Cranberry-derived proanthocyanidins prevent formation of
Candida albicans biofilms in artificial urine through biofilm- and
Hallie S. Rane1, Stella M. Bernardo1,2, Amy B. Howell3and Samuel A. Lee1,2*
1Section of Infectious Diseases, New Mexico Veterans Healthcare System, Albuquerque, NM, USA;2Division of Infectious Diseases,
University of New Mexico Health Science Centre, Albuquerque, NM, USA;3Marucci Center for Blueberry and Cranberry
Research and Extension, Rutgers University, Chatsworth, NJ, USA
*Corresponding author. Section of Infectious Diseases, New Mexico Veterans Healthcare System, Albuquerque, NM, USA.
Tel: +1-505-265-1711; Fax: +1-505-256-2803; E-mail: SamALee@salud.unm.edu
Received 22 May 2013; returned 8 August 2013; revised 6 September 2013; accepted 9 September 2013
prevention of bacterial UTIs, partially due to anti-adherence properties, but thereare limited data on their use for
cranberry-derived PACs on C. albicans biofilm formation in artificial urine.
trations and biofilm metabolic activity was assessed using the XTT assay in static microplate and silicone disc
tested, with a paradoxical effect observed at high concentrations in two clinical isolates. Further, cranberry PACs
were additive in combination with traditional antifungals. Cranberry PACs reduced C. albicans adherence to both
polystyrene and silicone. Supplementation of the medium with iron reduced the efficacy of cranberry PACs
Conclusions: These findings indicate that cranberry PACs have excellent in vitro activityagainst C. albicans biofilm
formation is due to anti-adherence properties and/or iron chelation.
Keywords: cranberry PACs, urinary tract infections, urinary catheters, Vaccinium macrocarpon Ait.
diduria is common in hospitalized patients and, although often
asymptomatic and of unknown clinical significance, candiduria is
still associated with increased morbidity and mortality.1,2In rare
morbidity and mortality.1–4Three major classes of antifungal
drugs are currently available for use against Candida infections,
but each class has notable limitations, particularly against candi-
duria.5Azoles are the most commonly used antifungal for the
treatment of C. albicans infections, but are fungistatic and poorly
active against biofilms.6–8Echinocandins are highly active
against C. albicans, but generally do not achieve clinically useful
tions, the mortality, morbidity and cost of care due to candiduria
tive treatments and preventive approaches is warranted.11
A major component of C. albicans pathogenesis is the ability to
form biofilms on both biotic and abiotic surfaces.8,12Unlike their
planktonic counterparts, fungal organisms embedded in biofilms
undergo phenotypic changes that protect them from the local
microenvironment and the host immune system. The formation
Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy 2013. This work is written by US Government
employees and is in the public domain in the US.
J Antimicrob Chemother 2014; 69: 428–436
doi:10.1093/jac/dkt398 Advance Access publication 10 October 2013
by guest on October 20, 2015
of biofilms is also associated with increased drug resistance. Al-
presenting with candiduria form strong biofilms in artificial urine
(AU) in vitro.6Further, C. albicans has been shown to form biofilms
in indwelling catheters in an in vivo murine model of catheter-
associated candiduria13and biofilms have been observed on
infected urinary catheters in vivo.14The initial stage of biofilm for-
process mediated both by abiotic factors, such as surface hydro-
sins and other cell-surface proteins.12Therefore, the inhibition of
adherence by C. albicans is a promising target for disrupting the
initial stages of biofilm formation in C. albicans.
Cranberry juice has been touted as a safe, alternative therapy
against bacterial urinary tract infections for centuries. Further,
the preventative value of cranberry juice against Escherichia coli
urinary tract infections has been demonstrated clinically15–17
and the anti-adherence quality of cranberry juice extract against
E. coli has been studied extensively.18–22Specifically, A-type
proanthocyanidins (PACs) isolated from cranberries have been
implicated in the anti-adherence properties of cranberry juice.18
The effect of cranberry PACs on E. coli adherence is not substrate
specific; cranberry PACs decrease E. coli adherence to uroepithelial
such as PVCand polytetrafluoroethylene.20Ingestionof cranberry
in vivo Caenorhabditis elegans model.21Interestingly, cranberry
PACs have both non-biospecific activity against adherence,
perhaps due to steric hindrance,20and biospecific activity against
adherence, including decreased expression of adhesion genes
PACs in the prevention of adherence in other species. A-type cran-
berry PACs have previously been reported to decrease C. albicans
adherence to oral epithelial cells and to reduce biofilm formation
and inflammatory responses in vitro.23
In order to determine the activity of cranberry PACs against
C. albicans urinary biofilms, we systematically assayed the effect
of serially increasing doses of cranberry PACs on biofilm formation
and mature biofilms in AU on both polystyrene and silicone
surfaces. To study the degree of strain-specific variation upon
treatment with cranberry PACs, we next studied the effect of
ing four C. albicans strains isolated from patients with candiduria.
