Eur Arch Paediatr Dent
DOI 10.1007/s40368-015-0180-1
SHORT COMMUNICATION
First-time isolation of Candida dubliniensis from plaque
and carious dentine of primary teeth
S. Kneist1 • A. Borutta2 • B. W. Sigusch3 • S. Nietzsche4 • H. Küpper1
M. Kostrzewa5 • A. Callaway6
Received: 3 November 2014 / Accepted: 4 March 2015
Ó European Academy of Paediatric Dentistry 2015
Abstract
Aim To determine those organisms of the genus Candida
associated with dental caries by investigating samples from
active carious lesions. Within the genus Candida, the
species Candida albicans and Candida dubliniensis are
capable of forming chlamydospores and germ tubes. Until
it became possible in 1995 to differentiate between the two
species taxonomically, C. dubliniensis was falsely identi-
fied as C. albicans. Whilst the importance of C. albicans
for rapidly progressing early childhood caries (ECC) has
been recognised, so far there have been only reports about
C. dubliniensis in connection with children/mothers who
have been infected with HIV or already developed AIDS.
In the present study, C. dubliniensis was for the first time
isolated from plaque and carious dentine of a healthy five-
year-old boy.
& S. Kneist
susanne.kneist@med.uni-jena.de
1
Biological Laboratory, Clinic for Prosthetic Dentistry and
Dental Materials, Centre of Dentistry, University Hospital,
Bachstraße 18, D-07740 Jena, Germany
2
WHO Collaborating Centre on Prevention of Oral Diseases at
the Centre for Dental, Oral and Maxillofacial Surgery,
University Hospital, Jena, Germany
3
Clinic of Conservative Dentistry Centre of Dentistry,
University Hospital, Jena, Germany
4
Centre of Electron Microscopy, University Hospital, Jena,
Germany
5
Bruker Daltonik GmbH, Bremen, Germany
6
Department of Operative Dentistry, University Medical
Centre, Mainz, Germany
•
Methods As part of the investigation, a number of sam-
ples were collected from individual children affected by
active dental caries. Amongst the samples, one in particular
indicated that Candida species might be involved. The
patient was a five-year-old boy with ECC of the primary
dentition, scheduled for restorative treatment under general
anaesthesia. Before treatment, a salivary, plaque (region of
54/55) and soft carious dentine sample from the tooth 51
was taken before extraction. The counts of yeasts, lacto-
bacilli (LB) and mutans streptococci were determined in
the samples.
Results The boy’s dmft was 11, which was dominated by
the d component. In the saliva of the boy, LB and mutans
streptococci (MS) were detected. In plaque and carious
dentine, MS and most interestingly C. dubliniensis were
present. The yeasts were visualised in carious dentine by
means of scanning electron micrographs.
Conclusions Plaque and carious dentine may be a further
habitat of C. dubliniensis.
Keywords Candida albicans  Candida dubliniensis 
Early childhood caries  Caries of primary teeth
Introduction
Since Candida dubliniensis was first described in 1995
(Sullivan et al. 1995), there have been 548 publications
dealing with the identification, occurrence and medical
importance of the yeast. C. dubliniensis can be found
worldwide and has been isolated mainly from patients
with HIV infections or AIDS, but have also been found in
non-human environmental sources such as excrements and
ticks from a seabird colony (Sullivan et al. 2004; Nunn
et al. 2007; Brenda et al. 2009). C. dubliniensis develops
123

in vivo rapidly a stabile resistance to fluconazole, which
could explain why this yeast species frequently appears as
opportunistic in the oral cavity of HIV-infected indi-
viduals and AIDS patients in addition to C. albicans.
Strains of C. albicans are characterised by an only phe-
notypic resistance to fluconazole, which is lost quickly
(Calvet et al. 1997; Moran et al. 1997). Moran et al.
(2012) attribute to C. dubliniensis a lower virulence than
to C. albicans and explained this by a lower tolerance of
the yeast to stress and the reduced formation of hyphae.
C. dubliniensis can only survive in ecological niches. In
dentistry, studies concerning C. dubliniensis have isolated
it from mothers and children and the periodontal pocket.
This short communication describes the first isolation of
Candida dubliniensis from plaque and carious dentine
from a healthy five-year-old child.
