Introduction
Diverse microbiomes inhabit the human oral cavity, which are responsible for various infectious diseases, including tooth decay, periodontitis, and endodontic infection ( Bhandary et al., 2024 ). Recent research also linked oral microorganisms with several systemic diseases, including type 2 diabetes ( Sanz et al., 2018 ), Alzheimer’s disease ( Wu et al., 2022 ), and cardiovascular disease ( Sanz et al., 2020 ). Tooth decay is one of the most prevalent oral diseases globally ( Peres et al., 2019 ). Early childhood caries ( Grier et al., 2021 ) is defined as decay of the primary teeth in children younger than 71 months, which affects 23% of children in the United States of America. Force (2021) and more than 60% in China ( Hu, Hong & Li, 2011 ). Early childhood caries (ECC) not only harms the physical and mental health of children but also causes a heavy burden to society as well ( Grier et al., 2021 ; Hemadi et al., 2017 ). The presence of dental caries can lead to serious oral problems and general health, including dental pain, limiting oral functions such as chewing/eating, impaired nutritional status, and sleep disorder. Severe ECC (S-ECC) is an aggressive form of ECC. Based on the definition of S-ECC by the American Academy of Pediatric Dentistry, any sign of smooth-surface caries in a child younger than three years of age, and children aged 3–5 years who have one or more cavitated lesions, caries-caused missing or filled smooth surface in primary anterior teeth or decayed, missing or filled surfaces greater than or equal to four (age of 3), five (age of 4) or six (age of 5) are diagnosed as S-ECC patients ( Anonymous, 2016 ).

The etiology of S-ECC is generally accepted as the pathogenic biofilms established as the consequence of complex, dynamic interactions among microorganisms, diet, and the host. The disease process was modulated through the polymicrobial interaction between pathogenic microorganisms and commensals within the highly structured biofilms ( Bowen et al., 2018 ). The latest research assigns that the microorganisms associated with caries include Streptococcus mutans , Lactobacillus , Actinomyces , Bifidobacterium , and Scardovia species. More than 700 species of microorganisms have been identified in the oral cavity by next-generation sequencing technologies, however, a part of them remian uncultivated ( Aas et al., 2005 ; Dewhirst et al., 2010 ; Paster et al., 2001 ). The 16S rRNA gene is the most commonly used bacterial taxonomy gene in culture-independent techniques to identify caries-associated pathogenic microorganisms ( Pace, Olsen & Woese, 1986 ). However, owing to Koch’s postulates (the ground rules to determine the pathogenic agent of the disease ( Fredricks & Relman, 1996 )), the genetic heterogeneity of bacterial species and the strong influence of the environment on phenotype, species-level identification may not be sufficient ( Wade, 2013 ). Hence, intensive studies of pure cultured fastidious bacteria may provide us with a new understanding of the role of microbiology in S-ECC.

Tian and his colleagues developed the SHI medium to sustain a higher diversity of the oral microbial community and more similar microbial profiles to the original saliva-derived oral microflora ( Tian et al., 2010 ). Using SHI medium, He et al. (2015) revealed the TM7 phylum as “microbial dark matter”, which was recalcitrant to be cultivated, making it one of the most enigmatic phyla known. Furthermore, its potential pathogenic associations with immune suppression ability was found.

Considering that SHI medium only displayed a certain level of selectivity of microorganisms, thus, in this study, we developed a novel nutrient-enriched microbiological medium (NEMM), to cultivate more oral bacteria, especially for uncultivated microorganisms, and make the preparation for further investigating the characterization of the microbiome in S-ECC.