTIVENESS OF STEVIA REBAUDIANA WHOLE LEAF EXTRACT
AGAINST THE VARIOUS MORPHOLOGICAL FORMS OF BORRELIA
BURGDORFERI IN VITRO
P. A. S. Theophilus, M. J. Victoria, K. M. Socarras, K. R. Filush, K. Gupta, D. F. Luecke, E. Sapi*
Department of Biology and Environmental Science, University of New Haven, West Haven, CT, USA
Received: September 7, 2015; Accepted: October 26, 2015
Lyme disease is a tick-borne multisystemic disease caused by Borrelia burgdorferi. Administering antibiotics is the primary treat-
ment for this disease; however, relapse often occurs when antibiotic treatment is discontinued. The reason for relapse remains un-
known, but recent studies suggested the possibilities of the presence of antibiotic resistant Borrelia persister cells and biofilms.
In this study, we evaluated the effectiveness of whole leaf Stevia extract against B. burgdorferi spirochetes, persisters, and bio-
film forms in vitro. The susceptibility of the different forms was evaluated by various quantitative techniques in addition to differ-
ent microscopy methods. The effectiveness of Stevia was compared to doxycycline, cefoperazone, daptomycin, and their combina-
tions. Our results demonstrated that Stevia had significant effect in eliminating B. burgdorferi spirochetes and persisters. Sub-
culture experiments with Stevia and antibiotics treated cells were established for 7 and 14 days yielding, no and 10% viable cells,
respectively compared to the above-mentioned antibiotics and antibiotic combination. When Stevia and the three antibiotics were
tested against attached biofilms, Stevia significantly reduced B. burgdorferi forms. Results from this study suggest that a natural
product such as Stevia leaf extract could be considered as an effective agent against B. burgdorferi.
Keywords: Borrelia burgdorferi, biofilms, persister cells, Stevia rebaudiana, antibiotic resistance
Abbreviations: ATCC – American type culture collection; BSK-H – Barbour–Stoner–Kelly H; CefP – cefoperazone; DapM –
daptomycin; DoxC – doxycycline; EPS – extracellular polymeric substances; Log phase – logarithmic phase; PBS – phosphate
buffered saline; PI – propidium iodide; PTLDS – post-treatment Lyme disease syndrome
Introduction
Lyme disease is a leading tick-borne multisystemic dis-
ease caused by the spirochete Borrelia burgdorferi. The
bacterium is transmitted by Ixodes ticks, which could feed
on white-footed mice, rodents, deer, and birds [1, 2]. In
the United States, there are approximately 300,000 people
diagnosed with Lyme disease each year [3]. The frontline
treatment for Lyme disease is antibiotics such as doxycy-
cline for adults and amoxicillin for children [4–8]. These
antibiotics are found effective in most cases of patients
diagnosed with Lyme disease [5–8]. However, accord-
ing to the Centers for Disease Control (CDC), approxi-
mately 10–20% of the Lyme disease patients treated with
anti biotics for a recommended 2 to 4 weeks experienced
symptoms of fatigue, pain or joint and muscle aches [9].
In some patients, the symptoms even lasted for more than
6 months [9]. This condition was termed as “post-treat-
ment Lyme disease syndrome (PTLDS)” or “chronic Lyme
disease” [9].
The mechanism associated with this condition in pa-
tients remains unclear. Though not proven, there are a cou-
ple of suggested explanations, such as the inability of the
immune system to completely clear B. burgdorferi persist-
ers [10], or due to the presence of antigenic debris, which
might cause immunological responses [11]. Another possi-
bility of Borrelia evading the host immune clearance after
antibiotic treatment is not well understood [12, 13].
Previous in vivo studies on mice, dogs, and nonhuman
primates have shown that B. burgdorferi could not be fully
eliminated by various antibiotics such as doxycycline, cef-
triaxone, and tigecycline. Also, a recent study had demon-
strated the presence of Borrelia DNA in mice following 12
months of antibiotic treatment [14]. However, the culturing
of viable organisms in Borrelia growth media could not be
achieved in these studies [14–17]. A recent study reported
the presence of Borrelia DNA from a patient with PTLDS
after antibiotic treatment [18]. Prospective clinical stud-
ies demonstrated no signifi cant effective antibiotic therapy
and failed to show evidence of the continued presence of
In vitro effect of Stevia on Borrelia burgdorferi
European Journal of Microbiology and Immunology
269
B. burgdorferi in patients with long-term symptoms [19,
20]. Other trials using prolonged intravenous ceftriaxone
treatment only improved fatigue symptoms [21]. In sum-
mary, fi ndings suggest that conventional treatments may
not completely eliminate Borrelia persisters.
The life style of B. burgdorferi is complex as it ex-
ists in different morphological forms like the spirochetes,
spheroplast (or L-form), round bodies, and biofi lms [7, 13,
22–25]. Several studies show that Borrelia can change into
round bodies when conditions become unfavorable such
as changes in temperature, high or low pH, starvation, an-
tibiotic exposure, and/or even an attack from the immune
system [7, 23–25]. Borrelia in these defensive forms be-
comes dormant, immobile, and remains in this morpho-
logical state until it fi nds favorable conditions to return to
its spirochete form [7, 22, 24, 26]. The most effective hid-
ing place proposed for B. burgdorferi is the recently sug-
gested biofi lm form [24]. Bacterial biofi lms are organized
communities of cells enclosed in a self-produced hydrated
polymeric matrix or extracellular polymeric substances
(EPS), which is a complex mixture of polysaccharides, lip-
ids proteins, nucleic acids, and other macromolecules [24,
27]. This unique matrix protects the underlying cells from
antimicrobial agents [24, 27]. Elimination of pathogenic
bacteria in their biofi lm form is very challenging since
these sessile bacterial cells can endure the host immune
responses and are much less susceptible to antibiotics or
any other biocides than their individual planktonic coun-
terparts [24, 27]. Biofi lm resistance is based upon multiple
mechanisms, such as phenotypic changes of cells form-
ing in the biofi lm, the expression of effl ux pumps, and the
presence of persister cells, which resist killing when ex-
posed to antimicrobial agents [28, 29].
