Clin Oral Invest DOI 10.1007/s00784-015-1696-9
ORIGINAL ARTICLE
Prevalence of Candida albicans and Candida dubliniensis in caries-free and caries-active children in relation to the oral microbiota—a clinical study A. Al-Ahmad 1 & T. M. Auschill 2 & R. Dakhel 1 & A. Wittmer 3 & K. Pelz 3 & C. Heumann 4 & E. Hellwig 1 & N. B. Arweiler 2
Received: 16 June 2015 / Accepted: 14 December 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Objectives The correlation between caries and the oral prevalence of Candida spp. in children is contradictory in literature. Thereby, authors focused on Candida albicans as the most isolated Candida species from the oral cavity. Therefore, the aim of the present study was to compare caries-free and caries-bearing children regarding their oral carriage of Candida spp. Material and methods Twenty-six caries-free (CF group) and 26 caries-active children (CA group) were included into this study. Three different types of specimens were assessed, saliva and plaque, and in the case of caries, infected dentine samples were microbiologically analyzed for aerobic and anaerobic microorganisms and their counts. Special attention was given to the differentiation between C. albicans and Candida dubliniensis. Additionally, different biochemical tests, VITEK 2 (VITEK®2, bioMérieux, Marcy-l’Etoile, France) and 16S and 18S ribosomal DNA (rDNA) sequencing, were applied for identification. Results The detection of C. albicans did not differ between the CF and CA groups. C. dubliniensis was never detected in any specimen of the CF group, but occurred in one quarter of
* N. B. Arweiler
[email protected]
1
Department of Operative Dentistry and Periodontology, Center for Dental Medicine, Albert-Ludwigs-University, Freiburg, Germany
2
Department of Periodontology, Philipps-University, Marburg, Germany
3
Institute of Medical Microbiology and Hygiene, Albert-Ludwigs-University, Freiburg, Germany
4
Department of Statistics, Ludwig-Maximilians-University, Munich, Germany
the CA group (27 % in plaque, 23 % in saliva), thus leading to a statistically significant difference between the two groups (p < 0.05). In six of these cases, C. dubliniensis was detected concomitantly in saliva and plaque and once only in plaque. CA group harbored statistically more Streptococcus mutans than the control group revealing a correlation between S. mutans and C. dubliniensis regarding the caries group. Conclusions This is the first study reporting a frequent detection of C. dubliniensis in caries-active children, which could have been underestimated so far due to difficulties in differentiation between this yeast species and C. albicans. Clinical relevance Microbiological diagnostic—especially of oral Candida species—is an important determinant for identifying etiological factors of dental caries in children. Keywords Caries-free and caries-active children . Candida spp. . Differentiation . Prevalence
Introduction Candida spp. (especially Candida albicans) are described, on the one hand, as innocuous commensal members of the human (oral) microbiota, but are also acknowledged, on the other hand, as microorganisms associated with harmful or even life-threatening diseases like neonatal candidiasis [1, 2], cancer, cystic fibrosis, radiotherapy, diabetes mellitus, or AIDS [3–7]. Analogous to other (oral) diseases, the mere colonization with bacteria does not necessary lead to infection or symptoms. The opportunistic pathogenicity of Candida spp. is dependent on predisposing factors (local and/or systemic) which affect the host (opportunistic infections). Candida species (yeasts) or other particularly opportunistic pathogens such as bacteria, viruses, and fungi can take advantage of certain
Clin Oral Invest
situations and do not usually cause diseases in a healthy host or with a healthy immune system. An immunodeficiency, however, presents an Bopportunity^ for the pathogen to infect. Already during delivery, a newborn’s skin becomes colonized with Candida spp. The source of infection seems to be the hospital staff [8]. It is obvious that these yeasts find good conditions to colonize especially in children. Aside from an outbreak of disease, these neonates carrying Candida spp. (as well as the staff) represent a source of infection [8]. Neonates with a low birth weight show a higher prevalence of colonization and infections with Candida spp. [8, 9], likely due to their immature immune systems [9, 10]. About 70 % of the population are carriers of Candida spp., but show no symptoms. While Candida spp. infections (such as angular cheilitis) are often seen in elderly or immunocompromised patients, oral candidiasis is also a frequent disease found in children and adolescents. In this context, Candida spp. display relevance from childhood until old age, i.e., thrush [1, 2], caries [11, 12], periodontitis [13, 14], infection of dental implants [15, 16], and denture stomatitis [17–19]. A preferred habitat seems to be the oral cavity and especially carious lesions [12]. One explanation is the property of C. albicans to excrete organic acids which could be more potent than lactic acid in decreasing the pH of an already intensely acidified environment. These acids together with other Bcaries bacteria^ (e.g., lactobacilli) can cause development and progression of caries—particularly dentine caries [20, 21]. So far, it is difficult to verify a clear positive association between C. albicans and children’s dental caries, since its significance as a caries pathogen has often been denied. Recent reviews do not exist and some studies could not find an association between caries and Candida spp. in saliva [22] or could not detect C. albicans in carious dentine [23]. Nevertheless, literature describing such an association is increasingly emerging [7, 24–29]. While in some studies, various yeasts or Candida spp. were investigated, other studies dealt specifically with C. albicans [24, 25, 27, 29]. A certain and, so far, neglected problem relates to the precise differentiation between C. albicans and Candida dubliniensis, which is only possible with the aid of specific diagnostic methods. Yeasts studied in dentistry research focused on C. albicans whereas other Candida species, particularly C. dubliniensis, were rarely mentioned. The question arises whether C. albicans was overestimated, while C. dubliniensis was overlooked. Concerning studies up to date, it cannot be excluded that oral isolates of C. dubliniensis had been falsely identified and were classified as C. albicans. This may partly explain the controversial available data. Recent extensive analysis of C. dubliniensis proteomics data revealed high virulence, drug resistance, pathogenesis, adaptability to host, and tolerance for host immune response for C. dubliniensis infections [30]. Furthermore, Priest and
Lorenz [31] analyzed the virulence of C. dubliniensis and showed that this Candida species was quite robust and has to be grouped with the most virulent species, namely C. albicans and Candida tropicalis. An additional aspect regarding the role of Candida species is their ability to form biofilms. It has been shown that both C. dubliniensis and C. albicans are capable of building biofilms in a similar manner [32]. These findings underline the clinical impact of having the ability to differentiate between C. dubliniensis and C. albicans gained from infection sites of the oral cavity. Thus, it was the purpose of the present study to assess the prevalence of Candida spp. and diverse caries-relevant and other oral microorganisms in caries-bearing children and in children without any caries experience by examining different substrata and differentiating C. dubliniensis from C. albicans.
