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Int Poster J Dent Oral Med 11 (2009), No. 2     15. June 2009

Int Poster J Dent Oral Med 2009, Vol 11 No 2, Poster 445

Candida Dubliniensis in Dental Plaque of ECC Affected Children

Language: English
 

Authors:
Dr. Filip Ruzicka, Veronika Hola, Prof. Martina Kukletová, Dr. Jarmila Kuklová,
Faculty of Medicine, Masaryk University and St. Ann's Faculty Hospital, Brno
Marie Horka,
Institute of Analytic Chemistry Academy of Sciences, Brno
Lenka Zackova,
Department of Pedodontics, Children's Teaching Hospital, Brno

Date/Event/Venue:
May 29th - June 1st, 2008
9th Congress of the European Academy of Paediatric Dentistry
Dubrovnik, Croatia
 

Introduction

The yeasts of Candida genus represent an important part of the normal oral microflora, including dental plaque. Their ability to produce organic acids during the fermentation of carbohydrates partakes in the cariogenic effect of dental plaque. The most frequent yeast in the oral cavity is Candida albicans but Candida dubliniensis is isolated relatively often from this location as well, especially in immunocompromised HIV+ patients. Its prevalence is highly variable and depends on the region, nationality, age, clinical status, etc.
 

Objectives

The study aims to evaluate the occurrence of C.dubliniensis in the dental plaque samples obtained from the children affected by Early Childhood Caries (ECC), and to assess reliability of the methods used for the differentiation between C.dubliniensis and C.albicans.
 

Material and Methods

Used strains:
The dental plaque samples (352) were obtained from children affected by Early Childhood Caries (ECC) and from caries-free children. The samples were cultivated on Sabouraud Agar (both HiMedia, Mumbai, India) at 30 oC for 24-72 hours. The identification of isolated strains was performed using typical growth on CHROM Agar (CHROM Agar, France), by conventional morphological analysis, especially micromorphology on rice agar, and by commercially available biochemical kits CANDIDA Test 21 (Pliva-Lachema) and/or ID 32C system (BioMérieux, Marcy-l'Etoile, France). As controls the strains C.albicans CCM 8261, CCM 8320 (Czech Collection of Microorganisms) and C. dubliniensis CCY29-177-1 (Culture Collection of Yeasts) were used.

Differentiation between C. dubliniensis and C.albicans:
CHROM Agar:cultivativation at 30° C for 48 hours; C. albicans forms light-green colonies (Fig. 1), C. dubliniensis creates dark-green colonies (Fig. 2).
Growth at 45° C: C. dubliniensis, contrary to C. albicans, fails to grow on Sabouraud agar at 45° C.
Staib agar: The strains were inoculated on Staib agar (50g of Guizotia abyssinica seed (pulverized), 1g of glucose, 1g of KH2PO4, 1g of creatinine, and 15g of agar per litre) and incubated at 30° C for 72 h. C. dubliniensis strains form rough colonies with abundant hyphae and chlamydospores on Staib agar (Fig. 6), whereas C. albicans isolates form smooth colonies without hyphae and chlamydospores (Fig. 5).
Chlamydospores formation on rice agar: C. albicans forms chlamydospores separately (Fig. 3); C. dubliniensis forms clusters or grapes of chlamydospores (Fig. 4).
Latex agglutination test to identify C. dubliniensis: BICHRO-Dubli (Fumoze Diagnostics)

Fig. 1: Fig. 1-6 Growth on CHROMagar and Staib agar Fig. 2
Fig. 3 Fig. 4
Fig. 5
 
Fig. 6
 

PCR-RFLP
The 50 µL PCR mixture contained HotStart Master Mix 1x (Quiagen, Hilden, Germany), 15 pmol UNF1 primer (5'ttg ata tgc tta agt tca gcg g 3'), 15 pmol UNF2 primer (5'ttg ata tgc tta agt tca gcg g 3'), 100µmol of dUTP (Sigma), 0,5 U of uracil-DNA-glycosylase (Sigma) for the elimination of cross-contamination and 5µL of isolated DNA sample. PCR reaction was performed in a thermocycler PTC-200 (BioRad).
Restriction analysis of amplified PCR products was performed by Sau3AI (New England BioLabs, Beverly, MA, USA). The product of PCR and restriction fragments were separated on 3% agarose gel with ethidium bromide, visualised by UV transiluminator (312 nm) and analysed by ULTRA LUM (Ultra-Lum, Inc., Clermont, CA, USA) gel detection system.