Recently, we provided evidence that B. burgdorferi is
capable of forming biofi lms in vitro [24]. The aggregation
of spirochete and round body forms with several differ-
ent protective layers which makes up the biofi lm and ex-
tracellular polymeric substances (EPS) is proposed to be
a signifi cant factor in antibiotic resistance [24]. It is also
reported that the biofi lms have higher population of the
persister cells, which could lead to the antimicrobial resis-
tance portrayed by these forms [24, 29].
Our previously published results on the in vitro effects
of doxycycline on B. burgdorferi showed that different
morphological forms have unique sensitivities to antimi-
crobial agents [7]. A recent study by J. Feng et al. showed
that doxycycline was effective in reducing the spirochetes
but not the persisters of B. burgdorferi [30, 31]. Studies
have also showed that doxycycline and amoxicillin could
eliminate the spirochetal form of B. burgdorferi, but the
dormant persisters/biofi lm-like aggregates/microcolonies
were not susceptible to these antibiotics [7, 31]. There-
fore, there is an urgent need to fi nd effective agents, which
can target/eliminate all the morphological forms of Bor-
relia.
Natural antimicrobial agents, which have been used for
thousands of years, have been shown to be effective against
various pathogens [32]. Many in vitro and clinical stud-
ies have demonstrated their effectiveness not only against
B. burgdorferi but also against many other pathogens
[33–38]. Stevia rebaudiana which belongs to the Astera-
ceae family is typically referred to as honey leaf or sweet
leaf, and due to its natural sweetness, it is used as a natural
substitute to synthetic sweetener [39–41]. The leaf extract
of Stevia possesses many phytochemicals, which include
austroinullin, -carotene, dulcoside, nilacin, rebaudi ox-
ides, ribofl avin, steviol, stevioside, and tiamin with known
antimicrobial properties against many pathogens [40, 42,
43]. The role of these compounds is mainly to protect the
plant from microbial infection and adverse environmental
conditions [38–43]. Stevia is also well known in tradition-
al medicine for its use in treatment of many diseases like
diabetes, high blood pressure, and weight loss [44, 45]. In
a few clinical studies, it is reported that the phytochemical
stevioside reduces blood pressure in patients experienc-
ing mild hypertension and reduces blood glucose levels in
type 2 diabetic patients [44, 45]. It was also demonstrated
that the patients did not encounter any adverse effects from
the use of stevioside [44, 45].
Considering the effectiveness of Stevia leaf extract in
laboratory and clinical studies, we evaluated the antimi-
crobial potential of Stevia (whole leaf extracts) against the
Lyme disease causing pathogen, B. burgdorferi, in a goal
to eliminate all the different morphological forms in vitro.
To effectively evaluate the whole Stevia leaf extract, we
compared the antimicrobial effect of Stevia with antibi-
otics (doxycycline, cefoperazone, daptomycin) and their
combination, which were recently found effective against
Borrelia persisters.
Materials and methods
Bacterial culture conditions and media requirements
Low passage isolates ( 8) of B. burgdorferi strain B31
were obtained from the American Type Culture Col-
lection (ATCC 35210) and were cultured in Barbour–
Stoner–Kelly H (BSK-H) media (Sigma, St Louis, MO)
supplemented with 6% rabbit serum (Pel-Freez®, Rogers,
AR). The antibiotic free cultures were maintained in ster-
ile 15 ml glass tubes and incubated at 33 °C with 5% CO2.
For logarithmic (log) phase, 1 × 105 cells/ml were seeded
in glass tubes and allowed to grow for 5 days. The ef-
fectiveness of the antimicrobial agents on the logarithmic
phase was tested by inoculating 1 × 105 spirochetes/ml
from the fi ve-day grown culture (containing only spiro-
chetes) in 90 l of BSK-H media in 96-well tissue culture
plates (BD Falcon, Franklin Lakes, NJ), which were in-
cubated for 5 days at 33 °C with 5% CO2. The cells were
treated with antimicrobial agents for 3 days after a two-
day incubation period. Likewise, for the stationary phase,
1 × 105 cells/ml were seeded in glass tubes and allowed to
grow for 7 days. The effectiveness of the antimicrobial
agents was tested by inoculating 1× 106 spirochetes/ml in
90 l of BSK-H media in a 96-well tissue culture plate,
P. A. S. Theophilus et al.
European Journal of Microbiology and Immunology
270
which was incubated for 8 days at 33 °C with 5% CO2.
The persister cells in stationary phase were treated with
antimicrobial agents for 3 days after 5 days of incubation.
Biofi lms were generated by inoculating 5 × 106 cells/ml
of Borrelia spirochetes in 1 ml of BSK-H media in four-
well chamber slides (Thermo Scientifi c, Waltham, MA) or
plastic/collagen-coated tissue culture 48-well plates (BD
Falcon), which were incubated for 7 days at 33 °C with
5% CO2. The treatment regime for the biofi lms was the
same as that for the stationary phase.
Compounds and antibiotic preparation
Different Stevia extracts manufactured by Nutramedix®,
Now®, Sweet leaf®, and Truvia® were purchased from
health food stores in the USA and were labeled randomly
as Stevia A, B, C, and D. The extracts A, B, and C were
formulated by standard alcohol extraction method where-
as extract D was purchased in a powder form dissolved
in distilled water. Stevioside (Sigma) was prepared in
0.001% DMSO and further diluted in 1× phosphate buff-
ered saline (PBS, 0.1 M, pH 7.4 from Sigma). The antibi-
otics doxycycline, cefoperazone, and daptomycin (Sigma)
at a concentration of 10 g/ml were prepared in PBS. All
antimicrobial agents were sterilized using a 0.2-m fi lter
unit (EMD Millipore, Billerica, MA). The antibiotic solu-
tions were aliquoted and stored at minus 20 °C.
Determining the protein concentrations
of the antimicrobial agents
Since the concentration of the antimicrobial components
in the Stevia extracts A, B, C, and D were unknown, the
concentration units used initially involved the use of part
of solute per 50 parts of solution (1:50 dilution). The pro-
tein content of antimicrobial agents was determined by
Bradford assay (Sigma) using serial dilutions of 2 mg/ml
of bovine serum albumin stock (Sigma) in PBS pH 7.4 as
a standard. Bradford reagent was added to the antimicro-
bial agents according to the manufacturer’s instructions
and incubated for 10 min at room temperature with gentle
shaking. The absorbance was measured at 595 nm using a
BioTek spectrophotometer.