Materials and methods Children groups involved in the study Before starting the investigation, the study had been approved by the local ethical committee (EC) of the Albert-LudwigUniversity of Freiburg, Germany (approval no. 427/08). Children (female and male) were recruited in the children’s course (Bchildren consulting hours,^ Department of Conservative Dentistry, Dental School, University of Freiburg, Germany) after their parents or guardians had received an oral and written description of the study and had signed a written consent. The children had to be in good general health (especially no signs of candidiasis or thrush) and had not used antibiotics during the past 30 days. Children were screened and categorized into two different groups: (1) caries-free children without any caries experience (DMFT = 0) (CF group) and (2) children with at least one tooth with active caries in dentine (DMFT ≥ 1) (CA group). Subjects were recruited until 26 children per group were achieved. Specimen for microbiological analysis When children appeared for assessment (in the afternoon), they had to refrain from toothbrushing for the last 6 h to standardize plaque biofilm age. Three different types of substrates (plaque, saliva, and in case of caries a dentine specimen) were assessed. Saliva samples were gathered in both groups from the floor of the mouth and from the left and right side of the buccal mucosa by means of sterile cotton pellets (Roeko, Coltene/ Whaledent, Langenau, Germany). The average weight of the cotton carrier was assessed, and based on this mean volume, the total number of bacteria per milliliter of saliva could be calculated. Pellets were placed in 0.75 ml of reduced transport fluid (RTF) [33] and immediately frozen at −80 °C until
Clin Oral Invest
microbiological analyses. The corresponding results are given in counts (CFU) per milliliter of saliva. Supragingival dental plaque was collected in both groups with sterile wooden toothpicks (Til, no. 2610799) from the buccal surface of the upper deciduous molars or premolars. The sampled plaque was transferred into 0.75 ml RTF and stored at −80 °C. The corresponding results are given in counts (CFU) per gram. To determine the bacterial counts per gram, the average weight of plaque and dentin samples were calculated in relation to the fluid. In the CA group, infected carious dentine was taken at the center of the carious lesion with sterile excavators (Hu-Friedy EXC17WH, USA). The infected dentine samples were also transferred and frozen in 0.75 ml RTF. The corresponding results are given in counts (CFU) per gram wet weight of dentine. Microbiological assessments The culture method was performed as described elsewhere [34–36]. The vials containing the samples in RTF were thawed at 36 °C in a water bath and vortexed for 30–45 s. To isolate and identify the microorganisms, 500 μl of the undiluted sample and serial dilutions thereof were cultivated. Serial dilutions (10−1 to 10−6) were prepared in peptone yeast medium (PY) containing cysteine hydrochloride [22]. Each dilution was plated on yeast-cysteine blood agar plates (HCB), on Columbia blood agar plates (CBA), on Sabouraud dextrose agar, and on bile esculin plates. HCB agar plates were used to cultivate anaerobic bacteria at 37 °C for 10 days (anaerobic jar, GENbox BioMérieux® sa, Marcy-l’Etoile, France). CBA agar plates were incubated at 37 °C and 5– 10 % CO2 atmosphere for 5 days to cultivate aerobic and facultative anaerobic bacteria. Bile esculin agar plates were used to cultivate Enterococcus faecalis at 37 °C and 5–10 % CO2 atmosphere for 2 days. Sabouraud dextrose agar was used to cultivate yeasts. Colony types were noted and counted to calculate the number of colony forming units (CFU) per milliliter (in case of saliva) or in grams (in case of plaque or infected dentine samples) of the original sample. All colony types were sub-cultivated to obtain pure cultures. Gram stains were prepared and bacterial cell morphology was determined using light microscopy (Axioscope; Zeiss, Jena, Germany; ×1000 magnification). The biochemical identification of anaerobic microorganisms was performed by routine anaerobic methods, including commercial tests (rapid ID 32 A; bioMérieux, Marcy-l’Etoile, France; rapid ANA II; Innovativ Diagnostic Systems, Innogenetics, Heiden, Germany). Both tests use conventional and chromogenic substrates for differentiation. To identify the aerobic and facultative anaerobic microorganisms, biochemical characteristics were analyzed with commercially available tablets and API 20 Strep (bioMérieux,
