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Pathogenic characteristics of Candida albicans

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DOI: 10.1016/j.micpath.2017.06.036

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 Microbial Pathogenesis 110 (2017) 128e134

Contents lists available at ScienceDirect

Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath

Pathogenic characteristics of Candida albicans isolated from oral

cavities of denture wearers and cancer patients wearing oral
prostheses
J.V. Mothibe, M. Patel*
Division of Oral Microbiology, Department of Oral Biological Sciences, School of Oral Health Sciences, Faculty of Health Sciences, University of the
Witwatersrand, Private Bag 3, Wits, 2050 Johannesburg, South Africa

a r t i c l e i n f o a b s t r a c t

Article history: Candida albicans cause opportunistic infections including oral candidiasis in immunocompromised pa-
Received 7 April 2017 tients. It has an ability to cause infection due to its virulence factors. This study investigated the path-
Received in revised form ogenic characteristics of C. albicans isolated from the oral cavities of healthy subjects and two vulnerable
5 May 2017
groups, denture wearers and cancer patients wearing oral prostheses.
Accepted 24 June 2017
Available online 24 June 2017
Oral rinse samples were collected and cultured for the quantitative and qualitative analysis of Candida.
Twenty strains of C. albicans isolated from the healthy individuals and denture wearers and, 14 strains
isolated from the cancer patients were selected and their pathogenic characteristics were measured. The
Keywords:
Candida
results of the study groups were compared using a Scheffe test for pairwise comparison and a chi square
Adherence test.
Germ tube Denture wearer and cancer patients with prostheses carried signicantly higher number (p < 0.01) and
Proteinase a variety of Candida than the normal individuals. Denture wearer and cancer patients carried several
Phospholipase Candida species. The adherence abilities (p 0.01) as well as phospholipase (p < 0.01) and proteinase
Cancer (p 0.03) production were signicantly higher in the strains from denture wearers. In addition, high
number of isolates from the denture wearers produced phospholipase and proteinase (85% and 80%
respectively) compared to the strains from normal subjects (25% and 60% respectively). Only the germ
tube formation and adherence ability were signicantly higher in the strains from the cancer patients
with prostheses (p 0.05 and p < 0.01 respectively).
In conclusion, during the commensal state, the increased expression of virulence factors in the denture
wearers suggests the readiness of these strains to cause infection in this group. The high number of
C. albicans and their increased adherence ability in the two study groups suggest that hygiene of oral
cavity and prostheses is important in the prevention of colonization of Candida and the development of
oral candidiasis.
2017 Published by Elsevier Ltd.

1. Introduction
Candida albicans is a commensal carried by humans in the oral
cavity, digestive system and vagina. It can cause supercial to sys-
temic opportunistic infection both in immunocompromised pa-
tients, hospitalized patients and otherwise healthy individuals. It
has an ability to cause infection due to its virulence properties or
pathogenic characteristics. These include the ability to adhere to
host tissues and prostheses (eg. dentures, indwelling devices),
* Corresponding author.
E-mail address: Mrudula.patel@wits.ac.za (M. Patel).
cause phenotypic switching and produce germ tubes for the tissue
invasion. In addition, they produce a range of tissue damaging
hydrolytic enzymes such as proteinases and phospholipases [1].
However, many Candida species other than C. albicans such as,
C. tropicalis, C. parapsilosis, C. krusei; C. kefyr, C. glabrata, and C.
guilliermondii, also exist on the human host and cause opportunistic
infections.
Denture-related stomatitis, an opportunistic infection caused by
C. albicans, affects approximately 50%e65% of denture wearers
[2,3]. Predisposing factors in denture-related stomatitis are trau-
matic occlusion, poor oral and denture hygiene, dryness of mouth,
systemic conditions, diabetes mellitus, immunodeciency and