In vitro antimicrobial susceptibility testing
of B. burgdorferi
The effective antibiotics and the antibiotic combination
identifi ed [30] were tested to compare and evaluate the
effectiveness of Stevia on B. burgdorferi. As a positive
control, doxycycline (10 g/ml) was used. As a negative
control, 1× PBS at pH 7.4 was used to dissolve antibiot-
ics and 25% alcohol (Fisher Scientifi c, Pittsburg, PA) was
used since all the Stevia extract contained the same alcohol
diluent.
SYBR Green I/PI assay
To evaluate live and dead cells, standard SYBR Green I/
propidium iodide assay (SYBR Green I/PI) was per-
formed as previously described [30, 31, 46, 47]. To
1 ml of sterilized distilled water, 10 l of SYBR Green I
(10,000× stock, Invitrogen, Grand Island, NY) and 30 l
of propidium iodide (Thermo Scientifi c) were briefl y
mixed. The staining mixture (10 l) was added to all the
wells containing B. burgdorferi and was incubated in the
dark for 15 min. The plates were measured using a fl uo-
rescent reader (BioTek FLx800) by setting the excitation
wavelength at 485 nm and the absorbance wavelength
at 535 nm (green emission) and 635 nm (red emission).
The standard equation was determined from 1 × 106 cells
(logarithmic phase), 5 × 106 cells (stationary phase), and
1 × 107 cells (7 days and 14 days subculture). A live and
dead population was prepared. For the dead cell popula-
tion, the cells were killed by adding 300 l of 70% iso-
propyl alcohol (Fisher Scientifi c). B. burgdorferi suspen-
sions at different ratios of live/dead cells (0:10, 2:8, 5:5,
8:2, 10:0) were added to the wells of the 96-well plate
accordingly. The staining mixture was added to each of
the fi ve samples, and the plate was read as above. Us-
ing least square fi tting analysis, the regression equation
was calculated between the percentage of live bacteria
and green/red fl uorescence ratios. The regression equa-
tion was used to calculate the percentage of live cells in
each sample of the screening plate. Also, images of the
treated sample were taken using fl uorescent microscopy
(Leica DM2500).
Total viable counts of B. burgdorferi
As a confi rmation test, the SYBR Green/PI stained cul-
tures were assessed for cell growth by directly counting
live and dead bacteria using a bacterial counting chamber
(Hausser Scientifi c, Horsham, PA) and fl uorescent micro-
scopy (Leica DM2500). As above, using least square fi tting
analysis, the regression equation was calculated between
the percentage of live bacteria and green/red fl uorescence
ratios. The regression equation was used to calculate the
percentage of live cells in each sample.
Long-term subculture experiments to assess viability
of treated B. burgdorferi stationary phase culture
To assess if the treated stationary phase cultures can re-
grow in fresh media, 96-well plates were fi lled with 100
l of fresh BSK-H media. From the treated stationary
phase cultures, 1:75 dilution of treated stationary phase
culture was added to a sterile 96-well plate (BD Falcon)
containing 100 l of fresh BSK-H media and incubated
for 7 days and 14 days without antimicrobial treatment.
Following incubation, the viability was assessed using the
SYBR Green I/PI assay and direct counting method.
In vitro effect of Stevia on Borrelia burgdorferi
European Journal of Microbiology and Immunology
271
Crystal violet biofilm assay
The effi cacy of the antimicrobial agents on biofi lms was
determined by quantifying the total biomass using crystal
violet staining. Following incubation, the media was slow-
ly discarded leaving behind the attached biofi lms on the
surface of the plate. The attached biofi lms were collected
by adding 500 l of PBS (0.1 M, pH 7.4) to the samples. All
centrifugation steps were performed at 12,000 × g at room
temperature. Biofi lms were pelleted for 5 min, and the su-
pernatant was discarded. A volume of 50 l (0.01%) crystal
violet (Sigma) was added to the pellet, and the mixture was
incubated for 15 min at room temperature. The unbound
stain was removed by pelleting the biofi lms for 5 min and
by discarding the supernatant. The pellet was washed with
200 l PBS and again pelleted for 5 min. The supernatant
was discarded in addition to adding 200 l of 10% acetic
acid (Sigma) to the pellet to release and dissolve the stain.
The samples were then incubated at room temperature for
15 min. Biofi lms were pelleted for 5 min prior to extracting
the crystal violet stain from the biofi lms, which were trans-
ferred to a 96-well plate, and absorbance was measured at
595 nm using a BioTek spectrophotometer.
Live/dead bacterial staining technique
To visualize the antimicrobial sensitivity of biofi lms, the
treated biofi lms were stained using SYBR Green (Invitro-
gen) and propidium iodide (Thermo Scientifi c). The stains
were prepared as per the SYBR Green I/PI assay proto-
col. To each well of the chamber slide, 5 l of the stain-
ing mixture was added and allowed to incubate in the dark
for 15 min. The media was removed carefully not to dis-
turb the attached biofi lm. Slides were cover slipped, and
images were taken using fl uorescent microscopy (Leica
DM2500).
Atomic force microscopy
To visualize the morphology of the antimicrobial treated
biofi lms grown on glass chamber slides (Thermo Scien-
tifi c), the media was carefully removed without disturbing
the biofi lm. Contact mode AFM imaging in air was per-
formed on a Nanosurf Easyscan 2 AFM (Nanosurf) using
SHOCONG probes (APPNANO). Images were processed
using Gwyddion software (Neas and Klapetek).
Statistical analysis
Statistical analysis was performed using two-tailed
Student’s t-test (Microsoft Excel, Redmond, WA) and
graphed using GraphPad Prism® 6.0 (La Jolla, CA). All
experiments were performed a minimum of three separate
times with at least three samples per experiment. Data was
normalized to the control and presented as the mean ± SD.