Marcy-l’Etoile, France). All tests were performed according to the manufacturers’ instructions. Isolates that could not be identified using the abovementioned methods were analyzed by universal bacterial PCR with the following primers: TP16U1 5′-AGAGTTTGATCMTGGCTCAG-3′ and RT16U6 5′-ATTGTAGCACGTGTGTNCCCC-3′ followed by sequencing. Sequencing was performed on a 3130 Genetic Analyzer (Applied Biosystems, Life Technologies GmbH, Darmstadt, Germany). The CHROM-agar (Becton Dickinson GmbH, Heidelberg, Germany) was used to detect all Candida species. C. albicans usually forms light green or light bluish green colonies whereas C. dubliniensis forms dark green colonies. However, the differences on CHROM agar cannot be easily distinguished. Furthermore, the rice agar was used to differentiate morphologically between C. albicans and C. dubliniensis. When incubated on rice agar at 25 °C for 24 h, C. dubliniensis forms pseudohyphae and some true hyphae with clusters of round blastoconidia at the septa. Additionally, pairs or small clusters of large, thick-walled terminal chlamydospores were formed by C. dubliniensis as opposed to C. albicans which usually produces terminal chlamydospores singly. To confirm the identification of both Candida species, the standard method VITEK 2 was used to biochemically differentiate between both species. The VITEK 2 yeast card to differentiate both Candida species includes 46 different biochemical reactions and is sufficient for the identification of C. albicans and C. dubliniensis. Nevertheless, sequencing the 18S ribosomal DNA (rDNA) genes of the biochemically identified Candida species was also performed as a reference method to ensure the correct identification by the other detection methods. After the 18S rDNA PCR (NL 1 GCA TAT CAA GSG GAG GAA AAG and NL 4 GGT CCG TGT TTC AAG ACG G), the PCR products were analyzed using a 3130 Genetic Analyzer (Applied Biosystems, Foster City, USA). The gained sequences were then analyzed by using the BLAST program from the NCBI (http://www.ncbi.nih.gov/BLAST). Sequence comparison to GenBank data entries served as a confirmation.
Statistical analysis and sample size calculation A sample size calculation was done on the logarithmic scale (log10) for C. albicans in the dental plaque. Assuming a mean difference of 4 between the CA and CF group and a standard error of 5, n = 25 per group were calculated to be necessary to achieve a power of 80 % (α = 0.05). Analysis was performed using SPSS 20 (IBM). Data sets with respect to the CFU in saliva, plaque, and dentine were presented descriptively for both groups as well as the presence of Candida and specific bacteria. Normal distribution of data sets was tested by using Kolmogorov Smirnov test and–since
109
108
109
107
1010 1010 107
2.07 × 1010 ± 2.92 × 5.32 × 1010 ± 7.60 × 1.44 × 107 ± 6.25 × 4/26; 15.4 % 1.26 × 107 ± 3.83 × 6/26; 23.1 % 3.58 × 109 ± 6.02 × 21/26; 80.8 % 1.09 × 108 ± 3.63 × 9/26; 34.6 % 6.21 × 108 ± 1.53 × 10/26; 28.5 % 105
105
105
102
106 Eubacterium spp.
104 Black pigmented
Streptococcus mutans
Candida dubliniensis
105
3.28 × 109 ± 3.28 × 109 5.11 × 109 ± 4.11 × 107 1.22 × 105 ± 4.90 × 105 3/24; 12.5 % – 0/24; 0 % 5.94 × 107 ± 2.06 × 107 3/24; 12.5 % 3.30 × 107 ± 9.91 × 107 7/24; 29.2 % 3.58 × 107 ± 9.9 × 107 5/24; 20.8 % 3.77 × 107 ± 4.44 × 5.01 × 107 ± 5.48 × 1.53 × 102 ± 6.21 × 4/24; 16.7 % – 0/24; 0 % 3.02 × 104 ± 1.48 × 1/24; 4.2 % 1.96 × 104 ± 7.47 × 3/24; 12.5 % 1.02 × 106 ± 3.73 × 6/24; 25 % Total aerobe counts Total anaerobes Candida albicans
107 107 102
Saliva Plaque Saliva
5.41 × 107 ± 8.06 × 1.02 × 108 ± 1.63 × 4.35 × 102 ± 2.13 × 4/26; 15.4 % 3.77 × 102 ± 9.16 × 6/26; 23.1 % 3.15 × 105 ± 9.84 × 4/26; 15.4 % 1.83 × 105 ± 7.18 × 5/26; 19.2 % 1.78 × 105 ± 4.58 × 6/26; 23.1 %
107 108 103
8.19 × 109 ± 9.53 × 109 2.23 × 108 ± 1.63 × 1010 4.62 × 105 ± 2.07 × 106 6/26; 23.1 % 1.56 × 106 ± 3.78 × 106 7/26; 26.9 % 5.22 × 108 ± 1.06 × 109 14/26; 53.8 % 4.02 × 108 ± 9.78 × 108 13/26; 50 % 3.57 × 108 ± 7.40 × 108 17/26; 65.4 %
Dentine Plaque CA group n = 26
While initially 26 children per group were recruited, two of the parents in the CF group have withdrawn their consent. Thus, 24 data sets in the CF group (DMFT = 0, age range 2–10, average age 5.24), and 26 data sets in the CA group (DMFT ≥ 1, age range 2–10, average age 6.17) could be analyzed. Table 1 summarizes the total aerobe counts and the total anaerobe counts as well as the oral microorganisms including C. albicans and C. dubliniensis in the different specimens of both groups. Table 2 displays the corresponding results of the statistical analysis. Accordingly, Table 3 shows data of additional microorganisms (bacteria) of the different specimen in both groups. With the exception of C. dubliniensis, no statistically significant differences between the groups were noted in saliva. In saliva and in the dental plaque, total aerobe and anaerobic counts did not statistically differ between the control and the test group. C. dubliniensis was never detected in the control specimen, but occurred in more than one quarter of the caries-active children in plaque (27 %) as well as in saliva (23 %) leading thus to a statistically significant difference between the two groups (saliva p = 0.013 and plaque p = 0.007; Table 2). In six of these cases C. dubliniensis was detected concomitantly in saliva and in plaque, once only in the plaque. Furthermore, in six cases, no C. albicans occurred when C. dubliniensis was foun d. No te worthy in the caries group c arrying C. dubliniensis, the burden of C. dubliniensis or C. albicans was very similar in saliva, and numbers of C. dubliniensis even exceeded those of C. albicans in plaque (Table 1). Compared to the bacterial counts, the proportion of yeasts was quite low ( 0.05). Similarly, a tenfold increase of S. mutans was seen in plaque, where 3 (13 %) children in the CF group harbored this bacterium compared to 14 (54 %) in the CA group. This difference came out to be significant (p = 0.003). In dentine, S. mutans could be detected in 21 children of the CA group.
