Download Candida albicans in oral biofilms could prevent caries

Pathogens and Disease, 74, 2016, ftw039
doi: 10.1093/femspd/ftw039
Advance Access Publication Date: 28 April 2016
Research Article
RESEARCH ARTICLE
Candida albicans in oral biofilms could prevent caries
Hubertine Marjoleine Willems† , Kevin Kos† , Mary Ann Jabra-Rizk
and Bastiaan P. Krom∗
Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam, University of Amsterdam and
VU University Amsterdam, Amsterdam, The Netherlands
∗
Corresponding author: Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU
University Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam, The Netherlands. Tel. +31-20-5980402; E-mail: [email protected]
†
These authors contributed equally
One sentence summary: While Candida albicans is commonly isolated from patients with caries, it is not per se a cariogenic species; it could prevent
caries eventually.
Editor: Thomas Bjarnsholt
ABSTRACT
Streptococcus mutans is a Gram-positive bacterium involved in development to caries, the most common infectious disease
of our time. Streptococcus mutans interacts with other microbes, like the fungus Candida albicans and both are commonly
isolated from patients with caries. Since the role of C. albicans in caries remains unknown, our aim was to unravel this using
an in vitro dual-species cariogenic oral biofilm model. Biofilms were grown for 24–72 h on glass cover slips or hydroxyapatite
(HA) disks to mimic the surface of teeth. Medium pH, lactic acid production capacity and calcium release from HA disks
were determined. All 24-h biofilms had external pH values below the critical pH of 5.5 where enamel dissolves. In contrast,
72-h dual-species biofilms had significantly higher pH (above the critical pH) and consequently decreased calcium release
compared to single-species S. mutans biofilms. Counter intuitively, lactic acid production and growth of S. mutans were
increased in 72-h dual-species biofilms. Candida albicans modulates the pH in dual-species biofilms to values above the
critical pH where enamel dissolves. Our results suggest that C. albicans is not by definition a cariogenic microorganism; it
could prevent caries by actively increasing pH preventing mineral loss.
Keywords: Candida albicans; oral biofilm; Streptococcus mutans; interspecies interaction
INTRODUCTION
Dental caries is the second most prevalent disease in humans,
only surpassed by the common cold (Islam, Khan and Khan
2007). It is the most dominant cause of tooth decay, which is often the result of extensive dental plaque (Islam, Khan and Khan
2007). Dental plaque is a biofilm consisting of multiple bacterial
and fungal species (Kuboniwa et al. 2012). The bacteria in the
mature biofilm ferment sugars into acids, most notably lactic
acid. Teeth covered with dental plaque are exposed to the lactic
acid, causing the calcium phosphate of the enamel to dissolve,
a process also known as demineralization (Barbieri et al. 2007).
Prolonged periods of demineralization triggers the development
of dental caries, where lesions and cavities in the teeth create
a portal for bacteria to enter the tooth tissues and eventually
the root (Jefferson 2004; Islam, Khan and Khan 2007; Metwalli
et al. 2013). When this condition stays untreated, it can eventually lead to teeth loss or even more severe problems, for example
endocarditis (Islam, Khan and Khan 2007; Metwalli et al. 2013).
Understanding the onset and progression of dental caries is important in tackling this problem. Increasing interest goes out to
a polymicrobial cause of dental caries, formulated in the ecological plaque hypothesis (Marsh 2003; Rosier et al. 2014), instead
of attributing the disease to a single bacterial species (Jenkinson
and Douglas 2002; Barbieri et al. 2007; Falsetta et al. 2014).
Received: 23 November 2015; Accepted: 25 April 2016
C FEMS 2016. All rights reserved. For permissions, please e-mail: [email protected]
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Streptococcus mutans is one of the main species involved in
caries. It is a Gram-positive, coccus-shaped bacterium (Madigan et al. 2012). Streptococcus mutans has several characteristics
which makes it capable of inducing caries. First, it is a facultative anaerobic bacterium, allowing survival in the oral cavity
where it is predominantly found in crevices and small fissures
(Islam, Khan and Khan 2007; Madigan et al. 2012). Second, it is
able to enhance its adhesion to teeth by producing the sticky
adhesive polysaccharide dextran. The dextransucrase derived
from S. mutans uses sucrose as a substrate. Streptococcus mutans and other microorganisms are held together by this sticky
material, an essential component of the mature biofilm matrix
(Madigan et al. 2012; Falsetta et al. 2014). In addition, S. mutans
is able to ferment several sugars, including sucrose, glucose,
dextrose and lactose, resulting in the production of lactic acid
(Madigan et al. 2012).
