Ethanol pretreatment and bile acid germinants help recover significant number of spore forming gut bacteria previously unculturable

Microbes are the dominant forms of life on Earth both in numbers (6 x 1030) and species. By mediating diverse biogeochemical processes ranging from photosynthesis to leaching of metals from minerals and methane biogenesis, they are essential to the proper functioning of the planet. Nevertheless, most of the microorganisms could not be cultivated in the laboratory using standard procedures such as growth on solid agar medium; thereby, resulting in only about 1% of all known microbes being recovered as pure culture on agar plates. Observations of the same environmental sample such as a water droplet under the optical microscope reveals significantly more microbes than those that could be cultured.


Known as the “Great Plate Count Anomaly” where the numbers and types of microbes detected (for example, through visual observation) significantly outnumbers those that could be cultured on the agar plate, many approaches, both culture based and culture-independent, have been proposed to lift the veil on the microbial dark matter. For example, one of the main culture-independent methodologies, 16S rRNA sequencing, was made more readily accessible through the provision of lower cost DNA sequencing machines as well as refinement of the metagenomics workflows and data analysis pipeline. But, culture based methods remain essential to understanding physiology and biochemical processes at the whole cell level.


In metagenomics, DNA from microbes present in an environmental sample such as seawater would be extracted and subjected to DNA sequencing through next-generation pyrosequencing methods where short reads of long strand DNA are sequenced, and subsequently, reconstructed into individual “genomes” through bioinformatics approaches. Hence, by comparing differences between the assembled genomes, it is possible to define the phylogeny between species and strains as well as quantify the number of individual microbial species present. But, these approaches suffer from possible bias in sequencing and the genome assembly process. Most fundamentally, while DNA sequencing or metagenomics offer a glimpse of the types of microorganisms present, they could not tell the physiological or metabolic characteristics of individual microbe in culture given that not all genes are expressed and the possibility of environment factors mediated modulation of gene expression (i.e., epigenetics).


Hence, the alternative thrust to identify microbes based on culture techniques remain requisite in the workflows of most microbial ecology studies. Through augmenting the growth medium with nutrients and vitamins as well as cofactors specific to environment from which the samples were obtained, microbiologists attempt to recreate the environmental cues important for prompting microbes in “hibernation” (e.g., microbes in stringent response or which are persisters) into vegetative growth. Another approach similar in concept but which differs in how the environmental cues are introduced to growth medium uses a semi-permeable membrane to separate microorganisms important to growth of another species. In this case, microbe A secretes cofactors or metabolic exchange factors that diffuses across the membrane into the chamber where microbe B reside. With the metabolic cues from the cofactors on hand, microbe B would be brought out of “hibernation” into vegetative growth; thereby, allowing it to be detected on solid medium.


Using a similar approach to solve the problem of inability to culture spore forming microbes known to exist in the gut microbiota metagenome, Lawley and coworkers in “Culturing of “unculturable” human microbiota reveals novel taxa and extensive sporulation”1 Link) managed to coax the growth of many spore forming anaerobic bacteria on a single medium, where without exertion of selection pressure, would be dominated by diverse species of Proteobacteria.


With YCFA as sole medium for cultivating gut microbes, more than 8 x 106 colonies would need to be individually isolated for species detection via biochemical assays or 16S rRNA sequencing. More importantly, microbes such as those of Proteobacteria that could be cultured relatively easily would dominate the types of microbes isolated on YCFA given that they have an advantage in nutrient utilization and growth over the microaerophiles and anaerobes as well as spore forming microbes, which are sensitive to oxygen and suffer from competitive disadvantage relative to Proteobacteria.


Thus, to probe microorganisms less competitive for nutrients in the gut microenvironment and which grows slower, specific selection pressure that prevents the growth of more easily culturable microbes from Proteobacteria need to be applied. With the objective of profiling spore forming microorganisms resistant to the common disinfectant ethanol, two selection pressures were applied in sequence by the authors to narrow down the subset of microorganisms culturable on YCFA growth medium. Note that YCFA growth medium is unable to allow the growth of all microorganisms present in the gut microbiota as is the case for all growth media. Its selection for use in the experiments is likely potentiated by its ability to allow the growth of many species of microbes.


Application of ethanol to the fecal materials from donors removed substantial numbers of ethanol sensitive microbes and provided room for the growth of less competitive microbes previously prevented from growing on YCFA agar due to poor access to nutrients. After the ethanol pretreatment step, the authors answered their hypothesized question on whether bile acids would serve as germinants for the growth of spore forming microbes such as the intestinal pathogenic bacterial species, Clostridium difficile, by testing the germination properties of three bile acids: taurocholate, glycocholate, and cholate.


Experiments revealed that taurocholate increased the cultivability of spore forming members of the commensal gut bacteria by between 8 and 70000 fold, while having no effect on the culture of non spore forming bacteria. In a similar vein, other bile acids have varying effect on improving the culturability of spore forming bacteria. Thus, bile acids, while a stimulant from the perspective of promoting the growth of spore forming microbes, also serve as a selection pressure against other non spore forming bacteria.


Metagenomic information highlighted 60% of the genera in the profiled gut microbiota comprised spore forming bacteria that potentially constituted about 30% of the total intestinal microbiota. Hence, without the help of selection pressure, principally ethanol pretreatment, most of the gut microbiota would not yield to culture, and thus, constitute as microbial dark matter, which prevent a more holistic understanding of the dynamics of how different species of gut microbes (spore forming or not) interact in the complex web of metabolite and energy flows that, overall, play critical roles in health and disease of the gut.


Overall, by illuminating a method through which difficult to culture spore forming bacteria could be cultivated on agar, and their physiology and phenotype examined more closely using a variety of biochemical and cell biological tools, more understanding of how spore forming bacteria contribute to ecosystem health in the gut as well as their roles in energy metabolism and dysbiosis could be deciphered.


Specifically, one intriguing question revolves on what roles spores play in digestion and energy flow in the gut since being dormant, they essentially do not participate in nutrient absorption and its conversion into metabolites suited both for gut absorption and utilization by other members of the gut microbiome. Additionally, without metabolic processes in action, wouldn’t spore forming bacteria be driven out of the gut microbiota as they could not reproduce in numbers comparable to those of Proteobacteria and Firmicutes? If the hypothesis is plausible, what are the factors that led to their continued and substantial presence in the gut microbiota?


Interested readers can download a pdf copy of this commentary from figshare:



  1. Browne, H. P. et al. Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation. Nature 533, 543–546 (2016).


Category: microbiology, biochemistry, genetics,

Tags: intestinal microbiome, energy metabolism, spore formers, selection pressure, metagenomics, 16S rRNA sequencing, next generation sequencing, viable but not cultured, Great plate count anomaly, microbial dark matter,


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