CF group n = 24
Results
Table 1
this was not the case—comparisons between groups were made by using Mann-Whitney U test. To measure the correlation between the parameters, the rank correlation coefficient of Spearman was used. It is invariant against monotone transformations (for example, logarithm) of the data.
Total counts (mean CFU ± standard deviation) and occurrence of Candida spp. and other oral microorganisms in the different substrates (given as a number out of total samples and percentage)
Clin Oral Invest
Clin Oral Invest Table 2 Statistical analysis in saliva and plaque between groups (CF and CA) concerning Candida spp. and other oral microorganisms (by MannWhitney U test)
Saliva
Plaque
p value
p value
Total aerobe counts
0.899
ns
0.230
ns
Total anaerobes Candida albicans
0.712 0.903
ns ns
0.003 0.384
* ns
Candida dubliniensis
0.013
*
0.007
*
Streptococcus mutans Black pigmented (Porphyromonas spp. and Prevotella spp.)
0.185 0.494
ns ns
0.003 0.045
* *
Eubacterium spp.
0.805
ns
0.002
*
ns nonsignificant *p < 0.05
The correlation analysis (rank correlation coefficient of Spearman) yielded a significant correlation between C. dubliniensis and S. mutans in plaque (p = 0.011) and saliva (p < 0.001) when regarding all subject independently of the groups. No significant correlations between C. albicans and C. dubliniensis or C. albicans and S. mutans could be found— nor in plaque nor in saliva (p > 0.05). When analyzing correlations within the CA and CF group, sample size was too small for a proper analysis; however, a significant correlation between C. dubliniensis and S. mutans in saliva (p < 0.001) could be seen for the CA group, which confirmed the results of the Boverall-analysis.^ Black pigmented (anaerobe) bacteria were found ten times more frequent in dental plaque in the CA group than in the CF; 29 % of these children harbored these bacteria in the CF group with 50 % carriage, which came out to be statistically significant (p = 0.045). Also, Eubacterium spp. were found to be ten times higher in dental plaque of the CA group compared to the control; 21 % of these CF children harbored this bacterium compared to the CA group with 65 % carriage, which was a significant difference (p = 0.002). In both study groups and in both specimens, saliva and plaque, the following bacteria appeared in more than 87 % and up to 100 % of cases: viridans streptococci, Actinomyces spp., Neisseria spp., Veillonella spp., Rothia spp./ Bacterionema spp., Capnocytophaga spp., and Fusobacterium nucleatum. Concerning the majority of bacterial groups and species tested, no statistically significant differences were found between the two test groups, the controls and caries-bearing children, neither in saliva nor in plaque (p > 0.05; Table 3).