Results
In this study, we compared and evaluated the antimicrobial
effect of S. rebaudiana whole leaf extract along with the
antibiotics doxycycline, cefoperazone, and daptomycin
on the different morphological forms (spirochetes, round
bodies, and biofi lms) of B. burgdorferi. We used a recently
developed high throughput screening method [30, 31, 46]
to assess the viability of the log phase and stationary phase
B. burgdorferi after the different antimicrobial treatments
(quantitatively and qualitatively) in conjunction with di-
rect counting methods as described in our previous pub-
lication [7]. Similar to recent studies, we have evaluated
both the log phase B. burgdorferi cultures which consist of
individual spirochetes, the stationary phase cultures which
consist of the persister cells, and the most resistant form
of B. burgdorferi, the attached surface biofi lms. Finally,
atomic force microscopy was used in conjunction with the
aforementioned methods, to visualize changes in the mor-
phology in attached biofi lms before and after antibiotic
treatments.
Preliminary screening of different Stevia extracts
Here, we fi rst evaluated four different commercially ob-
tained Stevia extracts (3 alcohol extracts and one extract
in powder form) and the purifi ed Stevioside on B. burg-
dorferi to fi nd the most effective agent for further studies
using the SYBR Green/PI assay. The protein concentra-
tion of the four Stevia leaf extracts was determined by a
standard Bradford assay and ranged from 1.2 g/ml to
1.9 g/ ml. Stevioside was tested at concentration from 100
to 1000 g/ml, concentration as suggested from a previous
study [42].
Fig. 1. Preliminary screening of four different extracts of Stevia
and Stevioside on the stationary phase of B. burgdorferi after a
three-day treatment using the SYBR Green/PI assay. n = 3 ± SD,
*p 0.05, **p 0.01 compared to the control
P. A. S. Theophilus et al.
European Journal of Microbiology and Immunology
272
For the prescreening experiments, we have pretested
all agents on the log and stationary phase Borrelia cul-
tures at different concentrations using the SYBR Green/
PI assay. Our results showed that Stevia A, Stevia B, and
Stevia C alcohol extracted agents had signifi cant effects
on the viability of Borrelia cells, but Stevia D, powdered
form, and Stevioside did not show any signifi cant effect
on both the log phase and the stationary phase cells. Fig-
ure 1 shows a representative experiment demonstrating
that the alcohol extracted based Stevia agents (A, B, C)
were the most effective against the Borrelia persisters,
while the powder form of Stevia leaf extract (D) and
Stevioside had no effect on those resistant cells. Fur-
thermore, Stevia A showed the most promising effect in
all experiments, and therefore, Stevia A was used in the
subsequent experiments on the different morphological
forms of B. burgdorferi.
Fig. 2. Susceptibility of log phase (5 days) and stationary phase (8 days) Borrelia burgdorferi to antimicrobial agents after a three-day
treatment determined by (A) SYBR Green I/PI assay, (B) direct counting of live and dead cells stained using a mixture of SYBR Green
I and propidium iodide using fluorescent microscopy. (C) Representative live/dead images of log and stationary phase B. burgdorferi
to antimicrobial agents taken at 200× magnification (Scale bar 100 m). Doxycycline (DoxC) was used as a positive control. 1×
PBS and 25% alcohol were used as negative controls, respectively. All antibiotics individually as well as in combination were used
at a concentration of 10 g/ml. Stevia A was used at a concentration of 1.2 g/ml. n = 3 ± SD, *p 0.05, **p 0.01 compared to the
control. n = 3 ± SD, +p 0.05, ++p 0.01 (doxycycline compared to Stevia A). n = 3 ± SD, *p 0.05, **p 0.01 (doxycycline compared
to Stevia A). Abbreviations: doxycycline – DoxC, cefoperazone – CefP, daptomycin – DapM
In vitro effect of Stevia on Borrelia burgdorferi
European Journal of Microbiology and Immunology
273
Stevia as a potential agent in eliminating different forms
of B. burgdorferi
To compare and evaluate the effectiveness of Stevia on
B. burgdorferi, recently identifi ed potential antibiotics and
their combinations were tested along with Stevia A extract
using the SYBR Green I/PI assay on both log and station-
ary phase cultures. Appropriate amounts of 1× PBS pH 7.4
and 25% alcohol were used as negative controls.
According to the SYBR Green I/PI assay and the live/
dead images, treatment with 1× PBS or 25% alcohol did
not signifi cantly reduce the viability of the spirochete rich
log phase culture and persisters rich stationary cultures
compared to the control (Fig. 2A, and 2C: panels i–iii and
ix–xi). Figure 2C: panels i–iii and ix–xi demonstrated that
the control B31 culture, 1× sterile PBS, and 25% alcohol
treated cultures comprised only live cells (green).
In the next set of experiments, we tested several previ-
ously reported effective antibiotics on B. burgdorferi such
as doxycycline, cefoperazone, and daptomycin individu-
ally and in combinations [30, 31].
Doxycycline, as reported earlier [30, 31] was signifi -
cantly able to reduce the viability of log phase B. burg-
dorferi by ~99% compared to the control (Fig. 2A, and
2C: panel iv). However, doxycycline treatment had no sig-
nifi cant effect on the cells in the stationary phase cultures
as observed by increasing proportion of viable cells after
antibiotic exposure compared to the control (Fig. 2A, and
2C: panel xii).
Cefoperazone at 10 g/ml, which was previously iden-
tifi ed as one of the drugs with high activity against Borre-
lia persisters [30, 31], signifi cantly reduced the log phase
viability of Borrelia by ~99% (Fig. 2A, and 2C: panel v)
but, in contrast, only reduced the stationary phase viability
by ~18% compared to the control (Fig. 2A). A large popu-
lation of live round body forms and a mixture of live and
dead spirochetal cells were observed in stationary phase
culture (Fig. 2C: panel xiii).
Daptomycin, one of the other drugs previously identi-
fi ed with potent activity against Borrelia persisters [30, 31],
also signifi cantly reduced the live spirochetal enriched log
phase culture by ~23%, but it was less sensitive in reduc-
ing viable cells in the stationary phase (~16% reduction
of live cells) compared to the control (Fig. 2A). Images
demonstrated that cells treated with daptomycin, both log
and stationary phase show more live cells than dead cells
(Fig. 2C: panels vi and xiv) compared to control.