The commensal fungus C. albicans is also a common colonizer of the oral cavity (Metwalli et al. 2013). However, in
immune-comprised individuals C. albicans is a possible opportunistic pathogen that is able to cause mucosal and systemic
infections (Metwalli et al. 2013). Candida albicans is able transform between a hyphal and a yeast form, each with different
characteristics (Sudbery 2011). In 97% of children with caries,
C. albicans can be isolated from the dental lesion (Klinke et al.
2011). However, it is the question whether this correlation is also
a causative factor for the pathogenesis of caries. Candida albicans does have traits that make it a possible cariogenic microorganism. Like S. mutans, C. albicans is extremely acid tolerant due
to an H+ ATPase, which actively pumps protons out of the cell
(Barbieri et al. 2007). Furthermore, C. albicans is able to efficiently
adhere to teeth enamel, a major component of teeth (Henriques,
Azeredo and Oliveira 2004).
Streptococcus mutans is highly prevalent in dental biofilms inhabited by C. albicans, making a symbiotic relationship likely
(Metwalli et al. 2013). In the past years, several studies investigated the relationship between these microorganisms (Barbieri et al. 2007; Jarosz et al. 2009; Metwalli et al. 2013; Falsetta et
al. 2014). The fungus is thought to provide adhesion sites for
S. mutans in the biofilm (Falsetta et al. 2014; Krom, Kidwai and
Ten Cate 2014), which is proposed to be facilitated by the dextran produced by S. mutans (Falsetta et al. 2014). Another possible binding mechanism is via direct adhesion, in which S. mutans adheres to C. albicans via one of the multiple adhesins on
the hyphal cell wall. It is already known that C. albicans adheres to Staphylococcus aureus and S. gordonii via the hyphal adhesin Als3p, the mechanism regarding S. mutans is yet unknown
(Silverman et al. 2010; Peters et al. 2012; Ricker, Vickerman and
Dongari-Bagtzoglou 2014). Besides providing adhesion, C. albicans is also able to use the lactic acid produced by S. mutans for
its own metabolism (Ene et al. 2012; Metwalli et al. 2013). This in
turn reduces the oxygen tension to preferred levels for S. mutans
and, finally, C. albicans provides growth stimulatory factors for S.
mutans (Metwalli et al. 2013).
We focused on the possible relationship between S. mutans
and C. albicans due to their prevalence and importance in caries
development (Barbieri et al. 2007; Klinke et al. 2011; Metwalli
et al. 2013; Falsetta et al. 2014). It is clear that the two species
have mutual interests in their association, due to their virulence factors and overlapping biochemical characteristics (Metwalli et al. 2013). We measured distinctive parameters related
to the pathogenesis of caries, which have not yet been investigated in this cross-kingdom association. These values include
pH, calcium release originating from hydroxyapatite (HA) disks,
lactic acid production and colony-forming units (CFUs), and will
be obtained from an in vitro biofilm model with supplemented
sucrose.
MATERIAL AND METHODS
Strains and growth conditions
Streptococcus mutans strain UA159 and C. albicans SC5314 were
used in this study. Strains were stored in glycerol stocks at
–80◦ C. Streptococcus mutans cultures were grown anaerobically
(80% N2 , 10% H2 , 10% CO2 ; Anoxomat AN2CTS, Mart Microbiology B.V.) in an anaerobic jar in brain-heart infusion (BHI, 35.9 g/L)
broth overnight at 37◦ C. Candida albicans cultures were grown
aerobically in BHI broth overnight at 30◦ C. Microscopic analysis showed that C. albicans was predominantly present in yeast
form under these conditions (data not shown).
In vitro biofilm model
The Amsterdam Active Attachment (AAA) model was used
for biofilm formation (Exterkate, Crielaard and Ten Cate 2010).
Briefly, the AAA model consists of a stainless steel lid on which
24 clamps are fixed. Each clamp can contain a different substrate
mimicking the tooth surface. Either glass coverslips (diameter 12
mm; Menzel, Braunschweig, Germany) or HA disks (HiMed, Old
Bethpage, NY) were used as substrata to grow biofilms. HA disks
were used to mimic the demineralization process of teeth. After
assembly, the AAA model was autoclaved to ensure sterility. The
lids were placed onto standard polystyrene 24-well plates (multiwell plates; Greiner Bio One, Alphen aan den Rijn, The Netherlands).
