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Clinical Implications of Oral Candidiasis: Host Tissue Damage and

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Clinical Implications of Oral Candidiasis: Host Tissue Damage and
Disseminated Bacterial Disease
Eric F. Kong,a,b Sona Kucharíková,c,d Patrick Van Dijck,c,d Brian M. Peters,b* Mark E. Shirtliff,e,f Mary Ann Jabra-Rizka,f
Department of Oncology and Diagnostic Sciences, Dental School, University of Maryland, Baltimore, Maryland, USAa; Graduate Program in Life Sciences, Molecular
Microbiology and Immunology Program, University of Maryland, Baltimore, Maryland, USAb; Department of Molecular Microbiology, VIB, KU Leuven, Belgiumc; Laboratory
of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Belgiumd; Department of Microbial Pathogenesis, Dental School, University of Maryland,
Baltimore, Maryland, USAe; Department of Microbiology and Immunology, School of Medicine, University of Maryland, Baltimore, Maryland, USAf

The clinical significance of polymicrobial interactions, particularly those between commensal species with high pathogenic po-
tential, remains largely understudied. Although the dimorphic fungal species Candida albicans and the bacterium Staphylococ-
cus aureus are common cocolonizers of humans, they are considered leading opportunistic pathogens. Oral candidiasis specifi-

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cally, characterized by hyphal invasion of oral mucosal tissue, is the most common opportunistic infection in HIVⴙ and
immunocompromised individuals. In this study, building on our previous findings, a mouse model was developed to investigate
whether the onset of oral candidiasis predisposes the host to secondary staphylococcal infection. The findings demonstrated that
in mice with oral candidiasis, subsequent exposure to S. aureus resulted in systemic bacterial infection with high morbidity and
mortality. Histopathology and scanning electron microscopy of tongue tissue from moribund animals revealed massive C. albi-
cans hyphal invasion coupled with S. aureus deep tissue infiltration. The crucial role of hyphae in the process was demonstrated
using a non-hypha-producing and a noninvasive hypha-producing mutant strains of C. albicans. Further, in contrast to previ-
ous findings, S. aureus dissemination was aided but not contingent upon the presence of the Als3p hypha-specific adhesion. Im-
portantly, impeding development of mucosal C. albicans infection by administering antifungal fluconazole therapy protected
the animals from systemic bacterial disease. The combined findings from this study demonstrate that oral candidiasis may con-
stitute a risk factor for disseminated bacterial disease warranting awareness in terms of therapeutic management of immuno-
compromised individuals.

A considerable number of infectious diseases involve multiple

microbial species coexisting and interacting in a host (1, 2). In
these infections, the presence of one microorganism predisposes
the host to colonization by others and in additive polymicrobial
infections two or more nonpathogenic microorganisms together
can cause disease (1, 3). Adherence of organisms to surfaces and to
each other is a prerequisite for establishing concurrent infections,
which often stem from the formation of polymicrobial biofilms
that form on biotic or abiotic surfaces (2, 4–6). In the oral cavity,
the oral mucosa serves as an important barrier to the myriad of
microbial species present in this complex environment. However,
the interactions between the various species can be synergistic in
that the presence of one microorganism generates a niche for
other microorganisms (1, 5, 7–11).
Candida albicans is an opportunistic fungal species commonly
colonizing human mucosal surfaces as a component of the normal
microflora (12–14). However, when host defenses are weakened,
C. albicans can proliferate causing an array of infections ranging
from mucosal such as oral candidiasis (OC) to systemic infections
that are often life-threatening (12, 15–17). Therefore, candidal
infections are unsurprisingly often endogenous occurring when
there is a disruption in host environment (13). Specifically, OC is
considered an AIDS-defining opportunistic infection with up to
80% of HIV⫹ individuals suffering recurrent episodes during the
course of their HIV disease progression (15, 18).
The ability of this highly adaptable fungal pathogen to transi-
tion from commensal to pathogen is primarily the result of its
ability to morphologically switch between yeast and hyphal forms
(14, 19, 20). In fact, the majority of C. albicans infections are as-
sociated with its ability to form biofilms where adhesion of yeast
cells to the substrate is followed by proliferation and hypha for-
mation resulting in a network of cells embedded in extracellular
polymeric matrix (21–23). In the oral cavity, C. albicans coexists
and forms tight associations with various commensal bacterial spe-
cies forming complex mucosal biofilms, a phenomenon known to
play an important factor in C. albicans colonization (2, 5, 10, 11,
24–26). Thus, when Candida infections arise, they often occur in
association with bacteria (27). Among the bacterial species, Staph-
ylococcus aureus specifically is a precarious opportunistic patho-
gen implicated in a variety of diseases, ranging from minor skin
infections to more serious invasive diseases (28–30). However, it
Received 28 October 2014 Returned for modification 11 November 2014
Accepted 13 November 2014
Accepted manuscript posted online 24 November 2014
Citation Kong EF, Kucharíková S, Van Dijck P, Peters BM, Shirtliff ME, Jabra-Rizk MA.
2015. Clinical implications of oral candidiasis: host tissue damage and
disseminated bacterial disease. Infect Immun 83:604 – 613. doi:10.1128/IAI.02843-
14.
Editor: G. S. Deepe, Jr.
Address correspondence to Mary Ann Jabra-Rizk, mrizk@umaryland.edu.
* Present address: Brian M. Peters, Department of Oral and Craniofacial Biology,
Dental School, Louisiana State University Health Sciences Center, New Orleans,
Louisiana, USA.
E.F.K. and S.K. contributed equally to this article.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/IAI.02843-14.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
doi:10.1128/IAI.02843-14

