Biology subjects

Comandatore, F.

Publications and source records attributed to Comandatore, F..

16 recordsLinked to original sources

bioRxiv2026

Food-Allergic Children Harbour a Reduced Oral Bacterial Load, Giving Misleading 16S rRNA Amplicon Profiles

BackgroundFood allergies have become a major public health concern in Western countries due to their rising prevalence. The microbiota is increasingly recognized as a key factor in their pathogenesis. Using V3-V4 16S rRNA amplicon sequencing, our group previously reported alterations in the oral microbiota of children with food allergies compared to controls. Given growing awareness of biases associated with relative-abundance analyses in 16S rRNA studies, we re-analyzed the salivary DNA samples from our earlier work. MethodsTotal bacterial loads were quantified by qPCR using universal 16S rRNA primers, enabling the estimation of absolute abundances for each taxon in each sample.These data were then used to reassess key microbial community features and to directly compare conclusions derived from absolute versus relative abundance approaches. ResultsAllergic children exhibited a marked reduction in salivary bacterial load, a feature not detected in the original relative-abundance analysis. Incorporating absolute abundances also partially reshaped the previously defined taxonomic pictures. Similar distortions affected functional predictions, such as Short Chain Fatty Acids (SCFA)-producing capacity. ConclusionsOur data confirm that variations of the bacterial load across samples hinder efforts to associate microbiota features and disease phenotypes. We identify reduced oral bacterial load as a previously unrecognized characteristic of children with food allergies. This observation aligns with reports in other immune-mediated disorders, suggesting that overall microbiota depletion may represent a defining and potentially diagnostically feature of disease-associated ecosystems.

microbiology↗

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

Does the host drive Wolbachia gene expression?

Wolbachia pipientis is an obligate intracellular bacterium, associated with several arthropods and filarial nematodes. Wolbachia establishes strict symbiotic relationships with its hosts, with the consequent loss of many genes and regulatory regions. Despite this, experimental studies show that Wolbachia gene expression is coordinated to host needs, but the mechanism is still unknown. The first published RNA-Seq study on Wolbachia evidenced a strong differential expression of a DNA methyltransferase (MTase). In bacteria, this enzyme methylates either adenines or cytosines on specific motifs, contributing to the regulation of gene expression. In this work, we tested the hypothesis that the activity of MTase modulates the expression of Wolbachia genes. We first determined the methylation motif of the Wolbachia MTase by expressing it in Escherichia coli. Surprisingly, the experiment revealed that the Wolbachia MTase methylates both adenine and cytosine, without recognising highly specific motifs. Then, re-analysing data from six RNA-Seq studies, we found that the nucleotide content of Wolbachia genes correlates with their expressions, with a pattern compatible to be a consequence of the DNA methylation. Lastly, we identified MTase as the Wolbachia gene with the most conserved binding site for the Ccka/CtrA signalling transduction system, a mechanism likely involved the host-bacterium communication. Overall, these findings suggest a cascade mechanism in which the host activates the Wolbachia Ccka/CtrA signalling system, thus inducing the expression of the MTase gene. Then, the subsequent DNA methylation will affect the expression of several Wolbachia genes on the basis of their cytosine and adenine content.

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↗

bioRxiv2023

On the forces shaping diversity and adaptation in the opportunistic pathogen Serratia marcescens

Bacterial species often comprise well-separated lineages, likely emerged and maintained by genetic isolation and/or ecological divergence. How these two evolutionary actors interact in the shaping of bacterial population structure is currently not fully understood. In this study, we investigated the genetic and ecological drivers underlying the evolution of Serratia marcescens, an opportunistic pathogen with high genomic flexibility and able to colonise diverse environments. Comparative genomic analyses revealed a population structure composed of five deeply-demarcated genetic clusters with open pan-genome but limited inter-cluster gene flow, partially explained by Restriction-Modification (R-M) systems incompatibility. Furthermore, a large-scale research on hundred-thousands metagenomic datasets revealed only a partial ecological separation of the clusters. Globally, two clusters only showed a peculiar gene composition and evident ecological adaptations. These results suggest that genetic isolation preceded ecological adaptations in the shaping of the species diversity, suggesting an evolutionary scenario for several bacterial species.

