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Biofilm formation by oral clinical isolates ofCandida species
Luis Octavio Sanchez-Vargas a,*, Deyanira Estrada-Barraza b,Amaury J. Pozos-Guillen c, Raimundo Rivas-Caceres d
aOral Microbiology, Pathology and Biochemical laboratory, Faculty of Stomatology, University Autonomous of San Luis
Potosı, MexicobBiomedical Sciences Institute, University Autonomous of Juarez City, Chihuahua, MexicocBasic Science laboratory, Faculty of Stomatology, University Autonomous of San Luis Potosı, MexicodDepartment of Chemical-Biological Sciences. Biomedical Sciences Institute, University Autonomous of Juarez City,
Chihuahua, Mexico
1
a r c h i v e s o f o r a l b i o l o g y x x x ( 2 0 1 3 ) x x x – x x x
a r t i c l e i n f o
Article history:
Accepted 5 June 2013
Keywords:
Candida species
Biofilm
Clinical isolates
Diabetes mellitus 2
Denture wearers
a b s t r a c t
We have conducted a longitudinal study to quantify biofilms in oral clinical isolates of
Candida species (spp.) from adults with local and systemic predisposing factors for candidi-
asis. A total of 69 yeast isolates from 63 Mexican patients were evaluated. These isolates (39
C. albicans, 15 C. tropicalis, 7 C. glabrata, 4 C. krusei, 1 C. lusitaniae, 1 C. kefyr, 1 C. guilliermondii
and 1 C. pulcherrima) were obtained from two clinical sites: 62.3% (n = 43) from the oral
mucosa of totally and partially edentulous patients, and 37.7% (n = 26) from the oral mucosa
of diabetics. In addition, Candida ATCC strains were used as controls for each experiment.
The kinetics of biofilm formation were measured by 2,3-bis(2-methoxy-4-nitro-5-sulfophe-
nyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide [XTT] reduction; each isolate
was tested at 6, 12 and 24 h. Biofilm formation is dependent on the Candida spp. and its
clinical origin. On average, the oral isolates of C. glabrata are strong biofilm producers,
whereas C. albicans and C. tropicalis are moderate producers. The most common species in
our population was C. albicans. While the kinetics of C. albicans biofilm formation varies
between oral isolates, it generally maintains steady growth from 2 to 48 h, when it reaches
its maximum growth.
# 2013 Published by Elsevier Ltd.
Available online at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
1. Introduction
The pathogenic yeast Candida, the most prevalent fungal
species in the human oral cavity, grows in diverse environ-
mental conditions. Its conversion from commensalism to
parasitism and exuberant growth is usually associated with
intraoral environmental changes (e.g., unhygienic prostheses,
xerostomia) or systemic factors such as diabetes mellitus type
* Corresponding author at: Laboratorio de Microbiologıa, Patologıa y BioqLuis Potosı, Av. Dr. Manuel Nava No. 2, Zona Universitaria, C.P. 78290
E-mail addresses: [email protected], [email protected]
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0003–9969/$ – see front matter # 2013 Published by Elsevier Ltd.http://dx.doi.org/10.1016/j.archoralbio.2013.06.006
2 (DM2) and immunodeficiency. C. albicans and, to a lesser
extent, other Candida spp. are commonly found in the oral
cavities of adults and children. They are recovered from the
dentition, tongue, cheeks, palatal mucosa, restorative materi-
als and prostheses. They are also found in root caries2 and in or
adjacent to infected gingival crevices.3 In healthy, dentulous
persons, Candida seldom causes disease. Candida in the oral
cavity serves as a reservoir for inoculation and infections
elsewhere in the body.4 When Candida penetrates the
uımica, Facultad de Estomatologıa, Universidad Autonoma de San, San Luis Potosı, S.L.P., Mexico. Tel.: +52 4441540940.