Growth of biofilms
The AAA model was inoculated with 1.5 mL BHI broth supplemented with 10% fetal bovine serum (FBS) to stimulate hyphal
formation (Gow 1997), containing either S. mutans UA159 (OD600
of 0,1) or C. albicans SC5314 (OD600 of 0,2) alone or with a mix
of both species. The inoculation medium for the dual-species
biofilms was a 20-fold diluted overnight culture of S. mutans
UA159 and C. albicans SC5314 in BHI broth supplemented with
10% FBS. The model was subsequently incubated aerobically for
6 h at 37◦ C. After this initial inoculation period, the lid was transferred to a new plate containing fresh medium supplemented
with 0.2% sucrose (without bacteria) and incubated for another
16 h, after which medium was refreshed every 24 h up to a total
of 72 h of biofilm formation.
pH measurement of spent medium
Upon every medium refreshment, the pH of the spent medium
of biofilms grown on HA disks for 24, 48 and 72 h was measured
R
using a PHM220 Lab digital pH meter (Meterlab
, Radiometer
Analytical). After every measurement, the electrode was rinsed
with 70% ethanol.
Determination of CFUs
After 24, 48 and 72 h of biofilm growth, disks were removed from
the lid and transferred into 2 mL phosphate-buffered saline. The
biofilms were dispersed using a sonicator (1 pulse/s, intensity
40% for 2 min), and a 10-fold serial dilution was made and plated
on BHI agar for total counts. To quantify C. albicans SC5314, the
plates were incubated aerobically at 37◦ C for 24 h (5% CO2 ) while
Willems et al.
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quantification of S. mutans UA159 was achieved by anaerobic incubation at 37◦ C for 24 h (10% CO2 , 10% H2 and 80% N2 ). To quantify colonization in dual-species biofilms, plates were incubated
both aerobically and anaerobically.
Acid production assay
At the end of the biofilm formation period (24, 48 and 72 h),
the lactic acid production capacity of the biofilms was determined as described previously (Janus et al. 2015) with minor adjustments. Briefly, the biofilms were placed in a new plate containing 1.5 mL/well buffered peptone water with 0.2% sucrose.
The model was incubated aerobically for 3 h at 37◦ C. Biofilms
were removed and the remaining liquid was heat inactivated
and stored at –20◦ C. The amount of lactic acid formed during
this period was subsequently analyzed by a colorimetric assay
(Van Loveren, Buijs and ten Cate 2000; Exterkate et al. 2014).
Figure 1 . pH values measured in spend BHI medium of different biofilms, after
24, 48 and 72 h of aerobic (5% CO) growth in BHI (0.2% sucrose) medium (mixed
biofilms represent 46 means, and both S. mutans and C. albicans biofilms represent 29 means). Values are mean ± SD (S. mutans biofilms represent 6 means,
mixed biofilms represent 14 means). Asterisk indicates a significant difference
between mixed biofilms and single-species biofilms.
Calcium quantification
Calcium release from HA disks during biofilm growth was determined using atomic absorption spectroscopy as described previously (ten Cate, Exterkate and Buijs 2006). In short, 200 μl of the
spent medium were taken in duplicate and diluted with 3 mL 3.6
mM La(NO3 )3 in 0.05 mol/L HCL. Atomic absorption spectroscopy
(Perkin Elmer 372) at 423 nm was used to quantify calcium in the
spent medium. The coefficient of variation of the calcium analysis was approximately 2%. The trends in daily calcium loss were
evaluated, as were the data accumulated over the biofilm formation period.
Statistical analysis
All data are expressed as mean ± SD. Data were analyzed using
the paired t-test for paired observations. Individual data were
analyzed using the unpaired t-test. Differences were considered
significant if P< 0.05.
Figure 2. CFU counts of biofilms, after 24, 48 and 72 h of growth. Values are mean
CFU (five single experiments) ± SD. Asterisk indicates a significant difference
between mixed biofilms and single-species biofilms.
RESULTS
pH of the spent medium
Since acidification is a key process leading to tooth demineralization, the pH of the medium of the three biofilms (C. albicans and S. mutans single biofilms and the mixed-species biofilm)
was determined (Fig. 1A). Candida albicans does not influence the
pH at any of the measured time points, compared to the pH of
fresh medium (pH = 7.2). In contrast, S. mutans acidified the environment (pH = 4.7) at all measured time points. After 24 h of
growth, pH values of dual-species biofilms were comparable to
the S. mutans biofilms. However, after 48 h, the pH was less acidic
(P = 0.0002), and this trend continued into the 72-h time point
(P = 4.7E-12), where the average pH was 5.54, which is above
the critical demineralization zone of pH 5.3–5.5 (Matsui and
Cvitkovitch 2010).