http://dx.doi.org/10.1016/j.micpath.2017.06.036
0882-4010/ 2017 Published by Elsevier Ltd.
 J.V. Mothibe, M. Patel / Microbial Pathogenesis 110 (2017) 128e134
nutritional deciencies [4,5]. High percentages of cancer patients
on cancer therapy also carry Candida in their oral cavities and are at
a greater risk of developing oral candidiasis and both increases with
cancer therapy. In patients with all types of cancers, the prevalence
of oral colonization with Candida has been shown to be 48% before
treatment and during treatment it increased to 72% [6]. This
increased colonization affects the risk of oral infection develop-
ment [7]. Prevalence of oral candidiasis is highest in patients with
head and neck cancers in comparison to patients with solid tumors
and hematological malignancy [8]. Up to 73% of patients with head
and neck cancers carry Candida in their oral cavities [9]. There is
limited information available on the status of Candida carriage and
virulence in patients with oral prostheses and cancer. This study
investigated the pathogenic characteristics of C. albicans isolated
from the oral cavities of normal subjects, denture-wearers and
cancer patients wearing oral prostheses.
2. Methods
2.1. Study population
A total number of 103 subjects were screened for Candida car-
riage. The HIV status of all the subjects was not known. The control
group consisted of 49 healthy staff members at the Oral Microbi-
ology laboratory and National Health Laboratories Services in
Charlotte Maxeke Johannesburg Academic Hospital. None of these
subjects wore dentures or had systemic illness. The denture
wearing group consisted of 35 patients with complete dentures
having no signs and/or symptoms of denture related stomatitis.
They were either attending clinics at Wits Oral and Dental Hospital
or living in Golden Acres, Sandringham Retirement Home. The third
group consisted of 19 patients with oral cancer and oral prostheses
attending Wits Oral and Dental Hospital. Nine patients had head
and neck cancers and related prostheses, whereas the rest of the
patients had solid tumors of other organs and they wore dentures.
Demography and brief history was collected.
All the subjects agreed to participate in the study and signed
consent forms. Ethical clearance was obtained from the Human
Research Ethics Committee, University of Witwatersrand, Johan-
nesburg (clearance number is M110320).
2.2. Isolation and identication of Candida species
Oral rinse samples were collected using the technique described
by Samaranayake et al., 1986 [10]. Each subject was requested to
rinse the mouth for 60 s with 10 mL of sterile distilled water and
expectorate the rinse into a sterile universal bottle. Both denture
wearers and cancer patients retained their prostheses during
sample collection. Oral rinse samples were diluted to 1:10 and
1:100, and 100 mL of undiluted and diluted samples were spread on
CHROMagar Candida agar and incubated aerobically at 37  C for
48 h. The number of colonies on the CHROMagar plates was
counted and expressed as colony forming units per milliliter (cfu/
mL) of oral rinse. Single yeast colonies with different colours were
subcultured on Sabouraud dextrose agar until pure cultures were
obtained and the colonies were identied using API 20C AUX kit
(Biomerieux, France).
Twenty strains of C. albicans were selected from normal subjects
and denture wearers groups, and 14 from cancer patients with
prostheses group. The virulence factors, such as ability to adhere to
the oral epithelial cells and form germ tube, and the production of
proteinase and phospholipase of these strains were examined.
129
2.3. Germ tube formation
Germ tube formation was investigated using a technique
described by Mackenzie, 1962 [11]. A 0.5 mL sterile horse serum
was inoculated with a loopful of culture of C. albicans. The mixture
was incubated for 2 hours, aerobically at 37  C. A drop of sus-
pension was placed on a glass slide, covered with a coverslip and
examined microscopically using X40 objective for the production of
germ tubes. Cells that produced a germ tube at least twice the
length of the cell were considered positive. Fifty yeast cells of each
isolate were examined for germ tube formation and the percentage
was calculated. These experiments were performed once per strain.
2.4. Adherence assay
Adherence assays were performed using a technique described
by Ghannoum and Elteen, 1986 [12] with minor modication. The
C. albicans cultures were inoculated into Sabouraud dextrose broth
(Oxoid, Hampshire, England) and incubated at 37  C for 24 h while
shaking at 60 rpm. Yeast cells were harvested by centrifugation
(5000 rpm, 15 min), washed three times with sterile distilled water
by repeated centrifugation, resuspended in 2 mL sterile distilled
water and adjusted to the concentration of 105 cells/mL using he-
mocytometer. Buccal epithelial cells (BECs) were collected from the
mouth of healthy (non-carrier of Candida-investigator J. V.
Mothibe) volunteer by gently rubbing the oral mucosal surface of
the cheek with a sterile swab. The swab was then immersed in 4 mL
sterile distilled water. The cell suspension was centrifuged
(5000 rpm for 15 min), then the cell pellet was washed three times
and resuspended into 2 mL sterile distilled water. Two milliliters of
the cells and 2 mL of yeast cell suspensions were mixed in sterile
screw-capped bottle, incubated for 3 h at 37  C while shaking at
60 rpm. The mixture was ltered through a nylon mesh with 20 mm
pores (Nucleopore GMBh, Germany) to remove non-adherent yeast
cells. The BECs on the lter were washed twice with 5 mL sterile
distilled water and nally suspended in 2 mL of sterile distilled
water. Glass slide were prepared, air-dried, heat xed and Gram
stained. Adherence was determined microscopically by counting
the number of adherent yeast cells per hundred BECs. These ex-
periments were performed once per strain.
2.5. Proteinase activity
Proteinase production was detected using the modied method
described by Aoki et al., 1990 [13]. A suspension of fresh C. albicans
culture containing 107 cfu/mL cells was prepared and 20 mL was
inoculated onto the four sterile paper discs placed on the Yeast
Carbon Base- Bovine Serum Albumin (YCB-BSA) medium plates.
The medium contained 1.5% Columbia agar, 1.17% Yeast Carbon Base
powder, 0.2% Bovine Serum Albumin (BSA) and 0.2% glucose. The
plates were incubated aerobically at 37  C for fourteen days. The
diameter of the colonies was measured and recorded. Plates were
then stained by ooding with 0.5% Coomasie brilliant blue R250
(Pierce Biotechnology) for 20 min at room temperature. Plates were
destained three times for 20 min at 37  C using a destaining solu-
tion (45 ml Ethanol, 45 ml dH2O, 10 ml Acetic acid) and once with
distilled water. The diameter of the colonies was measured before
staining and the diameter of the clearing zones were measured
after staining. Proteinase activity (Pr) was expressed as the ratio of
diameter of colony to total diameter of colony plus clearing zone (in
mm). The Pr value was taken as the average of the four measure-
ments. When the Pr equaled 1, no proteinase activity was detected
in the strain. Thus, a low Pr value indicated high enzyme
production.
130 J.V. Mothibe, M. Patel / Microbial Pathogenesis 110 (2017) 128e134

2.6. Phospholipase activity Table 1

Candida count (cfu/mL) in the colonized study groups.