Materials and methods
In a more general study on Candida species a five-year-
old-boy was identified who was a foster child of well-
educated parents. The child was never breast fed and
previously received many ‘‘sweets’’ from his biological
mother. No fluoride tablets or table salt were used. The
child was frequently ill and was treated with antibiotics.
The foster parents visited a dentist and received oral
hygiene instructions for the primary dentition from the
paediatrician. The foster mother was of the opinion that
decayed primary teeth needed to be treated, that it was
inevitable that children would develop carious lesions,
there was a relation between diet and dental health, and
good or bad teeth were inherited. The child’s teeth were
brushed before bedtime by the foster mother ‘‘sometimes
with, sometimes without’’ toothpaste; ‘‘sometimes with,
sometimes without resistance’’; daily up to 5 min.
Dental assessment
The boy had 20 teeth with a dmft value of 11, defined as
d1–4 mft-index (d1 = initial caries lesion, d2 = caries le-
sion restricted to enamel, d3 = caries lesion extending into
the first half of the dentine, d4 = caries lesion extending to
the pulp) (World Health Organization 1997). Six of the
primary teeth (51, 52, 61, 62 and 74, 84) could no longer be
restored and were extracted. The mother consented to the
participation of the child in the subsequent clinical-mi-
crobiological investigation in accordance with the Decla-
ration of Helsinki (64th WMA general assembly, October
2013, Fortaleza, Brazil).
123
Eur Arch Paediatr Dent
Clinical-experimental and microbiological
methods
Before treatment under general anaesthesia, a salivary
sample was taken with an Eppendorf pipette from the floor
of the oral cavity, a plaque sample (region of 54/55) with
dental floss, and soft carious dentine was sampled with an
excavator from tooth 51 before extraction.
The dentine and plaque samples were suspended in 1 ml
of sterile saline, mixed (Vortex-Schüttler Vibrofix, Staufen,
Germany) and sequentially diluted to 10-4; the saliva sample
was diluted to the same degree. Of the dilutions, 0.1 ml each
was plated in triplicate on Sabouraud agar (Merck KGaA,
Darmstadt, Germany), Rogosa agar (BD, Heidelberg, Ger-
many) and Mitis-Salivarius agar (BD, Heidelberg, Germany)
with bacitracin (MSB) (Gold et al. 1973), and incubated for
7 days aerobically (Sabouraud agar) at 28 ± 2 °C or
anaerobically (Rogosa agar, MSB) at 35 ± 2 °C. From agar
plates containing 100–150 colonies, up to 10 colonies were
chosen for identification, validated by Gram staining, sub-
cultured on the same agar from which they were isolated and
stored till further analyses. After identification, the colony
forming units (CFU) were determined for the yeasts, lacto-
bacilli (LB) and mutans streptococci.
The yeasts were identified by means of MALDI-TOF
mass spectrometry (Microflex LTTM, MALDI Biotyper 3.0,
Bruker Daltonik, Bremen, Germany) as previously pub-
lished (Hof et al. 2012). A database with 4110 entries was
used for comparison with a log (score) between 0 und 3.0.
A log (score) [1.69 indicates a close relationship to the
genus, whereas a log (score) [1.99 defines the threshold
for a match on species level. Lactobacilli were identified by
species-specific polymerase chain reaction (PCR) as re-
cently described in detail for L. casei, L. paracasei, L.
rhamnosus and L. gasseri (Callaway et al. 2013).
Mutans streptococci from MSB were identified accord-
ing to their characteristic colony morphology and by spe-
cies-specific PCR for S. mutans (Rupf et al. 1999) and S.
sobrinus (Yoshida et al. 2003).
Scanning electron microscopy
The six extracted teeth were visually analysed by scanning
electron microscopy (SEM). The teeth were fixed in 2.5 %
glutaraldehyde for 30 min, passaged three times for 30 min
in cacodylate buffer, dehydrated in a graded ethanol series
(30, 50, 70 %) 30–60 min, coated with a gold layer of
25–30 nm thickness (Sputter Coater BAL-TEC SCD 005,
BAL-TEC AG, Balzers, Liechtenstein) and visually anal-
ysed using a scanning electron microscope (LEO 1450
VP, LEO Elektronenmikroskopie GmbH, Oberkochen,
Eur Arch Paediatr Dent
Germany) first at a low magnification to gain an overall
view and for more details at magnifications of 1000x,
2000x and 4500x.
