Construction of a versatile in vitro cultivation screening platform using human oral microbiota

Abstract

We developed a simulation model of human oral microbiota using Bio Palette oral medium (BPOM) containing 0.02% glucose and lower bacterial nitrogen sources, derived from saliva and dental plaque. By decreasing the concentration of Gifu anaerobic medium (GAM) from 30 to 10 g L^-1 , we observed increased ratios of target pathogenic genera, Porphyromonas and Fusobacterium from 0.5% and 1.7% to 1.2% and 3.5%, respectively, in the biofilm on hydroxyapatite (HA) discs. BPOM exhibited the higher ratios of Porphyromonas and Fusobacterium, and amplicon sequence variant number on HA, compared with GAM, modified GAM and basal medium mucin. Mixing glycerol stocks of BPOM culture solutions from four human subjects resulted in comparable ratios of these bacteria to the original saliva. In this simulation model, sitafloxacin showed higher inhibitory effects on P. gingivalis than minocycline hydrochloride at a low dosage of 0.1 μg mL^-1 . Probiotics such as Streptococcus salivarius and Limosilactobacillus fermentum also showed significant decreases in Porphyromonas and Fusobacterium ratios on HA, respectively. Overall, the study suggests that BPOM with low carbon and nutrients could be a versatile platform for assessing the efficacy of antibiotics and live biotherapeutics in treating oral diseases caused by Porphyromonas and Fusobacterium.

FIGURE 1

Visual overview of an in vitro human oral microbiota simulation model. Each vessel set up hydroxyapatite discs in it and contained an agitated autoclaved medium, kept at 37°C and purged with a gas mixture.

FIGURE 2

16S rRNA sequencing was employed to investigate bacterial shifts at the genus level in saliva and dental plaque (origin: healthy volunteer) cultured in liquid media and biofilms grown on hydroxyapatite discs. The research utilized four media types: Gifu anaerobic medium (GAM), modified GAM (MGAM), Basal medium mucin (BMM)‐based media and Bio Palette oral medium (BPOM). Genera with lower abundance (<1.0%) and lower similarity (<97%) were included as ‘Others’ and ‘Unclassified Bacteria,’ respectively. Porphyromonas and Fusobacterium were identified using blue and red arrows, respectively. The amplicon sequence variant number and Shannon index of each sample are also reported. These findings shed light on microbial diversity and community structure in different media types and provided insights for further research.

FIGURE 3

(A) The amplicon sequence variant (ASV) number, representing the total number of unique variants detected in the samples, was determined. (B) The Shannon index, which measures the diversity of bacterial communities, was calculated. Higher values indicate greater diversity. (C) The genus‐level distributions of sequences based on 16S rRNA genes were analysed in inoculums derived from saliva and dental plaque (V1, V2, V3 and P1), as well as in corresponding culture solutions (V1, V2, V3, P1 and Mix 1 and 2). Genera with an abundance below 1.0% and similarity below 97% were classified as ‘Others’ and ‘Unclassified Bacteria,’ respectively. Porphyromonas and Fusobacterium highlighted using blue and red arrows, respectively, were particularly interesting due to their known roles in oral health and disease.

FIGURE 4

(A) The amounts of Porphyromonas gingivalis on hydroxyapatite (HA) discs were measured using Mix‐1: Control (no antibiotics administered), minocycline hydrochloride at 0.1 and 1.0 μg mL⁻¹ and sitafloxacin at 0.1 and 1.0 μg mL⁻¹. Statistical differences between each group and the Control were calculated using Student's t test, and significance was defined as p < 0.01 (denoted by double asterisks). (B) Box‐and‐whisker plots were used to display the effects of isolated Streptococcus salivarius and Limosilactobacillus fermentum administration (or no administration, Control) on the levels of Porphyromonas and Fusobacterium. The inoculums were collected from three patients and three volunteers. Paired t‐tests were conducted to determine statistical differences between each group and the Control, and significance was defined as p < 0.05 (denoted by single asterisk).