Phospholipase production was detected using the egg yolk Candida colonised group No. of patients with range of Candida counts
media as described by Price et al., 1982 [14]. It contained 13 g (cfu/mL)
Sabouraud agar, 11.7 g NaCl, 0.11 g CaCl2 and 20 ml sterile egg yolk 1-100 (%) 101-1000 (%) >1000 (%)
emulsion. A suspension of fresh C. albicans culture containing
Normal subjects 14 (66.66) 7 (33.34) 0
107 cfu/mL cells was prepared and 20 mL was inoculated onto the n 21
four blank paper discs placed on the egg yolk agar plates, incubated Denture wearers 1 (3.85) 10 (38.46) 15 (57.69)
aerobically for four days at 37  C. The diameters of colony (A) and n 26
Cancer patients 1 (6.25) 3 (18.75) 12 (75)
the colony plus precipitation zone (B) were measured. The phos-
n 16
pholipase activity of the isolates was considered positive when a
precipitation zone was visible around the growth. The phospholi-
pase activity (Pz) was expressed as Pz A/B (in mm). The phos-
pholipase activity of each isolate was recorded as the average of the cancer patients (75%) with prostheses carried higher numbers of
four measurements. Pz of 1 indicated no phospholipase activity. Candida in their oral cavity (>1000 cfu/mL) than the denture
Lower values indicated higher production of phospholipase. wearers (57.69%).

2.7. Statistical analysis of data 3.4. Germ tube formation

Scheffe test for pairwise comparison and chi square test was
used to compare the Candida counts in these study groups. The
results of the virulence factors i.e. germ tube formation, adherence,
proteinase and phospholipase production by the strains of
C. albicans isolated from the 3 groups were compared using a
Kruskal-Wallis test for pairwise comparison and a chi square test.
3. Results
3.1. Demography and Candida carriage
The mean age of normal individuals, denture wearers and can-
cer patients with prostheses screened for the colonization were 34,
75 and 70 years respectively. The gender ratio in normal in-
dividuals, denture wearers and cancer patients with prostheses
was 9M:40F, 17M:18F and 6M:13F respectively. The carriage of
Candida species in the normal individuals, denture wearers and
cancer patients with prostheses was 42.86% (21/49), 74.29% (26/35)
and 84.21% (16/19) respectively. All though the groups were not
matched for the risk factors such as age, HIV status etc., the carriage
of Candida species between the normal subjects, denture wearers
and cancer patients with prostheses were signicantly different
(p < 0.01). The carriage rate was signicantly higher in the denture
wearers (p < 0.01) and cancer patients with prostheses (p < 0.01)
compared to the normal individuals.
3.2. Multiple Candida colonization
Several Candida species were isolated from the denture wearers
and cancer patients with prostheses, whereas normal subjects
yielded only C. albicans strains. Multiple Candida species were car-
ried by 61.54% and 56.25% of Candida colonised denture wearers
and cancer patients with prostheses respectively. Denture wearers
carried C. albicans (23), C. glabrata (13), C. tropicalis (3),
C. parapsilosis (1), C. guilliermondii (1), C. famata (2) and
C. dubliniensis (1). Cancer patients with oral prostheses carried
C. albicans (15), C. glabrata (8), C. tropicalis (2) and C. krusei (2).
Fig. 1 shows the germ tube formation results which are signi-
cantly high (p 0.05) in strains isolated from cancer patients
(57.17%) compared to the normal subjects (41.2%). There was no
signicant difference between the strains isolated from denture
wearers (43.2) and normal subjects (41.2). In addition, there was no
signicant difference between the results of denture wearers and
cancer patients with prostheses.
3.5. Adherence assay
Adherence of strains isolated from denture wearers and cancer
patients with oral prostheses was signicantly higher than strains
isolated from the normal individuals (Fig. 2). However, there was no
signicant difference between the results of denture wearers and
cancer patients.
3.6. Proteinase production (quantities)
Proteinase production of strains isolated from denture wearers
was signicantly high compared to the strains isolated from the
normal individuals, as well as strains isolated from cancer patients
with oral prostheses (Fig. 3). However, proteinase production of
strains isolated from cancer patients with prostheses was similar to
the strains isolated from the normal individuals.
3.7. Phospholipase production (quantities)
Phospholipase production of strains isolated from denture
wearers was signicantly high compared to the strains isolated