Results
Twenty yeast isolates originating from plaque or dentine
but not from saliva were sampled from Sabouraud agar and
identified as C. dubliniensis by mass spectrometry with a
log (score) of 2.1142 (±0.1467). They were present in
plaque with a log CFU of 2.6020 and in carious dentine
(tooth 51) with a log CFU of 1.5682.
In addition to the identification by culture methods in the
sample from carious dentine from tooth 51, C. dubliniensis
could also be visualised by means of scanning electron
microscopy (Figs. 1, 2, 3). Rogosa agar yielded only six
isolates originating from saliva, but none from plaque or
carious dentine. One isolate was identified as L. paracasei,
Fig. 1 (Tooth 51): a overview of the tooth; b detail from (a); c, d pseudomycelial growth of Candida dubliniensis (a) magnification 915 (b)
magnification 9500 (c) magnification 91000 (d) magnification 92000
and the remaining five as L. gasseri. With a log CFU of
0.7781, L. gasseri represented 83.3 % of the salivary LB
count. Four isolates from MSB originating from saliva, and
10 isolates each from plaque or dentine of the child were
identified as S. mutans. S. mutans was present in saliva with
a log CFU of 0.9965, in plaque with a log CFU of 4.5315
and in carious dentine with a log CFU of 3.9208.
Discussion
Early childhood caries is associated with transmission and
subsequent acquisition of cariogenic microorganisms, fos-
tered by a substrate of sugar-containing beverages (juices,
tea, etc.) from a nursing bottle, in older children also as
snacks of cariogenic foods (candy, chocolate, cakes,
cookies). The use of the rubber teat means the rinsing
function of the saliva becomes limited whilst sucrose was
continuously available, and the microbiological cycle of
123
 Eur Arch Paediatr Dent

Fig. 2 (Tooth 62): a overview of the tooth; b, c, d pseudomycelial growth (a) magnification 919 (b) magnification 91000 (c) magnification
92000 (d) magnification 94500

Fig. 3 (Tooth 74): a overview of the tooth; b detail from (a) (a) magnification 915 (b) magnification 92000

123
Eur Arch Paediatr Dent
glucan matrix formation, acid production and plaque ac-
cumulation became initiated. In the absence of an adequate
salivary flow, this process leads to the selection of an
aciduric and acidogenic plaque flora. This situation is as-
sociated with a worldwide increase in the prevalence of
ECC (Berkowitz et al. 2009; Parisotto et al. 2010); dif-
ferent acidogenic and aciduric microorganisms are impli-
cated in the progression of the disease (Kanasi et al. 2010).
Mainly small children and preschool children from socio-
economically disadvantaged sections of the society are
affected (Berkowitz et al. 2009). At the first dental visit, the
child was 4 years old; the foster mother had noticed
something ‘‘peculiar’’ with the teeth. The child now drank
milk and unsweetened tea, was offered for main meals and
one snack between meals. According to the judgement of
the examiner, two main meals and the snack between meals
were cariogenic. Approximately once per week, the child
was offered sweet spread, chocolate and sweet beverages,
and several times per week cookies, cakes, pastries, fruit
yoghurt and, puddings and sweets as a treat.
Also in the present case, S. mutans could be found in
plaque, saliva and dentine. Lactobacilli such as L. para-
casei and L. gasseri could only be detected in saliva. It was
shown in retrospect that initially L. rhamnosus, L. para-
casei subsp. paracasei, L. paracasei subsp. tolerans and L.
gasseri promoted the carious progression (Callaway et al.
2013). Byun et al. (2004) described subsequent pulpal in-
flammations associated with L. rhamnosus and L. gasseri in
carious dentine. In such an environment, also Candida
albicans develops luxuriously in ECC. Many authors in-
cluding most recently, Li et al. (2007), Raja et al. (2010)
and Yang et al. (2012) have already indicated a direct re-
lationship between the presence of C. albicans and the
occurrence and fast progression of ECC.
In situ studies by Gregoire et al. (2011) showed that
bacterial metabolites formed from sucrose adhere to the
fungal surfaces, favour the transport of S. mutans to the
enamel surface of the tooth and at the same time promote
the formation of a glucan-rich matrix. Glucan-mediated
interactions between fungi (yeasts) and bacteria explain the
rising numbers of S. mutans in the increasing extracellular
polysaccharide matrix in the plaque of small children with
ECC. According to in vitro studies by Waltimo et al. (2000)
yeasts can in addition actively invade the dentinal tubules.
