Using DGGE profiling to develop a novel culture medium suitable for oral microbial communities

Abstract

More than 700 bacterial species have been detected in the human oral cavity. They form highly organized microbial communities and are responsible for many oral infectious diseases, such as dental caries and periodontal disease. The prevention and treatment of these diseases require a comprehensive knowledge of oral microbial communities, which largely relies on culture-dependent methods to provide detailed phenotypic and physiological analysis of these communities. However, most of the currently available laboratory media can only selectively support the growth of a limited number of bacterial species within these communities, and fail to sustain the original oral microbial diversity. In this study, using denaturing gradient gel electrophoresis (DGGE) as an index to systematically survey and analyse the selectivity of commonly used laboratory media, we developed a new medium (SHI medium) by combining the ingredients of several selected media that can support different subpopulations within the original oral microbial community derived from pooled saliva. DGGE and 454 pyrosequencing analysis showed that SHI medium was capable of supporting a more diversified community with a microbial profile closer to that of the original oral microbiota. Furthermore, 454 pyrosequencing revealed that SHI medium supported the growth of many oral species that have not before been cultured. Crystal violet assay and the confocal laser scanning microscope analysis indicated that, compared with other media, SHI medium is able to support a more complex saliva-derived biofilm with higher biomass yield and more diverse species. This DGGE-guided method could also be used to develop novel media for other complex microbial communities.

Figure 1

PCR-DGGE analysis showing the bacterial profiles of saliva-derived overnight planktonic culture in different media. 1, PYG; 2, ASS; 3.columbia; 4, TYGVS; 5, TH; 6, BHI; 7, Original saliva; 8, LB; 9, CYE; 10, Chopped meat medium; 11, Cooked meat medium; 12, BMM; 13, Sheep blood supplemented NAM broth. Three replicates were performed and a representative gel image is shown.

Figure 2

The bacterial profile of original pooled saliva and the microbial profile of saliva-derived overnight planktonic culture in newly developed SHI medium. Three replicates were performed and a representative gel image is shown.

Figure 3

Comparison of original salivary and SHI medium cultivated salivary bacterial species distribution using 454 pyrosequencing. Taxonomic assignments of the sequences were made using the Ribosomal Database Project which classifies sequences to the genus level. The community profile of the dominant members from A) the original pooled saliva and B) the in vitro grown community in SHI medium.

Figure 4

Comparison of dominant sequences falling within the Streptococcus genus for the original saliva and the in vitro SHI medium cultivated community. For this analysis, all Streptococcus 16S rRNA gene sequences of cultured and uncultured strains were downloaded from RDP. Each 454 sequence that was classified as belonging to the Streptococcus genus, was assigned to the best matching Streptococcus RDP sequence. A total of 295 and 296 unique sequences for Streptococcus were observed for the original saliva and in vitro grown sample respectively. Total Streptococcus sequences matching the cultured and uncultured strains for each sample are shown in the inset.

Figure 5

The crystal violet assay to assess overnight biofilm formation of saliva-derived microbes cultivated in BHI, TH or SHI medium. Three replicates were performed for each assay. Average values ± SD are shown. indicates significant difference (P< 0.05) between the two values.

Figure 6

Comparison of saliva-derived biofilms grown in TH or SHI medium visualized by CLSM. Z-stack image of saliva-derived biofilm (live/dead stained) grown in (A) TH medium, and (B) SHI medium. Biovolume of biofilms formed using different media was calculated (C) using the software COMSTAT. Image of five randomly chosen spots were collected for each experimental sample, representative images are shown. Error bar in the calculated biovolume was standard deviation. Scale bar = 50 μm.