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FIG 1 Infection timeline. A timeline for infection and antifungal therapy model beginning with immunosuppression the day prior to infection with C. albicans
until animals are euthanized 5 days after C. albicans infection is shown.
primarily exists as a commensal organism with approximately
30% of healthy humans colonized in their nasopharynx or on their
skin. Although colonization itself is a harmless state, colonized
individuals are at risk of endogenous infection when S. aureus
enters otherwise sterile sites via wounds or indwelling medical
devices. With the emergence of methicillin-resistant S. aureus
(MRSA), this ubiquitous pathogen is becoming an even greater
therapeutic challenge (31–33).
Our in vitro studies have demonstrated that S. aureus exhibits
high affinity to the C. albicans hyphae as these species coexist in
biofilm and identified the C. albicans hyphal specific adhesin
Als3p to be involved in the coadherence process (34, 35). Further,
our previous in vivo studies have indicated that the interaction of
these species may carry important clinical implications (36, 37). In
fact, several studies have reported the coisolation of these diverse
species from a multitude of diseases such as periodontitis, denture
stomatitis, cystic fibrosis, keratitis, ventilator-associated pneumo-
nia, urinary tract catheters and burn wound infections (38–42).
Given the prevalence of OC as an opportunistic infection in
immunocompromised populations and the established clinical
similarity of OC in mice to that in the human host, we developed
a mouse model to assess whether OC predisposes to secondary
bacterial infection. Further, hypha-deficient mutant strains of C.
albicans were used to demonstrate the crucial role of hyphae in the
disease process and antifungal therapy was implemented as a
strategy to impede the development of bacterial dissemination.
MATERIALS AND METHODS
Ethics statement. All animal experiments were conducted at the AAA-
LAAC accredited Animal Facility of the University of Maryland in accor-
dance with the USA Animal Welfare Act as regulated by the U.S. Depart-
ment of Agriculture. Animal studies were approved by the University of
Maryland Animal Care and Use Committee (IACUC protocol 0814012).
This institution has an Animal Welfare Assurance on file with the Office of
Laboratory Animal Welfare, National Institutes of Health. The assurance
number is A-3199-01.
Strains and growth conditions. The C. albicans wild-type strain
SC5314 (43), the non-hypha-producing double mutant cph1⌬/efg1⌬
strain (HLC54) (44), the tup1⌬ mutant strain (BCa2-10) lacking the
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transcriptional repressor of filamentous growth and its parent strain
(SN250) (45, 46), the als3⌬ strain lacking the hyphal specific adhesin
Als3p (47, 48) and an MRSA strain (MRSA-M2) recently sequenced by
us (GenBank accession number AMTC00000000) (49) were used in
the present study. C. albicans strains were grown in yeast peptone dex-
trose broth (Difco Laboratories) overnight at 30°C, and cells were equili-
brated in fresh medium to an optical density at 600 nm of 1.0. S. aureus
cultures were grown overnight in Trypticase soy broth (TSB; Difco) at
37°C and then grown in fresh TSB to mid-log phase. The cells were har-
vested, washed with phosphate-buffered saline (PBS), and then sus-
pended in Hanks’ balanced salt solution (Fisher Scientific), and suspen-
sions were maintained at 30°C. C. albicans was used at a final cell density of
2 ⫻ 107 cells/ml, and S. aureus was used at a final cell density of 1 ⫻ 106
cells/ml.
Animals. Three-month-old pathogen-free female C57BL/6 mice
(Charles River Laboratories) were used in these studies. Mice were housed at
a maximum of five animals per cage. To suppress bacterial flora and allow C.
albicans colonization, mice received ampicillin at a dose of 0.5 mg/ml in
their drinking water 2 days prior to infection and throughout the study.
Infection model. The coinfection mouse model was performed in
accordance with an established model of OC (50) with modification.
Time-line of infection and treatment is illustrated in Fig. 1. Mice were
rendered susceptible to candidiasis by subcutaneous administration
(0.2 ml) of cortisone acetate (225 mg/kg of body weight) in the dorsum
of the neck every other day starting 1 day before infection (total of
three injections). Animals were divided into groups, with five mice in
each group: mono- or coinfected groups with WT or mutant strains
and groups with or without therapy. On the day of infection, mice were
anesthetized by intraperitoneal injections (0.5 ml) of Tribromoetha-
nol (Sigma-Aldrich; 250 mg/kg of body weight). While under anesthe-
sia, the animals were placed on heating pads (Braintree Scientific)
maintained at 37°C. Anesthetized animals were weighed and then
orally inoculated by placing calcium alginate swabs (Fisher Scientific)
saturated for 5 min with C. albicans yeast cell suspension sublingually for
50 min (Fig. 2A). Animals were placed in a supine position on isothermal
pads and monitored until they recovered from anesthesia. Two days after
C. albicans infection, the animals were similarly infected with S. aureus cell
suspension; however, in addition, S. aureus was also added to drinking
water at a final cell density of 106 cells/ml for the duration of experiments.
Animals were monitored daily for any developing clinical signs of distress,
and those showing signs of severe disease or loss of significant body weight
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FIG 2 Mouse infection model. (A) Anesthetized mice were infected sublingually using swabs saturated with C. albicans cell suspensions. (B) At 3 days
postinfection with the wild-type strains, white lesions can be seen on the surface of the tongue, and at the day animals are euthanized, advanced oral candidiasis
characterized by white plaques is seen covering the tongue and other oral surfaces (red arrows). (C) A similar clinical picture is seen in animals infected with the
als3 strain (red arrows). (D) No lesions, however, were visible in mice infected with C. albicans receiving fluconazole therapy.