(L.O. Sanchez-Vargas).
ation by oral clinical isolates of Candida species. Archives of Oral Biology
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epithelium and invades the host tissues, septicemia and
systemic infections may result. These infections are difficult
to treat with antifungals and therefore have a high reported
mortality (40%).5,6 The most common Candida systemic
infections, in addition to septicemia, are catheter-related,
intra-abdominal and urinary tract infections. Candidemia is a
leading cause of morbidity and mortality in both the
immunocompetent and immunocompromised, critically ill
patients.7,8 Diabetics are especially predisposed to oral
diseases as candidiasis, which is associated with poor
glycemic control and therapeutic dentures. This predisposi-
tion also contributes to xerostomia, which may be due to
increased glucose levels in the oral fluids or immune
dysregulation. Wearing complete dentures is also a risk factor
because they can promote Candida colonization, candidal
biofilm and oral candidiasis.9 Candida infections are common-
ly associated with biofilms on mucosa and on the plastic
surfaces of indwelling devices. This biofilm consists of matrix-
enclosed, micro-colonies of yeast, hyphae and pseudohyphae
arranged in a complex structure.10 Because biofilm is
inherently resistant to antifungals, the affected devices
generally need to be removed.11,12
Candida pathogenicity has been attributed to several
factors, including adhesion to medical devices or host cells,
biofilm formation and secretion of hydrolytic enzymes
[proteases, phospholipases and haemolysins].13 Among clini-
cal Candida strains, biofilm formation is variable and depends
on the Candida spp.14,15; thus, the increased attention to
biofilm formation by oral isolates of Candida spp. is justified.
The availability of a rapid, inexpensive and reproducible
method of quantifying biofilm formation is essential for
evaluating biofilm generation. Development of biofilm on a
surface is better studied by quantifying the biofilm mass at
different time points. Methods such as counting colony-
forming units (CFU), spectrophotometric analysis and the 2,3-
bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)car-
bonyl]-2H-tetrazolium hydroxide (XTT) colorimetric reduction
assay have been employed to quantify Candida biofilms.16 The
objectives of our study were to quantify biofilms by XTT
reduction assay of oral clinical isolates of Candida spp. and to
determine the kinetics of formation from adults with local and
systemic predisposing factors for candidiasis. We also
assessed the possible influence of those important predispos-
ing factors on colonization, infection and the kinetics of
biofilm formation.
2. Materials and methods
2.1. Clinical isolates
A total of 69 yeast isolates from 63 Mexican patients were
evaluated, with the species identity and clinical origins of each
having been previously described.17 All adults included had
local (denture wearers) and systemic (DM2) predisposing
factors for candidiasis. A total of 69 yeast isolates (39 C.
albicans, 15 C. tropicalis, 7 C. glabrata, 4 C. krusei, 1 C. lusitaniae, 1
C. kefyr, 1 C. guilliermondii and 1 C. pulcherrima) were obtained
from various clinical sites. Twenty-six isolates (39.2%) were
from the oral mucosa of diabetics, and 43 (58.2%) were from
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the oral mucosa of total and partial edentulous denture
wearers. Reference Candida strains (C. albicans ATCC 90028, C.
krusei ATCC 6258, C. glabrata ATCC 2001 and C. tropicalis ATCC
0750) were used as controls in each experiment.
All yeasts were identified by their growth on CHROMagar1
Candida (CHROMagar, Paris, France), chlamydospore produc-
tion, morphology on cornmeal agar (Difco, Detroit, MI, USA)
and PCR with specific primers for the ARNr ITS1, ARNr ITS2,
and topoisomerase II genes. Assimilation profiles were
determined by ID 32C test (bioMerieux, Marcy l‘Etoile, France).
Prior to biofilm formation testing, each original isolate was
subcultured on Sabouraud dextrose agar and CHROMagar1
Candida to ensure purity and viability. All confirmed organ-
isms in our strain collection were stored in containers with
distilled water at room temperature.