evolutionary biology↗

bioRxiv2023

P-DOR, an easy-to-use pipeline to reconstruct outbreaks using pathogen genomics

Bacterial Healthcare Associated Infections (HAIs) are a major threat worldwide, which can be counteracted by establishing effective infection control measures, guided by constant surveillance and timely epidemiological investigations. Genomics is crucial in modern epidemiology but lacks standard methods and user-friendly software, accessible to users without a strong bioinformatics proficiency. To overcome these issues we developed P-DOR, a novel tool for rapid bacterial outbreak characterization. P-DOR accepts genome assemblies as input, it automatically selects a background of publicly available genomes using k-mer distances and adds it to the analysis dataset before inferring a SNP-based phylogeny. Epidemiological clusters are identified considering the phylogenetic tree topology and SNP distances. By analyzing the SNP-distance distribution, the user can gauge the correct threshold. Patient metadata can be inputted as well, to provide a spatio-temporal representation of the outbreak. The entire pipeline is fast and scalable and can be also run on low-end computers. Availability and implementationP-DOR is implemented in Python3 and R and can be installed using conda environments. It is available from GitHub https://github.com/SteMIDIfactory/P-DOR under the GPL-3.0 license.

microbiology↗

bioRxiv2022

The origin and evolution of mitochondrial tropism in Midichloria bacteria

Midichloria are intracellular bacterial symbionts of ticks. Some representatives of this genus have the unique capability to colonize mitochondria in the cells of their hosts. Hypotheses on the nature of this interaction have proven difficult to test, partly due to a lack of data. Indeed, until now, mitochondrial tropism information and genomes were available only for symbionts of three and two tick host species, respectively. Here we analyzed the mitochondrial tropism of three additional Midichloria and sequenced nine novel genomes, showing that the tropism is pnon-monophyletic, either due to losses of the trait or multiple parallel acquisitions. Comparative genome analyses support the first hypothesis, as the genomes of non-mitochondrial symbionts appear to be reduced subsets of those capable of colonizing the organelles. We detect genomic signatures of mitochondrial tropism, showing a set of candidate genes characteristic of the strains capable of mitochondrial colonization. These include the type IV secretion system and the flagellum, which could allow the secretion of unique effectors, direct interaction with, or invasion of the mitochondria. Other genes, including putative adhesion molecules, proteins possibly involved in actin polymerization, cell wall and outer membrane proteins, are only present in mitochondrial symbionts. The bacteria could use these to manipulate host structures, including mitochondrial membranes, in order to fuse with the organelles or manipulate the mitochondrial network.

evolutionary biology↗

bioRxiv2021

A dual endosymbiosis drives nutritional adaptation to hematophagy in the invasive tick Hyalomma marginatum

Many animals are dependent on microbial partners that provide essential nutrients lacking from their diet. Ticks, whose diet consists exclusively on vertebrate blood, rely on maternally inherited bacterial symbionts to supply B vitamins. While previously studied tick species consistently harbor a single lineage of those nutritional symbionts, we evidence here that the invasive tick Hyalomma marginatum harbors a unique dual-partner nutritional system between an ancestral symbiont, Francisella, and a more recently acquired symbiont, Midichloria. Using metagenomics, we show that Francisella exhibits extensive genome erosion that endangers the nutritional symbiotic interactions: Its genome includes folate and riboflavin biosynthesis pathways but deprived functional biotin biosynthesis on account of massive pseudogenization. Co-symbiosis compensates this deficiency since the Midichloria genome encompasses an intact biotin operon, which was primarily acquired via lateral gene transfer from unrelated intracellular bacteria commonly infecting arthropods. Thus, in H. marginatum, a mosaic of co-evolved symbionts incorporating gene combinations of distant phylogenetic origins emerged to prevent the collapse of an ancestral nutritional symbiosis. Such dual endosymbiosis was never reported in other blood feeders but was recently documented in agricultural pests feeding on plant sap, suggesting that it may be a key mechanism for advanced adaptation of arthropods to specialized diets.

evolutionary biology↗

bioRxiv2021

One Health or Three? Transmission modelling of Klebsiella isolates reveals ecological barriers to transmission between humans, animals and the environment

The Klebsiella group is highly diverse both genetically and ecologically, being commonly recovered from humans, livestock, plants, soil, water, and wild animals. Many species are opportunistic pathogens, and can harbour diverse classes of antimicrobial resistance (AMR) genes. K. pneumoniae is responsible for a high public-health burden, due in part to the rapid spread of health-care associated clones that are non-susceptible to carbapenems. Klebsiella thus represents a highly pertinent taxon for assessing the risk to public health posed by animal and environmental reservoirs. Here we report an analysis of 6548 samples and 3,482 genome sequences representing 15 Klebsiella species sampled over a 15-month period from a wide range of clinical, community, animal and environmental settings in and around the city of Pavia, in the northern Italian region of Lombardy. Despite carbapenem-resistant clones circulating at a high frequency in the hospitals, we find no genotypic or phenotypic evidence for non-susceptibility to carbapenems outside of the clinical environment. The non-random distribution of species and strains across sources point to ecological barriers that are likely to limit AMR transmission. Although we find evidence for occasional transmission between settings, hierarchical modelling and intervention analysis suggests that direct transmission from the multiple non-human (animal and environmental) sources included in our sample accounts for less than 1% of hospital disease, with the vast majority of clinical cases originating from other humans.