were euthanized. Animals were euthanized 5 days postinfection with C.
albicans by CO2 inhalation, followed by cervical dislocation, and then
weighed. The tongues and kidneys were harvested, weighed, homoge-
nized, and cultured in triplicate on bacterial and yeast chromogenic media
CHROMagar (DRG International, Inc.). The plates were incubated for 48
h at 37°C, and viable counts were enumerated and expressed as the log
CFU/g of tissue.
Antifungal therapy. In order to demonstrate that S. aureus does not
disseminate in the absence of OC, some animals were given daily intra-
peritoneal injections (0.2 ml) of fluconazole (West-Ward Pharmaceutical
Corp.; 16 mg/kg of body weight) beginning the day of C. albicans infection
until they were euthanized, and then the tissue was harvested and pro-
cessed as described. Control animals were treated with sterile PBS. Exper-
iments were performed to demonstrate lack of any effect for fluconazole
on S. aureus colonization.
Histopathology of infected tongue tissue. One half of harvested
tongues were fixed and processed for histopathology. In addition, kidneys
from mice with severe disease were also subjected to histopathology. The
tissue was embedded in paraffin and sectioned, the sections were depar-
affinized with xylene and stained with periodic acid-Schiff (PAS) or Gram
stain for better visualization of bacteria, and the slides were examined
using light microscopy. In order to rule out the gastrointestinal tract as a