A recent study reported that the combination of doxy-
cycline, cefoperazone, and daptomycin successfully elim-
inated both spirochetes and persisters [30]. Our data from
this study also confi rmed the effectiveness of these three-
antibiotic combination on B. burgdorferi by signifi cantly
eliminating the live log phase spirochetes (100% effect)
and also signifi cantly reduced the viability of the station-
ary phase culture by ~86% (Fig. 2A, and 2C: panels vii
and xv).
In these experiments, we also tested potential anti-
microbial agent Stevia A as an individual agent and com-
pared its effectiveness to the individual antibiotics and the
three-antibiotic combination. In Fig. 2A, and 2C: panels
viii and xvi, Stevia A signifi cantly eliminated the log phase
spirochetes and the stationary phase persisters compared
to the control (100% effect for both). The log phase and
the stationary phase culture treated with Stevia A had only
dead cells (Fig. 2C: panels viii and xvi).
In Fig. 2A, the effectiveness of doxycycline and the
three-antibiotic combination was compared with Stevia A.
A signifi cant elimination (p value 0.01) in the persisters
was found with Stevia A compared to doxycycline.
Direct counting confirms the effectiveness of Stevia A
on the log phase spirochetes and stationary phase rich
persisters
To further validate the effectiveness of the antimicrobial
agents evaluated using the SYBR Green I/PI assay, direct
counting was performed as described previously [7, 31].
According to the direct counting method, the nega-
tive controls, 1× sterile PBS and 25% alcohol, did not
signifi cantly reduce the number of viable cells in both
the log phase and stationary phase compared to the con-
trol (Fig. 2B). While doxycycline signifi cantly reduced
the log phase viability of Borrelia by ~98%, it did not re-
duce the stationary phase viability compared to the control
(Fig. 2B). Cefoperazone was also signifi cantly able to re-
duce the viability of log phase Borrelia by ~99% (Fig. 2B),
whereas cefoperazone in the stationary phase cultures was
less effective (~32% reduction) in eliminating the persist-
ers as observed by increasing proportion of remaining vi-
able cells after antibiotic exposure compared to the control
(Fig. 2B, and 2C: panel xiii). Daptomycin, signifi cantly
reduced the live spirochetal enriched log phase culture by
~44% and also signifi cantly reduced the stationary phase
by ~42% compared to the control (Fig. 2B). The three-
antibiotic combination of doxycycline, cefoperazone, and
daptomycin signifi cantly eliminated the log phase culture
and also the stationary phase culture by ~84% compared to
the control (Fig. 2B). The potent antimicrobial agent, Ste-
via A, signifi cantly eliminated the log phase spirochetes
and signifi cantly reduced the persisters by ~94% (Fig. 2B).
In Fig. 2B, the effectiveness of doxycycline and the
three-antibiotic combinations was compared with Ste-
via A. A signifi cant reduction (p value 0.01) in the per-
sisters was found with Stevia A compared to doxycycline.
Effectiveness of Stevia A after a 7-day and 14-day
subculture of antimicrobial treated B. burgdorferi
In the above-mentioned experiments, we have demon-
strated the effectiveness of Stevia A on both spirochetes
and the persisters of Borrelia and provided data that its
signifi cant effect is very comparable to the three-antibiotic
combination reported previously [31]. To confi rm the ef-
fectiveness of Stevia on the persisters, we performed a
P. A. S. Theophilus et al.
European Journal of Microbiology and Immunology
274
7-day and 14-day subculture experiment in fresh BSK-H
medium to observe if the persisters, if any, still left in the
culture, could regrow in fresh medium assayed by the
SYBR green I/PI and the direct counting methods.
As expected, the treatment with negative controls, 1×
PBS and 25% alcohol, was able to successfully repopulate
the media with predominating green live cells similar to
the untreated B31 control after a 7- and 14-day subcul-
ture (Fig. 3A, and 3C: panels i–iii and ix–xi). The three-
antibiotic combination and cefoperazone resulted in 5–7%
of viable cells after a 7-day subculture, but there was a
signifi cant 14% increase in the number of viable cells
treated with cefoperazone and a 11% increase in viable
cells treated with the three-antibiotic combination after a
14-day subculture (Fig. 3A, and 3C: panels v, vii, xiii, and
xv). There was a signifi cant 13% and 53% increase in vi-
able cells from the sample treated with doxycycline after
a 7-day subculture and a 14-day subculture, respectively
(Fig. 3A, and 3C: panels iv and xii). The treatment with
daptomycin, however, produced predominating live cells
Fig. 3. A 7-day and 14-day subculture of 8-day-old Borrelia burgdorferi stationary phase culture treated with antimicrobial agents
determined by (A) SYBR Green I/PI assay and direct counting of live and dead cells stained using a mixture of SYBR Green I and
propidium iodide using fluorescent microscopy. (B) Representative live/dead images of a 7-day-old subculture on stationary phase
B. burgdorferi treated with antimicrobial agents taken at 200× magnification (Scale bar 100 m). Doxycycline (DoxC) was used as
a positive control. 1× PBS and 25% alcohol were used as negative controls respectively. n = 3 ± SD, *p 0.05, **p 0.01 compared
to the control. n = 3 ± SD, +p 0.05, ++p 0.01 (doxycycline compared to Stevia A). n = 3 ± SD, *p 0.05, **p 0.01 (doxycycline
compared to Stevia A). Abbreviations: doxycycline – DoxC, cefoperazone – CefP, daptomycin – DapM
In vitro effect of Stevia on Borrelia burgdorferi
European Journal of Microbiology and Immunology
275
after a 7-day and 14-day subculture (Fig. 3A, and 3C: pan-
els vi and xiv). Interestingly, there was no regrowth with
the sample treated with Stevia A as there were only dead
cells (100% elimination) after a 7-day subculture, and only
a 10% increase in viable cells was observed after a sub-
culture for 14 days (Fig. 3A, and 3C: panels viii and xvi).
In Fig. 3A, the effectiveness of doxycycline and the
three-antibiotic combination after a 7-day and a 14-day
subculture was compared with Stevia A. A signifi cant re-
duction (p value 0.05) in the regrowth of Borrelia was
found with Stevia A compared to doxycycline after a 14-
day subculture.
