1802333 (1 of 12) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proanthocyanidin Interferes with Intrinsic Antibiotic
Resistance Mechanisms of Gram-Negative Bacteria
Vimal B. Maisuria, Mira Okshevsky, Eric Déziel, and Nathalie Tufenkji*
Dr. V. B. Maisuria, Dr. M. Okshevsky, Prof. N. Tufenkji
Department of Chemical Engineering
3610 University Street, Montreal, Quebec H3A 0C5, Canada
Prof. E. Déziel
531 boul. des Prairies, Laval, Québec H7V 1B7, Canada
The ORCID identiﬁcation number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/advs.201802333.
cells, or formation of bioﬁlm, and emer-
gence of antibiotic-resistant pathogens
recalcitrant to treatment due to acquired
resistance. Here, we report that a puri-
ﬁed cranberry proanthocyanidin (cPAC)
fraction potentiates the activity of a broad
range of antibiotic classes against the
opportunistic pathogens Escherichia coli,
Proteus mirabilis, and Pseudomonas aer-
uginosa. cPAC was an effective potentiator
against diverse bacterial lifestyles normally
tolerant to antibiotics, such as bioﬁlm bac-
teria, dormant cells, and in experimental
models of chronic infections. Remark-
ably, when combined with tetracycline,
cPAC was able to completely prevent
the evolution of resistance in E. coli and
P. aeruginosa. These results suggest that in
combination with antibiotic therapy, cPAC
has the potential to decrease the spread of
antibiotic resistance and prolong the effec-
tiveness of currently available drugs.
Opportunistic pathogens colonize sur-
faces in healthcare settings, on indwelling medical devices,
and on living tissues, leading to infections that must be treated
with antibiotics. Two major factors complicate the effective-
ness of antibiotic treatments: i) antibiotic-resistant bacteria
and ii) the formation of antibiotic-tolerant bioﬁlms. The
latter sessile bacteria can withstand antibiotic doses up to thou-
sands of times higher than their planktonic counterparts,[2–4]
due largely to the presence of tolerant and persister cells that
remain dormant within the bioﬁlm, ready to reinitiate growth
when antibiotic concentrations decrease. Prolonged antibiotic
treatment and high antibiotic doses necessitated by the recalci-
trant nature of such infections put patients’ health at risk and
create a strong selective pressure for the evolution of antibiotic
resistance.[2,3,5] In order to decrease the occurrence of antibiotic
resistance and mitigate negative side effects brought on by high
antibiotic doses, novel approaches to enhance the effectiveness
of currently available antibiotics represent an attractive alterna-
tive to the search for new antibiotics. Exploiting natural mole-
cules with antibiotic-potentiating activities provides one such
The American cranberry (Vaccinium macrocarpon L.) fruit
and its derivatives have long been anecdotally reported as a
natural remedy for urinary tract infections.[6,7] cPAC are con-
densed tannins that can hinder bacterial attachment to cel-
lular or biomaterial surfaces,[8–11] impair bacterial motility,[12–17]
induce a state of iron limitation, and interfere with quorum
sensing. Studies suggest that consumption of cranberry
Antibiotic resistance is spreading at an alarming rate among pathogenic
bacteria in both medicine and agriculture. Interfering with the intrinsic
resistance mechanisms displayed by pathogenic bacteria has the
potential to make antibiotics more effective and decrease the spread of
acquired antibiotic resistance. Here, it is demonstrated that cranberry
proanthocyanidin (cPAC) prevents the evolution of resistance to tetracycline
in Escherichia coli and Pseudomonas aeruginosa, rescues antibiotic efﬁcacy
against antibiotic-exposed cells, and represses bioﬁlm formation. It is shown
that cPAC has a potentiating effect, both in vitro and in vivo, on a broad
range of antibiotic classes against pathogenic E. coli, Proteus mirabilis,
and P. aeruginosa. Evidence that cPAC acts by repressing two antibiotic
resistance mechanisms, selective membrane permeability and multidrug
efﬂux pumps, is presented. Failure of cPAC to potentiate antibiotics against
efﬂux pump-defective mutants demonstrates that efﬂux interference is
essential for potentiation. The use of cPAC to potentiate antibiotics and
mitigate the development of resistance could improve treatment outcomes
and help combat the growing threat of antibiotic resistance.
The global spread of antibiotic resistance is undermining dec-
ades of progress in ﬁghting bacterial infections. Due to the
overuse of antibiotics in medicine and agriculture, we are on
the cusp of returning to a pre-antibiotic era in which minor
infections can once again become deadly. Countering the fall
in antibiotic efﬁcacy by improving the effectiveness of currently
available antibiotics is therefore an important goal. Antibiotic
efﬁcacy is limited by the expression of intrinsic tolerance mecha-
nisms such as production of antibiotic-tolerant and/or persister
© 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA,
Weinheim. This is an open access article under the terms of the Creative
Commons Attribution License, which permits use, distribution and re-
production in any medium, provided the original work is properly cited.
Adv. Sci. 2019, 6, 1802333
1802333 (2 of 12) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
can prevent bacterial infections,[20–25] and one reports the
combined effect of bulk cranberry derivatives (devoid of the
proanthocyanidin fraction) and
-lactam antibiotics against
a Gram-positive bacterium. It has also been suggested that
cPAC can inhibit bioﬁlm formation and potentiate gentamicin
against P. aeruginosa; however, the potential for cPAC to
interfere with the evolution of resistance to antibiotics or rescue
the effectiveness of antibiotics has never been investigated. Fur-
thermore, no studies have explored the activity spectrum or the
molecular mode of action of speciﬁc cranberry-derived fractions
such as proanthocyanidins for the treatment of bacterial infec-
tions. Here, we report the ability of cPAC to thwart the evolu-
tion of resistance to tetracycline in E. coli and P. aeruginosa, and
demonstrate the broad spectrum, antibiotic-potentiating activity
of cPAC against various pathogenic Gram-negative bacteria,
both in vitro and in vivo. We also show that cPAC is able to
repress two important intrinsic antibiotic resistance mecha-
nisms: selective membrane permeability and multidrug efﬂux
2.1. cPAC Potentiates a Broad Range of Antibiotics In Vitro
We conducted screening assays with cPAC in combination with
several classes of clinically approved antibiotics belonging to
the World Health Organization’s list of essential medications.