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FIG 3 Tissue microbial recovery from mono- and coinfected mice with or without fluconazole therapy. At 5 days postinfection with C. albicans, mice were
euthanized, and tissue samples were harvested and assessed for microbial burden. (A) Tongue fungal burden. A high level of C. albicans colonization was seen in
mice infected with the wild-type alone (WT) or in combination with S. aureus (WT⫹SA). In contrast, no C. albicans was recovered from mice infected with the
efg1/cph1 strain with some recovery from mice coinfected with S. aureus. On the other hand, mice infected with the asl3 strain with or without S. aureus recovery
of als3 was comparable to that for the wild type. (B) Recovery of S. aureus from tongues. S. aureus was recovered from the majority of mice infected with S. aureus
alone (SA) or in combination with the efg1/cph1 strain. However, significantly higher level of colonization was noted in mice coinfected with wild-type C. albicans
(WT⫹SA) compared to infection with the efg1/cph1 strain. (C) C. albicans and S. aureus recovery from kidneys of coinfected animals. The microbial presence in
kidneys was assessed as an indication of systemic disease. S. aureus was consistently recovered from the kidneys of all mice coinfected with wild-type strain
(WT⫹SA) concomitant with clinical signs of disease. Similarly, although not consistent, S. aureus was recovered from kidneys of mice coinfected with the als3
strain (als3⫹SA). No organisms were recovered from the kidneys of all other groups, i.e., animals monoinfected or coinfected with C. albicans mutants. (D) C.
albicans and S. aureus recovery from the tongues of fluconazole-treated and untreated animals. No C. albicans was recovered from any of the mice receiving
fluconazole therapy with (CA/SA/F) or without (CA/F) S. aureus. However, S. aureus was recovered from coinfected fluconazole treated (SA/CA/F) mice
although at significantly lower levels than the coinfected group not treated with fluconazole (SA/CA). Five animals were included in each experimental and
control group, and all experiments were performed on at least three separate occasions. Since some groups served as controls for subsequent experiments, higher
number of animals were tested, and therefore more data points were available for some experimental groups. Error bars represent standard error of the means;
P values of ⬍0.05 (*) were considered significant. NS, not significant.

portal of entry for S. aureus dissemination, sections from the small and
large intestines were also harvested and processed.
SEM of infected tongue tissue. For scanning electron microscopy
(SEM) analysis, tongue tissue was fixed in 2% paraformaldehyde–2.5%
glutaraldehyde and, after washing steps with PBS, postfixed with 1%
osmium tetroxide, rinsed with PBS, and dehydrated using a series of
washes with ethyl alcohol (30 to 100%). Samples were dried by critical
point drying using an Autosamdri-810 (Tousimi), mounted on alumi-
num stubs, sputter coated with 10 to 20 nm of platinum/palladium,
and imaged with a Quanta 200 scanning electron microscope (FEI Co.,
Hillsboro, OR).
Data analysis. The database for statistical analysis was built up by
incremental addition of experimental data. In order to determine the
optimal variables and conditions (infectious doses, endpoint of infection,
etc.) and standardize the model, initial experiments were performed using
small groups of animals. For the main experiments, a total of 11 experi-
mental groups were tested, each including five randomly selected mice for
each strain and experimental condition analyzed. All experiments were
performed on at least three separate occasions, and averages were used to
present the data. Since the experiments were dynamic (systemic infection,
testing mutant strains, fluconazole therapy, etc.), they were conducted
sequentially in phases, and several experimental sessions were performed
to complete the database. Since some groups served as controls for subse-
quent experimental sets (such as coinfected), the number of animals in
these groups was substantially higher than a total of 15 mice. Therefore,
more data points for these groups were included in the analysis.
All statistical analysis was performed using GraphPad Prism 5.0 soft-
ware. A Kruskal-Wallis one-way analysis of variance test was used to com-
pare differences between multiple groups, and Dunn’s multiple-compar-
ison test was used to determine whether the difference between two
samples was statistically significant. A Student unpaired t test was used to
compare differences between two samples (Fig. 3D). P values of ⬍0.05
were considered significant.
RESULTS
Tissue microbial burden. To determine whether OC constitutes a
risk factor for the development of secondary bacterial infection, 5