3.3. Candida counts

The mean SD Candida counts in the colonized normal subjects,

denture wearers and cancer patients were 326.05 549.4,
18616.5 25967 and 25329 45842.8 cfu/mL respectively. The
Candida counts were signicantly higher in the two study groups
than the normal subjects (p < 0.01). Therefore, the Candida counts Fig. 1. Germ tube formation by C. albicans isolated from normal subjects, denture
were categorized as shown in Table 1. The results showed that wearers and cancer patients with oral prostheses.
 J.V. Mothibe, M. Patel / Microbial Pathogenesis 110 (2017) 128e134
Fig. 2. Adhesion to oral epithelial cells by C. albicans isolated from normal subjects,
denture wearers and cancer patients with oral prostheses.
Fig. 3. Proteinase production by C. albicans isolated from normal subjects, denture
wearers and cancer patients with oral prostheses. Pr: ratio of the colony diameter and
the clear zone, therefore lower values indicate greater enzyme activity and 1 indicate
no enzyme production.
from the normal individuals (p < 0.01) as shown in Fig. 4. However,
there was no signicant difference found between the results of
strains isolated from normal subjects and cancer patients with
prostheses (p > 0.05), as well as between denture wearers and
cancer patients with prostheses groups.
3.8. Number of C. albicans strains that exhibited pathogenic
characteristics
All the strains produced germ tube and showed adherence
ability (Fig. 5). Proteinase was produced by 60%, 80% and 64.29% of
the C. albicans strains isolated from normal subjects, denture
wearers and cancer patients with prostheses respectively with no
signicant difference between each other. Phospholipase was
produced by 25%, 85% and 57.14% of the C. albicans strains isolated
from normal subjects, denture wearers and cancer patients with
oral prostheses respectively. Signicantly high number of strains
from denture wearers and cancer patients produced phospholipase
compared to the strains from normal individuals (p > 0.01).
4. Discussion
The virulence factors expressed by Candida may vary, depending
on the type, stage and site of infection, and the nature of the host's
immunity [15]. Germ tube formation, the production of hydrolytic
enzymes and ability to adhere to epithelial cells are known to
131
Fig. 4. Phospholipase production by C. albicans isolated from normal subjects, denture
wearers and cancer patients with oral prostheses Pz: ratio of the colony diameter and
the precipitation zone, therefore lower values indicate greater enzyme activity and 1
indicate no enzyme production.
Fig. 5. Number of C. albicans strains that exhibited pathogenic characteristics.
contribute in the pathogenesis of C. albicans [16,17]. This host-
Candidal interaction is best studied in the host which can be an
animal. Nevertheless, some of these virulence factors are measur-
able in the laboratory environment [18e20] therefore, virulence
factors of C. albicans isolated during commensal state were
measured in this study and compared between the study groups.
This study has shown that although the adherence ability of strains
isolated from denture wearers and cancer patients with oral pros-
theses was higher, compared to the strains from normal in-
dividuals, signicantly high number of strains from denture
wearers produced phospholipase. In addition, these strains also
produced high quantities of phospholipase and proteinase
compared to the strains from normal individuals and cancer pa-
tients with prostheses. These results suggest that strains isolated
from denture wearers had more potential or readiness to cause
infection. Due to the high quantities and many enzyme producing
strains, collectively these enzymes may accelerate the development
of infection in this study group.
The adhesion of Candida cells to host mucosal surface is a vital
prerequisite for successful colonization and infection [21].
C. albicans adheres to host cells more than any other species of
Candida. In this study, C. albicans strains isolated from the denture
wearers as well as cancer patients had signicantly higher level of
adherence to buccal epithelial cells than strains isolated from
normal subjects. Similarly, C. albicans isolated from the HIV positive
patients and patients with diabetes mellitus have shown increased
132 J.V. Mothibe, M. Patel / Microbial Pathogenesis 110 (2017) 128e134
adherence ability [22,23]. Adherence allows yeast cells the chance
to proliferate and colonize the host, with or without signs or
symptoms of infection [17]. The balance between colonization and
overt infection may be tipped toward infection in compromised
individuals by changes in the expression of adherence ligands and
receptors. The host defense against Candida infection depends on
the early activation of innate immunity cells such as neutrophils,
followed by a specic immune response that is mediated by lym-
phocytes [24e26]. The counts and the adherence ability were
increased in the C. albicans isolated from denture wearers and
cancer patients with prostheses, suggesting that this early activa-