CFU counts
Streptococcus mutans–C. albicans mixed biofilms showed an increased amount of S. mutans CFU counts at every time point
(Fig. 2). CFU counts of S. mutans biofilms were increased after 72
h of biofilm growth (P = 8.53E-05). Both 48 and 72 h old mixedspecies biofilms harbored more viable S. mutans cells compared
to single-species S. mutans biofilms (P = 2.44E-05 and 9.00E-06).
Figure 3. Average lactic acid production for different biofilms. Biofilms were incubated in BPW medium (0.2% sucrose) for 3 h. Values are mean lactic acid ± SD.
Asterisk indicates a significant difference between mixed biofilms and singlespecies biofilms (mixed biofilms represent 18 means, and both S. mutans and C.
albicans biofilms represent 8 means).
An increase in C. albicans CFU counts was observed over time, but
mixed biofilms did not harbor more C. albicans cells than singlespecies C. albicans biofilms (data not shown).
Lactic acid production
To investigate whether the increase in cariogenic organisms in
the mixed-species biofilm has consequences for the cariogenic
potential of the biofilm, the amount of lactic acid produced
by the biofilm was analyzed (Fig. 3). Candida albicans biofilms
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Figure 4. Average calcium release originating from HA disks exposed to dualspecies and single-species biofilms. (A) Average calcium release exposed to dualspecies biofilm matched with corresponding pH values. (B) Average calcium release from HA disks exposed to single-species S. mutans. Asterisk indicates a
significant difference between 72 h over 48 h and 48 h over 24 h biofilms. Values represent mean calcium concentration and pH ± SD (panel A represents 9
means, panel B represents 11 means).
did not produce lactic acid. Lactic acid production of S. mutans
biofilms significantly increased as the biofilms aged (P < 0.05).
The mixed-species biofilms produced increasingly more acid in
the 3-h period as compared to S. mutans biofilms (24 h; P = 0.029,
48 h; P = 0.024, 72 h; P = 8.4E-05).
Calcium measurements
Demineralization was quantified by measuring the calcium released from HA disks exposed to different biofilms. Figure 4A
shows changes in pH and calcium release during growth of
mixed-species biofilms. In time, the pH of the culture medium
increases, as calcium release decreases (24 h vs 48 h; P = 0.015,
48 h vs 72 h; P = 0.004). This descending trend is not observed
in S. mutans biofilms, where a shift in pH was also not observed
(Fig. 4B). HA disks exposed to C. albicans grown biofilms showed
very little demineralization (data not shown).
DISCUSSION
Several studies have described C. albicans as an organism capable
of synergizing the onset and progression of caries induced by S.
mutans (Jarosz et al. 2009; Metwalli et al. 2013; Falsetta et al. 2014).
This study performed with an in vitro biofilm provides an alternative view. We show that the presence of C. albicans in a dualspecies biofilm with S. mutans does not synergize the cariogenic
capacity in terms of acidity and calcium release. Moreover, the
presence of C. albicans appears to decrease the cariogenic potential of the biofilm. We showed that mixed biofilms become less
acidic over time, likely due to the presence of C. albicans. After 72
h of growth in the presence of C. albicans, the average pH remains
above the critical demineralization zone of 5.3–5.5 (Matsui and
Cvitkovitch 2010). This indicates an effect of C. albicans on pH
levels. Since continuous exposure to low pH levels results in the
development of caries, this data might suggest a positive role of
C. albicans in preventing caries.
Lactic acid production was increased in our mixed biofilms,
showing that C. albicans does not inhibit the acid production by
S. mutans. Therefore, it is reasonable to think that C. albicans influences the pH in an independent fashion of S. mutans. There
are several possible explanations for this observation. Candida
albicans biofilms did not produce lactic acid, indicating that C.
albicans does not metabolize the available sucrose and other sugars into lactic acid. According to known metabolic pathways, C.
albicans lacks L-lactate dehydrogenase, the enzyme responsible
for converting pyruvate into lactic acid (Laboratories Kanehisa
2014). The end product of the metabolization of sucrose by C. albicans is ethanol (Laboratories Kanehisa 2014). However, ethanol
does not influence the pH of the medium. A study by Ene et al.