Direct microscopic counting confi rmed the SYBR
Green I/PI assay and showed that drug-free controls and
the negative controls (1× PBS and 25% alcohol) grew well
in the 7-day and 14-day subcultures (Fig. 3B). Samples
treated with doxycycline, cefoperazone, and the three-anti-
biotic combination produced ~2% viable cells after 7 days
(Fig. 3B). Cefoperazone and the three-antibiotic combina-
tion produced ~1% viable cells after a 14-day subculture
(Fig. 3B). However, there was an increase in viable cells
by 68% treated with doxycycline after a 14-day subculture
(Fig. 3B). Daptomycin treated cells were able to recover
better than the other antibiotic counterparts with regrowth
similar to the drug-free control in the 7- and 14-day sub-
cultures (Fig. 3B). Samples treated with Stevia A after a
7-day subculture showed no signs of regrowth, and only
1% of viable cells were observed after a 14-day subculture
(Fig. 3B).
In Fig. 3B, the effectiveness of doxycycline and the
three-antibiotic combination in the direct counting meth-
od, after a 7-day and a 14-day subculture, was compared
with Stevia A. Signifi cant reduction (p value 0.01) in the
regrowth of Borrelia was found with Stevia A compared to
doxycycline after a 14-day subculture.
Stevia A significantly reduced Borrelia biofilms grown
on plastic and collagen surfaces
Multiple morphological forms of B. burgdorferi, i.e, spi-
rochetal form, round bodies, and biofi lms have been ob-
served to have different antimicrobial susceptibilities [7].
In our previous publication, we characterized the antibiot-
ics sensitivity of the biofi lm form of B. burgdorferi and
found that it is the most resistant form [7]. In this study, to
evaluate the effect of antimicrobial agents on Borrelia bio-
fi lms grown on plastic and collagen surfaces, we stained
the antimicrobial treated biofi lms with crystal violet to
quantify the effectiveness of the different antimicrobial
agents.
Biofi lms grown on plastic surface and treated with
negative controls, 1× PBS or 25% alcohol, did not show
reduction in the biofi lm masses compared to the untreated
biofi lm control (Fig. 4). Biofi lms treated with doxycy-
cline, cefoperazone, daptomycin, and the three-antibiotic
combination showed signifi cant increase in Borrelia bio-
fi lm mass compared to the drug-free control (Fig. 4). The
treatment with Stevia A, however, signifi cantly reduced
Borrelia biofi lms on plastic by ~40% compared to the
control (Fig. 4).
Biofi lms grown on collagen-coated surface show that
the treatment with negative control, 1× PBS or 25% al-
cohol, did not show reduction in the biofi lm masses com-
pared to the control (Fig. 4). Treatments with doxycycline
and daptomycin did not have any signifi cant effect on
Borrelia biofi lm mass (Fig. 4). There was an increase in
Borrelia biofi lms grown on collagen when treated with
cefoperazone, and in contrast, there was a 10% reduction
observed in these biofi lms when treated with the three-
antibiotic combination compared to the control (Fig. 4).
In addition, biofi lms treated with Stevia A also showed
signifi cant reduction on collagen by ~34% compared to
the control (Fig. 4). In order to assess the effectiveness of
doxycycline and the three-antibiotic combination to Stevia
A, the results in Fig. 4 showed that there was a signifi cant
reduction in the total Borrelia biomass grown on plastic
and collagen surfaces by Stevia A compared to doxycy-
cline and the three-antibiotic combination (p value 0.01).
Stevia A decreases viability in attached Borrelia biofilms
In order to directly observe the viability of attached bio-
fi lms after antimicrobial treatment, we stained the treated
biofi lms using a mixture of SYBR Green I and propidium
iodide (live/dead stains). The Borrelia biofi lms treated
with 1× PBS and 25% alcohol were large and compact,
mostly staining green, depicting that the biofi lm was live
Fig. 4. Susceptibility of Borrelia burgdorferi biofilms grown
on plastic and collagen-coated surface to antimicrobial agents
after a three-day treatment determined by crystal violet assay.
Doxycycline (DoxC) was used as a positive control. 1× PBS and
25% alcohol were used as negative controls, respectively. All
antibiotics individually as well as in combination were used at a
concentration of 10 g/ml. Stevia A was used at a concentration
of 1.2 g/ml. n = 3 ± SD, *p 0.05, **p 0.01 compared to the
control. n = 3 ± SD, +p 0.05, ++p 0.01 (doxycycline compared
to Stevia A). n = 3 ± SD, •p 0.05, ••p 0.01 (doxycycline
compared to Stevia A). Abbreviations: doxycycline – DoxC,
cefoperazone – CefP, daptomycin – DapM
P. A. S. Theophilus et al.
European Journal of Microbiology and Immunology
276
(Fig. 5: panels ii and iii). The biofi lms treated with cefo-
perazone were signifi cantly smaller compared to the bio-
fi lms treated with doxycycline, and both were green with
small red spots indicating that they were alive (Fig. 5: pan-
els iv and v). The biofi lms treated with daptomycin were
large and compact and stained mostly green with live cells
(Fig. 5: panel vi), whereas the biofi lms treated with the
antibiotic combination were made up with a mixture of
live and dead cells with live cells predominating (Fig. 5:
panel vii). Borrelia biofi lm treatment with Stevia A stained
predominantly red, depicting that the biofi lm had mainly
dead spirochetes and round bodies (Fig. 5: panel viii). The
Fig. 5. Representative live/dead images of Borrelia biofilms treated with different antimicrobial agents followed by staining with
SYBR Green I and PI dye mixture taken at 200× magnification. Doxycycline (DoxC) was used as a positive control. 1× sterile PBS
and 25% alcohol were used as negative controls, respectively. All antibiotics individually as well as in combination were used at a
concentration of 10 g/ml. Stevia A was used at a concentration of 1.2 g/ml. Scale bar 100 m. Abbreviations: doxycycline – DoxC,
cefoperazone – CefP, daptomycin – DapM
Fig. 6. Representative atomic force microscopy images showing the ultrastructural details of Borrelia biofilm before and after treatment
with antimicrobial agents. The preparations of B. burgdorferi strain B31 biofilms on chamber slides are described in Materials and
methods section. All biofilms were scanned at 0.4 Hz using contact mode and the individual Z ranges (height) are indicated next to
each panel by means of a scale. The images were scanned using the Nanosurf Easyscan 2 software, and the images were processed
using Gwyddion software. All antibiotics individually as well as in combination were used at a concentration of 10 g/ml. Stevia A
was used at a concentration of 1.2 g/ml. Abbreviations: doxycycline – DoxC, cefoperazone – CefP, Daptomycin – DapM
In vitro effect of Stevia on Borrelia burgdorferi
European Journal of Microbiology and Immunology
277
morphology of the Stevia A treated biofi lm was small and
loosely packed compared to the biofi lms treated with anti-
biotics (Fig. 5: panel viii).