tion of innate immunity may have been compromised. In cancer
patients, cancer therapy causes myelosuppression and damage to
the mucosal barrier which is known to increase the adherence
property of these organisms [7].
Wearing prostheses was the common factor in denture wearers
and the cancer patients for Candida carriage. Acrylic dentures are
known to act as a reservoir for microorganisms, including Candida
and therefore, play an important role in the development of
Candidiasis. Silicone and PMMA made prostheses obturators are
also known to become colonized with Candida [27]. In addition,
pores, cracks and structural defects allow penetration and there-
fore, persistence of Candida into these prostheses [28]. This ex-
plains the high counts found in the oral cavities of these two
groups.
Germ tube formation is the initial stage in the yeast-hyphal
transition. The presence of hyphae seems to facilitate the candi-
dal colonization on the mucosal surfaces and raises the C. albicans
counts [29]. All the strains of C. albicans isolated from all the study
subjects produced germ tube. Quantitative analysis showed that
there was only a marginal difference in the germ tube formation
between the strains from cancer patients and normal individuals.
Similar results were obtained by Ramla et al. (2015) who compared
germ tube formation in C. albicans isolated from cancer patients on
treatment and normal individuals [20]. Antineoplastic drugs have
been reported to either increase the hyphal formation [30] or
decrease the germ tube formation [31] depending on the type of
drugs. Although germ tube formation is important in the disease
process, all the virulence factors are not required at all the stages of
infection [32]. Nevertheless, germ tube formation is important in
the oral and vaginal infections and in some other invasive
infections.
Phospholipases are a group of enzymes that have the ability to
hydrolyze one or more ester linkages in glycerophospholipids. They
are considered putative virulence factors because they are associ-
ated with adhesion to epithelial cells, host cell penetration, inva-
sion of epithelial cells, and interaction with host signal transduction
pathways [33,34]. Experiments involving the disruption of a gene
that encodes for a phospholipase has shown a decrease in virulence
and a reduced ability of C. albicans to penetrate host cells. This
suggests that phospholipases plays a role in causing damage to host
cells [35,36]. In this study, phospholipase production was signi-
cantly high (quantity) in 85% of strains of C. albicans isolated from
denture wearers compared to the normal subjects and cancer pa-
tients with prostheses. High quantities of phospholipase have been
found in the strains isolated from the patients with stomatitis [37].
Furthermore, Marco-Arias et al. (2009) has reported that phos-
pholipase producing strains were signicantly higher in patients
with Newton's type II and III classied denture stomatitis compared
to the commensal state [38]. Increase in phospholipase production
in these strains from denture wearers, as also shown in our study,
suggests the readiness of these strains to cause infection. Slight
changes in the risk factors would tip the balance between the
commensalism and infection. Low levels of phospholipase pro-
duced by fewer strains of C. albicans isolated from the cancer
patients cannot be explained. However cancer drugs are known to
increase or decrease fungal growth, development and virulence
[39].
Secreted aspartic proteinases (SAPs) are secreted by pathogenic
species of Candida, including C. albicans during infection. They are
responsible for the adhesion, tissue damage and invasion of host
immune responses. In the present study, strains isolated from
denture wearers produced signicantly higher amount of protein-
ase compared to the normal subjects and cancer patients. This once
again suggests that these strains have greater potential to cause
infection. In the literature, results of proteinase activity are
controversial. It has been reported that strains of C. albicans isolated
from patients with denture stomatitis produce higher amounts of
proteinase compared to the colonizing strains isolated from the
denture wearers [40,41]. In contrast, Marcos-Arias et al. (2009)
showed no difference in the proteinase production by strains of
C. albicans isolated from the denture wearers with and without
stomatitis [38]. In C. albicans, 10 Secretory Aspartic Proteases (SAPs)
have been identied but their role in the commensal state is not
known. These SAPs are produced at different stages of infections
and the production is also associated with the morphological state
of C. albicans. Studies have reported that SAPs 1, 2 and 3 are
expressed by only the yeast phase of C. albican, whereas SAPs 4, 5
and 6 are expressed in the hyphal phase. In addition, environmental
factors and host factors also inuence the production of these en-
zymes [32]. An in vitro study has shown that some of the anticancer
drugs and irradiation can increase the proteinase production in
C. albicans [42]. However, no difference in the proteinase was found