2.2. Growth conditions
Each isolate was propagated in yeast peptone dextrose (YPD)
medium (1% w/v yeast extract, 2% w/v peptone, 2% w/v
dextrose) in conical screw-cap tubes (Falcon #2095,
17 mm � 120 mm; Becton Dickinson, Franklin Lakes, NJ,
USA). Twenty millilitres of medium were inoculated from
YPD agar plates containing fresh growths of each isolate. The
tubes were incubated overnight at 36 8C (�1 8C) in an orbital
shaker at 95 rpm (New Brunswick Scientific, Edison, NJ, USA);
each culture grew to the budding-yeast phase under these
conditions. Cells were harvested and washed three times with
sterile phosphate buffer (2.7 mM potassium chloride and
137 mM sodium chloride, pH 7.4; Sigma–Aldrich, St. Louis,
USA). The cells were resuspended in RPMI-1640 supplemented
with L-glutamine and buffered with morpholinepropanesul-
fonic acid (MOPS) (Sigma–Aldrich, St. Louis, USA). The cells
were adjusted to the cellular density (OD600 nm = 0.8–1.0)
equivalent to 1–5 � 106 cells/ml, which was determined by
counting in a haemocytometer and by spectrophotometry.
This concentration was selected because optimal biofilm
formation occurs at this particular cell density.18 The
standardized cell suspension was used immediately.
2.3. Biofilm assays and measurement of biofilm formation
Biofilm was measured by XTT reduction assay,19 which has
excellent repeatability and consistency,16 using pre-sterilized,
polystyrene, flat-bottomed, 96-well microtitre plates that had
the highest repeatability in previous assays (Costar, EIA/RIA
plate, with low evaporation lid, high binding; Corning, NY,
USA). Every experiment was performed in triplicate to confirm
the results, and each isolate was tested in a series of seven
wells in microtitre plates. To measure the kinetics of biofilm
formation, each isolate was tested at 6, 12 and 24 h. The
kinetics of biofilm formation by C. albicans was tested at, 2, 4, 6,
8, 12, 24 and 48 h. The means and standard deviations were
determined for three independent experiments. The Multis-
kan system (Microplate Reader; Thermo Fisher Scientific,
Austin, TX, USA) system was used to measure growth kinetics
on the surface. Biofilms were formed with standardized cell
suspensions (100 ml of a suspension containing 1–5 � 106 cells/
ml in RPMI-1640/L-glutamine buffered with MOPS) (Sigma–
Aldrich, St. Louis, USA) that were placed in selected wells of
ation by oral clinical isolates of Candida species. Archives of Oral Biology
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microtitre plates and incubated for 6–24 h at 37 8C (the biofilm
maturation and complexity were described previously).19,20
After biofilm formation, the medium was aspirated, and the
non-adherent cells were removed by gentle washing three
times in sterile PBS. A growth and a negative control were
included in each assay.
XTT (Sigma, USA) was prepared as a saturated solution of
0.5 g/l in Ringer’s lactate. This solution was filter-sterilized
through a 0.22 mm-pore filter, aliquoted, and stored at -80 8C.
Prior to each assay, an aliquot of stock XTT was thawed and
diluted in menadione (Sigma, 10 mM, prepared in acetone) to a
final concentration of 1 mM. A 100-ml aliquot of XTT/menadi-
one was added to each pre-washed biofilm sample and to
control wells. The plates were incubated in the dark for 1 h at
37 8C, and the colorimetric change (a reflection of the
metabolic activity of the biofilm) in the solution was measured
with a microtitre plate reader (Multiskan system, Microplate
Reader; Thermo Fisher Scientific, Austin, TX, USA). The
absorbance values for all new wells were read at 490 nm.
