Discussion
Due to different nutritional requirements, factors produced by other microbes, strict inter-species interactions, slow growth, competition/inhibition and dormancy in diversified bacterial species ( Overmann, Abt & Sikorski, 2017 ; Tidjani Alou et al., 2020 ), developing a medium for cultivating microbiota, especially oral microbial flora, has always been a challenge. In previous studies, SHI medium has been widely used for oral bacteria research, but it was originally designed to support different sub-populations within the original oral microbial community derived from pooled saliva ( Edlund et al., 2013 ; Tian et al., 2010 ). Specific nutrient or chemical requirements may be necessary for the growth of some fastidious bacteria. Some studies regarding functional alteration associations with S-ECC indicated that carbohydrate metabolism, amino acid metabolism, and metabolism of cofactors and vitamins are the major pathways of KEGG in caries and healthy groups ( Tang et al., 2022 ; Wang et al., 2019 ). Thus, we designed a novel bacterial medium attempting to culture the fastidious bacteria, which were nutritionally demanding and were difficult to grow on ordinary medium, especially for dental plaque in a diseased state of S-ECC.

We designed NEMM medium based on S-ECC bacterial metabolism and the role of nutrients in bacterial culture and proliferation. Bacteria must obtain nutrients from their surrounding environment to survive. Carbohydrates are the primary energy and carbon sources for most bacteria during growth in laboratory media. In bacteria, proteins in the outer membrane surrounding the cell actively transport carbohydrates and trace nutrients like iron into the cell’s interior ( Zinke et al., 2024 ). In this study, NEMM meets the needs of previous studies’ findings that multiple pathways involving carbohydrate metabolisms are obviously up-regulated in the S-ECC group compared to caries free group ( Tang et al., 2022 ; Wang et al., 2019 ). It should be emphasized that the upregulation of carbohydrate transport and metabolism in NEMM might reflect more bacteria with higher cariogenic potential.

In the metabolism of S-ECC, several pathways involving carbohydrate metabolism were deferentially regulated ( Tang et al., 2022 ; Wang et al., 2019 ). Dextrose and corn starch served as carbon sources. β -Nicotinamide adenine dinucleotide (NAD), thiamin pyrophosphate (TPP) and L-glutamine play key roles in glycolysis. As a cofactor in over 300 redox reactions, NAD (NAD(+)) and its reduced form NADH (reduced nicotinamide-adenine dinucleotide) play a critical role in cellular metabolism due to their function ( Zhou et al., 2011 ). TPP, an active form of thiamine (Vitamin B1), is an essential molecule for all living organisms ( Goyer, 2017 ). TPP acts as a co-factor for key enzymes of glycolysis such as pyruvate dehydrogenase, transketolase, and pyruvate decarboxylase ( Llavero-Pasquina et al., 2022 ; Strobbe & Van Der Straeten, 2018 ). Many bacteria rely on the TPP to lose the ability to produce thiamine de novo ( Llavero-Pasquina et al., 2022 ).

Cofactors and vitamins are vital for bacteria growth and function as well. Glutamine comprises more than 50% of the body’s free amino acid pool and is a precursor for the synthesis of nucleic acids and glutathione ( Heys & Ashkanani, 1999 ). Glutamine might initiate the signaling pathways related to the metabolism of nitrogenous compounds in bacteria ( Dai et al., 2013 ). A study demonstrated that L-glutamine supplements in basal salt medium with carbon and nitrogen sources along with requisite inoculum size yielded the best quantities ( Patel et al., 2021 ). Cobalamin (vitamin B12) has regulatory involvement in gene expression, microbial folate, ubiquinone, and methionine processes ( Romine et al., 2017 ; Rosnow et al., 2018 ), suggesting a requirement for this co-factor. Cobalamin was demonstrated to be the essential nutrilite ( Lochhead & Thexton, 1951 ) because of the lack of the ability to biosynthesize cobalamin for most bacteria ( Lu et al., 2020 ). Recent research reported that Porphyromonas gingivalis growth relies on vitamin B12 ( Saiki et al., 2022 ).

Adenine and guanine have various functions in bacteria. Kiyoshi and colleagues added adenine to culture Clostridium saccharoperbutylacetonicum strain N1-4 at 37 °C. The addition of adenine exhibited the maintenance of cell viability originating from the maintenance of ATP levels ( Kiyoshi et al., 2017 ). Torino et al. (2005) compared the growth and extracellular polysaccharides(EPS) production by Lactobacillus helveticus ATCC 15807 in the different media, and found that the addition of p-amino benzoic acid did not affect either growth or the EPS production, while guanine displayed a dual effect, a decrease in cell growth and an increase in EPS formation.

To date, although nearly 1/4 of bacteria are still uncultured in the oral cavity, researchers have known the existence of these uncultured bacteria through next-generation sequencing, third-generation sequencing or metagenomics technologies. However, some species were likely present at a low abundance in the inoculum and were therefore not detected via sequencing. Cultivating these “dark matter” in S-ECC plaque will help to explore their contributions to health and their role in disease etiology, and pave the way for prevention of dental caries. In this study, we combined 16S rRNA gene sequencing and culturomics to prove that NEMM can support the development of more bacteria from S-ECC dental plaque and help to cultivate uncultured or targeted bacteria.

For example, Veillonella , which belongs to Gram-negative obligate anaerobic coccus, is recognized to play an important role in the S-ECC-causing process and identified as a candidate biomarker in S-ECC with low abundance or without S. mutans ( Gross et al., 2012 ; Zhang et al., 2022 ). The potential biomarkers of different groups were presented by LEfSe. Among these significantly different genera, Veillonella was one of the most specific biomarkers in aNEMM group. Moreover, we exhibited the changing process of bacterial community structure through 16S rRNA gene sequencing and culturomics. It indicated a better capture ability for Veillonella before the first day in aNEMM group. Growth development and bacterial activity are important aspects that need to be addressed. It will be helpful for isolating targeted strains at proper time points. The relative abundance of the bacterial genus shifted in biofilms over 28 days of growth in NEMM and SHI medium. It is likely that inhibition of metabolism, due to low pH and possibly carbohydrate limitation, starts to impact growth of some community members and leads to a shift in the community structure. Vagococcus , which is uncommon in the oral bacteria community, was found enriched in NEMM and SHI medium. Actually, there has been some articles that reported their existence in the oral cavity such as Vagococcus fluvialis ( Al-Ahmad et al., 2008 ). We can rule out the possibility of medium contamination from Fig. S7 . Therefore, we speculate some of them may come from the food eaten such as milk or food additives or non-food substances such as soil and toys contaminated by bacteria entering the children’s mouths through accidental events ( Wullschleger et al., 2018 ).