in the C. albicans isolated from normal individuals and cancer pa-
tients on cancer therapy [20]. Similarly, in our study, although the
cancer treatment regime was not recorded, fewer isolates (64%)
from cancer patients produced low level of proteinase compared to
the isolates from the denture wearers (80%). Proteinase results are
debatable because the detection tests performed in the laboratory
environment are not uniform across the laboratories and they are
not very accurate. Molecular techniques with the detection of gene
expression may prove to be more accurate.
Fifty eight percent of denture wearers and 75% of cancer pa-
tients carried more than 1000 cfu/mL of Candida, which suggests
that the immune response, environmental and physiological factors
may have played a signicant role. Secreted IgA is very important in
host defense against oral candidiasis and may regulate the oral
Candida population [43]. And cancer patients are known to have
reduced secretory immunoglobulin A [44]. Abaci et al. (2010) have
reported that when the count of Candida species in saliva
is  400 cfu/mL, then frequency of development of denture-related
stomatitis increases [45]. In our study group, 96% of colonized pa-
tients carried 100 cfu/mL of which 58% of patients carried
>1000 cfu/mL which suggest that these patients are at high risk of
oral candidiasis development.
Although C. albicans was the predominant species isolated from
the study groups, the role of non - C. albicans yeasts in the patho-
genesis of oral candidiasis cannot be ignored. They have become
clinically important. C. glabrata was the second most common
Candida species isolated from denture wearers and cancer patients
with prostheses and are known to cause oral candidiasis in cancer
patients [46]. Oral prostheses, including dentures, are mostly pre-
pared using acrylic resin which also allows adherence of C. glabrata
and this may have been the reason why this species was isolated
from both study groups [47]. In addition, C. glabrata has developed
resistance to widely used antifungal uconazole and relatively
newly developed antifungal echinocandins [48,49]. These results
suggest that isolation and identication of Candida species in can-
cer patients is very important for the appropriate treatment which
can be instituted without a delay.
 J.V. Mothibe, M. Patel / Microbial Pathogenesis 110 (2017) 128e134
The reason for the difference in the hydrolytic enzyme results of
denture wearers and cancer patients cannot be explained when the
prostheses were common in both the groups. Larger sample size,
data regarding other factors that can affect virulence and more
sensitive techniques for the proteinase production would have
explained these results. Nevertheless, in the denture wearers, high
quantities of enzymes produced by many of the strains collectively
may accelerate the development of infection. It also suggests that
hygiene of the oral cavity, dentures and the prostheses is important
in the reduction of number of Candida, hence the prevention of oral
candidiasis. Proper cleaning and regular use of mild antimicrobial
mouth rinses can keep the number of Candida low. Although
chlorhexidine has a good antifungal effect, it cannot be used
regularly due to the side effects. Triclosan containing mouth rinses,
which are milder, are known to reduce the number of Candida in
the oral cavities of HIV patients [50], therefore, can be used
regularly.
In conclusion, many denture wearers and cancer patients with
prostheses carried high number of Candida in their oral cavities.
Although the adherence to oral epithelial cells was signicantly
increased in strains from denture wearers and cancer patients with
prostheses compared to the strains from normal subjects, many
strains of C. albicans from denture wearers produced high quanti-
ties of phospholipase and proteinase. These results suggest that
strains isolated from denture wearers had more potential or read-
iness to cause infection. Due to the high quantities and many
enzyme producing strains, collectively these enzymes may accel-
erate the development of infection in this study group. Therefore,
hygiene of the oral cavity, dentures and the prostheses is important
in the reduction of number of Candida, hence the prevention of oral
candidiasis.
Conict of interest
None to declare.
Acknowledgements
We thank the National Health Laboratory Services, South Africa
for the nancial support.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.micpath.2017.06.036.
References
[1] L.P. Samaranayake, Essential Microbiology for Dentistry, Harcourt Publishers
Limited, London, 2002, pp. 60e145.
[2] A.M. Darwazeh, S. Al-Refai, S. Al-Mojaiwel, Isolation of Candida species from
the oral cavity and ngertips of complete denture wearers, J. Prosthet. Dent. 8
(4) (2001) 420e423.
[3] A. Akpan, R. Morgan, Oral candidiasis, Postgrad. Med. J. 78 (2002) 455e459.
[4] A.V. Naik, R.C. Pai, A study of factors contributing to denture stomatitis in a
north Indian community, Int. J. Dent. 2011 (2011) 589064, http://dx.doi.org/
10.115/2011/589064. Published online.