(2012) showed that C. albicans is able to grow using lactic acid as
a carbon source. Furthermore, lactic acid is the most preferred
source of carbon in hypoxic conditions for the fungus (Ene et al.
2012). Since lactic acid is present in the growth medium, produced by S. mutans, it is likely that C. albicans uses the available
lactic acid as an energy source. It is assumable that saccharolytic
bacteria quickly metabolize all available sucrose (0.2% sucrose is
not an excessive amount) into lactic acid, forcing C. albicans to
rely on lactic acid as a carbon source. The resulting acid elimination by C. albicans causes the environment to become less acidic.
This effect is enlarged as S. mutans increases in numbers, since
sucrose is then consumed even faster. At an early time point
when sucrose is not limited, C. albicans favors the consumption
of sucrose over lactic acid, producing ethanol. When sucrose is
limited, C. albicans is obliged to consume the lactic acid.
Mixed-species biofilms harbored more viable S. mutans cells
than S. mutans biofilms, but this was not observed for C. albicans.
The increase in S. mutans could be explained by the presence of
C. albicans. The fungus provides new adhesion sites for S. mutans
cells via dextran on the hyphal body, which might not have been
able to attach to the substrate otherwise (Falsetta et al. 2014).
The increased CFU counts of S. mutans in dual-species biofilms
might also be explained by the shift in extracellular pH to a more
convenient level, as the age of the biofilm increases.
Mixed-species biofilms have increased CFU counts of acidproducing species, and this is consistent with our findings in
lactic acid production. We show that lactic acid production of
mixed biofilms is increased compared to S. mutans biofilms.
Since C. albicans does not produce lactic acid, the lactic acid measured for dual-species biofilms originates solely from S. mutans
in these biofilms. The increase in lactic acid production by S. mutans cells in mixed biofilms implies that the presence of C. albicans drives the mixed biofilm to a more cariogenic nature than
S. mutans biofilms. However, despite the results of increased lactic acid production and increased acid-producing species, mixed
biofilms eventually turn out to be less cariogenic, likely due to
the role of C. albicans. Dual-species biofilms contain lower concentrations of acid after 48 and 72 h of growth, and calcium release as a result of demineralization is also decreased. This indicates that the HA disks were less damaged, due to the lower acid
concentration that these HA disks were exposed to. In S. mutans
biofilms, high amounts of calcium were released from HA disks,
Willems et al.
Figure 5. Difference in pH of biofilms grown on HA disks or glass slides. Values
represent mean pH of 44 HA disks and 8 glass slides ± SD.
caused by the continuous highly acidic environment. This process is causing the destruction of teeth in vivo (Islam, Khan and
Khan 2007).
A study by Falsetta et al. (2014) implied that the formed acid
is more concentrated within the biofilm. To investigate this, we
measured the pH directly in the spend medium, as well as indirectly in the biofilm, showing no difference in pH measured
in spend medium and within the biofilm. It could be argued that
when HA demineralizes phosphate is released into the medium,
acting as a buffer, affecting the pH. However, the same experiments were performed on glass slides with similar outcome,
showing that the possible release of phosphate into the medium
does not affect the pH (Fig. 5). In the same study they found that
in their in vivo rat model, S. mutans grows more rapidly in the
presence of C. albicans (Falsetta et al. 2014). This is in contrast
with our data. An explanation for this might be that in our in
vitro model host immune factors do not play a role and we solely
investigate the interaction between the two species.
In summary, dual-species biofilms show clear differences in
pH, lactic acid-producing potential, calcium release and CFUs,
when compared to either single-species biofilms. These findings
suggest that C. albicans is not by definition a cariogenic organism
in this dual-species biofilm. Moreover, the presence of C. albicans
in an S. mutans biofilm appears to decrease the cariogenic potential of the biofilm in terms of acidity and demineralization,
while lactic acid production and the growth of acid-producing
species are not inhibited. Therefore, we speculate that C. albicans
metabolically drives alkalization within the biofilm by consuming lactic acid, which in turn, decreases demineralization of HA
disks. Altogether, these findings provide new knowledge about
this recently identified cross-kingdom interaction, which may
be relevant in understanding and preventing the development
of caries.
ACKNOWLEDGMENTS
BPK is supported by a grant from the University of Amsterdam
for research into the focal point ‘Oral Infections and Inflammation’.
Conflict of interest. None declared.
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