Checkerboard microdilution analysis was performed using
pathogenic strains of E. coli, P. mirabilis, and P. aeruginosa.
We determined whether cPAC was able to potentiate the effec-
tiveness of antibiotics by calculating the fractional inhibitory
concentration index (FICI). Figure 1A–C shows positive and/
or negative interactions between cPAC and antibiotics against
bacterial pathogens. FICI values of ≤0.5 (indicated by the gray
zone) demonstrate that cPAC potentiated the effectiveness of
sulfamethoxazole (SMX), nitrofurantoin (NIT), gentamicin
(GEN), kanamycin (KAN), tetracycline (TET), and azithromycin
(AZT) to inhibit the growth of E. coli CFT073, P. mirabilis
HI4320, and P. aeruginosa PA14 using up to 98% less antibiotic
than that required in the absence of cPAC. cPAC also poten-
tiated trimethoprim (TMP) and fosfomycin (FOS) activities to
inhibit the growth of P. mirabilis HI4320; 81% and 98% less
antibiotic were needed, respectively, than in the absence of
cPAC. In the case of P. aeruginosa strain PAO1, cPAC enhanced
the efﬁciency of the antibiotics SMX, FOS, NIT, GEN, KAN,
and AZT (Figure S1A, Supporting Information). The fact that
cPAC potentiates a given antibiotic against one strain but not
another (e.g., cPAC potentiates FOS against P. mirabilis HI4320
but not against P. aeruginosa PA14 or E. coli CFT073) provides
evidence that the effect is speciﬁc and that cPAC is not simply
inactivating the antibiotic. It is not surprising that the FICI
values for the two P. aeruginosa strains were different for some
antibiotics as they had different MICantibiotic values.
At concentrations required to potentiate antibiotic efﬁ-
cacy, cPAC alone had no detectable growth inhibition activity
against all four pathogenic strains (Figures S1B and S2A–C,
Supporting Information). Given the ability of cPAC to poten-
tiate TMP or SMX alone, we investigated the interaction of
cPAC with co-trimoxazole (the combination of SMX and TMP,
commonly used to treat urinary tract infections and bacterial
dysentery). cPAC enhanced the synergistic efﬁcacy of co-
trimoxazole, reducing the MIC up to 64-fold against P. mira-
bilis HI4320. In the case of P. aeruginosa PA14, combination of
cPAC with co-trimoxazole decreased the MIC by 32-fold, which
is signiﬁcantly more effective than the potentiating combina-
tions of cPAC with TMP or SMX alone (Figure S3, Supporting
Information). The fact that cPAC potentiates antibiotics, but
does not act as an antibiotic on its own, suggests that treatment
with cPAC is unlikely to create selective pressure for the evolu-
tion of resistance.
2.2. cPAC Prevents Re-Activation of Antibiotic-Exposed Cells
To investigate the inhibitory activity of cPAC against antibiotic-
exposed bacteria, a modiﬁed disk-diffusion test was performed.
Figure 2A shows that following treatment with the bacterio-
static antibiotic tetracycline (Step 1: application of TET antibi-
otic disk), bacteria in the growth-inhibition zone were able to
recover when a new disk impregnated with glucose replaced the
TET disk (at Step 2). An analysis of bacterial re-activation based
on the occurrence of colonies inside a typical inhibition/clear
zone shows that the degree to which cells “re-activate” to form
colonies differs depending on the presence or absence of cPAC.
Replacement with a glucose-only disk (at Step 2) enhanced the
re-activation of cells (i.e., bacterial lawn in previous inhibition
zone), while a disk with a combination of cPAC and glucose
showed no re-activation of antibiotic-exposed cells. There were
no colonies observed close to the cPAC-only disk, and the size
of the clear zone with the cPAC-only disk (at Step 2) was similar
to the TET disk (Step 1) and the glucose+cPAC disk (Step 2).
Since cPAC alone did not inhibit bacterial growth of this, or
any other tested strains (Figures S1B,C and S2A–F, Supporting
Information), it is probable that the clear zones around the
cPAC-only and the glucose+cPAC disks result from synergy of
cPAC with TET remnants, which is in agreement with a slightly
smaller diameter, since lower concentrations of cPAC and TET
should be present at the distal edge of the zone. Similar effects
were observed with minocycline (MIN; Figure S4, Supporting
Information) against E. coli CFT073. These results suggest that
cPAC can prolong the efﬁcacy of remnant antibiotic against
antibiotic-exposed cells even after treatment has ceased.
