Supplementary Information for
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Vancomycin and maltodextrin affect structure and activity of Staphylococcus aureus
biofilms
Mia Mae Kimco1, Erhan Atci1, Qaiser Farid Khan1, Abdelrhman Mohamed1, Ryan S. Renslow3,
Nehal Abu-Lail1, Boel A. Fransson2, Doug Call2, and Haluk Beyenal1,*
1
The Gene and Voiland School of Chemical Engineering and Bioengineering, 2Department of
Veterinary Clinical Sciences, and Paul G. Allen School for Global Animal Health, Washington
State University, Pullman, WA, 3 Environmental Molecular Sciences Laboratory, Pacific
Northwest National Laboratory, Richland, Washington 99352, USA
Running title: Vancomycin and maltodextrin affects biofilms
*
Corresponding author:
Email: beyenal@wsu.edu ; Telephone: +1-509-335-6607 ; Fax: +1-509-335-4806
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Flat plate flow cell
P
Peristaltic
Pump
Feed
Waste
CLSM
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Stepper motor
controller
Stepper
motor
ADC
Dissolved oxygen
microelectrode
Relative distance
Concentration
Electrometer/ammeter
Biofilm
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Data from the
computer
Distance
from the
bottom
Dissolved
oxygen
concentration
B
Figure SI1. A) Flow cell experimental setup. B) Experimental setup used to measure dissolved
oxygen depth profiles.
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A
B
Figure SI2. A) Raw intensity image of a maltodextrin solution in a 5-mm NMR tube. Diffusion
tensor imaging was used to determine the absolute and relative diffusion coefficients. B) Relative
diffusivity of vancomycin in the presence of various concentrations of maltodextrin.
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Figure SI3 shows the average diffusion distance (ADD) of biofilms under various treatment
conditions with maltodextrin and vancomycin. The ADD of biofilm in the absence of any
treatment decreased to 81.3±14% of that of the initial biofilm after 8 hours. This decrease,
however, is statistically insignificant (P = 0.25). ADD also decreased when the biofilm was
challenged with 2 mM vancomycin: it went down to 89.4±17% during the 8-hour treatment. This
decrease too was found to be statistically insignificant (P = 0.56). Thus, ADD did not change
when biofilm was treated with 2 mM vancomycin.
When biofilm was challenged with 10 mM maltodextrin, ADD increased by 1.97±20% during 8
hours of exposure (Figure SI3). We found that this increase was statistically insignificant (P =
0.93). The ADD of biofilm was reduced significantly (P = 0.01) after it was challenged with
20 mM maltodextrin. About 52.3±11% of the initial biofilm remained after 8 hours. Similarly,
the ADD of biofilm challenged with 30 mM maltodextrin decreased to 79.9±6.5% of that of the
initial biofilm. Overall, concentrations of maltodextrin higher than 10 mM decreased ADD in
biofilms.
Finally, a significant decrease in ADD was observed after the biofilm was challenged with a
combination of 10 mM maltodextrin and 2 mM vancomycin (Figure SI3). About 37.4±0.81% of
the initial biofilm was left after 8 hours. In contrast, ADD was only reduced to 95.4±34% after
the biofilm was treated with a combination of 20 mM maltodextrin and 2 mM vancomycin. We
found that this decrease was statistically insignificant (P = 0.90). When biofilm was challenged
with a combination of 30 mM maltodextrin and 2 mM vancomycin, we observed a decrease in
ADD: 78.6±16% remained. These findings show that maltodextrin and vancomycin in
combination decrease the ADD of biofilms.
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1.4
1.2
Relative ADD
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tia
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ilm
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ntr
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2m
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MD
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MD
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Treatment
Figure SI3. Average diffusion distance (ADD) of biofilms under various treatment conditions
with maltodextrin (MD) and vancomycin (V). The concentrations used were as follows:
2900 μg/mL V (2 mM), 36000 μg/mL MD (10 mM), 72000 μg/mL MD (20 mM), and
108000 μg/mL MD (30 mM). The bars depict the results for the initial biofilm (before treatment)
and after 8 hours of exposure to treatments. The data are means from 10 images taken for each of
3 biological replicated biofilms. The error bars represent the standard errors of the means
calculated from the triplicate measurements Stars indicate significant statistical differences from
the initial biofilm.
Figure SI4 shows the average biomass thickness of biofilms under control and various treatment
conditions. The biomass thickness of control biofilm decreased significantly (P = 0.0098) after 8
hours without any treatment. The thickness declined to 60.4±5.6% of that of the initial biofilm.
This can be explained by possible substrate depletion in the reactor and the dispersion of outer
cells of biofilm. Thus, we proceeded to compare the thickness of the control biofilm to that of the
treated biofilms. When biofilm was challenged with 2 mM vancomycin, the average biomass
thickness decreased to 63.3±10% during the 8-hour exposure.
The average biomass thickness of biofilm treated with 10 mM maltodextrin was reduced to
47.4±4.2% of the initial value after 8 hours. This change was statistically insignificant (P = 0.13)
when compared to that of the control biofilm. When biofilm was challenged with 20 mM
maltodextrin, the average biomass thickness decreased to 39.5±4.9%. A similar change was
found when biofilm was treated with 30 mM maltodextrin: the average biomass thickness
decreased to 42.5±3.2% after 8 hours. In general, maltodextrin diminished the biomass thickness
of biofilms (Figure SI4.).
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When biofilm was challenged with a combination of 10 mM maltodextrin and 2 mM
vancomycin, the average biomass thickness decreased to 37.1±1.5% of that of the initial biofilm.
We also found this change to be statistically significant (P = 0.02). A change in average biomass
thickness was found when biofilm was treated with a combination of 20 mM maltodextrin and
2 mM vancomycin: 82.5±12% of it remained after 8 hours. Although there was a reduction in
average biomass thickness, we found this change to be statistically insignificant (P = 0.07).
Finally, when biofilm was treated with a combination of 30 mM maltodextrin and 2 mM
vancomycin, the biomass thickness decreased to 71.5±11% of that of the initial biofilm. Like that
for the combination of 20 mM maltodextrin and 2 mM vancomycin, we found this change to be
insignificant (P = 0.18). Overall, the combination of 10 mM maltodextrin and 2 mM vancomycin
effectively reduced the average biomass thickness of biofilm while combinations of 2 mM
vancomycin with higher maltodextrin concentrations did not (Figure SI4.). However, decrease in
biofilm thickness was observed for all treatments, indicating the possible involvement of a
detachment period in biofilms and hydrodynamic effects from introducing treatments.
Relative Average Biofilm Thickness
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ol
ntr
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MD
MD
mM
mM
mM
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30
mM
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MD
Treatment
Figure SI4. Average biomass thickness of biofilms under various treatment conditions with
maltodextrin (MD) and vancomycin (V). The concentrations used were as follows: 2900 μg/mL
V (2 mM), 36000 μg/mL MD (10 mM), 72000 μg/mL MD (20 mM), and 108000 μg/mL MD
(30 mM). The bars depict the results for the initial biofilm (before treatment) and after 8 hours of
exposure to treatments. The data are means from 10 images taken for each of 3 biological
replicated biofilms. The error bars represent the standard errors of the means calculated from the
triplicate measurements. Stars indicate significant statistical differences from the control biofilm.
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