As it was seen in the scanning electron micrographs of the
carious dentine in this study, they probably also transport
mutans streptococci in carious dentine, as the latter adhere
to the mycelium and this way they can be transported to-
wards the pulp. Beighton et al. (2004) in their analysis
concerning deprived children aged 3–4 years found that
those children with ECC were particularly at risk, when also
tested positive for yeasts (C. albicans). There were 17
countries in this multicentre study.
Candida dubliniensis has so far not been detected in
plaque or carious dentine of primary teeth. The microbial
counts were with numbers of below 100 CFU relatively
low, but according to the studies by Klinke et al. (2009)
they probably contribute to the rapid progression of the
caries process. These authors determined after all the dry
weight of a colony of C. albicans to be 17.4 pg, in contrast
to S. mutans (0.41 pg) and Lactobacillus spec. (3.5 pg).
When these numbers are applied to findings about the oc-
currence of yeasts, mutans streptococci and LB in plaque
and carious dentine in children with ECC, it becomes clear
that ultimately yeasts dominate the biomass in spite of
lower CFU and are in the presence of a continuous sugar
supply and at low pH values characterised by a strong acid
formation. The five-year-old boy was frequently ill and was
treated with antibiotics. Whether the biological mother of
the boy served as the source for C. dubliniensis and whe-
ther C. dubliniensis was able to spread opportunisticly due
to the antibiotic treatment cannot be answered here.
Until now, C. dubliniensis had only been found in the
oral cavities of immunosuppressed children (for example—
Melo et al. 2009; Mokaddas et al. 2010; Domaneschi et al.
2011) and in a teenager with denture stomatitis (Mosca
et al. 2005), and in children with mucocutaneous can-
didiasis (Bhai et al. 2014). Patients with ECC are, however,
often malnourished and immunosuppressed, a situation
favourable to an infection by Candida species.
Conclusions
In children with caries of primary teeth, in addition to C.
albicans for therapeutic reasons the presence of C.
dubliniensis should also be considered.
Conflict of interest The authors declare that there is no conflict of
interest. Dr. M. Kostrzewa is an employee of the company Bruker
Daltonik GmbH, Bremen, Germany, the manufacturer of the mass
spectrometry system used in this work.
References
Beighton D, Brailsford S, Samaranayake LP, et al. A multi-country
comparison of caries-associated microflora in demographically
diverse children. Community Dent Health. 2004;21(suppl 1):
96–101.
Berkowitz RJ, Koo H, McDermott MP, et al. Adjunctive
chemotherapeutic suppression of mutans streptococci in the
setting of severe early childhood caries. J Public Health Dent.
2009;69:163–7.
Bhai N, Tendolkar U, Baradkar V, Mathur M, Kulkarni M. Paediatric
oropharyngeal and cutaneous candidiasis with special reference
to Candida dubliniensis. J Med Microbiol. 2014;63(4):518–21.
Brenda A, McManus BA, Sullivan DJ, et al. Genetic differences
between avian and human isolates of Candida dubliniensis.
Emerg Infect Dis. 2009;15(9):1467–70.
123

Byun R, Nadkarni MA, Chhour KL, Martin FE, Jacques NA, Hunter
N. Quantitative analysis of diverse Lactobacillus species present
in advanced dental caries. J Clin Microbiol. 2004;42:3128–36.
Callaway A, Kostrzewa M, Willershausen B, et al. Identification of
lactobacilli from deep carious lesions by means of species-
specific PCR and MALDI-TOF mass spectrometry. Clin Lab.
2013;59:1373–9.
Calvet HM, Yeaman MR, Filler SG. Reversible fluconazole resistance
in Candida albicans: a potential in vitro model. Antimicrob
Agents Chemother. 1997;41:535–9.
Domaneschi C, Massarente DB, de Freitas RS, et al. Oral colonization
by Candida species in AIDS pediatric patients. Oral Dis.
2011;17(4):393–8.
Gold O, Jordan HV, van Houte J. A selective medium for
Streptococcus mutans. Arch Oral Biol. 1973;18:1356–64.
