Adapted protocol for Saccharibacteria co-cultivation: two new members join the club of Candidate Phyla radiation

The growing application of metagenomics to different ecological and microbiome niches in recent years has enhanced our knowledge of global microbial biodiversity. Among these abundant and widespread microbes, Candidate Phyla Radiation or CPR have been recognised as representing a large proportion of the microbial kingdom (> 26%). CPR are characterised by their obligate symbiotic or exo-parasitic activity with other microbial hosts, mainly bacteria. Currently, isolating CPR is still considered challenging for microbiologists. The idea of this study was to develop an adapted protocol for the co-culture of CPR with a suitable bacterial host. Based on various sputa, we tried to purify CPR (Saccharibacteria members) and to cultivate them with pure hosts. This protocol was monitored by real-time PCR quantification using a specific system for Saccharibacteria designed in this study, as well as by electron microscopy and sequencing. We succeeded in co-culturing and sequencing a complete genome of two new Saccharibacteria species: Candidatus Minimicrobia naudis and Candidatus Minimicrobia vallesae. In addition, we noticed a decrease in the Ct number of Saccharibacteria, and a significant multiplication through their physical association with Schaalia odontolytica strains in the enriched medium that we developed. This work may help bridge gaps in the genomic database by providing new CPR members and, in the future, their currently unknown characteristics may be revealed. IMPORTANCEIn this study, the first real-time PCR system has been developed. This technique is able to quantify specifically Saccharibacteria members in any sample of interest in order to investigate their prevalence. In addition, another easy, specific and sensitive protocol has been developed to maintain the viability of Saccharibacteria cells in an enriched medium with their bacterial host. The use of this protocol subsequently facilitates studying the phenotypic characteristics of CPR and their physical interactions with bacterial species, as well as the sequencing of new genomes to improve the current database.