A seminal culturing of the “unculturable” intestinal microbiota reveals sporulation influences microbiota spread and persistence in humans. Large-scale genome sequencing, computational modeling, and phylogenetic analysis influenced the culturing framework’s effectiveness.
Humans have unique intestinal microbiota containing about 100-100 species of compositionally diverse bacteria (Qin et al. 2010). Researchers considered intestinal bacteria unculturable because there was a gap in scholarly evidence to support the isolation of intestinal bacteria in the laboratory (Stewart 2012). Many functions and phenotypes of the intestinal bacteria remain unknown (Walker et al. 2014). Ethanol treatment to induce spore formation supports intestinal bacteria culturing through large-scale genome sequencing, computational modeling, and phylogenetic analysis (Browne et al. 2016).
Browne et al. (2016) recruited six healthy human participants for the study. The researchers collected the participants’ fecal samples and utilized an integrated metagenomic sequencing and bacterial culturing technique to identify their resident bacterial communities. They profiled the bacterial species existing in the acquired samples and compared them to those grown on agar plates as distinct colonies. The two samples had a strong positive correlation confirmed by Spearman’s coefficient of 0.75 at the species level. The cultured bacterial community and the original fecal sample shared about 93% of raw reads when sequenced.
Comparing the inclusive catalog derived from 318 individuals’ intestinal microbiota revealed that Browne et al.’s cohort represented 39.4 percent of the genes in the comprehensive database. The researchers detected the presence of 73.5 percent of the 741metagenomic species that were derived computationally in the cultured samples. They realized that a single growth medium could aid in culturing a significant percentage of the fecal microbiota bacteria.
Browne et al. (2016) realized that culturing required matching more than 8×106 unique colonies from YCFA agar plates with the metagenomic sequencing’s sensitivity detection. They established a culturing method that could facilitate novel bacteria isolation and identification from the gastrointestinal tract. The study’s hypothesis was: “Sporulation is an unappreciated basic phenotype of the human intestinal microbiota that may have a profound impact on microbiota spread and persistence in humans” (Browne et al. p. 543).
Browne et al. (2016) used ethanol treatment to select spores from ethanol-sensitive cells and a mixed population of spores. They utilized the mixed culture and metagenomic workflow to process fecal samples. Principal component analysis results showed that ethanol treatment interfered with the composition of culturable bacteria, easing their isolation. The investigators prepared ethanol-treated and non-ethanol-treated conditions and picked approximately 2000 unique bacterial colonies from both conditions. They conducted full-length gene sequencing based on the 16S ribosomal RNA and archived unique taxa for future phenotypic analysis.
Browne et al. (2016) identified 66 ethanol-resistant bacteria species distributed two new candidates and five known families. After sequencing the 234 archived ethanol-sensitive and ethanol-resistant bacteria genomes, the researchers assembled and annotated the outcomes to define genetic pathways supporting germination and sporulation in intestinal microbiota. They provided oxygen exposure to both non-spore-forming and spore-forming bacteria to understand if spore formation facilitates longer environmental survival. The facultative anaerobe and the spore-forming remained viable over the 21-day exposure period. Exposure to disinfectant ethanol and digestive bile acids revealed the dominance of spore-forming bacteria. Spore formers germinated, in response to the digestive bile acids, increasing their culturability. Moreover, the authors confirmed that spore formers comprise 60% of the bacteria in the intestinal microbiota. The findings suggest that spore-forming bacteria such as C.difficile are resilient, remain alive for many days after shedding, and could be highly transmissible in the local environment.
Browne et al. (2016) furthered Lagier et al.’s (2012) and Goodman et al.’s (2011) studies, and provided a framework that unlocked phenotypic characterization of the human intestinal microbiota based on phenotypic culturing. The framework applied large-scale sequencing, computational modeling, and phylogenetic analytics. Future investigations need to utilize standard approaches and commercially available medium with high-performance ratings for intestinal microbiota culturing. The approaches can involve single colony isolation, genome sequencing, and phylogenetics for characterization (Ito et al. 2018).
A 97% similarity sequence defined the 7549 operational taxonomic units (OTUs) generated across samples (Browne et al. 2016). While there were distinct differences between the spore-forming and the other group of non-spore-forming bacteria, additional media was needed to identify the most abundant OTU. Future studies need to report the most abundant OTUs and their distinctive features (Lau et al. 2016).
Institutional laboratories can replicate effective culturing frameworks to provide new knowledge about natural organisms. Researchers need to provide evaluations on the potential scientific returns on culturing investments. Availability of funding can support the identification of new isolation techniques to support the investigation of the many uncultured species (Thrash 2019).
While Browne et al. (2016) revealed the possibilities of intestinal bacteria culturing, future mechanistic studies need to leverage additional species. The approach can increase the evidence-based application of intestinal microbiota in disease and human health studies (Almeida et al. 2016). The interrelations between host and microbes are linked with some degrees of medical importance. The framework developed by Browne et al. (2016) creates opportunities for researchers to classify and understand the critical biological roles of microbial species.
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