AFM analysis shows loose morphology with biofilms
treated with Stevia A
The atomic force microscopic images, which are 3D ren-
dered and digitally colored for improved visualization,
show the ultra structural features in the biofi lms before
and after treatment with antimicrobial agents. The red
color shows the highest peak in the biofi lms, which mainly
indicates the potential presence of biofi lm EPS matrix. The
drug-free control had a very compact and rigid structure
with notable EPS buildup (Fig. 6: panel i). The biofi lms
treated with doxycycline, cefoperazone, daptomycin, and
the three-antibiotic combination were similar to the con-
trol having compact structure with more potential layers
of EPS (Fig. 6: panels ii–v). Interestingly, Stevia treated
biofi lms have a very loose structure with the EPS layer
almost not formed (Fig. 6: panel vi).
Discussion
In this study, we evaluated the antimicrobial potential
of whole leaf Stevia extracts against the Lyme disease
causing pathogen, B. burgdorferi. We compared the
antimicrobial effect of Stevia with antibiotics (doxycy-
cline, cefoperazone, daptomycin) and their combination,
which were recently found effective against Borrelia
persisters [31]. For this study, we have chosen to utilize
a novel quantitative method developed by J. Feng et al.
combined with our previously reported direct counting
method [7, 30, 31, 46]. Findings from this study show
that Stevia whole leaf extract, as an individual agent,
was effective against all known morphological forms of
B. burgdorferi.
In the preliminary screening experiment using differ-
ent extracts of Stevia, we demonstrated a signifi cant vari-
ation in the effectiveness of the alcohol extracts of Stevia
(Stevia A, Stevia B, and Stevia C) on Borrelia persisters
compared to the powder form of Stevia (Stevia D) and
purifi ed Stevioside which did not show any effect (Fig. 1).
We also found that one of the Stevia compounds, Stevia
A, signifi cantly eliminated Borrelia persisters compared
to other Stevia extracts (Fig. 1). This might be explained
by the reported variations observed in the different phy-
tochemical concentrations of different Stevia extracts,
which resulted from the growing conditions and agri-
cultural practices followed [47, 48]. Since Stevia A was
effective in eliminating Borrelia persisters, Stevia A was
chosen for further investigating the effectiveness on the
different morphological forms of B. burgdorferi.
In the next set of experiments, we compared the ef-
fectiveness of Stevia A to the antibiotics and the three-
antibiotic combinations found effective in recent reports
by J. Feng et al., [31] fi rst to Borrelia spirochetes and then
the persisters using the quantitative method developed by
J. Feng et al. and E. Sapi et al. We observed that Stevia
A, even at a lower concentration (1.2 g/ml), could sig-
nifi cantly eliminate the viability of early log phase and
stationary cultures of B. burgdorferi using the SYBR
Green I/ PI method (Fig. 2A, and 2C: panels viii and xvi)
which was also confi rmed using the direct counting meth-
od (Fig. 2B, and 2C: panels viii and xvi). According to
a report by J. Feng et al. (2015), daptomycin and cefo-
perazone, although effective against Borrelia persisters,
could not completely eliminate the microcolony form of
B. burgdorferi [30]. It was also observed from previous
in vivo and in vitro studies that doxycycline was effec-
tive in eliminating the spirochetes but not the persisters
of Borrelia [7, 16, 17, 30, 31]. However, J. Feng et al.
also showed that the three-antibiotic combination (doxy-
cycline, cefoperazone, and daptomycin) was very potent
in eliminating the stationary phase rich persisters of Bor-
relia [30]. In this study, we provided evidence that Stevia
A, as an individual agent, was capable of eliminating the
spirochetes and the persisters of Borrelia similar to the
reported three-drug combination treatment. Our data also
showed that the antibiotics in combination on the persist-
ers of Borrelia was indeed consistent with the previous
study [30]; this result further confi rms the effectiveness
of Stevia A.
In our previous studies, we characterized the biofi lm
form of B. burgdorferi in vitro, and showed that it rep-
resents the most antibiotic resistant form [24]. It is also
reported that biofi lms attached to a surface are protected
from antibiotics or other antimicrobial agents [28, 29]. In
this study, we also evaluated the effect of all antimicrobial
agents on attached Borrelia biofi lms. Our results showed
that Stevia A is very effective, in reducing attached Bor-
relia biofi lm mass on both plastic and collagen coated sur-
faces by ~40% (Fig. 4) whereas the individual antibiotics
actually induced the size of the biofi lm mass (Fig. 4).
It was previously reported that certain antibiotics
could indeed promote bacterial biofi lm formation [49].
One potential explanation is that cells in biofi lms are
capable of protecting themselves from unfavorable an-
tibiotic environment [24, 28] by developing several de-
fensive mechanisms such as poor antibiotic penetration,
phenotypic changes of cells forming in the biofi lm, the
expression of effl ux pumps, and the presence of persister
cells, which resist dying when exposed to antimicrobial
agents [28, 29]. Persisters are a subpopulation of highly
resistant cells, which are found among the normal cell
population, and they are dormant and highly protected
[28]. Studies show that small fraction of cells in biofi lms
were unaffected after prolonged antibiotic treatment [29,
53].