The absorbance values for the controls were then subtracted
from the values for the test wells to eliminate spurious results
due to background interference. The biofilm was examined by
light microscopy with an inverted microscope (Zeiss, Ober-
kochen, Germany). The kinetics of C. albicans oral isolate
biofilm formation was evaluated via non-invasive technical
Confocal Scanning Laser Microscopy (CSLM) on discs 0.5 cm in
diameter made of polyethylene material in the 96-well plates
(Costar, EIA/RIA plate, with low evaporation lid, high binding;
Corning, NY, USA). Briefly, discs were carefully placed on the
bottom of a 12-well plate (Corning, NY, USA) and, 1 ml of a
Candida cell suspension was pipetted into each well of the
plate to submerge the coupons. After incubation at 37 8C for
different periods of time, the biofilm was washed with PBS
three times and incubated with LIVE/DEAD fluorescent stain
(Molecular Probes, LIVE/DEAD1 Yeast Viability Kit, Eugene,
OR). Biofilms were incubated with FUN 1 cell stain for 30 min in
the dark at 30 8C before the CSLM examinations. The stained
biofilm samples were observed using an argon ion laser with a
Leica DMI 4000B laser-scanning confocal microscope. The
images were processed for display using the LAS AF Lite
software (Leica Microsystems, Wetzlar, Germany).
2.4. Statistical analysis
For continuous variables, Student’s t and ANOVA tests were
used to determine differences in means and percentages.
Differences between categorical variables were evaluated by
the chi-square test. Values of P < 0.05 were considered
statistically significant by a two-tailed test. For statistical
analysis, we used SPSS version 11 for Windows.
Table 1 – XTT-based classification of Candida spp. biofilm form
Group Biofilm formation
I Not producer
II Weakly producer
III Moderately producer
IV Strongly producer
Please cite this article in press as: Sanchez-Vargas LO, et al. Biofilm form(2013), http://dx.doi.org/10.1016/j.archoralbio.2013.06.006
3. Results
A total of 69 yeast isolates from 63 Mexican patients were
evaluated, the species identify and clinical origins of each
having been previously described.17 Each isolate was tested
using seven replicates on three different occasions, including
a positive and negative control for growth; similar results were
obtained in all experiments (P < 0.0001).
3.1. Comparison of Candida spp. biofilms in differentstages of maturation
All the oral isolates of Candida spp. tested showed different
abilities to form biofilms. The validity of this approach was
tested within the experiment replicates and between inde-
pendent experiments, and only those with standard devia-
tions less than 0.05 were considered for further analysis. Based
on these results, we categorized the ability to form biofilms
using the XTT reduction technique and expressed the values
in OD490 nm (Table 1).
C. albicans was associated with both strong and moderate
production of biofilms between 6 and 24 h of development.
The isolates of non-albicans Candida species were similar but
had significant differences in growth (Student’s t-test, P = 0.04)
at 6 h. At 24 h, strong biofilm producers were frequently
observed in isolates of C. glabrata (7 of 7; 100%), C. albicans (16 of
40; 40%) and C. krusei (1 of 4; 25%). Most C. albicans strains were
moderate biofilm producers (OD490 nm 0.41–0.77); in other
species, we observed moderate biofilm production in C.
tropicalis (12 of 15; 80%) and C. krusei (3 of 4; 75%).
At 12 h, there were 7 (17.5%) C. albicans and 7 (100%) C.
glabrata strong biofilm producers. There were 3 (75%) C. krusei
moderate producers and 1 (25%) poor producer. At 6 h, C.
albicans had five (12.5%) strong producers; C. tropicalis had 2
(13.3%) high producers. C. glabrata had 3 (42.8%) strong
producers and 4 (57.1%) moderate producers. C. krusei had 4
(100%) moderate producers.
The average reduction of soluble formazan dye (XTT) by
Candida spp. isolates was measured at OD490 nm at different
maturation stages (6, 12 and 24 h); this assay highlighted the
differences in biofilm formation between species and between
isolates from different patients (Fig. 1).
Table 2 shows the comparative analysis of species
between groups according to the origin of the isolates. It
was observed that 50% of the C. albicans isolates from DM2
patients were strong producers at 24 h of maturation, but
those from denture wearers remained moderate biofilm
producers over different time points. C. tropicalis from a
DM2 patient was a moderate producer at 6, 12 and 24 h, while
ation.