Capillary isoelectric focusing
Capillaries: Fused silica 0.1 mm I. D., 350 mm length, 200 mm to the detection window.
High-voltage supply: (-) 20kV.
Detection: on-column UV-Vis detector (λ = 280 nm)
Electrolyte solution: 3% (v/v) ethylalcohol, 0.3 %(w/v) PEG 10000 dissolved in 40 mM NaOH (catholyte) and 100 mM H3PO4 (anolyte).

Fig. 7: Capillary isoelectric focusing
 

Results

We isolated 110 yeasts of Candida genus in 308 dental plaque samples from children affected by ECC and from caries-free children. 101 isolates grew as green colonies on chromogennic medium in CHROMagar Candida and formed pseudomycelium and chlamydospores on rice agar. These strains were considered C. albicans/dubliniensis. Phenotypic methods (chlamydospore arrangement, characteristic growth on CHROMagar Candida and Staib Agar, growth at 45° C and latex agglutination) were used for the differentiation between C. dubliniensis and C. albicans.

n = 101 CHROMagar Growth at 45°C Staib agar Chlamydospores
0 +/− + 0 +/− + 0 +/− + 0 +/− +
Agglutination 0 76 7 2 65 14 6 61 24 0 74 11 0
+ 3 4 9 0 2 14 0 0 16 0 4 12
Table 1: Phenotypic methods
 

Identification of C. dubliniensis was confirmed by means of genotypic method (PCR-RFLP) (16).
Simultaneously, the group of 31 C. albicans strains was examined using this method.
Control strains C. albicans and C. dubliniensis were also included.

n = 49 Agglutination
+ 0
PCR-RFLP + 17 0
0 0 32
Table 2: Genotypic method
 

Capillary isoelectric focusing C. albicans and C. dubliniensis, pH gradient 2.0 - 4.7
The difference isoelectric points of C. dubliniensis (12) and C. albicans (16) strains was determined by means of Capillary Isoelectric Focusing (CIEF). All the C. dubliniensis strains focused near pI value 2.8, while the pI values of C. albicans strains were near 2.6.

Fig. 8: Capillary isoelectric focusing
 

Conclusions

All the C. dubliniensis isolates originated from the dental plaque of ECC affected children. The reliability of simple phenotypic methods used for the differentiation between C. dubliniensis and C. albicans is limited. Therefore, these methods are suitable only for screening clinical strains. PCR-RFLP and latex agglutination are more reliable in comparison with the above mentioned method, but they are more expensive. Therefore they are suitable for the confirmation of results.

We have found that pI of C. dubliniensis differs from pI of C. albicans. Therefore CIEF is a suitable method for the differentiation between these species and isoelectric point is a useful criterion for the differentiation between these two similar species.

Acknowledgement
This work was supported by the Grant Agency AV CR, No. AX00310701 and by Project 1M0528.
 

Literature

  1. Sullivan D, Coleman D. Candida dubliniensis: characteristics and identification: J Clin Microbiol 1998, 36, pp. 329-34.
  2. Moran GP, Sullivan DJ, Henman MC, McCreary CE, Harrington BJ, Shanley DB, Coleman DC. Antifungal drug susceptibilities of oral Candida dubliniensis isolates from human immunodeficiency virus (HIV)-infected and non-HIV-infected subjects and generation of stable fluconazole-resistant derivatives in vitro: Antimicrob Agents Chemother 1997, 41, pp. 617-23.
  3. Jabra-Rizk MA, Brenner TM, Romagnoli M, Baqui AA, Merz WG, Falkler WA Jr, Meiller TF. Evaluation of a reformulated CHROMagar Kandida: J Clin Microbiol 2001, 39, pp. 2015-6.
  4. Sahand IH, Moragues MD, Robert R, Quindos G, Ponton J. Evaluation of Bichro-Dubli Fumouze to distinguish Candida dubliniensis from Candida albicans: Diagn Microbiol Infect Dis 2006, 55, pp. 165-7.
  5. Staib P, Morschhäuser J. Chlamydospore formation on Staib agar as a species-specific characteristic of Candida dubliniensis: Mycoses 1999, 42, pp. 521-4.
  6. Mähnss B, Stehr F, Schäfer W, Neuber K.Comparison of standard phenotypic assays with a PCR method to discriminate Candida albicans and C. dubliniensis: Mycoses 2005, 48, pp.55-61.
     

This Poster was submitted by Dr. Jarmila Kuklová.
 

Correspondence address:
MUDr. Jarmila Kuklová
Faculty of Medicine, Masaryk University and St. Ann's Hospital
Stomatological Clinic
Pekarská 53
656 91 Brno
Czech Republic
00420 543183430