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Title: In vitro modelling of mixed species oral biofilms using a matrix perfusion system and analysis of community biogenesis of volatile compounds
Author: Taylor, Benjamin John
Awarding Body: University of the West of England, Bristol
Current Institution: University of the West of England, Bristol
Date of Award: 2012
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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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available