2.3. cPAC Thwarts Evolution of Antibiotic Resistance
To understand the role of cPAC in effectively inhibiting the re-
activation of antibiotic-exposed cells, we analyzed the ability of
cPAC to suppress evolution of resistance in E. coli CFT073 and
P. aeruginosa PA14. As shown in Figure 2B,C, sequential pas-
saging on TET alone for 21 days resulted in 128-fold and 32-fold
increases in MIC for E. coli CFT073 and P. aeruginosa PA14,
respectively, while cPAC prevented the evolution of resistance
in both strains when co-administered with TET. cPAC alone did
not promote resistance in either strain. This result shows that
cPAC can suppress the emergence of antibiotic resistance in
Adv. Sci. 2019, 6, 1802333
1802333 (3 of 12) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.4. cPAC Potentiates In Vivo Activity of SMX
Our next goal was to investigate the potential of cPAC to
enhance the efﬁcacy of antibiotics against bacterial infections
in vivo. To this end, we used the model host Drosophila mela-
nogaster infected with P. aeruginosa PA14, in which cPAC or
SMX was administered alone or in combination. The median
survival of the insects following infection was 138 h in the
absence of treatment, but more than 225 h when cPAC and
SMX were combined (Figure 3A). Survival of ﬂies following
combination therapy was signiﬁcantly (
2 = 3.88, df = 1,
P < 0.05) higher than survival with SMX treatment alone. The
median survival time of infected D. melanogaster treated with
cPAC alone or SMX alone was 202 and 178 h, respectively
(Figure 3A), which is not signiﬁcantly (P > 0.05) different from
the survival of untreated ﬂies infected with P. aeruginosa PA14.
The survival of uninfected D. melanogaster was similar to treat-
ment with cPAC or SMX alone (Figure S5A, Supporting Infor-
mation), which indicates that cPAC (at 50 µg mL−1) or SMX
(at 256 µg mL−1) alone is safe for this animal.
To conﬁrm that cPAC is able to potentiate antibiotics in more
than one host, we also used the greater wax moth (Galleria
mellonella) larvae killing model, in which cPAC or SMX was
administered alone, or in combination, to larvae infected with a
lethal dose of P. aeruginosa PA14. The median survival of infected
and untreated larvae was 21 h, but increased to 47 h with the
Adv. Sci. 2019, 6, 1802333
Figure 1. Potentiating interaction of cPAC with antibiotic results in growth inhibition. MICs were determined for the combination of cPAC with each
antibiotic in vitro. Fractional inhibitory concentration index (FICI) for each combination are shown for A) E. coli CFT073, B) P. mirabilis HI4320, and C) P.
aeruginosa PA14. A FICI of ≤0.5 is indicated by the gray shaded area. TMP: trimethoprim; SMX: sulfamethoxazole; FOS: fosfomycin; NIT: nitrofurantoin;
GEN: gentamicin; KAN: kanamycin; TET: tetracycline; AZT: azithromycin.
1802333 (4 of 12) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
cPAC and SMX combination treatment. This is a signiﬁcantly
2 = 14.3, df = 1, P < 0.005) longer survival time than the 36 h
median survival time with SMX treatment alone (Figure 3B).
The median survival of infected larvae with cPAC treatment
alone was similar to the treatment of SMX alone (Figure 3B),
which is in contrast to cPAC’s inability to act as an antibiotic at
this concentration in vitro, or in the ﬂy feeding model. As with
the D. melanogaster feeding assay, there was no difference in sur-
vival curves of uninfected G. mellonella larvae with or without
treatment of cPAC or SMX alone (Figure S5B, Supporting Infor-
mation). These results conﬁrm that cPAC potentiates the activity
of SMX in vivo, at the tested concentration of 50 µg mL−1 cPAC.
Adv. Sci. 2019, 6, 1802333
Figure 2. Synergistic effect of cPAC with TET for the inhibition of growth re-activation of antibiotic-exposed cells and prevention of the evolution of
resistance. A) Detection of growth re-activation of antibiotic-exposed E. coli CFT073 cells using a modiﬁed disk-diffusion assay. Step 1: a TET antibi-
otic disk was placed on top of MHB-II agar. The dashed lines mark the diameter of the clear zone surrounding the TET disk. Step 2: the TET disks
are replaced with a glucose alone, cPAC+glucose, or cPAC alone disk on the MHB-II agar plate. The diameter of the clear zone (CZ) and no colony
formation inside the clear zone surrounding the cPAC+glucose disk indicate no re-activation of antibiotic-exposed cells after disk replacement at Step
2 and colonies inside the inhibition zone (indicated by black arrow) surrounding the glucose disk indicate re-activation of antibiotic-exposed cells.
The cPAC-only disk prevented re-activation of growth, most likely because of synergy with TET remnants. The images shown are only representative
images of three independent experiments. Disk diameter: 6 mm. Emergence of antibiotic resistance in B) E. coli CFT073 and C) P. aeruginosa PA14
during 21 serial passages in the presence of sub-MIC levels of TET compared to 400 µg mL−1 cPAC alone or its combination with 100 µg mL−1 cPAC.
1802333 (5 of 12) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.5. cPAC in Combination with an Antibiotic Impairs Bioﬁlm
To explore whether cPAC can impair the formation of bioﬁlms
during antibiotic treatment, monoculture bacterial bioﬁlms
were grown in microtiter plates in the presence of different con-
centrations of cPAC and SMX. As shown in Figure 4A–C, cPAC
decreased bioﬁlm formation of E. coli CFT073, P. mirabilis
HI4320, and P. aeruginosa PA14. cPAC alone at 100 µg mL−1
showed signiﬁcant (P < 0.05) inhibition of monoculture bio-
ﬁlms, which is consistent with other studies showing that cPAC
or cranberry extracts can decrease bacterial adhesion.[8–11]
Importantly, cPAC in combination with SMX had a signiﬁcant
(P < 0.01) inhibitory effect on the development of bioﬁlms in a
dose-dependent manner (Figure 4A–C; Figure S1D, Supporting
Information). Untreated control bioﬁlms were composed of
viable cells (Figure 4D, in green) attached to the surface and
forming dense microcolonies (Figure 4Di). SMX alone at tested
concentrations had minimal effect on bioﬁlms. However,
treatment with cPAC and SMX in combination resulted in a
decrease in the total density of attached biomass and viability
when compared to untreated bioﬁlms (Figure 4E,F). Interest-
ingly, the majority of the bacterial biomass was dead when
treated with 512 µg mL−1 SMX in combination with cPAC
(Figure 4Dviii,ix). If we presume that the few cells left alive in
the presence of 512 µg mL−1 SMX and 50 µg mL−1 cPAC are
dormant, capable of reactivating when the antibiotic threat has
passed and forming a new bioﬁlm, then increasing the concen-
tration of cPAC to 100 µg mL−1 was sufﬁcient to eradicate these
surviving cells. Because a combination of cPAC and SMX is
more effective against bioﬁlms than either compound is alone,
these observations demonstrate that cPAC acts synergistically
with SMX to reduce the ability of bacteria to form bioﬁlms, and
supports the hypothesis that cPAC contributes to the impair-
ment of dormant antibiotic-exposed cells.
