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Oral malodour (Halitosis) is a socially excluding condition which can result in low self esteem and continual worry. Oral malodour results from the putrefactive action of mainly Gram- negative anaerobic bacteria resident on the tongue dorsum biofilm degrading proteinacious substrates from food debris, shedded epithelial cells and stagnant saliva. The gasses suspected in contributing the majority to oral malodour are the volatile sulphur compounds (VSC), hydrogen sulphide (H2S) and methylmercaptan; with H2S forming the majority of the bouquet of oral malodour. Sorbarod tongue biofilms were prepared by inoculating a tongue dorsum scrape sample onto a sorbarod cellulose matrix which were perfused with 1/5th strength brain heart infusion media (plus supplements) until biofilm growth rates reached the quasi-steady state. A portable sulphide gas chromatograph was calibrated using diluted 2000 ppb H2S and used to calculate generation of H2S from the biofilm by using a gas syringe dilution method. Initial model development experiments were conducted to validate the sorbarod perfusion system. Sorbarod biofilms reached quasi-steady state growth rate and H2S generation rate after 48 h of media perfusion. Comparisons between air and anaerobic gas (10% CO2 in N2) perfused biofilms showed a significant difference (p<0.05, unpaired t-test) in the recovery of facultative/strict anaerobes, H2S producing species from eluate samples and generation of H2S in gas phase. No significant difference (p>0.05, unpaired t-test) was seen in the recovery of Fusobacteria/Veillonella species in eluate recovery, or between any group in biofilm recovery. A perfusion media flow rate experiment on three sorbarod biofilms matured to steady state at a 20 ml/h flow rate and then perfused at 10, 40 or 100 ml/h for 48 h. H2S analysis conducted at 0, 24 and 48 h showed an increase in H2S generation as flow rate increased from 20 ml/h to 40 and 100 ml/h and a H2S decrease as flow rate decreased to 10 ml/h. Extrapolation of gas phase flow rates to 'average' breathing rates (6 Llmin) revealed that in vitro biofilm H2S concentration was similar to the oral cavity concentration found in the volunteer donating the sample. Pulsing in vitro tongue biofilms with metronidazole (25 mg/ml) significantly reduced recovery of strict an aerobes in eluate (p=0.0084) and reduced the generation of H2S. Pulsinq with vancomycin (20 mg/ml) significantly reduced facultative anaerobic recovery in eluate (p=0.0051). Metronidazole and vancomycin showed no significant reduction (p>0.05) in strict and facultative anaerobes (respectively) in the total biofilm. Sorbarod biofilms developed over a pH range (pH 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0) revealed the greatest yields of facultative and strict an aerobes were supported between pH 6.5 and 7.0 with greater proportions of Gram- negative organisms supported between pH 7.0-8.0 and greater proportions of Gram-positive organisms supported between pH 5.5-6.0. In addition, community level physiological profiling also demonstrated the greatest metabolic diversity at pH 6.5 and 7.0 (H=1.99 and 1.98 respectively). Qualitative and quantitative 16S rRNA analysis by the Human Oral Microbe Identification Microarray (HOMIM) revealed the majority of suspected malodour producing organisms was supported between pH 6.5 and 8.0. Analysis of H2S by Selected Ion Flow Tube Mass Spectrometry revealed biofilms supported at pH 7.5 generated the greatest concentrations of VSC. In addition, the specific activity for H2S generation also calculated that strict anaerobes supported at pH 7.5 generated the greatest amounts of H2S. The sorbarod perfusion system demonstrated the ability to support quasi-steady state in vitro tongue biofilms which responded to challenges in a similar way to findings conducted in vivo and was able to identify some of the effects of pH on microbial yields, metabolic diversity and generation of malodorous compounds.

The knowledge of the mechanisms giving rise to halitosis in the mouth is quite limited despite the social constraints the condition imposes and the attendant commercial interest that results. In part, this understanding is limited by the difficulties inherent in undertaking in vivo studies which are further exacerbated by the complexity of the systems under investigation, in particular, the influence of transport mechanisms such as salivary flow, pH, temperature and others had not been investigated. The research described here is based on a new approach to investigate the transport mechanisms contributing to oral malodour emanating from the tongu, dorsum as the main source of volatile compounds in the mouth (Yaegaki and Sanada 1992, Rosenberg and Leib 1995, De Boever and Loesche 1996).