OD 490 nm CFU Log10 cells/ml
�0.10 <0.1 � 108
0.11–0.40 0.1–0.75 � 108
0.41–0.74 0.76–2 � 108
�0.75 >2 � 108
ation by oral clinical isolates of Candida species. Archives of Oral Biology
Fig. 1 – Growth at different maturation stages (6, 12 and 24 h) of Candida biofilm formation, analyzed by reduction of soluble
formazan dye (XTT) by Candida spp. isolates (OD at 490 nm). Error bars indicate the standard deviation.
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C. tropicalis from denture wearers was moderate at 6 h
maturation and strong (25%) at 12 and 24 h. C. glabrata/DM2
were strong producers at all stages, and C. glabrata/denture
wearers were moderate (67%) at 6 h and strong producers
(100%) at 12 and 24 h. For C. krusei, which was only isolated
from a diabetic (n = 4), one isolate was a strong producer at
24 h, but the rest were moderate.
Only one isolate each of C. lusitaniae and C. kefyr from a
diabetic was tested, and these isolates were moderate
producers at all maturation times. C. guilliermondii and C.
pulcherrima were only isolated from denture wearers, and
these isolates were moderate producers in all cases. When
analyzed by categories, we observed that most isolates from
edentulous patients denture wearers produced moderate or
strong biofilms, and the association was significant (Chi-
squared test, P = 0.019).
All species showed increased capacity for biofilm forma-
tion at 24 h except C. glabrata, where the highest value was
observed at 12 h (OD490 nm = 0.994). This value was the highest
observed value of biofilm formation, with minor intra-species
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differences. All C. albicans biofilms developed with OD490 nm
average values of 0.546, 0.643 and 0.738 at 6, 12 and 24 h,
respectively. C. albicans/DM2 showed the highest intra-species
differences at 6 h (average OD490 nm 0.55, range 0.15–0.992),
12 h (average OD490 nm 0.740, range 0.435–1.141) and 24 h
(average OD490 nm 0.790, range 0.635–1.125). The average values
per time period were analyzed using ANOVA. The biofilm
production values per time period were not different between
species; the only significant differences in biofilm production
(P = 0.01) were between groups of C. albicans at 12 h.
3.2. Kinetics of biofilm formation in oral isolatesof C. albicans
Because C. albicans was the most prevalent species in both
groups, the biofilm production kinetics of all C. albicans isolates
(n = 39) were tested in sets of 8 wells per isolate in triplicate at
2, 4, 6, 8, 12, 24 and 48 h (Fig. 2). Biofilm formation started at 2 h
after incubation (OD490 nm 0.632) then decreased slightly at 4 h
(due to mating) before an increase at 6 h. At 8 h, there is again a
ation by oral clinical isolates of Candida species. Archives of Oral Biology
Fig. 2 – Biofilm formation kinetics by Candida albicans. After 2 h, biofilm formation is moderate, and this production is
sustained for 48 h, with a slight coupling at 8 h.
Table 2 – Biofilm production by various Candida species at different stages of maturation.