[5] L. Gendreau, Z.G. Loewy, Epidemiology and etiology of denture stomatitis,
J. Prosthodont 20 (2011) 251e260.
[6] R.V. Lalla, M.C. Latortue, C.H. Hong, A. Ariyawardana, S. D Amato-Palumbo,
D.J. Fischer, A. Martof, O. Nicolatou-Galitis, L.L. Patton, L.S. Elting,
F.K. Spijkervet, M.T. Brennan, Fungal infections section, oral care study group,
multinational association of supportive care in cancer (MASCC)/International
society of oral oncology (ISOO). A systemic review of oral fungal infections in
patients receiving cancer therapy, Support Care Cancer 18 (2010) 985e992.
[7] R. Bensadoun, L.L. Patton, R.V. Lalla, J.B. Epstein, Oropharyngeal candidiasis in
head and neck cancer patients treated with radiation: update 2011, Support
Care Cancer 19 (2011) 737e744.
[8] S. Schelenz, S. Abdallah, G. Gray, H. Stubbings, I. Gow, P. Baker, P. Hunter,
Epidemiology of oral yeast colonization and infection in patients with
133
hematological malignancies, head neck and solid tumors, J. Oral Pathol. Med.
40 (2011) 83e89.
[9] S.W. Redding, R.C. Zellars, W.R. Kirkpatrick, R.K. McAtee, M.A. Caceres,
Aw Fothergill, J.L. Lopez-Ribot, C.W. Bailey, M.G. Rinaldi, T.F. Patterson,
Epidemiology of oropharyngeal Candida colonization and infection in patients
receiving radiation for head and neck cancer, J. Clin. Microbiol. 37 (12) (1999)
3896e3900.
[10] L.P. Samaranayake, T.W. MacFarlane, P.J. Lamey, M.M. Ferguson, A comparison
of oral rinse and imprint sampling techniques for the detection of yeast,
coliform and Staphylococcus aureus carriage in the oral cavity, J. Oral Pathol. 15
(1986) 386e388.
[11] D.W.R. MacKenzie, Serum tube identication of Candida albicans, J. Clin.
Pathol. 15 (1962) 563e565.
[12] M. Ghannoum, K.A. Elteen, Correlative relationship between proteinase pro-
duction, adherence and pathogenicity of various strains of Candida albicans,
J. Med. Vet. Mycol. 24 (1986) 407e413.
[13] S. Aoki, S. Ito-Kuwa, Y. Nakamura, T. Masuhara, Comparative pathogenicity of
wild-type strains and respiratory mutants of Candida albicans in mice, Zen-
tralbl Bakteriol. 273 (1990) 332e343.
[14] M.F. Price, I.D. Wilkinson, L.O. Gentry, Plate method for detection of phos-
pholipase activity in Candida albicans, Sabouraudia 20 (1982) 7e14.
[15] V. Mohan das, M. Ballal, Proteinase and phospholipase activity as virulence
factors in Candida species isolated from blood, Rev. Iberoam. Micol. 25 (2008)
208e210.
[16] K. Haynes, Virulence of Candida species, Trends Microbiol. 9 (2001) 591e596.
[17] R.A. Calderone, W.A. Fonzi, Virulence factors of Candida albicans, Trends
Microbiol. 9 (2001) 327e335.
[18] E. Pinto, I.C. Ribeiro, N.J. Ferreira, C.E. Fortes, P.A. Fonseca, M.H. Figueiral,
Correlation between enzyme production, germ tube formation and suscepti-
bility to uconazole in Candida species isolated from patients with denture-
related stomatitis and control individuals, J. Oral Pathol. Med. 37 (10)
(2008) 587e592.
[19] J.P. Lyon, M.A. de Resende, Correlation between adhesion, enzyme production
and susceptibility to uconazole in Candida albicans obtained from denture
wearers, Oral Surg. Oral Pathol. Oral Radiol. Endod. 102 (5) (2006) 632e638.
[20] S. Ramla, V. Sharma, M. Patel, Inuence of cancer treatment on the Candida
albicans isolated from the oral cavities of cancer patients, Support Care Cancer
24 (6) (2016) 2429e2436, http://dx.doi.org/10.1007/s00520-015-3035-8.
Epub 2015 Dec 5.
[21] R.D. Cannon, A.K. Nand, H.F. Jenkinson, Adherence of Candida albicans to
human salivary components adsorbed to hydroxylapatite, Microbiol 141
(1995) 213e219.
[22] S.P. Sweet, S. Cookson, S.J. Challacombe, Candida albicans isolates from HIV-
infected and AIDS patients exhibit enhanced adherence to epithelial cells,
J. Med. Microbiol. 43 (1995) 452e457.
[23] M. Manfredi, M.J. McCullough, Z.M. Al-Karaawi, P. Vescovi, S.R. Porter, In vitro
evaluation of virulence attributes by diabetes mellitus, Oral Microbiol.
Immunol. 21 (3) (2006) 183e189.
[24] T.H. Gasparoto, C.E. de Oliveira, N.A. Vieira, V.C. Porto, C.T. Gasparoto,
A.P. Campanelli, V.S. Lara, The pattern recognition receptors expressed on
neutrophils and the associated cytokine prole from different aged patients
with Candida-related denture stomatitis, Exp. Gerontol. 47 (2012) 741e748.
[25] M.G. Netea, L. Maro di, Innate mechanisms for recognition and uptake of
Candida species, Trends Immunol. 31 (2010) 346e353.
[26] M. Schaller, U. Boeld, S. Oberbauer, G. Hamm, B. Hube, H.C. Korting, Poly-
morphonuclear leukocytes (PMNs) induce protective Th1-type cytokine
epithelial responses in an in vitro model of oral candidosis, Microbiol 150
(2004) 2807e2813.
[27] R.A. Depprich, J.G. Handschel, U. Meyer, G. Meissner, Comparison of preva-
lence of microorganisms on titanium and silicone/polymethyl methacrylate
obturators used for rehabilitation of maxillary defects, J. Prosthet. Dent. 99
(2008) 400e405.
[28] R.T. Glass, J.W. Bullard, C.S. Hadley, E.W. Mix, R.S. Conrad, Partial spectrum of
microorganisms found in dentures and possible disease implications, J. Am.
Osteopath Assoc. 101 (2001) 92e94.