In addition, we tested cranberry PAC treatment in combination
with traditional antifungals for activity against C. albicans biofilm
To gain further insight into the mechanism of activity, we assayed
C. albicans adherence to both polystyrene and silicone surfaces
in response to cranberry PACs. Finally,we tested whether iron che-
lation could account for cranberry PAC activity against C. albicans
Isolation of cranberry-derived PACs
Cranberry PACs were isolatedfrom cranberryfruit (Vaccinium macrocarpon
Ait.) using solid-phase chromatography as previously described.18Briefly,
cranberry fruit was homogenized with 70% aqueous acetone, filtered
and thepulpdiscarded.Toremoveacetone, thecollectedextractwascon-
water, applied to a pre-conditioned C-18 solid-phase chromatography
column and washed with water to remove sugars, followed by acidified
aqueous methanol to remove acids. The fats and waxes retained on the
C-18 sorbent were discarded. The polyphenolic fraction containing antho-
cyanins, flavonol glycosides and PACs (confirmed using reverse-phase
HPLC with diode array detection) was eluted with 100% methanol and
dried under reduced pressure. This fraction was suspended in 50% EtOH
and applied to a pre-conditioned Sephadex LH-20 column washed with
aqueous acetone and monitored using diode array detection at 280 nm.
The absence of absorption at 360 and 450 nm confirmed that anthocya-
nins and flavonol glycosides were removed. Acetone was removed under
reduced pressure and the resulting purified PAC extract freeze-dried. The
presence of A-type linkages and concentration of PACs present in the
extract were confirmed using electrospray mass spectrometry,13C-NMR,
matrix-assisted laser desorption/ionization time-of-flight mass spectrom-
etry and acid-catalysed degradation with phloroglucinol.18,24,25
Assays for biofilm activity
For initial studies of the activity of cranberry PACs against mature biofilms
and against biofilm formation (i.e. biofilm prevention), C. albicans SC5314
was used as a reference isolate (gift from W. Fonzi, GeorgetownUniversity,
Washington DC, USA).26To characterize the degree of strain-specific vari-
ation, C. albicans ATCC 10231, ATCC 14053 and ATCC 24433 strains and
two C. albicans fks1 mutant strains (strains 42379 and 53264) character-
ized byechinocandin resistance were also studied.27,28Clinical urinary iso-
NM VA Healthcare System, Albuquerque, NM, USA). C. albicans biofilm for-
mation in polystyrene 96-well microtitre plates and the XTT assay, used
to determine the metabolic activity of the biofilms, were performed as
described previously,29with slight modifications. Briefly, each strain was
grown in yeast extract/peptone/dextrose (‘YPD’) broth with 0.008%
uridine overnight (308C, 250 rpm). Cells were harvested via centrifugation,
for 24 h. The biofilms were then washed with PBS three times to remove
non-adherent cells. Next, 100 mL of cranberry PACs serially diluted in AU
to concentrations of 4–1024 mg/L was added to biofilm-containing
with cranberry PACs, all biofilms were again washed three times with PBS
and theXTTassay was usedto determinethemetabolicactivityof thebio-
films.29The opticaldensity (OD) at 490 nm was measured via spectropho-
tometry on a BioTek ELx808 microplate reader (BioTek Instruments, Inc.,
was expressed as a percentagerelativeto themetabolicactivityof the un-
treated control. Each experiment was performed in triplicate or quadrupli-
controls using one- or two-wayanalyses of variance and Tukey’s multiple-
comparison post-test. Differences were considered significant at P,0.05.
Statistical analyses were performed and graphs were produced using
GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA) and Micro-
softwExcel (Microsoft Corp., Redmond, WA, USA). The MIC was defined as
the concentration of cranberry PAC or traditional antifungal agent needed
to reduce the metabolic activity of the biofilms of the strain by 50%
(MIC50) and 80% (MIC80).
described above was followed, except that cells in AU were coincubated
Cranberry PACs prevent Candida albicans urinary biofilms
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Rane et al.
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