Candida species(total frequency)
Biofilm production Origin of clinical isolates
Diabetes 2 (n = 26) Denture wearers (n = 43)
Time hours, frequency (%) Biofilm production,frequency (%)
6 h 12 h 24 h 6 h 12 h 24 h
C. albicans (39) Weakly 3 (19) 5 (22) 1 (4)
Moderately 11 (69) 11 (69) 8 (50) 15 (65) 20 (87) 15 (65)
Strongly 2 (12) 5 (31) 8 (50) 3 (13) 2 (9) 8 (35)
Total 16 (100) 16 (100) 16 (100) 23 (100) 23 (100) 23 (100)
C. tropicalis (15) Weakly 1 (33)
Moderately 2 (67) 3 (100) 3 (100) 10 (83) 9 (75) 9 (75)
Strongly 2 (17) 3 (25) 3 (25)
Total 3 (100) 3 (100) 3 (100) 12 (100) 12 (100) 12 (100)
C. glabrata (7) Weakly
Moderately 4 (67)
Strongly 1 (100) 1 (100) 1 (100) 2 (33) 6 (100) 6 (100)
Total 6 (100) 6 (100) 6 (100)
C. krusei (4) Weakly 1 (25)
Moderately 4 (100) 3 (75) 3 (75)
Strongly 1 (25)
Total 4 (100) 4 (100) 4 (100)
C. lusitaniae (1) Moderately 1 (100) 1 (100) 1 (100)
C. kefir (1) Moderately 1 (100) 1 (100) 1 (100)
C. guilliermondi (1) Moderately 1 (100) 1 (100) 1 (100)
C. pulcherrima (1) Moderately 1 (100) 1 (100) 1 (100)
Bold values represent the estadio more frequently observed.
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considerable period of coupling with OD low values, which is
followed by a steady rise to a peak value at 48 h. As shown in
the graph in Fig. 2, the biofilm production increases from 2 to
48 h with intermittent periods of coupling, especially between
6 and 12 h. Non-invasive scanning laser confocal microscopy
was used to observe intact biofilms at different maturation
stages (Fig. 3).
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4. Discussion
We investigated biofilm formation by Candida isolates from the
oral soft tissues of adult patients with local (denture wearers)
and systemic (DM2) predisposing factors for candidiasis.17 In
an unhygienic oral environment, biomaterials may act as
ation by oral clinical isolates of Candida species. Archives of Oral Biology
Fig. 3 – Confocal laser scanning microscopic analysis of C. albicans biofilms. Scattered foci of growth, such as budding yeast,
was observed after 2 h. By 24 h, growth is considerable and uniform, and most of the structures correspond to
pseudohyphae. At 6 h, development is more uniform; yeasts are larger and show true hyphal filamentous structure.
Between 8 and 12 h, filamentous structures aggregate, consisting mainly of pseudohyphae, true hyphae and budding yeast.
There is important metabolic activity, evidenced by increased fluorescence in the biofilm cells. At 24 h, there is a complex,
mature biofilm, which the conglomerate overlaps and entangles for 48 h. The biofilm has greater maturity and structural
complexity; the fluorescence is variable, indicating that metabolic activity has diminished.
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reservoirs for opportunistic respiratory, systemic and dissem-
inated pathogens.21,22 Several different Candida species form
biofilms.23,24 Reference strains and isolates from dentures, soft
relined and oral tissue have been evaluated by culturing
methods, the SEM counting method, and other semi-quanti-
tative methods such as XTT reduction.1,25 We evaluated
biofilm production with the semi-quantitative and extensively
validated XTT reduction assay. Our data demonstrate that
biofilm formation is principally dependent on the species of
Candida. Although the species most common in our test
population was C. albicans, the biofilm production was highest
in C. glabrata isolates, followed by C. tropicalis, C. albicans and
C. krusei. The evaluation of biofilm formation in these species
can produce variable results. For example, saliva coatings
may affect clinical colonization patterns, and they should be
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studied further to evaluate the mechanism(s) involved.25,26 In
contrast to Hasan in India,15 we found that biofilm formation
by C. glabrata isolates was substantial; C. tropicalis has also
been reported to produce more biofilm.24 In contrast to other
reports, we have observed that C. glabrata was the most
frequent non-albicans Candida species, and these strains
showed higher colonization than C. tropicalis.27
Furthermore, we hypothesize that there are not only
conditions favouring colonization by Candida species but also
that Candida spp. have higher potential virulence and ability to
form biofilms. Isolates have been evaluated from the oral
mucosa of patients with clinical conditions that promote C.