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FIG 4 Histopathology images of tissue sections of tongues from coinfected animals. Tongues were also subjected for histopathology and examined by light
microscopy. (A) Representative image from PAS-stained section of tongues from mice coinfected with S. aureus and wild-type C. albicans demonstrating
extensive presence and penetration of epithelial tissue by the invasive C. albicans hyphae. (B) Upon magnification, massive amounts of S. aureus could be seen
on the outer epithelial layer intertwined with the invading hyphae with marked presence of host inflammatory cells. (C) Tissue sections were also stained with
Gram stain for better visualization of the bacteria. An image of tissue from within the epithelium shows bacterial cells adhering to penetrated hyphae. (D) Image
of Gram-stained kidney tissue sections from coinfected mice with systemic disease showing the presence of S. aureus cells. Arrows: white, hyphae; black, S. aureus.
Bar, 20 ␮m.

days following infection with C. albicans, mice were euthanized,
and tissue samples were harvested for assessment of oral coloni-
zation and systemic infection. Findings demonstrated that all
mice infected with the wild-type C. albicans strains (SC5314 and
SN250) developed clinical OC where, 3 days postinfection, white
lesions could be seen which rapidly progressed into overt candi-
diasis (white plaques) covering the tongue surface (Fig. 2B) with
high level of C. albicans recovered. In the group coinfected with S.
aureus and wild-type strains, in addition to OC, animals exhibited
symptoms indicative of severe morbidity, including weight loss,
hunch back, ruffled fur, and lethargy with a mortality rate of up to
20% and extensive recovery of both species from homogenized
tongues (see Fig. 3A and B). More significantly, S. aureus was
recovered from the kidneys of all moribund animals, and in some
(30%) C. albicans was also recovered from kidneys. In contrast,
mice infected with S. aureus or C. albicans individually, although
organisms were recovered from the tongues (Fig. 3B), animals did
not show any signs of systemic disease with no microbial presence
in kidneys. Interestingly, significantly higher level of S. aureus col-
onization was seen when C. albicans was present (Fig. 3B). Al-
though otherwise healthy, mice infected with C. albicans alone
exhibited some weight loss, likely due to interference of lesions on
ability to eat; however, in animals coinfected with both species, up
to 25% of loss in body weight was seen concomitant with high
morbidity and S. aureus presence in the kidneys.
In contrast to the wild-type C. albicans strains, mice infected
with the non-hypha-producing mutant strain (efg1/cph1) did not
develop any clinical disease, and no C. albicans was recovered
from the tongues (Fig. 3A). Importantly, no organisms were re-
covered from the kidneys from any of the mice in this group.
To provide more insight into the mechanism leading to S. au-
reus dissemination, two additional C. albicans mutant strains were
tested in the animal model in order to assess the importance of
hyphal tissue invasiveness for disseminated disease (tup1 mutant
strain) and to determine whether the staphylococcal dissemina-
tion is contingent upon the presence of the C. albicans hypha-
specific Als3 adhesin (als3 mutant strain). As expected, the find-
ings demonstrated that the tup1 did not colonize the oral cavity,
affirming the importance of hyphae not only in causing disease
but also in mediating colonization (data not shown). On the other
hand, animals infected with the als3 mutant developed OC (Fig.
2C), and the recovery of both species from the tongues was com-
parable to that observed with the parent strain (Fig. 3A and B).
However, in contrast to the parent strain, S. aureus was recovered
from the kidneys of 50% of mice coinfected with S. aureus and the
als3 mutant.