Discussion The present study aimed to assess the prevalence of Candida spp. concomitantly to numerous other microorganisms in
saliva and in the dental biofilm of children with and without caries experience. Moreover, the prevalence of Candida spp. in relation to the important group of microbiota in carious dentin was evaluated. To date, data regarding the complex assembly of the oral microbiota and the microorganisms of the oral biofilm of the juvenile oral cavity are quite rare. In most cases, only the occurrence of single bacterial species was elucidated [11, 22, 24, 27, 28]. The differentiation between C. dubliniensis and C. albicans was of specific interest. It was only quite late, in 1995, that C. dubliniensis was identified as a non-C. albicans fungal species (NCAC) [37]. The characteristics of C. dubliniensis are very similar to those of C. albicans, including germ tube and chlamydospores formation, which are features usually considered only in the identification of C. albicans. As a result, both species were wrongly categorized for a long time. One significant difference between C. albicans and C. dubliniensis is the inability of the latter to express β-glucosidase activity [38, 39]. Regarding the two fungal species, only C. dubliniensis showed a clear difference in occurrence between children with caries and children without caries experience. When focusing on discrimination between both Candida species, it is striking that the dental plaque in children of the CA group harbored either C. albicans or C. dubliniensis, while only one child harbored both species concomitantly. Moreover, in this group, the occurrence of C. albicans or C. dubliniensis in saliva was similar (low) (15 and 23 %, respectively), and the corresponding total count was low, too. However, due to the high proportion and frequency of Candida cells, the role of this species should not be neglected in caries research [40]. The same holds true for plaque, where a similar occurrence was noted. Analogous occurrence rates were also found in dentine, as well as similar counts of C. dubliniensis compared to C. albicans. A positive correlation with caries was found regarding C. dubliniensis, but not for C. albicans. All study findings underline an
8.34 × 108 ± 1.64 × 109 23/24; 95.8 % 2.97 × 108 ± 3.80 × 108 21/24;87.5 % 7.28 × 107 ± 2.88 × 108 2/24; 8.3 % 2.16 × 108 ± 5.79 × 108 15/24; 62.5 % 4.25 × 108 ± 6.06 × 108 22/24; 91.7 % 5.8 × 102 ± 2.8 × 103 1/24; 4.2 %
102
106
105
105
106
108
108
109
6.35 × 105 ± 1.61 × 9/24; 37.5 % 2.01 × 105 ± 7.36 × 8/24; 33.3 % – 0/24; 0 % 1.21 × 105 ± 5.91 × 1/24; 4.2 % 1.67 × 106 ± 3.72 × 19/24; 79.2 % 3.92 × 101 ± 1.92 × 1/24; 4.2 %
No statistically significant differences found between groups
Enterococcus faecalis
Veillonella spp.
Campylobacter (Wolinella spp.)
Parvimonas micra (Peptostreptococcus micros)
Fusobacterium nucleatum
Capnocytophaga spp.
Rothia/Bacterionema
Neisseria spp.
Actinomyces spp.
1.37 × 109 ± 1.40 × 24/24; 100 % 1.55 × 109 ± 1.62 × 24/24; 100 % 2.23 × 108 ± 3.35 × 24/24; 100 % 1.31 × 108 ± 1.78 × 23/24; 95.8 %
2.87 × 107 ± 3.30 × 107 24/24; 100 % 5.21 × 106 ± 8.05 × 10 24/24; 100 % 1.71 × 106 ± 3.69 × 106 24/24; 100 % 7.27 × 105 ± 1.49 × 106 21/24; 87.5 % 106
106
106
107
1.84 × 105 ± 7.11 × 105 6/26; 23.1 % 6.70 × 105 ± 2.21 × 105 8/26; 30.8 % 7.66 × 104 ± 3.55 × 105 3/26; 11.5 % 2.55 × 105 ± 6.52 × 105 6/26; 23.1 % 4.62 × 106 ± 8.87 × 106 15/26; 57.7 % 1.53 × 101 ± 7.8 × 101 1/26; 3.8 %
4.78 × 107 ± 7.95 × 26/26; 100 % 4.61 × 106 ± 9.68 × 25/26; 96.2 % 2.14 × 106 ± 4.01 × 24/26; 92.3 % 8.86 × 105 ± 2.25 × 21/26; 80.8 %
Saliva
Plaque
Saliva 109
Caries group n = 26
Caries-free group (control) n = 24
108
108
109
109
4.73 × 108 ± 5.28 × 108 24/26; 92.3 % 8.85 × 108 ± 1.27 × 109 25/26; 96.2 % 1.78 × 108 ± 7.25 × 108 4/26; 15.4 % 2.44 × 108 ± 3.5 × 108 20/26; 76.9 % 1.57 × 109 ± 2.91 × 109 23/26; 88.5 % — 0/26; 0 %
4.53 × 109 ± 8.32 × 26/26; 100 % 2.06 × 109 ± 2.14 × 26/26; 100 % 1.60 × 108 ± 2.18 × 25/26; 96.2 % 1.06 × 108 ± 1.39 × 23/26; 88.5 %
Plaque
Total counts (mean CFU ± standard deviation) of further oral microorganisms in the different substrates (given as a number out of total samples and percentage)
Viridans streptococci
Table 3
8.97 × 108 ± 3.47 × 13/26; 50 % 1.05 × 109 ± 1.82 × 13/26; 50 % 1.67 × 108 ± 5.71 × 3/26; 11.5 % 3.36 × 108 ± 7.63 × 11/26; 42.3 % 1.96 × 109 ± 3.47 × 21/26; 80.8 % 4.05 × 105 ± 2.07 × 1/26; 3.8 %
106
109
108
108
109
109
6.65 × 109 ± 1.52 × 1010 25/26; 96.2 % 2.12 × 109 ± 3.98 × 109 25/26; 96.2 % 2.40 × 108 ± 7.10 × 108 21/26; 80.8 % 5.20 × 108 ± 1.50 × 109 20/26; 76.9 %
Dentine
Clin Oral Invest
Clin Oral Invest