albicans and non-albicans Candida species growth. This obser-
vation is relevant because Candida spp. most often induce
pathology in immunocompromised persons or in those with
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impaired salivary function, e.g., diabetics. C. albicans from
patients with DM2 were major producers at 24 h of matura-
tion. C. tropicalis was a moderate producer at 6, 12 and 24 h, but
C. glabrata from diabetic persons was a strong producer in all
stages. C. krusei was only isolated from diabetics (n = 4), and
one isolate was a strong producer at 24 h. The oral mucosa
of diabetics may allow species such as C. glabrata and C. krusei
to develop as strong biofilm producers in these micro-
environments.9
In denture-wearing edentulous patients, Candida spp. often
cause denture stomatitis.28 Ramage et al.29 used scanning
electron microscopy (SEM) to show that Candida biofilms could
be visualized on denture samples obtained from patients with
denture stomatitis. Clinical isolates of C. albicans formed
biofilms in vitro, although the amount of biofilm varied for
different isolates recovered from the same patient. Suscepti-
bility testing indicated that the resulting biofilms showed
increased resistance to antifungal treatment,30 however, the
resistance is dependent on biofilm metabolic activity,31 of
species, strains and kind of antimycotics.32,33 Our results
suggest that C. albicans from denture wearers are moderate
biofilm producers during different periods of maturation. The
high intra-species differences are most likely due to variability
in ecological niches and the host conditions. Moreover, 100%
of C. glabrata isolates were strong producers at 12 and 24 h; this
species may easily develop processes that favour their
survival. C. tropicalis was moderate at 6 h maturation, and
only 25% of these isolates converted to strong producers at 12
and 24 h. Most isolates from edentulous denture wearers
produced moderate or strong biofilms (P = 0.019). These
isolates grow in adverse environments with factors that
favour biofilm production, such as, cell surface hydrophobicity
(an important predictor of the initial adhesion of the micro-
organism to the inert surface), surfaces modified with salivary
pellicle, the surface roughness of the prosthesis, the complex
relationship between oral microorganisms, the sucrose con-
centrations, the acid pH and the thermodynamic conditions in
the oral cavity. All of these factors affect the production and
interaction of adhesins in the salivary pellicle, which forms on
dentures and oral mucosa in edentulous persons.34,35 Howev-
er, the adherence of Candida spp. to dentures is similar to other
medical devices, such as voice prostheses,36 blood vessel and
urinary catheters 37 and heart valves.38
Oral isolates exhibit intra-species variability that may be
due to the physico-chemical microenvironment where the
isolates developed. The different stages of biofilm develop-
ment induce changes in regulatory mechanisms that govern
biofilm biosynthesis. Systemic conditions, such as DM2, or
local edentulism and prostheses condition this microenviron-
ment. In DM2, there are qualitative and quantitative altera-
tions in saliva. Glycosylated products in the oral environment
increase the metabolism of carbohydrates, which increases
biomass and presumably shortens the intermediate stage of
biofilm formation; we observed this effect in isolates that
achieved strong production within 24 h of training.39
In edentulous patients, prostheses cause multiple altera-
tions in the oral environment including changes in saliva,
phosphate deposits, calcium and protein in acrylic surfaces
contacting the epithelia, competition between bacteria,
decreased pH, and increased potential for oxide reduction.
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As a result, fungal species need rapid osmotic adaptation,
oxidative stress responses and metabolic changes that
promote biofilm maturation in less time. Consequently,
Candida isolates evolve from intermediate to strong produ-
cers with 12–24 h of training. We have observed this
phenomenon predominantly in C. glabrata and various
isolates of C. albicans.34,39
Interestingly, the newly discovered C. albicans mating
pathway might impact biofilm formation in some situations.
This study also provides experimental support for the
hypothesis that a biofilm is a mating-permissive environment,
even for widely separated mating partners.40
According to our calibration curves, CFU counting and XTT
reduction assays are consistent and similar to other studies in
their predictions of biofilm formation.41 The strong producer
category (OD490 nm value � 0.75) corresponds to >2 � 108 cells/
ml. However, the method requires an adequate standardiza-
tion for all parameters. The microplates used can reduce the
consistency of the results; we have observed that microplates
with low evaporation lids and high binding result in lower
intra-experiment and intra-isolate variation.