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FIG 5 Scanning electron micrographs of outer tongue epithelium from mice with oral candidiasis and systemic infection. (A) Low-magnification (⫻100)
overview image of an excised tongue with advanced OC showing the thick biofilm formed on the surface. (B) Magnified image (⫻2,000) of the surface of tongue
showing the massive matrix consisting of C. albicans hyphae invading the tissue with S. aureus intertwined with the hyphae. (C) Higher-magnification (⫻5,000)
image of the outer surface of the tongue showing the spiny layer of the tongue surface with hyphae penetrating the surface from the sublingual area where C.
albicans was inoculated. (D) Magnified image (⫻10,000) showing a significant gap in the tissue caused by hyphae invading from the sublingual area as it emerges
through the tongue surface.

Fluconazole treatment. Of the mice receiving antifungal ther-
apy, those infected with the wild-type strain and administered
fluconazole did not develop OC (Fig. 2D), and no C. albicans was
recovered from tongues with or without S. aureus coinfection (Fig.
3D). Similarly, significantly less S. aureus was recovered from the
tongues of the coinfected group treated with fluconazole com-
pared to those not treated (Fig. 3D). Notably, no organisms were
recovered from the kidneys of any of the animals receiving flu-
conazole.
Histopathology of infected tissue. In addition to assessing mi-
crobial burden, tissue harvested from animals was also processed
for histopathology in order to visualize the tissue invasion process.
Microscopic analysis of PAS-stained (Fig. 4A and B) and Gram-
stained (Fig. 4C) tissue sections revealed extensive fungal adher-
ence and hyphal penetration of the epithelial tissue with a massive
influx of inflammatory cells. Significantly, extensive and deep
tongue tissue infiltration of the typically noninvasive S. aureus was
seen (see Fig. S1 in the supplemental material) and, more impor-
tantly, in mice with severe disease S. aureus was also seen in stained
kidney sections (Fig. 4D). Similarly, based on histopathology
analysis of tongue tissue, the als3 strain exhibited invasive ability
comparable to that of the wild-type strain (see Fig. S2A in the
supplemental material). Although S. aureus could be seen within
the damaged epithelium, its association with the invasive hyphae
was to a lesser extent than that with the wild-type strain (see Fig.
S2B, C, and D in the supplemental material). Histopathology
analysis of gastrointestinal tissue sections indicated no signs of
hyphal invasion or pathology to the mucosal tissue (data not
shown).
SEM of infected tongue tissue. In addition, infected tongues
were also subjected to SEM analysis. Consistent with histopathol-
ogy, SEM analysis revealed a thick matrix covering the whole sur-
face of the tongue consisting of C. albicans hyphae, S. aureus, and
host cells (Fig. 5A and B). Upon higher magnification, C. albicans
hyphae could be seen penetrating the outer epithelial layer of the
tongue from the sublingual area where it was inoculated (Fig. 5C),
leaving gaps in the tissue at sites of emergence (Fig. 5D). In addi-
tion to the outer surface, tongues were also sectioned prior to
processing in order to visualize the subepithelial tissue (Fig. 6A,
red box). Analysis of the deep tissue area revealed extensive pres-

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FIG 6 Representative SEM images from within deep tissue of mice with oral candidiasis and systemic infection. (A) Lower-magnification (⫻200) image
demonstrating the massive biofilm matrix formed on the surface of the tongue with sloughing of tissue. (B) Image (⫻5,000) from within the tongue tissue (red
box) showing deep hyphal invasion and the associated presence of S. aureus. (C and D) High-magnification images (⫻8,000 and ⫻10,000, respectively) showing
hyphae penetrating crevices within the tissue, with an overwhelming amount of S. aureus adhering to tissue within the crevices.

