Comandatore, Francesco

Background Candidate Phyla Radiation (CPR) is a large monophyletic group encompassing about 25% of bacterial diversity. Among CPR, “Candidatus Saccharibacteria” is one of the most clinically relevant phyla. Indeed, it is enriched in the oral microbiota of subjects suffering from immune-mediated disorders and it has been found to have immunomodulatory activities. For these reasons, it is crucial to have reliable methods to detect and quantify this bacterial lineage in human samples, including saliva. Methods and results Four qPCR protocols for quantifying “Ca. Saccharibacteria” (one targeting the 23S rRNA gene and three the 16S) were tested and compared. The efficiency and coverage of these four protocols were evaluated in silico on large genomic datasets, and in vitro on salivary DNA samples, already characterized by amplicon sequencing on the V3-V4 regions of the 16S rRNA. In silico PCR analyses showed that all qPCR primers lose part of the “Ca. Saccharibacteria” genetic variability, even if the 23S qPCR primers matched more lineages than the 16S qPCR primers. In vitro qPCR experiments confirmed that all 16S-based protocols strongly underestimated “Ca. Saccharibacteria” in salivary DNA, while the 23S qPCR protocol gave quantifications more comparable to 16S amplicon sequencing. Conclusion Overall, our results show that the 23S-based qPCR protocol is more precise than the 16S-based ones in quantifying “Ca. Saccharibacteria”, although all protocols probably underestimate specific lineages. These results underline the current limits in quantifying “Ca. Saccharibacteria”, highlighting the needs for novel experimental strategies or methods. Indeed, the underestimation of “Ca. Saccharibacteria” in clinical samples could hide its role in human health and in the development of immune-mediated diseases.

AbstractBackgroundCandidate Phyla Radiation (CPR) is a large monophyletic group thought to cover about 25% of bacterial diversity. Due to peculiar characteristics and unusual 16S rRNA gene structure, they are often under-represented or lost in 16S rRNA-based microbiota surveys. Among CPR, “CandidatusSaccharibacteria” is a phylum experimentally found to modulate the immune response and enriched in the oral microbiota of subjects suffering from several immune-mediated disorders, e.g. food allergies, as reported by us in a previous work. Due to the growing evidence of “Ca. Saccharibacteria”’s role in clinical settings and in order to unravel its role in host physiology and pathology, it is crucial to have a reliable method to detect and quantify this lineage.Methods and ResultsFour qPCR protocols for quantifying “Ca.Saccharibacteria” (one targeting the 23S rRNA gene and three the 16S) were selected from the literature among the few available. Efficiency and coverage of primer pairs used in these protocols were preliminary evaluated viain silicoanalyses on the “Ca.Saccharibacteria” known taxonomic variability, and then testedin vitroon the salivary DNA previously investigated by 16S metagenomics in the food allergy study.In silicoanalyses evidenced that the 23S qPCR protocol covered more “Ca. Saccharibacteria” variability compared to the 16S-based ones, and that the 16S metagenomics primers were the most comprehensive. qPCR experiments confirmed that 16S-based protocols strongly underestimated “Ca. Saccharibacteria” while the 23S protocol was the only one to yield results comparable to 16S metagenomics both in terms of correlation and absolute quantification. However, only 16S metagenomics evidenced an expansion of “Ca. Saccharibacteria” in allergic subjects compared to controls, while none of the four qPCR protocols detected it.ConclusionThese results underline the current limits in experimentally approaching “Ca. Saccharibacteria”. To obtain a more realistic picture of their abundance within bacterial communities, and to enable more efficient taxonomic resolution, it is essential to find novel experimental strategies. This is a necessary premise for more targeted and systematic functional studies to clarify the role of “Ca. Saccharibacteria” and, generally, CPR bacteria, in maintaining the health of the host.