2.6. Mechanisms by Which cPAC Potentiates Antibiotic Activity
To identify the mechanism(s) of action by which cPAC potenti-
ates antibiotic activity, we quantiﬁed the changes in bacterial cell
outer membrane permeability using 1-N-phenylnapthylamine
(NPN) as an indicator, which revealed that cPAC increases
outer membrane permeability of bacterial cells (Figure 5A–C).
Investigations into efﬂux pump activity using ethidium bro-
mide (EtBr) as a ﬂuorescent indicator substrate showed that,
in contrast to the decay in ﬂuorescence observed in untreated
cells (Figure 5D–F), cells treated with cPAC, or the efﬂux pump
inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP),
remained ﬂuorescent over time due to a failure to pump out
EtBr (Figure 5D–F). These observations demonstrate that cPAC
is able to inhibit multidrug resistance efﬂux pumps. To further
investigate the interaction between cPAC and multidrug resist-
ance efﬂux pumps, we employed a systematic checkerboard
MIC analysis of P. aeruginosa PA14 efﬂux pump mutants.
The overexpression of efﬂux pump systems causes increased
resistance to antibiotics compared to wild-type isolates while
disruption of efﬂux pump protein components is associated
with increased intracellular antibiotic accumulation and antibi-
otic susceptibility. We show here that the ability of cPAC to
potentiate TET activity correlates with efﬂux pump activity. The
mutant strains with nonfunctioning efﬂux pumps were more
susceptible to TET compared to the wild-type strain, such that
addition of cPAC provided no further beneﬁt for the potency of
TET. Therefore, potentiation between cPAC and TET was not
observed in efﬂux pump-nonfunctioning mutants (Figure 5G).
This lack of TET potentiation correlates well with TET potentia-
tion by cPAC in the case of wild type P. aeruginosa PA14, which
has functioning efﬂux pumps (Figure 1C). Interestingly, cPAC
caused no signiﬁcant damage to bacterial cell membranes
(Figure 5H), compared to signiﬁcant (P < 0.05) membrane dis-
ruption by cetyl trimethylammonium bromide (CTAB), a cell
Adv. Sci. 2019, 6, 1802333
Figure 3. In vivo synergistic effect of cPAC with antibiotic for the protection of insect models. A) In vivo synergy between sulfamethoxazole (SMX) and
cPAC was tested in a D. melanogaster ﬂy feeding model. Flies (N = 30 per experimental group) were infected orally with P. aeruginosa PA14 cells and
maintained on agar containing SMX in combination with cPAC. Results represent measurements from experiments performed in triplicate (*, P < 0.05,
log-rank (Mantel–Cox) test). B) In vivo synergy between SMX and cPAC was tested in a G. mellonella larvae infection model. G. mellonella larvae were
infected with a lethal dose of P. aeruginosa PA14 cells. These infected G. mellonella larvae (N = 20 per experimental group) were injected a second time
at the same infection site with cPAC or SMX, alone or in combination at 3 h post. Results represent measurements from two independent experiments
performed in duplicate (*, P < 0.005, log-rank (Mantel–Cox) test).
1802333 (6 of 12) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
membrane–disrupting agent, suggesting that outer membrane
permeabilization and target speciﬁc efﬂux pump inhibition are
achieved by cPAC without altering cell membrane integrity.
2.7. cPAC Interacts with Efﬂux Pump Components In Silico
To understand how cPAC is able to inhibit the activity of
efﬂux pumps, we performed in silico docking analyses using
the efﬂux pump protein complexes AcrAB–TolC of E. coli, and
MexAB–OprM of P. aeruginosa, with the A-type dimeric cPAC
molecule as the test ligand. Ligand-binding domains of AcrAB–
TolC and MexAB–OprM efﬂux pump components exhibit
sufﬁcient space to accommodate the cPAC molecule with an
average volume of 497.1 Å3 (Table S1, Supporting Informa-
tion). We tested structural in silico interactions of cPAC with
the exit duct, adapter, and transporter components of the efﬂux
pumps (Figure 6A; Figure S6A–G, Supporting Information).
Molecular docking of cPAC with the exit duct complex predicts
that cPAC favorably binds at the equatorial domain of the TolC
Adv. Sci. 2019, 6, 1802333
Figure 4. Effect of cPAC alone and in combination with SMX on bioﬁlm formation of A) E. coli CFT073, B) P. mirabilis HI4320, and C) P. aeruginosa
PA14. The graphs present normalized bioﬁlm levels (OD570/cell OD600) at subinhibitory concentrations of SMX. Statistically signiﬁcant differences are
indicated for each sample treated with cPAC and SMX compared to the control (sample treated with the corresponding concentration of SMX alone)
(**, P < 0.01; *, P < 0.05; two-way ANOVA) and for samples treated with cPAC plus SMX compared to the sample treated with the same concentration
of cPAC without SMX (*, P < 0.05; two-way ANOVA). D) Confocal microscopy images of P. aeruginosa PA14 bioﬁlms grown with or without cPAC and/
or SMX at sub-MICs. Green color cells represent viable cells with intact membranes and red color cells are dead cells. Each representative image was
selected from experiments performed in triplicates. E) Bioﬁlm biomass as a percentage of untreated biomass, and F) percentages of live cells present
in each treatment were quantiﬁed from confocal microscopy images. In all confocal microscopy images, red and green axis lengths are 100 µm, and
the blue z-axis length is 10 µm.
