Maatouk, M. | Search bioRxiv

bioRxiv2021

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.

microbiology↗

bioRxiv2021

Small and equipped: the rich repertoire of antibiotic resistance genes in Candidate Phyla Radiation genomes

Microbes belonging to Candidate Phyla Radiation (CPR) have joined the tree of life as a new unique branch, thanks to the intensive application of metagenomics and advances of sequencing technologies. Despite their ultra-small size, reduced genome and metabolic pathways which mainly depend on symbiotic/exo-parasitic relationship with their bacterial host, CPR microbes are abundant and ubiquitous in almost all environments and are consequently survivors in highly competitive circumstances within microbial communities. They have been eventually identified by 16S rRNA analysis and represent more than 26% of microbial diversity. CPR microbes were able to survive in this context, although their defence mechanisms and phenotypic characteristic remain, however, poorly explored. Here, we conducted a thorough in-silico analysis on 4,062 CPR genomes to test whether these ultrasmall microorganisms might encode for antibiotic resistance (AR)-like enzymes. We used an adapted AR screening criteria with an exhaustive consensus database and complementary steps conferring their resistance functions. We conclude by reporting the surprising discovery of rich reservoir of divergent AR-like genes (n= 30,545 HITs, mean=7.5 HITs/genome [0-41] encoding for 89 AR enzymes, distributed across the 13 CPR phyla, and associated with 14 different chemical classes of antimicrobials. However, most HITs found (93.6%) were linked to glycopeptide, beta-lactams, macrolide-lincosamide-streptogramin, tetracycline and aminoglycoside resistance. Moreover, a distinct AR profile was discerned between the microgenomates group and Candidatus Parcubacteria, and between each of them and other CPR phyla. CPR cells seem to be active players during microbial competitive interactions and are well-equipped for the microbial combat in different habitats, supporting their natural survival/persistence and continued existence.

microbiology↗