The obvious question on how Stevia could affect the
highly resistant Borrelia biofi lm warrants further investi-
gation. In a study using a sugar alcohol, it was reported
that xylitol acts as an antiplaque agent by disrupting the
formation of biofi lms in the oral cavity [54]. In another
P. A. S. Theophilus et al.
European Journal of Microbiology and Immunology
278
study, they showed that xylitol affects the production of
adhesive polysaccharides of Streptococcus mutans [55].
It was previously shown that sugars prime the uptake of
antibiotics in Staphylococcus aureus and Escherichia coli
[56]. Based on these previous fi ndings, we hypothesize
that Stevia could act as a sugar derivative, which might
prime the uptake of the phytochemicals responsible for
the antimicrobial effect and, thereby, disrupt the biofi lm
structure. In support to our hypothesis, we also showed
that the ultrastructure of Stevia A-treated biofi lm has very
loose morphology with large shallow pits compared to the
compact structure of biofi lms treated with doxycycline,
cefoperazone, daptomycin, and the antibiotic combina-
tion (Fig. 6: panels ii–vi).
To further confi rm the effectiveness of Stevia, we per-
formed long-term subculture experiments using the an-
timicrobial treated, stationary phase predominated with
Borrelia persisters by transferring a population of the
treated cells into fresh culture medium to observe if vi-
able cells can regrow after a 7-day and a 14-day period in
the absence of antimicrobial agent. The natural antimicro-
bial agent (Stevia A) was shown to be effective with no
regrowth of viable cells after a 7-day subculture and with
only 10% increase in viable cells after a 14-day subcul-
ture. The effects of the three-antibiotic combination, when
compared to Stevia A, regrew with 5% and 11% of viable
cells as detected using the SYBR Green I/PI method and
confi rmed by fl uorescent microscopy (Fig. 3A, and 3B:
panels vii, viii, xv, and xvi). We also observed that Bor-
relia treated with doxycycline and cefoperazone, which
did not show any signifi cant effect against the persisters
(Fig. 2A, 2B, and 2C: panels xii and xiii), recovering with
only 13% and 6% viable cells after a 7-day subculture
(Fig. 3A, and 3C: panels iv and v). One possible explana-
tion for this phenomenon could be that antibiotic sensitiv-
ity might have been restored when bacteria are dispersed
from a biofi lm during favorable conditions [28].
Stevia leaf extract is a widely used sugar substitute
[39–41, 57]; however, recent studies show that one of
the major glycosides, stevioside, could have antimi-
crobial effect against Bacillus cereus, Bacillus subtilis,
Klebsiella pneumoniae, and Pseudomonas aeruginosa
[42]. These antimicrobial studies used a high concentra-
tion of purifi ed stevioside, and in our study, we achieved
similar antimicrobial effect against Borrelia by using a
lower concentration of the whole leaf extract. Our data
with purifi ed Stevioside did not show any signifi cant an-
timicrobial effect on Borrelia spirochetes and persisters
compared to the whole leaf extracts of Stevia (Fig. 1).
This fi nding suggests that other components within the
Stevia whole leaf extract could have antimicrobial activ-
ity against B. burgdorferi, which are yet to be identifi ed in
future studies. In a good agreement with our fi ndings, Ste-
via leaf extract has also demonstrated antimicrobial activ-
ity against pathogens such as E. coli, S. aureus, Vibrio
mimicus, Salmonella typhimurium, S. mutans, Bacillus
subtilis, Shigella dysenteriae, and Vibrio cholera [38–42].
The next question is whether Stevia could be safely used
as a therapeutic agent. Toxicological studies have shown
that Stevia does not have mutagenic, teratogenic, or car-
cinogenic effects [57], and recent studies demonstrated
its safety at high dietary intake levels [57–59]. In a study
examining the mutagenicity of Stevioside and Steviol, it
was noted that Stevioside at 10 mg/ml did not induce any
mutation in S. typhimurium [60]. Apart from these stud-
ies, there are two important clinical studies based on the
glycoproteins present in Stevia. In a randomized, double-
blinded study on Chinese men and women experiencing
mild hyper tension, it was reported that the glycoprotein
stevioside decreased the systolic and diastolic blood pres-
sure and also improved quality of life without causing any
adverse effects compared to the placebo [44]. In another
study, the acute effects of stevioside in type 2 diabetic pa-
tients were analyzed [45]. Compared to the control group,
stevioside reduces postprandial blood glucose levels in
type 2 diabetic patients [45]. It was noted that both these
studies used an encapsulated powdered form of stevioside
and whole leaf extract that had been taken orally [44, 45].
It was also observed that one of the clinical studies used a
whole leaf preparation, which contained 91% stevioside,
4% rebaudioside A, and 5% of other derivatives of stevio-
side [45]. The outcome from these clinical studies dem-
onstrates that the patients did not encounter any adverse
effects from the use of stevioside [44, 45]. Although the
safeness of Stevia is widely studied, more in vivo studies
are warranted before Stevia could be used as an antimi-
crobial agent for any infectious diseases.
Our future goal is to further investigate the indi-
vidual components of whole leaf Stevia extract against
B. burgdorferi and to identify the most effective compo-
nent responsible for its signifi cant antimicrobial effect.
In conclusion, the overall antimicrobial effectiveness of
Stevia A extract on the different morphological forms of
B. burgdorferi was comparable to the combination of cer-
tain antibiotics. Although the results of this preliminary
study cannot be extrapolated directly to clinical practice,
further follow-up studies are necessary which can address
the safeness of Stevia and to further identify the most ef-
fective component(s) against Borrelia.
Acknowledgements
The authors thank Arun Timmaraju MS for his valuable
inputs and his review of the manuscript. This work was
supported by grants from the University of New Haven,
Joshua Research Foundation, Tom Crawford’s Leader-
ship Children Foundation, National Philanthropic Trust,
Alyssa Wartman Funds to E.S., and postgraduate fel-
lowships from NH Charitable Foundation to P.A.S.T.
and K.M.S. We also thank the Lymedisease.org, the
Schwartz Research Foundation for the donation of the
Atomic Force and the Leica DM2500 microscopes as
well as the Hamamatsu ORCA Digital Camera and to
the Global Lyme Alliance for the AFM supporting com-
puter.
In vitro effect of Stevia on Borrelia burgdorferi
European Journal of Microbiology and Immunology
279
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