1802333 (7 of 12) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Sci. 2019, 6, 1802333
Figure 5. Mechanisms of antibiotic potentiation by cPAC. cPAC-mediated NPN uptake in A) E. coli CFT073, B) P. mirabilis HI4320, and C) P. aeruginosa
PA14. Bacterial cells were pretreated with cPAC or gentamicin (Gen) for NPN uptake. Control represents NPN ﬂuorescence in the presence of only cPAC
without bacteria. Inhibition of multidrug efﬂux pump by cPAC or the known efﬂux pump inhibitor CCCP in D) E. coli CFT073, E) P. mirabilis HI4320, and F) P.
aeruginosa PA14. Control represents untreated bacteria. G) MICs were determined for the combination of cPAC with TET in vitro. FICI values for cPAC+TET
combination are shown for efﬂux pump mutants of P. aeruginosa PA14: mexL− (overexpressing MexJK-OprM), nfxB− (overexpressing MexCD-OprJ),
nfxC− (overexpressing MexEF-OprN), nalC− (overexpressing MexAB-OprM), mexA− (nonfunctioning MexAB-OprM), and oprM− (nonfunctioning
MexAB-OprM, MexJK-OprM, and MexXY-OprM). A FICI index of ≤0.5 is indicated by the gray shaded area. H) Effect of cPAC on cell membrane
integrity. Cells of each strain were pretreated separately with cPAC or the cell membrane disrupting agent CTAB. The ratio of green to red ﬂuorescence
was normalized to that of the untreated control and expressed as a percentage of the control. All assays were repeated independently three times
(* P < 0.05; Student’s t-test).
1802333 (8 of 12) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
exit duct (Figure 6A) and coiled-coil domain of the OprM exit
duct (Figure S6A–C, Supporting Information). The molecular
docking analyses also predict that cPAC can form hydrogen
bonds with amino acid residues (N274, A279, and R620) pre-
sent in the distal substrate-binding pocket of AcrB that are
critical for antibiotic/substrate binding, transportation to the
Adv. Sci. 2019, 6, 1802333
Figure 6. Molecular docking analysis of cPAC in the AcrAB–TolC efﬂux pump. A) Complete side view with ribbon representation of docked complexes of
efﬂux pump proteins with A-type cPAC molecule (a dimeric form of epicatechin), visualized along the Gram-negative cell membrane plane (OM, outer
membrane; PP, periplasmic space; and IM, inner membrane). The inset views show the electron density map (2F0–Fc) of cPAC in binding sites of
multidrug efﬂux pump exit duct, adapter, and transporter proteins. The amino acid residues around each binding site are depicted and all possible
hydrogen bonds are shown using green lines. The ribbon representation of tripartite efﬂux pump components are color-coded: TolC exit duct (pink),
AcrA adapter (gold) and AcrB transporter (blue). B) The AcrB monomer with inset views shows docking of cPAC molecule (salmon) to the distal
binding pocket in the co-crystal structure of AcrB–MIN complex (yellow color carbons represent MIN molecule as found in the highest resolution
crystal structure, PDB 4U8Y). C) Molecular docking analysis of different efﬂux pump substrates (yellow) binding at the distal binding pocket of AcrB
transporter. D) The inset views show the optimum binding position of cPAC (salmon) and substrate (yellow) at the distal binding pocket of AcrB. The
docking models of nonpotentiated antibiotics are highlighed with a dashed box.
1802333 (9 of 12) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Sci. 2019, 6, 1802333
exit duct, and proper functioning of the entire efﬂux pump
assembly (Figure 6A). cPAC showed the highest afﬁnity for
AcrB and MexB efﬂux pump components, with −9.9 and
−9.6 kcal mol−1 binding energies, respectively (Table S1,
A diverse range of antibiotics are pumped out by AcrAB–
TolC. However, only the co-crystal structures of MIN and dox-
orubicin binding to the active site of AcrB are reported. To
compare the binding of cPAC to that of a well-characterized
efﬂux pump substrate, we docked cPAC to the distal binding
pocket of the AcrB periplasmic porter domain co-crystallized
with MIN, and found that cPAC and MIN bound to the same
furrow of the distal substrate binding pocket (Figure 6B). We
separately examined the predicted docking of the AcrB sub-
strates FOS, TMP, NIT, TET, SMX, and GEN in the absence and
presence of cPAC (Figure 6C,D). All tested substrates bound to
the AcrB–cPAC complex, speciﬁcally, in the same furrow of the
distal binding pocket in which MIN binds (Figure 6C). Inter-
estingly, the binding position of FOS and TMP was unaffected
by the presence of cPAC (Figure 6D), which is in agreement
with the inability of cPAC to potentiate FOS or TMP against
E. coli in vitro (Figure 1A). In contrast, we observed different
binding conformations of the potentiated antibiotics NIT, TET,
SMX, and GEN in the presence of cPAC compared to that in
the absence of cPAC (Figure 6C,D). This suggests that binding
of cPAC in the distal binding pocket leads to efﬂux inhibition
by interfering with the preferred binding position of the poten-
tiated antibiotics. Finally, we conﬁrmed that the binding confor-
mation of cPAC in the distal binding pocket of AcrB is similar
to that of known efﬂux pump inhibitors (Figure S7A–F, Sup-
porting Information), which supports the hypothesis that cPAC
acts as a potent efﬂux pump inhibitor.