Introduction : Les déterminants génétiques du statut en folate (MTHFR, MTR, NOS3, SHMT1 et 2, MTHFD1 et 2, MTHFS, SLC19A1, ABCB1, FOLR2 et FOLH1) sont variablement associés au risque primaire de fentes chez les sujets hispaniques, irlandais, norvégiens et polonais. Ce risque est associé aux déterminants génétiques de la vitamine B12 (TCN2) en Italie et au métabolisme de la choline (BHMT, BHMT2 et PCYT1A) en Pologne. Des polymorphismes génétiques du gène CBS, une enzyme majeure de la voie de la transulfuration, y sont associés en Italie. But de l'étude : Nous avons étudié les déterminants génétiques des fentes labio-maxillo-palatines en France en lien avec le métabolisme des monocarbones par SNP Array. Méthodes : Notre étude cas-témoins porte sur 250 enfants, leurs mères (200), et 521 contrôles appareillés. Une analyse multiplex de 384 SNPs (77 gènes sélectionnés dans la base NCBI et à partir d'études in silico), a été réalisée par technologie Golden Gate Illumina (BeadExpress Reader, San Diego, California). Toutes les analyses ont été réalisées en modèles additifs, dominants et récessifs. Résultats : En analyse univariée chez les mères en modèle additif, rs2124459 CBS, rs35789560 FPGS, rs234709 CBS, rs4816 PCMT1, rs737865 COMT, rs4820889 TCN2, rs13043752 AHCY, rs2168781 SHMT1 et rs1801229 CUBN sont associés au risque primaire de fentes. Après correction de Bonferroni, seul CBS rs2124459 1552+1199A-->G atteint la significativité dans le modèle additif avec P= 0,009 (OR (Dd) versus (dd) = 0,46 (0,26 to 0,81) et (DD) versus (Dd) = 0,51 (0,26 to 0,97)). En modèle dominant le P= 0,09 (OR (DD, Dd) versus (dd) = 0,40 (0,23 to 0,69). Chez les enfants, CBS rs2124459 est associé au risque de fentes dans les modèles additif et dominant, avec des P=0.026 et P=0.011respectivement. Discussion et conclusion: CBS est très fortement associée au risque primaire de fentes chez les mères et les enfants. Ces résultats montrent une mise en jeu prépondérante de la voie de la transulfuration dans l'étiologie des fentes en France / Background : Monocarbons determinants are involved in the etiology of cleft lip with or without cleft palate, based on ethnical background and periconceptional mother's diet. Genetic polymorphisms of actors of folate and monocarbon availability (MTHFR, MTR, NOS3, SHMT1 and 2, MTHFD1 and 2, MTHFS, SLC19A1, ABCB1, FOLR2 and FOLH1) are in association with the primary risk of oro-facial cleft in contrasting ways in Hispanic, non-Hispanic-white, Irish, Norwegian or Polish subjects. The actors of B12 availability like TCN2 are only involved in Italian subjects. Cholin metabolism (BHMT, BHMT2 and PCYT1A) is reported in Polan. CBS, actor of transulfuration pathway?s, is in association with cleft risk in Italian studies. Aim : This study is built to determine the primary risk of cleft (OCF) in association with single nucleotid polymorphisms of the actors of folate, B12 and choline availability, monocarbons metabolism and transulfuration pathway?s in France. Methods : We performed a case-control study of 250 children and their mothers (200), and 521 matches controls. A multiplex analysis of 384 SNPs (77 genes), selected by NCBI databases research's and in silico studies was performed, using GoldenGate Illumina technology (BeadExpress Reader, San Diego, California) with appropriate genotyping controls. Genotypes with call rate under 0.95 and SNPs with Hardy-Weinberg disequilibrium linkage were excluded. SNP association analyses were performed applying additive, dominant and recessive models to the data. Results :In univariate analysis of mothers versus controls for additive model 9 SNPs were found in association with primary risk of cleft, concerning rs2124459 CBS ; rs35789560 FPGS ; rs234709 CBS ; rs4816 PCMT1 ; rs737865 COMT ; rs4820889 TCN2 ;rs13043752 AHCY ; rs2168781 SHMT1 ; rs1801229 CUBN. After multiple testing correction, only CBS rs2124459 1552+1199A-->G reached statistical significance in additive models, with P= 0.009 in the additive model (OR (Dd) versus (dd) = 0.46 (0.26 to 0.81) and (DD) versus (Dd) = 0.51(0.26 to 0.97)). P= 0.09 in the dominant model (OR (DD, Dd) versus (dd) = 0.40 (0.23 to 0.69), respectively. In patients, CBS rs2124459 was significantly associated with a reduced risk of OFC in both additive and dominant models, with respectively P=0.026 and P=0.011. Discussion and conclusion : This study reported an intronic SNP from CBS not previously described, with a strong association with primary risk of cleft in mothers and child. These results argues to the predominant way of transulfuration in etiology of oro-facial cleft in France