ence of both hyphae and S. aureus (Fig. 6B). Importantly, upon
higher magnification, significant amount of S. aureus was seen
within the tissue in some cases associated with the hyphae (Fig. 6C
and D). Figure 7 is a false-colored image for better visualization of
the deep copenetration of C. albicans hyphae and S. aureus in
coinfected mice.
DISCUSSION
The human mouth with its diverse niches and ample supply of
nutrients is undoubtedly conducive for the unrestricted forma-
tion of natural microbial biofilms, such as those found on the oral
mucosal tissue, where a multitude of microbial species coexist (10,
51). Mucosal biofilms have been implicated in a wide variety of
infections, and it is therefore imperative that we understand the
implications of the interactions between colonizing microbial spe-
cies as they coexist on host tissue surfaces (7).
Oral candidiasis is perhaps the most common oral mucosal
infection particularly in immunocompromised individuals. Al-
though C. albicans is commonly encountered as a commensal in
healthy individuals, this opportunistic pathogen is capable of in-
vading virtually any site of the human host, from superficial sites
to deep tissues and organs. C. albicans and S. aureus are frequent
colonizers of human mucosal surfaces; using confocal fluorescent
and atomic force microscopy, our previous in vitro studies re-
vealed a unique mixed biofilm architecture wherein S. aureus as-
sociated closely with the hyphae (34, 52). However, in order to
investigate the clinical implications of this interaction during co-
colonization of host mucosal tissue, a suitable animal model is
needed.
In the present study, at 3 days after oral infection with C. albi-
cans, localized lesions consistent with OC were readily seen on the
tongues rapidly progressing into overt candidiasis. Given the mas-
sive hyphal invasion and deep infiltration of S. aureus observed
upon microscopic analysis of tongues, it was not surprising that
the mice with OC became moribund within 2 days of exposure to
S. aureus, indicating that S. aureus rapidly gained entry into the
circulatory system.
The ability of C. albicans to switch morphology between a yeast
and hyphal form is central to its pathogenesis (19). Where the
yeast form is associated with systemic disease the hyphae are adept
at causing mucosal infections such as OC and Candida vaginitis,
and therefore mutant strains of C. albicans that do not produce