Tetracycline resistance is a well-documented phenomenon
caused by efﬂux pump activity and the evolution of acquired
antibiotic resistance. We discovered that in the presence of
cPAC, the acquisition of resistance in E. coli and P. aeruginosa
following TET treatment is completely abrogated. cPAC’s ability
to interfere with intrinsic resistance mechanisms is therefore
able to suppress the typically inevitable, long-term evolution of
acquired antibiotic resistance.
We have shown that cPAC potentiates the in vitro activity of
a range of antibiotic classes against the opportunistic human
pathogens E. coli, P. mirabilis, and P. aeruginosa. This potentia-
tion also occurs in vivo, at least with the antibiotic SMX. cPAC
is an especially promising antibiotic-potentiating agent because
it does not exhibit antimicrobial activity of its own. Agents
that do not negatively affect the viability of bacterial patho-
gens are less likely to promote resistance than conventional
antibiotics[32–34] and are, therefore, especially well suited for
combination treatment with antibiotics. Accordingly, we have
not observed any evolution of resistance to cPAC.
Bioﬁlms can lead to chronic bacterial infections and are
commonly associated with antibiotic treatment failure.[35,36]
The presence of persister and antibiotic-tolerant cells is closely
linked to bioﬁlm formation, and also plays an important role in
the recalcitrance of chronic infections. The antiquorum sensing
activity of a cranberry proanthocyanidin fraction alone and
in combination with ciproﬂoxacin has been reported. As
quorum sensing is required for normal bioﬁlm formation, this
agrees with our observations that cPAC in combination with
antibiotic substantially represses bioﬁlm formation. Further-
more, we propose that it is the ability of cPAC to target not only
actively metabolizing cells, but also dormant antibiotic-exposed
cells that enable cPAC to potentiate antibiotic efﬁcacy against
The intrinsic antibiotic resistance mechanisms of bacte-
rial cells are naturally occurring phenomena found in bacte-
rial species that complicate antibiotic treatments. For instance,
P. mirabilis has intrinsic resistance to NIT and TET, and
P. aeruginosa has intrinsic resistance to multiple antibiotic
classes including aminoglycosides,
-lactams, quinolones, and
polymyxins.[38,39] The intrinsic antibiotic resistance mechanisms
include selective outer membrane permeability, poor antibiotic
transport, and active multidrug efﬂux.[40,41] Among resistance–
nodulation–division family efﬂux pumps, AcrAB–TolC[42,43] and
MexAB–OprM are well known for their importance in bac-
terial survival and intrinsic antibiotic resistance. Our in silico
analysis predicts that the A-type dimeric cPAC molecule (which
is the most common terminating unit of the cPAC fraction)
can occupy the ligand-binding pocket of the AcrB transporter
in E. coli and MexB transporter of P. aeruginosa.[43,44,46] Mole-
cular docking calculations indicate that of all the antibiotics
tested against E. coli, the two nonpotentiated antibiotics adopt
the same position in the distal binding pocket in the presence
and absence of cPAC. In contrast, the potentiated antibiotics
are predicted to take a different binding position in the pres-
ence of cPAC, which would hinder their efﬂux from the cell.
Exposure to cPAC enhanced membrane permeabilization and
decreased efﬂux pump activity in all tested wild-type bacteria
in vitro. The failure of cPAC to potentiate antibiotic activity in
efﬂux pump-defective mutants supports a model where the spe-
ciﬁc effect cPAC has on efﬂux is essential for the observed anti-
biotic potentiating activity. Inactivation of efﬂux pump activity
has previously been shown to have a negative impact on bioﬁlm
formation and pathogenicity in Gram-negative bacteria.
This is consistent with our observations that in the presence of
cPAC, bioﬁlm formation is decreased. Overall, this study pro-
vides a proof of concept and a starting point for investigating
the molecular mechanism of the reported efﬂux pump inhibi-
tion in bacteria by cPAC.
Insect animal models have a relatively evolved antimicro-
bial defense system and are thus often used to generate infor-
mation relevant to the mammalian infection process.[49,50]
In both infection models used in this study, cPAC at a dose
of 50 µg mL−1 was sufﬁcient to potentiate the activity of the
tested antibiotic and signiﬁcantly increase survival rates of
the animals during infection with P. aeruginosa. These in vivo
studies provide a promising outlook for the potential future
development of cPAC as an antibiotic-potentiating agent in
higher organisms; however, a few studies report on the safety
of cPAC to human cells or its bioavailability and the rate
of clearance in animals. Thus, further work is needed to
verify the efﬁcacy and safety of the combination treatment in
an in vivo mammalian (e.g., mouse) model. Encouragingly,
1802333 (10 of 12) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Sci. 2019, 6, 1802333
our data show that cPAC is able to enhance the efﬁcacy of
a broad spectrum of antibiotics. The ability to potentiate the
action of antibiotics in a patient could improve treatment
outcomes and hinder the emergence of antibiotic-resistant
4. Experimental Section
Bacterial Strains, Growth Conditions, and Cranberry Proanthocyanidin:
Opportunistic bacterial pathogens were used in this study: E. coli strain
CFT073 (ATCC 700928), P. mirabilis HI4320, P. aeruginosa PAO1 (ATCC
15692), and P. aeruginosa PA14 (UCBPP-PA14). Mutant strains of P.
