Biofilm formation by oral clinical isolates of Candida species

AOB-3018; No. of Pages 9

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]

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

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

http://dx.doi.org/10.1016/j.archoralbio.2013.06.006

mailto:[email protected]

mailto:[email protected]


http://www.sciencedirect.com/science/journal/00039969


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 x2


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


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



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 3


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


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



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.



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


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



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.



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).


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



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.



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


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





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.


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





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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Biofilm formation by oral clinical isolates of Candida species

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