Gregoire S, Xiao J, Silva BB, et al. Role of glucosyltransferase B in
interactions of Candida albicans with Streptococcus mutans and
with an experimental pellicle on hydroxyapatite surfaces. Appl
Environ Microbiol. 2011;77(18):6357–67.
Hof H, Eigner U, Maier T, Staib P. Differentiation of Candida
dubliniensis from Candida albicans by means of MALDI-TOF
mass spectrometry. Clin Lab. 2012;58:927–31.
Kanasi E, Dewirst FE, Chalmers NI, et al. Clonal analysis of the
microbiota of severe early childhood caries. Caries Res.
2010;44:485–97.
Klinke T, Kneist S, de Soet JJ, et al. Acid production by oral strains of
Candida albicans and lactobacilli. Caries Res. 2009;43:83–91.
Li Y, Ge Y, Saxena D, Caufield PW. Genetic profiling of the oral
microbiota associated with severe early childhood caries. J Clin
Microbiol. 2007;45(1):81–7.
Melo NR, Taguchi H, Culhari VP, et al. Oral candidiasis of HIV-
infected children undergoing sequential HIV therapies. Med
Mycol. 2009;47(2):149–56.
Mokaddas E, Burhamah MH, Khan ZU, Ahmad S. Levels of (1 ? 3)-
b-D-glucan, Candida mannan and Candida DNA in serum
samples of pediatric cancer patients colonized with Candida
species. BMC Infect Dis. 2010;10:292. doi:10.1186/1471-2334-
10-292.
Moran GP, Sullivan DJ, Henman MC, et al. Antifungal drug
susceptibility of oral Candida dubliniensis isolates from HIV-
infected and non-HIV-infected subjects and generation of stable
123
Eur Arch Paediatr Dent
fluconazole-resistant derivatives in vitro. Antimicrob Agents
Chemother. 1997;41:617–23.
Moran GP, Coleman CD, Sullivan DJ. Candida albicans versus
Candida dubliniensis: why is C. albicans more pathogenic? Int J
Microbiol. 2012; 2012:205921. doi:10.1155/2012/205921.
Mosca CO, Moragues MD, Brena S, Rosa AC, Pontón J. Isolation of
Candida dubliniensis in a teenager with denture stomatitis. Med
Oral Patol Oral Cir Bucal. 2005;10(1):28–31.
Nunn MA, Schäfer SM, Petrout MA, Brown JRM. Environmental
source of Canida dubliniensis. Emerg Infect Dis. 2007;13(5):
747–50.
Parisotto TM, Steiner-Oliveira C, Silva CM, Rodrigues LK, Nobre-
dos-Santos M. Early childhood caries and mutans streptococci: a
systematic review. Oral Health Prev Dent. 2010;8:59–70.
Raja M, Hannan A, Ali K. Association of oral candidal carriage with
dental caries in children. Caries Res. 2010;44:272–6.
Rupf S, Kneist S, Merte K, Eschrich K. Quantitative determination of
Streptococcus mutans by using competitive polymerase chain
reaction. Eur J Oral Sci. 1999;107:75–81.
Sullivan DJ, Westerneng TJ, Haynes KA, Bennett DE, Coleman DC.
Candida dubliniensis sp. nov.: phenotypic and molecular
characterisation of a novel species associated with oral candi-
dosis in HIV-infected individuals. Microbiology. 1995;141:
1507–21.
Sullivan DJ, Moran GP, Pinjon E, et al. Comparison of the
epidemiology, drug resistance mechanisms, and virulence of
Candida dubliniensis and Candida albicans. FEMS Yeast Res.
2004;4(4–5):369–76.
Waltimo TM, Ørstavik D, Sirén EK, Haapasalo MP. In vitro yeast
infection of human dentin. J Endod. 2000;26(49):207–9.
World Health Organization. Oral health surveys, basic methods.
WHO 1997; 4th edn. Geneva, Switzerland.
Yang QX, Zjang Q, Lu LY, et al. Genotypic distribution of Candida
albicans in dental biofilm of Chinese children associated with
severe early childhood caries. Arch Oral Biol. 2012;57:1048–53.
Yoshida A, Suzuki N, Nakano Y, Kawada M, Oho T, Koga T.
Development of a 50 nuclease-based real-time PCR assay for
quantitative detection of cariogenic dental pathogens Strepto-
coccus mutans and Streptococcus sobrinus. J Clin Microbiol.
2003;41(9):4438–41.