aeruginosa PA14 were used in this study: mexL− (overexpressing MexJK-
OprM), nfxB− (overexpressing MexCD-OprJ), nfxC− (overexpressing
MexEF-OprN), nalC− (overexpressing MexAB-OprM), mexA−
(nonfunctioning MexAB-OprM), and oprM− (nonfunctioning MexAB-
OprM, MexXY-OprM, and MexJK-OprM). Pure stock cultures were
maintained at −80 °C in 30% (v/v) frozen glycerol solution. Starter
cultures were prepared by streaking frozen cultures onto lysogeny broth
agar (LB broth contained 10 g L−1 tryptone, 5 g L−1 yeast extract, and
5 g L−1 NaCl, supplemented with 1.5% (w/v) agar (Fisher Scientiﬁc,
ON, Canada)). After overnight incubation at 37 °C, a single colony was
inoculated into 10 mL of Mueller–Hinton broth adjusted with Ca2+ and
Mg2+ (MHB-II; Oxoid, Fisher Scientiﬁc Canada) and the culture was
incubated at 37 °C on an orbital shaker at 200 rpm for a time length
speciﬁc to each experiment. LB broth was used for bacterial culture in all
experiments unless otherwise speciﬁed. The puriﬁed cranberry-derived
proanthocyanidin (cPAC, 93% proanthocyanidins, 7% anthocyanins,
and ﬂavonoids monomers) was obtained from Ocean Spray Cranberries
Inc. The supplier prepared the sample according to well-established
methods[11,57] by enriching from cranberry fruit juice extract. A dry
powder of cPAC was solubilized in deionized water and sterilized by
ﬁltration (0.22 µm polyvinylidene ﬂuoride (PVDF) membrane ﬁlter).
Determination of MICs: MICs were determined by preparing twofold
serial dilutions of cPAC, and antibiotics in MHB-II broth. A range of
concentrations of the antibiotics (0.0003–1024 µg mL−1) was chosen
due to their known potency against all four bacterial strains. Bacterial
growth was assessed by i) monitoring the cell growth (observed as a
pellet and turbidity) in the wells ii) monitoring the optical density of
the cell suspension in each well at 600 nm (OD600), and iii) using the
resazurin microtiter plate assay. Additional details can be found in the
In Vivo Infection Assay Using D. Melanogaster Flies: Fruit ﬂies
(D. melanogaster) were infected orally in ﬂy feeding assay in which ﬂies
were anesthetized, starved of food and water for several hours, and
separated into vials containing ﬁlter paper disks inoculated with freshly
grown P. aeruginosa PA14, as well as 5% sucrose agar (sterile) with and
without 50 µg mL−1 cPAC alone or in combination with 256 µg mL−1
SMX. Post infection mortality of ﬂies was monitored daily for 14 days,
with each treatment tested twice in triplicate. Additional details can be
found in the Supporting Information.
In Vivo Infection Assay Using Galleria Mellonella Larvae: Twenty
G. mellonella larvae were injected with active cultures of P. aeruginosa
per treatment. All injected larvae were incubated in Petri dishes at 28 °C
under 30% relative humidity in the dark, and the number of dead larvae
was scored daily post infection. A larva was considered dead when it
displayed no vital signs in response to touch, followed by increased
melanization. Additional details are given in the Supporting Information.
Bioﬁlm Assays: Bioﬁlm formation was quantiﬁed using the standard
microtiter plate model and crystal violet staining, the details of which
are described in Appendix S1 (Supporting Information). For bioﬁlm
imaging and analysis, bioﬁlms were grown for 16 h at 37 °C under
static conditions, after which planktonic cells were removed by rinsing.
The cell-membrane impermeable ﬂuorescent stain TOTO-1 (Thermo
Fisher) and membrane permeable stain SYTO 60 (Thermo Fisher)
were added to ﬁnal working concentrations of 2 × 10−6 and 10 × 10−6 m,
respectively. Bioﬁlms were imaged as z-stacks using a 63× objective
on a Zeiss 800 confocal laser scanning microscope and rendered such
that living cells are colored green, and dead cells are colored red as
described in the Supporting Information.
Modiﬁed Disk-Diffusion Assay: A modiﬁed disk-diffusion assay was
used to detect recovery of antibiotic-exposed cells. This modiﬁed disk-
diffusion assay was conducted in two steps, where Step 1 was similar
to classical disk-diffusion assay and Step 2 was slightly modiﬁed.
Brieﬂy, an overnight bacterial culture (MHB-II broth, 37 °C, 200 rpm)
was diluted into fresh MHB-II broth to 106 CFU mL−1 and plated on
MHB-II agar plate. Step 1: commercially available TET 30 µg or MIN
30 µg antibiotic disk (Thermo Scientiﬁc Oxoid, Fisher Scientiﬁc,
Canada) was placed on top of the inoculated agar surface and the
plate was incubated at 37 °C for 18 h. Custom disks were prepared
using sterile blank disks (Thermo Scientiﬁc Oxoid, Fisher Scientiﬁc,
Canada) supplemented with 400 µg glucose or 400 µg cPAC with or
without 400 µg glucose and left to dry at room temperature. Step 2:
The antibiotic disk was carefully replaced with custom disks containing
glucose or cPAC or their combination, and the plate was incubated at
37 °C for an additional 18 h.
Emergence of Resistance Analysis: For characterization of in vitro
resistance evolution by standard sequential passaging technique,[63,64]
E. coli CFT073 and P. aeruginosa PA14 cells were grown to
exponential phase in MHB-II at 37 °C. MICs were determined by
preparing twofold serial dilutions in 96-well microtiter plates. Each
well was inoculated with the desired bacterial strain, and the plate was
incubated at 37 °C for 18 h under static conditions. Bacterial growth
was assessed by i) monitoring the cell growth (observed as a pellet
and turbidity) in the wells and ii) monitoring the optical density
of the cell suspension in each well at 600 nm (OD600nm). Bacterial
suspension from sub-MIC (0.5 × MIC) of TET or cPAC or cPAC+TET
combination was used to prepare the inoculum for the next day MIC
experiment by diluting it to a ﬁnal concentration of ≈106 CFU mL−1
in MHB-II. This was repeated for 21 passages, and the ratio of the
MIC obtained during each day relative to the MIC at 0 day (ﬁrst
time exposure) was determined. The data were expressed as relative
fold increase in MIC with each day or passage. Experiments were
performed with biological replicates.
