Development of anaerobic culturing methods to study the evolution and transmission of the human intestinal microbiota

The human gastrointestinal tract is colonised by a diverse range of health-associated bacteria, in addition to other microorganisms, termed the intestinal microbiota. Sequence-based, culture-independent approaches have revolutionised this field of study, however, due to the perception that these bacteria are largely unculturable, in vitro phenotypic analysis has been hindered. In this study, an anaerobic culturing workflow was developed which revealed that the majority of these bacteria can be cultured using one growth medium. In total, 137 characterised and novel bacterial species were isolated and whole-genome sequenced. Inter-host transmission of the intestinal microbiota may represent a means to maintain a diverse assortment of commensal bacteria within individuals, yet it remains a poorly understood process. Some anaerobic pathogens utilise resilient aero-tolerant spores to survive externally and to facilitate transmission to new hosts. To investigate if commensal spore-formers utilise similar mechanisms, a phenotypic screen was incorporated into the culturing workflow to target spore-forming bacteria. This resulted in the isolation of 66 phylogenetically diverse, spore-forming species which, through subsequent phenotypic characterisation are shown to be specialised for host-to-host transmission and intestinal colonisation. Further phylogenetic and evolutionary analysis revealed body-site associated, loss of sporulation has occurred in different taxa. Also, loss of sporulation is associated with features of host-adaptation that are not present in spore-formers such as a smaller genome size and loss of genetic redundancy. This suggests that the human intestinal microbiota is populated by commensal bacteria that have evolved to engage in opposing lifestyles, either orientated towards inter-host dispersal or within-host adaptation. This study demonstrates the intestinal microbiota is not unculturable. In addition, commensal microbial transmission may be more prevalent than once thought as a significant proportion of these bacteria can survive outside of a host through the use of spores that are intrinsically resistant to environmental stresses.