Modelling the effects of pH on tongue biofilm using a sorbarod biofilm perfusion system

Abstract

The pH of the tongue biofilm is likely to influence microbial composition and ecology with consequent effects on the metabolic activities and generation of volatile sulfur compounds (VSC) and other malodour gasses. The aim of this study was to identify the effects of pH on the development of biofilms and hydrogen sulfide production using an in vitro tongue-derived biofilm model. Community level physiological profiling (CLPP) was employed to examine the influence of pH on the collective metabolic fingerprint of each tongue-derived biofilm. A sorbarod perfusion system (n = 6 sorbarods) was inoculated from a single suspension of tongue scrape sample and mixed community tongue-derived biofilms were grown at pH 5.5, 6.0, 6.5, 7.0 7.5 and 8.0. Biofilms were perfused with medium for 120 h and gas phase samples (n = 4 per biofilm) removed and analysed with a portable sulfide gas chromatograph before being sacrificed into 10 ml sterile PBS-diluent and cells suspended by vortex mixing. Further ten-fold dilutions were made (down to 10−7) and dilutions plated out onto selective (fastidious anaerobic agar (FAA) + 0.0025% vancomycin) and non-selective (FAA) media for enumeration of strict and facultative anaerobes respectively. Biofilm suspensions were also mixed with Biolog inoculation fluid and distributed into 96 wells of Biolog AN plates for CLPP. Tongue biofilms developed at pH 7.5 produced significant (p < 0.05) concentrations of H2S (≈52.2 ± SEM 5.6 µg H2S per ml biofilm gas phase) followed by tongue biofilm developed at pH 7.0 and 8.0 (≈43.2 ± SEM 3.5 and ≈ 39.6 ± SEM 7.3 µg H2S per ml biofilm gas phase respectively). Tongue biofilm developed at pH 6.0 and 6.5 produced approximately 21.5 ± SEM 2.3 and 37.1 ± SEM 1.7 µg H2S per ml biofilm gas phase respectively and tongue biofilm developed at pH 5.5 produced approximately 0.19 ± SEM 0.09 µg H2S per ml biofilm gas phase. Highest numbers of strict and facultative anaerobes were recovered from biofilms at pH 6.5 (1.10 × 1012 and 2.07 × 1012 cfu ml−1 respectively), with a reduced number recovered from pH values above and below this range. CLPP and similarity index revealed biofilms at pH 6.5 and 7.0 most similar (Sj = 78%) and most diverse in terms of metabolic activity. The biofilm at pH 5.5 was the least related to all others and least diverse. The sorbarod perfusion system, in conjunction with H2S analysis and CLPP, enables some of the physiological and ecological effects of pH at a local level within the biofilm on H2S production to be identified.