association of C. dubliniensis with children having caries or caries experience. So far, similar findings are missing in literature. Only one recent study [41] emphasized the value of C. dubliniensis and its isolation from plaque and carious dentine of primary teeth. The authors underlined the difficulties of identification and separation between both species suggesting that a misidentification could not be excluded in earlier studies, which is in line with the present findings. No further studies concerning C. dubliniensis in childhood caries could be found in literature. All available former studies describe intensively the role of C. albicans in the childhood. For example, Starr et al. [42] reported a high prevalence of up to 47 % of C. albicans in the oral cavity of children. Furthermore, the authors emphasize that the occurrence of C. albicans remained albeit treatment and regular dental care. However, Starr et al. [42] discussed the difficulties to distinguish between C. albicans and C. dubliniensis and justified classifying both species as C. albicans. This emphasizes the need to separate both of these species by studying their relevance to caries in the childhood. Moreover, the study of Starr et al. [42] led to the suggestion that the role of C. dubliniensis was underestimated so far, as revealed by our present study. In another study [27], a high prevalence of C. albicans was also shown. The authors found a correlation between caries lesions and a higher prevalence of C. albicans and suggested a role of C. albicans in caries development. Again, no differentiation between C. dubliniensis and C. albicans was conducted, a fact which allows the assumption that the role of C. dubliniensis was also underestimated in this aforementioned study. This assumption could be substantiated by the findings that C. dubliniensis was the most frequent nonalbicans Candida in AIDS patients [43]. Moreover, Shen et al. [44] showed that C. dubliniensis comprised approximately up to 14 % of yeasts found in root caries lesions in elderly. The authors emphasized that C. dubliniensis was not reported previously as a germ associated with human caries. Linossier et al. [45] assumed a coaggregation of S. mutans with C. albicans in salivary samples of children with Down syndrome. The authors suggested that C. albicans integrates in the dental plaque by adhesion to the polysaccharides produced by S. mutans. Similar suggestions were made for the association between C. albicans and Streptococcus gordonii [46]. However, as mentioned above, all authors focused on C. albicans while C. dubliniensis was not mentioned. The detection of S. mutans in the present study was a supporting factor for the incidence of C. dubliniensis and could be for incidence of C. albicans, too. The occurrence of all other microorganisms, particularly S. mutans, is in line with a plethora of studies. The prevalence of S. mutans in the saliva of the caries group was 15 % and higher than in the controls (4 %), but did not reach statistical significance. However, a significant difference was evaluated in the dental plaque. Clearly, a frequent and regular dental
hygiene, which is assumed in children without any caries experience, prevents adhesion or a severe colonization of S. mutans on the dental surfaces even though S. mutans is prevalent in their saliva. A further interesting output of the present study was the high prevalence of Eubacterium spp. in the CA group. This is in contrast with the findings of Gross et al. [47] who reported a decrease of Eubacterium spp. in caries lesions in young permanent teeth. However, these authors used only 16S rRNA gene analysis to describe the microbial populations of caries lesions while the present study cultivated the germs and used 16S rRNA only for confirmation, which minimizes the risk of overestimation. A comparison to the results of Gross et al. [47] is limited to the fact that the authors studied the microbiota only of permanent teeth. Nevertheless, the high prevalence of Neisseria spp. in dentin samples of the caries group is in accordance with the results reported by Gross et al. [47]. It seems that the characteristics of C. albicans related to cariogenicity and its preferred habitat in caries lesions given by Klinke et al. [40] are also transferable to or—in the present study—only applicable for C. dubliniensis which only occurred in caries group. Obviously, our data support the findings that this microorganism has a high acid tolerance and enables acidification by excreting organic acids thus favoring a milieu for other bacteria like S. mutans, black pigmented bacteria or eubacteria. In conclusion, the present study revealed for the first time a high prevalence of C. dubliniensis in children with caries suggesting a role of this yeast in caries development and progression as well as a correlation with S. mutans. Nevertheless, after the application of Auxacolor TM, Klinke et al. [48] managed to detect only C. albicans but no C. dubliniensis in saliva and plaque of children with early childhood caries. To confirm a possible underestimation of C. dubliniensis in literature so far as suggested by our results, clinical studies using additional methods such as Auxacolor TM (BioRaD, Munich, Germany) have to be conducted along with the established techniques applied in the present study.
Acknowledgments Bettina Spitzmüller is acknowledged for the skillful technical laboratory assistance during the experiments. Special thanks are also given to Kristina Schmidt for helping with the English edits of the final version of the article.
Compliance with ethical standards All procedures performed in this clinical study were approved by the research committee of the AlbertLudwig-University of Freiburg, Germany (approval no. 427/08), and in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Conflict of interest The authors declare that they have no competing interests. Funding The study received no funding.
Clin Oral Invest Informed consent Written informed consent was obtained from all individual participants and their parents prior to their inclusion in the study.
16.
17.
18.
References 1.
2.
3.
4.
5.
6.
7.
8. 9.
10.
11.
12.
13.
14.
15.