Membrane Permeabilization and Membrane Integrity Assays: The
outer membrane permeabilization activity of cPAC was determined by
the NPN (Sigma-Aldrich, Canada) assay, with some modiﬁcations.
Overnight bacterial cultures were diluted 1:1 in MHB-II medium
to a ﬁnal volume of 10 mL, with or without supplementation of
cPAC or GEN (positive control), and grown to an OD600 of 0.5–0.6
(37 °C, 200 rpm). The cells were harvested, washed, resuspended
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer
containing 1 × 10−3 m N-ethylmaleimide (NEM; Sigma-Aldrich,
Canada). Aliquots were mixed with NPN to a ﬁnal concentration of
10 × 10−6 m (in cell suspension), and ﬂuorescence was measured using
the microplate reader (excitation: 350 nm; emission: 420 nm). The
BacLight kit (L-13152; Invitrogen, Life Technologies Inc., Canada) was
used to assess cell membrane damage, with ﬂuorescence readings
from samples normalized to the values obtained from the untreated
control to determine the ratio of membrane-compromised cells to
cells with intact membranes. Details can be found in the Supporting
EtBr Efﬂux Assay: To assess the effect of cPAC on the inhibition of
the proton motive force–driven multidrug efﬂux pump, an EtBr efﬂux
assay was performed. An overnight-grown culture of each strain
was diluted 1:100 in MHB-II broth to a ﬁnal volume of 10 mL and was
grown to an OD600 of 0.8–1.0 (at 37 °C, 150 rpm). Cells were loaded in
polystyrene microcentrifuge tubes (2 mL) and mixed with 5 × 10−6 m EtBr
and cPACs at 25% of their MIC or a 100 × 10−6 m concentration of the
proton conductor CCCP (Sigma-Aldrich Canada) as a positive control.
Replica tubes that did not receive cPACs or proton conductor served as
negative controls. The tubes were incubated for 1 h (37 °C, 150 rpm).
The inoculum then was adjusted to an OD600 of 0.4 with MHB-II broth
containing 5 × 10−6 m EtBr, and 2 mL of aliquot of this mixture was
1802333 (11 of 12) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Sci. 2019, 6, 1802333
pelleted (5000 × g, 10 min, 4 °C). The pellets were incubated on ice
immediately, resuspended in 1 mL of MHB-II, and aliquoted (200 µL)
into a polystyrene 96-well, black, clear-bottom plate (Corning, Fisher
Scientiﬁc Canada). EtBr efﬂux from the cells was monitored at room
temperature using the microplate reader (excitation wavelength 530 nm;
emission wavelength, 600 nm). Readings were taken at 5 min intervals
for 1 h to monitor efﬂux pump activity. The background ﬂuorescence of
the medium was subtracted from all measurements, and the assay was
repeated independently in triplicate.
In Silico Docking Analysis: 3DLigandSite server was used to predict
ligand binding sites on targeted protein structures using an automated
method by searching a structural library to identify homologous
structures with bound ligands. These bound ligands were
superimposed onto the targeted protein structure to predict a ligand-
binding site. In silico docking was performed using the Autodock Vina
tool without the incorporation of water molecules. In silico docking
analysis by Autodock Vina was compared with SwissDock web service
(http://www.swissdock.ch/) to conﬁrm the accuracy and robustness of
predicted docking complexes. The ligand docking methods are described
in the Supporting Information.
Statistical Analysis: Where indicated, a two-tailed Student’s t-test
(P < 0.05) was used to determine whether the presence of cPAC
resulted in a signiﬁcant difference compared to levels for the control.
A two-way analysis of variance (ANOVA), followed by Sidak’s multiple
comparison, was used for bioﬁlm assays to analyze statistical
signiﬁcance of the difference in biomass. Fruit ﬂy survival curves were
prepared using GraphPad Prism 6 (GraphPad Software, Inc., San Diego,
CA) to perform a statistical log–rank (Mantel–Cox) test. Throughout the
text, all of the changes (increase or decrease) reported were statistically
signiﬁcant. Statistically nonsigniﬁcant values were not mentioned in
Full Methods: Detailed procedures of all methods are available in the
Supporting Information is available from the Wiley Online Library or
from the author.
The authors thank C. Khoo (Ocean Spray Cranberries Inc.) for providing
the puriﬁed cPAC sample; M.-C. Groleau for technical assistance with
in vivo experiments and helpful discussions; E. Curling, J. Johnson,
and P. Sundaram for technical assistance in preliminary screening for
antibiotic potentiation in vitro; and the Advanced Bioimaging Facility at
McGill University for providing access to their confocal microscope. This
work was supported by the Natural Sciences and Engineering Research
Council of Canada (NSERC), and Ocean Spray Cranberries Inc. N.T.
holds the Canada Research Chair in Biocolloids and Surfaces, and E.D.
holds the Canada Research Chair in Sociomicrobiology. V.B.M., E.D., and
N.T. conceived the experiments. V.B.M. conducted in vitro, in vivo, and
in silico docking experiments and analyzed the results. M.O. conducted
confocal laser scanning microscopy and bioﬁlm image analysis. V.B.M.
wrote the manuscript with revisions by M.O., N.T., and E.D.
Conﬂict of Interest
Authors Nathalie Tufenkji and Vimal B. Maisuria have applied for a
patent (WO 2017/096484) on the use of cranberry derived phenolic
compounds as antibiotic synergizing agent against pathogenic bacteria.
The patent application is presently under review by the USPTO, with
both authors listed as inventors. All authors declare that they have no
other conﬂicts of interest.
anti-bioﬁlm, antimicrobial, efﬂux pump, multidrug resistance,
Received: December 25, 2018
Revised: April 3, 2019
Published online: May 28, 2019
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