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FIG 7 False-colored SEM image. In order to better visualize the coinvasion of
C. albicans hyphae and S. aureus into the subepithelial tissue, a captured image
was false colored using Adobe Photoshop. The micrograph shows the invasive
hyphae (purple) within the tissue, with infiltrated S. aureus adhering to the
hyphae and the surrounding tissue.
hyphae are not capable of causing mucosal infections (44, 46, 48).
The importance of the hypha-mediated tissue damage manifested
as OC in the development of secondary bacterial infection was
clearly established when a mutant strain of C. albicans unable to
form hyphae (efg1/cph1) and a strain producing noninvasive hy-
phae (tup1) failed to induce staphylococcal infection. Interest-
ingly, however, although the mutant strain was not efficient at
colonizing the oral cavity by itself, some recovery was noted in
coinfected mice, indicating that the presence of S. aureus aided the
mutant in colonization. Along these lines, it was also interesting
that although the C. albicans was not recovered from the kidneys
of any of the monoinfected mice, in some cases, C. albicans dis-
seminated in some coinfected mice. Although it is not clear
whether S. aureus contributed to C. albicans dissemination, it is
noteworthy that a study by Xu et al. (53) demonstrated that mu-
cosal commensal bacteria, namely, Streptococcus oralis, modified
C. albicans virulence, suggesting pathogenic synergy. Whether a
similar process occurs between C. albicans and S. aureus, however,
remains to be investigated.
The dissemination of S. aureus was clearly via the extensive
tissue damage caused by the invading hyphae. In fact, in some of
our SEM images red blood cells could be seen around the hyphae
and S. aureus as they coinvaded the tissue (data not shown), indi-
cating that the process of invasion caused injury to the vascular
system, thereby allowing S. aureus to gain entry. Adherence of S.
aureus to the C. albicans biofilm matrix also likely played a key
role in the process by enhancing its colonization and persis-
tence on the tissue within the biofilm hyphal matrix as indi-
cated by the significantly higher level of S. aureus recovery in the
presence of C. albicans. In fact, in some images of infected tissue,
infiltrating bacterial cells could be seen adhered to the hyphae.
Since we have previously shown that the Als3p hyphal adhesin
February 2015 Volume 83 Number 2 Infection and Immunity
Oral Candidiasis and Systemic Bacterial Infection
plays a role in S. aureus adherence to the hyphae, the als3 mutant
strain was also used to coinfect a group of mice. Although our in
vitro adherence studies demonstrated diminished S. aureus adher-
ence to the hyphae, in the animal model the level of S. aureus
recovery from tongues was comparable to that with the wild type.
However, S. aureus was only recovered from the kidneys of 50% of
the mice coinfected with the als3 mutant strain. Interestingly, in
our previous study using a different model, animals were only
directly inoculated once with S. aureus simultaneously with C.
albicans to investigate whether Als3p is crucial during the early
stages of colonization prior to development of OC (37). In con-
trast to the findings from the present study, Als3p was found to be
necessary for S. aureus to disseminate (37). Combined, these find-
ings are in line with the hypothesis that the process of C. albicans-
mediated staphylococcal dissemination is multifactorial and OC
Downloaded from http://iai.asm.org/ on January 15, 2015 by guest
development prior to bacterial exposure is sufficient to mediate
systemic disease, and although not contingent upon, the process is
likely augmented by the ability of S. aureus to adhere to the inva-
sive hyphae via Als3p.
The azole fluconazole is the most commonly used antifungal
agent for the prevention and treatment of OC (22, 54). Therefore,
to further affirm the significant clinical implications of OC, a
group of animals were administered daily fluconazole treatments.
In contrast to the untreated group, those receiving therapy did not
develop OC or systemic disease. Notably, significantly less S. au-
reus was recovered from the treated group, highlighting the im-
portance of enhanced colonization and persistence of biofilm-
associated microbial communities. Our findings are in line with
those from a recent study by Roux et al. (55), who, using a rat
model, showed that C. albicans airway colonization favors the de-
velopment of bacterial pneumonia. Importantly, and similar to
our findings, antifungal treatment decreased airway fungal colo-
nization and in turn the risk of bacterial pneumonia. These find-
ings are significant since they not only solidify the importance of
implementing antifungal therapy upon the indication of OC but
also suggest that antibiotic therapy may be similarly warranted in
immunocompromised individuals, including HIV⫹ patients, who
are prone to recurrent episodes of OC.
It is important to note, however, that although individuals with
immune suppression are the most vulnerable, other mundane fac-
tors are also considered risk factors for candidiasis; these factors
include steroid and antibiotic usage, hormonal imbalances, nutri-
tional deficiency, age, diabetes, metabolic disorders, and denture
usage, among others (13, 56, 57). In fact, denture stomatitis stem-
ming from the adherence of C. albicans to denture material, fol-
lowed by hyphal infiltration of the denture-associated palatal tis-
sue, is prevalent in approximately 70% of denture wearers (58).
Therefore, although our model is an immunocompromised
model, it is conceivable to speculate that under certain conditions,
potentially other individuals with OC could also be predisposed to
secondary bacterial infections.
In the case of S. aureus, nasal carriage is a well-defined risk
factor of infection with this bacterium, supported by the fact that
in most of the cases strains isolated from colonization and infec-
tion sites are indistinguishable (59). Therefore, given the demon-
strated propensity of staphylococci to disseminate in our animal
model, it is feasible to speculate that a similar phenomenon may
take place in a susceptible human host as these species cocolonize
the oral and nasal mucosa. It is important to note, however, that
although we focused here on C. albicans and S. aureus, C. albicans
iai.asm.org 611
Kong et al.
coexists in the oral cavity with more than 700 different bacterial
species, so it is also conceivable that similar interactions with clin-
ical relevance may occur between C. albicans and other species.
As our knowledge of interspecies interactions on mucosal
niches expands, the role of C. albicans in pathogenesis might prove
to be more intricate than currently recognized. The identification
of oral candidiasis as a risk factor for disseminated bacterial dis-
ease carries serious clinical implications warranting awareness in
terms of therapeutic management, particularly in immunocom-
promised individuals who experience recurrent episodes of oral
candidiasis.
ACKNOWLEDGMENTS
This study was supported by the National Institutes of Health grant
DE14424 to M.A.J.-R., by the Interuniversity Attraction Poles Programme
initiated by the Belgian Science Policy Office (P.V.D. and M.A.J.-R.), by
Flemish Science Foundation (FWO) grant WO.026.11N (P.V.D. and
M.A.J.-R.), and by FWO postdoctoral fellowships awarded to S.K.
We thank Scott Filler for his input and assistance. We also thank Ru-
Ching Hsia and the University of Maryland Core Imaging Facility for
assistance with electron microscopy, Shariq Khan and Christina Tsui for
their contributions, and Vincent Bruno for providing C. albicans strains.
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