Bliss JM, Wong AY, Bhak G, Laforce-Nesbitt SS, Taylor S, Tan S, Stoll BJ, Higgins RD, Shankaran S and Benjamin DK, Jr. (2012) Candida virulence properties and adverse clinical outcomes in neonatal candidiasis. J Pediatr 161:441–447.e2. doi:10.1016/ j.jpeds.2012.02.051 Leibovitz E (2012) Strategies for the prevention of neonatal candidiasis. Pediatr Neonatol 53:83–89. doi:10.1016/j.pedneo.2012.01. 004 Gammelsrud KW, Sandven P, Hoiby EA, Sandvik L, Brandtzaeg P, Gaustad P (2011) Colonization by Candida in children with cancer, children with cystic fibrosis, and healthy controls. Clin Microbiol Infect 17:1875–1881. doi:10.1111/j.1469-0691.2011.03528.x Grotz KA, Genitsariotis S, Vehling D, Al-Nawas B (2003) Longterm oral Candida colonization, mucositis and salivary function after head and neck radiotherapy. Support Care Cancer 11:717– 721. doi:10.1007/s00520-003-0506-0 Nagao M, Saito T, Doi S, Hotta G, Yamamoto M, Matsumura Y, Matsushima A, Ito Y, Takakura S, Ichiyama S (2012) Clinical characteristics and risk factors of ocular candidiasis. Diagn Microbiol Infect Dis 73:149–152. doi:10.1016/j.diagmicrobio.2012.03.006 Rautemaa R, Ramage G (2011) Oral candidosis—clinical challenges of a biofilm disease. Crit Rev Microbiol 37:328–336. doi: 10.3109/1040841x.2011.585606 Siahi-Benlarbi R, Nies SM, Sziegoleit A, Bauer J, Schranz D, Wetzel WE (2010) Caries-, Candida- and Candida antigen/ antibody frequency in children after heart transplantation and children with congenital heart disease. Pediatr Transplant 14:715–721. doi:10.1111/j.1399-3046.2008.01115.x Sharp AM, Odds FC, Evans EG (1992) Candida strains from neonates in a special care baby unit. Arch Dis Child 67:48–52 Marodi L, Johnston Jr RB (2007) Invasive Candida species disease in infants and children: occurrence, risk factors, management, and innate host defense mechanisms. Curr Opin Pediatr 19:693–697. doi:10.1097/MOP.0b013e3282f1dde9 Hacimustafaoglu M, Celebi S (2011) Candida infections in nonneutropenic children after the neonatal period. Expert Rev AntiInfect Ther 9:923–940. doi:10.1586/eri.11.104 de Carvalho FG, Silva DS, Hebling J, Spolidorio LC, Spolidorio DM (2006) Presence of mutans streptococci and Candida spp. in dental plaque/dentine of carious teeth and early childhood caries. Arch Oral Biol 51:1024–1028. doi:10.1016/j.archoralbio.2006.06. 001 Hossain H, Ansari F, Schulz-Weidner N, Wetzel WE, Chakraborty T, Domann E (2003) Clonal identity of Candida albicans in the oral cavity and the gastrointestinal tract of pre-school children. Oral Microbiol Immunol 18:302–308 Sardi JC, Duque C, Mariano FS, Peixoto IT, Hofling JF, Goncalves RB (2010) Candida spp. in periodontal disease: a brief review. J Oral Sci 52:177–185 Waltimo TM, Sen BH, Meurman JH, Orstavik D, Haapasalo MP (2003) Yeasts in apical periodontitis. Crit Rev Oral Biol Med 14: 128–137 Leonhardt A, Renvert S, Dahlen G (1999) Microbial findings at failing implants. Clin Oral Implants Res 10:339–345
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33. 34.
Pye AD, Lockhart DE, Dawson MP, Murray CA, Smith AJ (2009) A review of dental implants and infection. J Hosp Infect 72:104– 110. doi:10.1016/j.jhin.2009.02.010 Bilhan H, Sulun T, Erkose G, Kurt H, Erturan Z, Kutay O, Bilgin T (2009) The role of Candida albicans hyphae and Lactobacillus in denture-related stomatitis. Clin Oral Investig 13:363–368. doi:10. 1007/s00784-008-0240-6 Gendreau L, Loewy ZG (2011) Epidemiology and etiology of denture stomatitis. J Prosthodont 20:251–260. doi:10.1111/j.1532849X.2011.00698.x Salerno C, Pascale M, Contaldo M, Esposito V, Busciolano M, Milillo L, Guida A, Petruzzi M, Serpico R (2011) Candidaassociated denture stomatitis. Med Oral Patol Oral Cir Bucal 16: e139–e143 Klinke T, Guggenheim B, Klimm W, Thurnheer T (2011) Dental caries in rats associated with Candida albicans. Caries Res 45:100– 106. doi:10.1159/000324809 Szabo B, Majoros L, Papp-Falusi E, Szabo Z, Szabo J, Marton I, Kelentey B (2014) Studies on the possible aetiological role of different Candida species in pathogenesis of dentine caries by monitoring the calcium release from tooth particles. Acta Microbiol Immunol Hung 61:11–17. doi:10.1556/AMicr.61.2014.1.2 Peretz B, Mazor Y, Dagon N, Bar-Ness Greenstein R (2011) Candida, mutans streptococci, oral hygiene and caries in children. J Clin Pediatr Dent 36:185–188 Maijala M, Rautemaa R, Jarvensivu A, Richardson M, Salo T, Tjaderhane L (2007) Candida albicans does not invade carious human dentine. Oral Dis 13:279–284. doi:10.1111/j.1601-0825. 