Effects of Arginine on Microbial Ecological Shifts in Oral Microcosms.

Introduction: The oral cavity is a complex ecosystem, and its highly diverse microbial community comprises bacteria, Archaea, fungi, viruses, and protozoa. In a healthy host, there is a balance between the microbes and the host itself. Perturbations such as frequent exposure to fermentable carbohydrates, may disturb this balance, resulting in an ecological shift towards a diseased state. Potential prebiotics such as the amino acid arginine, may have a beneficial effect by increasing the environmental pH, thus inhibiting microbial shifts. The aim of the present study was to assess the effects of an arginine supplement on oral microcosms under highly cariogenic growth conditions. Methods: The eight-station multi-plaque artificial mouth (MAM) model was inoculated with plaque-enriched saliva from a healthy volunteer. Four of the stations received defined mucin medium (DMM) supplemented with arginine (1.6% w/v) (DMM+ARG). The remaining four stations were exposed to DMM without arginine (DMM-ARG). To mimic cariogenic challenges, all stations received eight 6 min sucrose (10% w/v) pulses a day at 2 h intervals. Each day contained a 'resting period' of medium alone (DMM+ARG or DMM-ARG) lasting 10 h. Organic acid anions (formate, succinate, acetate, lactate, propionate, and butyrate) in resting and fermenting biofilms were measured using capillary ion electrophoresis. The ammonium concentration in the biofilms was measured using an enzymatic ammonium assay. The biofilms were sampled at 14 time points (t) divided over 22 days, including eight time points where the biofilms were 'at rest', four time points following a 6 min sucrose pulse, and two time points following an arginine (8% w/v) pulse. Extracted DNA from the biofilms was used to assess Candida abundance through RT-PCR and to obtain a bacterial ecological profile through denaturing gradient gel electrophoresis (DGGE). Results: Biofilms exposed to arginine had a higher ammonium concentration compared to those not receiving additional arginine (2.96±0.45 and 2.04±0.44 mM/ml). The acid profiles showed that the DMM-ARG biofilms had produced significantly more butyrate and acetate (0.58±0.55 and 1.06±1.02 mmol/mg protein) than the DMM+ARG biofilms (0.17±014 and 0.60±0.33 mmol/mg protein), irrespective of the metabolic state. Candida abundance increased with time and was generally higher in the DMM-ARG biofilms, (t1: DMM+ARG 2.1x10³ CFU/ml, DMM-ARG 6.3x10³ CFU/ ml; t14: DMM+ARG 2.0x104 CFU/ml, DMM-ARG 2.7x105 CFU/ml). Additionally, DGGE of the DMM-ARG and DMM+ARG biofilms resulted in clearly distinguishable profiles for both conditions. Discussion: Arginine reduced the acid challenge induced by sucrose, inhibiting outgrowth of Candida and preventing a shift in microbial composition towards a more aciduric and acidogenic community. The actual compositional changes will be investigated using next generation sequencing approaches in the near future. Based on these preliminary findings, arginine has a strong potential in stabilizing the oral ecosystem during cariogenic perturbations. [ABSTRACT FROM AUTHOR]