The kinetics of biofilm formation by oral isolates of Candida
spp. are not well understood. We agree with other research-
ers41,42 that the analyses of growth kinetics and the architec-
ture of biofilms – including molecular genomics and
proteomics – are important for understanding and interpret-
ing their behaviour. The kinetics of biofilm production of the
different species tested (three points, 6, 12 and 24 h) are
consistent in their average values, although there are
significant intra-species differences for C. albicans and C.
glabrata. In particular, some isolates of C. glabrata behaved as
strong producers after 6 h training. In contrast to other
studies,43 C. albicans produced better biofilms, and C. glabrata
seemed to be the better biofilm former according to the XTT
reduction assay.
We evaluated the kinetics of biofilm formation by C. albicans
(the most frequent species in our population) from 2 to 48 h.
There was steady growth between 2 and 6 h, a short period of
coupling between 6 and 8 h and continued steady growth up to
48 h, when it reached its maximal cell density. Our results are
consistent with the previous report that C. albicans maintains
steady biofilm production up to 72 h, as measured by the XTT
reduction assay and other methods.44 Ramage19 observed
steady biofilm growth starting at 2 h, with a slight coupling at
8 h and maximum growth at 48 h. These results suggest that
the clinical origin and host niche of an isolate influence its
biofilm formation and thereby increase the virulence of
C. albicans.
Although there are increasing numbers of biofilm studies
using the XTT assay, some pitfalls of the method have also
been noted. Because there are inter-species and inter-strain
variations in the ability to metabolize XTT, one has to be
cautious when comparing the growth kinetics of different
species based on XTT readings.41,45 When we evaluated the
kinetics of C. albicans by confocal microscopy, the highest
structural maturation of biofilm architecture was observed at
48 h (Fig. 2), although for some isolates, increased metabolic
activity was observed by the XTT assay between 12 and 24 h.
Thus, biofilm kinetics are variable and dependent on both the
species and its clinical origin. Elderly edentulous denture
ation by oral clinical isolates of Candida species. Archives of Oral Biology
a r c h i v e s o f o r a l b i o l o g y x x x ( 2 0 1 3 ) x x x – x x x8
AOB-3018; No. of Pages 9
wearers, patients with DM2 or other debilitating diseases and
users of acrylic prosthetics have significant risk of virulent oral
yeast infections. The resulting biofilm formation increases the
susceptibility of these patients to septicemia, especially when
these patients undergo a procedure or are hospitalized. The
degree of biofilm formation depends on the species and the
clinical origin. It may be that the cell wall proteins mediating
the adherence of Candida to host cells or inert materials are
also responsible for the formation and growth of biofilms.
These adhesins enable Candida to form biofilms in different
physiological niches and in many different forms of candidia-
sis.18 The oral isolates of C. glabrata are average to strong
biofilm producers, whereas C. albicans and C. tropicalis are
moderate producers. Although the kinetics of C. albicans
biofilm formation vary between oral isolates, there is generally
steady growth from 2 h to maximum growth at 48 h. Our data
also confirm previous findings that Candida strains vary in
their ability to form biofilms.41 Therefore, future studies of
Candida biofilm growth kinetics should consider strain origins
and alterations to their expression patterns and metabolism,
and these experiments should employ analytical techniques
and direct methodology with novel tools such as CSLM,
quantification systems and different strategies46 for elucidat-
ing the mechanisms that regulate the formation of Candida
biofilms combine tools from biology, chemistry, nanoscience,
material science and physics.
Funding
This work was supported by grant no. CHIH-2009-C02-125216
mixed fund of the State Government of Chihuahua and the
National Council for Science and Technology [Consejo
Nacional de Ciencia y Tecnologıa -CONACYT], Mexico.
Competing interests
None declared.
Ethical approval
In this study we used strains from previous studies, was not
required an approval from the ethics committee.
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