Biology subjects

D'Auria, E. C.

Publications and source records attributed to D'Auria, E. C..

3 recordsLinked to original sources

bioRxiv2025

A Simple and Cost-Effective Protocol for Enriching 'Ca. Saccharimonadia' from Human Saliva to Enable Efficient Genome Sequencing

Background"Candidatus Saccharimonadia", a lineage within the Candidate Phyla Radiation (CPR), is characterized by small cell size, reduced genomes, and an epibiotic lifestyle dependent on bacterial hosts. Despite their presence in diverse environments, including the human oral microbiota, their role in human health remains largely unexplored. Observational studies have linked increased "Ca. Saccharimonadia" abundance in human saliva with inflammatory conditions such as periodontitis and inflammatory bowel disease, while recent in vivo evidence suggests potential anti-inflammatory properties. Investigating "Ca. Saccharimonadia" is challenging due to their fastidious nature, making standard culturing methods impractical. Current genomic studies rely on either co-cultivation with bacterial hosts or shotgun metagenomics, both requiring advanced technical expertise and costly resources. To address this limitation, we developed an affordable and efficient protocol for enriching "Ca. Saccharimonadia" from human saliva samples, facilitating genome sequencing. Our protocol, termed the "Ca. Saccharimonadia" Enrichment (SE) protocol, consists of two mandatory phases (detachment from host bacteria and weight/size-based separation) along with an optional DNA degradation phase. We validated this method on two human saliva samples. ResultsAfter SE protocol, the two saliva showed a substantial increase in "Ca. Saccharimonadia" DNA concentration. The enriched samples enabled high-quality metagenome-assembled genomes (MAGs) to be obtained via shotgun metagenomics. ConclusionsThis study presents a cost-effective and scalable approach to studying "Ca. Saccharimonadia", overcoming prior limitations and expanding opportunities for future research on their role in human health and disease.

microbiology↗

bioRxiv2024

RecA is a reliable marker for bacterial taxonomy, even in the Candidate Phyla Radiation

Culture-independent approaches are commonly used to characterise the taxonomic composition of bacterial communities. Among these approaches, the amplicon-based metagenomics relies on specific genetic markers, such as the 16S rRNA gene, while the shotgun metagenomics annotates the whole bacterial DNA. Despite the 16S being the gold standard marker, studies highlighted its inefficiency in characterising and quantifying divergent bacterial groups such as the Candidate Phyla Radiation. On the other hand, shotgun metagenomics is highly informative and accurate but it is more expensive and requires computational resources and time. In this study, we propose RecA as a pan-bacterial genetic marker, particularly suitable for the Candidate Phyla Radiation. Indeed, we found that applying a Random Forest machine learning model on RecA amino acid sequences provides an accurate and fast taxonomic annotation across the whole bacterial tree of life. Ultimately, we produced Forestax, a tool for the characterisation and quantification of bacterial communities in metagenomics data, on the basis of RecA sequences. The analyses showed that RecA-based metagenomics has a taxonomic accuracy comparable to other multi-gene approaches, reinforcing RecA as a powerful marker for taxonomic annotation in bacteria. In perspective, RecA could be considered as a broad-spectrum marker for amplicon-based studies to overcome the limits of 16S rRNA.

microbiology↗

bioRxiv2024

Unraveling and quantifying "Candidatus Saccharibacteria": in silico and experimental evaluation of V3-V4 16S rRNA metagenomics and qPCR protocols

BackgroundCandidate 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, "Candidatus Saccharibacteria" 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 via in silico analyses on the "Ca. Saccharibacteria" known taxonomic variability, and then tested in vitro on the salivary DNA previously investigated by 16S metagenomics in the food allergy study. In silico analyses 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.

microbiology↗