[29] H. Bilhan, T. Sulun, G. Erkose, H. Kurt, Z. Erturan, O. Kutay, T. Bilgin, The role of
Candida albicans hyphae and Lactobacillus in denture-related stomatitis, Clin.
Oral Invest. 13 (4) (2009) 363e368, http://dx.doi.org/10.1007/s00784-008-
0240-6. Epub 2008 Dec 20.
[30] N.M. Moussa, M.A. Ghannoum, P.A. Whittaker, M.S. el-Ezaby, S. Quraman,
Effects of cisplatin and two novel palladium complexes on Candida albicans,
Microbios 62 (252e253) (1990) 165e178.
[31] G.A. Land, K.L. Hulme, W.L. Chafn, The effect of selected antineoplastic agents
on the morphology of Candida albicans 5865, Can. J. Microbiol. 26 (7) (1980)
812e818, 10.1139/m80-140.
[32] J.R. Naglik, S.J. Challacombe, B. Hube, Candida albicans secreted aspartyl pro-
teinases in virulence and pathogenesis, Microbiol. Mol. Biol. Rev. 67 (3) (2003)
400e428.
[33] M. Schaller, H.C. Korting, W. Schafer, J. Bastert, W. Chen, B. Hube, Secreted
aspartic proteinase (SAP) activity contributes to tissue damage in a model of
human oral candidosis, Mol. Microbiol. 34 (1999) 169e180.
[34] N.N. Mishra, T. Prasad, N. Sharma, A. Payasi, R. Prasad, D.K. Gupta, R. Singh,
Pathogenicity and drug resistance in Candida albicans and other yeast species,
Acta Microbiol. Immunol. Hung. 54 (3) (2007) 201e235.
[35] S.D. Leidich, A.S. Ibrahim, Y. Fu, C. Jessup, J. Vitullo, W. Fonzi, F. Mirbod,
134 J.V. Mothibe, M. Patel / Microbial Pathogenesis 110 (2017) 128e134
S. Nakashima, Y. Nozawa, M.A. Ghannoum, Cloning and disruption of caPLB1,
a phospholipase B gene involved in the pathogenicity of Candida albicans,
J. Biol. Chem. 278 (1998) 26078e26086.
[36] M.S.A. Khan, I. Ahmad, F. Aqil, M. Owais, M. Shahid, J. Musarrat, Virulence and
pathogenicity of fungal pathogens with special reference to Candida albicans,
in: Ahmad, et al. (Eds.), Combating Fungal Infections, Chapter 2, Springer-
Verlag, Berlin Heidelberg, 2010, http://dx.doi.org/10.1007/978-3-642-12173-
9_2.
[37] T. Kadir, B. Gumr, B. Uygun-Can, Phospholipase activity of Candida albicans
isolated from patients with denture stomatitis: the inuence of chlorhexidine
gluconate on phospholipase production, Arch. Oral Biol. 52 (7) (2007)
691e696.
[38] C. Marcos-Arias, E. Eraso, L. Madariaga, J.M. Agurre, G. Quindo  s, Phospholipase
and proteinase activities of Candida isolates from denture wearers, Mycoses
54 (2009) e10ee16.
[39] S.C.-A. Chen, R.E. Lewis, D.P. Kontoyiannis, Direct effects of non-antifungal
agents used in cancer chemotherapy and organ transplantation on the
development and virulence of Candida and Aspergillus species, Virulence 2 (4)
(2011) 280e295, http://dx.doi.org/10.4161/viru.2.4.16764. Published online
Jul-Aug 2011.
[40] C.Y. Koga-Ito, J.P. Lyon, V. Vidotto, M.A. de Resende, Virulence factors and
antifungal susceptibility of Candida albicans isolates from oral candidosis
patients and control individuals, Mycopathologia 4 (2006) 219e223.
[41] O. Abaci, Investigation of extracellular phospholipase and proteinase activities
of Candida species isolated from individuals' denture wearers and genotypic
distribution of Candida albicans strains, Curr. Microbiol. 62 (2011) 1308e1314.
[42] E. Ueta, T. Tanida, K. Yoneda, T. Yamamoto, T. Osaki, Increase of Candida cell
virulence by anticancer drugs and irradiation, Oral Microbiol. Immunol. 16 (4)
(2001) 243e249.
View publication stats
[43] S.J. Challacombe, Immunologic aspects of oral candidiasis, Oral Surg. Oral Med.
Oral Pathol. 78 (2) (1994) 202e210.
[44] T. Shpitzer, G. Bahar, R. Feinmesser, R.M. Nagler, A comprehensive salivary
analysis for oral cancer diagnosis, J. Cancer Res. Clin. Oncol. 133 (9) (2007)
613e617.
[45] O. Abaci, A. Haliki-Uztan, B. Ozturk, S. Toksavul, M. Ulusoy, H. Boyacioglu,
Determining Candida spp. incidence in denture wearers, Mycopathologia 169
(5) (2010) 365e372.
[46] S.W. Redding, W.R. Kirkpatrick, B.J. Coco, L. Sadkowski, Aw Fothergill,
M.G. Rinaldi, T.Y. Eng, T.F. Patterson, Candida glabrata oropharyngeal candi-
diasis in patients receiving radiation treatment for head and neck cancer,
J. Clin. Microbiol. 40 (5) (2002) 1879e1881.
[47] C.A. Zamperini, L. Carneiro Hde, E.C. Rangel, N.C. Cruz, C.E. Vergani,
A.L. Machado, In vitro adhesion of Candida glabrata to denture base acrylic
resin modied by glow-discharge plasma treatment, Mycoses 56 (2) (2013)
134e144, http://dx.doi.org/10.1111/j.1439-0507.2012.02223.x. Epub 2012 Jul
19.
[48] M.A. Pfaller, M. Castanheira, S.R. Lockhart, A.M. Ahlquist, S.A. Messer,
R.N. Jones, Frequency of decreased susceptibility and resistance to echino-
candins among uconazole-resistant bloodstream isolates of Candida glabrata,
J. Clin. Microbiol. 50 (4) (2012) 1199e1203.
[49] S. Silva, M. Negri, M. Henriques, R. Oliveira, D.W. Williams, J. Azeredo, Candida
glabrata, Candida parapsilosis and Candida tropicalis: biology, epidemiology,
pathogenicity and antifungal resistance, FEMS Microbiol. Rev. 36 (2012)
288e305.
[50] M. Patel, J.A. Shackleton, M.M. Coogan, J. Galpin, Antifungal effect of mouth
rinses on oral Candida counts and salivary ow in treatment-nave HIV-
infected patients, AIDS Patient Care STDS 22 (8) (2008) 613e618, http://
dx.doi.org/10.1089/apc.2007.0160.