2006.01279.x Akdeniz BG, Koparal E, Sen BH, Ates M, Denizci AA (2002) Prevalence of Candida albicans in oral cavities and root canals of children. ASDC J Dent Child 69(289–92):235 Moalic E, Gestalin A, Quinio D, Gest PE, Zerilli A and Le Flohic AM (2001) The extent of oral fungal flora in 353 students and possible relationships with dental caries. Caries research 35:149– 55. doi:47447 Raja M, Hannan A, Ali K (2010) Association of oral candidal carriage with dental caries in children. Caries Res 44:272–276. doi:10.1159/000314675 Rozkiewicz D, Daniluk T, Zaremba ML, Cylwik-Rokicka D, Stokowska W, Pawinska M, Dabrowska E, Marczuk-Kolada G, Waszkiel D (2006) Oral Candida albicans carriage in healthy preschool and school children. Adv Med Sci 51(Suppl 1):187–190 Signoretto C, Burlacchini G, Faccioni F, Zanderigo M, Bozzola N, Canepari P (2009) Support for the role of Candida spp. in extensive caries lesions of children. New Microbiol 32:101–107 Yang XQ, Zhang Q, Lu LY, Yang R, Liu Y, Zou J (2012) Genotypic distribution of Candida albicans in dental biofilm of Chinese children associated with severe early childhood caries. Arch Oral Biol 57:1048–1053. doi:10.1016/j.archoralbio.2012.05.012 Kumar K, Prakash A, Tasleem M, Islam A, Ahmad F, Hassan MI (2014) Functional annotation of putative hypothetical proteins from Candida dubliniensis. Gene 543:93–100. doi:10.1016/j.gene.2014. 03.060 Priest SJ, Lorenz MC (2015) Characterization of virulence-related phenotypes in Candida species of the CUG clade. Eukaryotic Cell 14:931–940. doi:10.1128/ec.00062-15 Alnuaimi AD, O'Brien-Simpson NM, Reynolds EC, McCullough MJ (2013) Clinical isolates and laboratory reference Candida species and strains have varying abilities to form biofilms. FEMS Yeast Res 13:689–699. doi:10.1111/1567-1364.12068 Syed SA, Loesche WJ (1972) Survival of human dental plaque flora in various transport media. Appl Microbiol 24:638–644 Al-Ahmad A, Pelz K, Schirrmeister JF, Hellwig E, Pukall R (2008) Characterization of the first oral vagococcus isolate from a root-
Clin Oral Invest filled tooth with periradicular lesions. Curr Microbiol 57:235–238. doi:10.1007/s00284-008-9182-0 35. Anderson AC, Hellwig E, Vespermann R, Wittmer A, Schmid M, Karygianni L, Al-Ahmad A (2012) Comprehensive analysis of secondary dental root canal infections: a combination of culture and culture-independent approaches reveals new insights. PLoS One 7: e49576. doi:10.1371/journal.pone.0049576 36. Schirrmeister JF, Liebenow AL, Pelz K, Wittmer A, Serr A, Hellwig E, Al-Ahmad A (2009) New bacterial compositions in root-filled teeth with periradicular lesions. J Endod 35:169–174. doi:10.1016/j.joen.2008.10.024 37. Sullivan DJ, Westerneng TJ, Haynes KA, Bennett DE, Coleman DC (1995) Candida dubliniensis sp. nov.: phenotypic and molecular characterization of a novel species associated with oral candidosis in HIV-infected individuals. Microbiology 141(Pt 7): 1507–1521 38. Schoofs A, Odds FC, Colebunders R, Ieven M, Goossens H (1997) Use of specialised isolation media for recognition and identification of Candida dubliniensis isolates from HIV-infected patients. Eur J Clin Microbiol Infect Dis 16:296–300 39. Sullivan D, Haynes K, Bille J, Boerlin P, Rodero L, Lloyd S, Henman M, Coleman D (1997) Widespread geographic distribution of oral Candida dubliniensis strains in human immunodeficiency virus-infected individuals. J Clin Microbiol 35:960–964 40. Klinke T, Kneist S, de Soet JJ, Kuhlisch E, Mauersberger S, Forster A, Klimm W (2009) Acid production by oral strains of Candida albicans and lactobacilli. Caries Res 43:83–91. doi:10.1159/ 000204911 41. Kneist S, Borutta A, Sigusch BW, Nietzsche S, Kupper H, Kostrzewa M, Callaway A (2015) First-time isolation of Candida
dubliniensis from plaque and carious dentine of primary teeth. Eur Arch Paediatr Dent. doi:10.1007/s40368-015-0180-1 42. Starr JR, White TC, Leroux BG, Luis HS, Bernardo M, Leitao J, Roberts MC (2002) Persistence of oral Candida albicans carriage in healthy Portuguese schoolchildren followed for 3 years. Oral Microbiol Immunol 17:304–310 43. Owotade FJ, Patel M, Ralephenya TR, Vergotine G (2013) Oral Candida colonization in HIV-positive women: associated factors and changes following antiretroviral therapy. J Med Microbiol 62: 126–132. doi:10.1099/jmm.0.047522-0 44. Shen S, Samaranayake LP, Yip HK, Dyson JE (2002) Bacterial and yeast flora of root surface caries in elderly, ethnic Chinese. Oral Dis 8:207–217 45. Linossier A, Vargas A, Villegas R, Chimenos E (2002) Quantitative relationship between salivary level of Streptococcus mutans and Candida albicans in children with Down's syndrome. Med Oral 7: 284–292 46. Holmes AR, Gopal PK, Jenkinson HF (1995) Adherence of Candida albicans to a cell surface polysaccharide receptor on Streptococcus gordonii. Infect Immun 63:1827–1834 47. Gross EL, Leys EJ, Gasparovich SR, Firestone ND, Schwartzbaum JA, Janies DA, Asnani K, Griffen AL (2010) Bacterial 16S sequence analysis of severe caries in young permanent teeth. J Clin Microbiol 48:4121–4128. doi:10.1128/jcm.01232-10 48. Klinke T, Urban M, Luck C, Hannig C, Kuhn M, Kramer N (2014) Changes in Candida spp., mutans streptococci and lactobacilli following treatment of early childhood caries: a 1-year follow-up. Caries Res 48:24–31. doi:10.1159/000351673