microbiology↗

bioRxiv2021

Hypervariable-Locus Melting Typing (HLMT): a novel, fast and inexpensive sequencing-free approach to pathogen typing based on High Resolution Melting (HRM) analysis

ObjectivesSubspecies pathogen typing is a pivotal tool to detect the emergence of high-risk clones in hospital settings and to limit their spreading among patients. Unfortunately, the most used subspecies typing methods (i.e. Pulsed-field Gel Electrophoresis - PFGE, Multi-Locus Sequence Typing - MLST and Whole Genome Sequencing - WGS) are too expensive and time consuming to be suitable for real-time surveillance. Here we present Hypervariable-Locus Melting Typing (HLMT), a novel subspecies typing approach based on High Resolution Melting (HRM) analysis, which allows pathogen typing in a few hours and with [~]5 euros per sample. MethodsHLMT types the strains by clustering them using melting temperatures (HLMT-clustering) and/or by assigning them to Melting Types (MTs) on the basis of a reference dataset (HLMT-assignment). We applied HLMT (clustering and typing) to 134 Klebsiella pneumoniae strains collected during outbreaks or surveillance programs in four hospitals. Then, we compared HLMT typing results to PFGE, MLST and WGS. ResultsHLMT-clustering distinguishes most of the K. pneumoniae high-risk clones with a sensitivity comparable to PFGE and MLST. It also drawed surveillance epidemiological curves comparable to those obtained by MLST, PFGE and WGS typing. Furthermore, the results obtained by HLMT-assignment were coherent to MLST for 96% of the typed strains with a Jaccard index of 0.912. ConclusionsHLMT is a fast and scalable method for pathogen typing, suitable for real-time hospital microbiological surveillance. HLMT is also inexpensive and thus it is applicable to infection control programs in low-middle income countries.

microbiology↗

bioRxiv2020

Phylogenomics reveals that Asaia symbionts from insects underwent convergent genome reduction, preserving an insecticide-degrading gene

The mosquito microbiota is composed by several lineages of microorganisms whose ecological roles and evolutionary histories have yet to be investigated in depth. Among these microorganisms, Asaia bacteria play a prominent role, given its abundance in the gut, reproductive organs and salivary glands of different mosquito species, while its presence has also been reported in several other insects. Notably, Asaia has great potential as a tool for the control of mosquito-borne diseases. Here, we present a wide phylogenomic analysis of Asaia strains isolated from different species of mosquito vectors and from different populations of the Mediterranean fruit fly (medfly) Ceratitis capitata, an insect pest of worldwide economic importance. We show that phylogenetically distant lineages of Asaia experienced independent genome reductions, despite following a common pattern, characterized by the early loss of genes involved in genome stability. This result highlights the role of specific metabolic pathways in the symbiotic relationship between Asaia and the insect host. Finally, we discovered that all but one of the Asaia strains included in the study possess the pyrethroid hydrolase gene. Phylogenetic analysis revealed that this gene is ancestral in Asaia, strongly suggesting that it played a role in the establishment of the symbiotic association between these bacteria and the mosquito hosts. We propose that this gene from the symbiont contributed to initial pyrethroid resistance in insects harboring Asaia, also considering the widespread production of pyrethrins by several plants. ImportanceWe have studied genome reduction within several strains of the insect symbiont Asaia, isolated from different species/strains of mosquito and medfly. Phylogenetically distant strains of Asaia, despite following a common pattern involving the loss of genes related to genome stability, have undergone independent genome reductions, highlighting the peculiar role of specific metabolic pathways in the symbiotic relationship between Asaia and its host. We also show that the pyrethroid hydrolase gene is present in all the Asaia strains isolated except for the South American malaria vector An. darlingi, for which resistance to pyrethroids has never been reported, suggesting a possible involvement of Asaia in determining the resistance to insecticides.

microbiology↗

bioRxiv2020

Repeatability and reproducibility of the wzi High Resolution Melting-based clustering analysis for Klebsiella pneumoniae typing

AimsHigh Resolution Melting (HRM) is a fast closed-tube method for nucleotide variant scanning applicable for bacterial species identification or molecular typing. Recently a novel HRM-based method for Klebsiella pneumoniae typing has been proposed: it consists of an HRM protocol designed on the capsular wzi gene and an HRM-based algorithm of strains clustering. In this study, we evaluated the repeatability and reproducibility of this method. Methods and ResultsWe performed HRM typing of a set of K. pneumoniae strains, on three different instruments and by two different operators. The results showed that operators do not affect measured melting temperatures while different instruments can. Despite this, we found that strains clustering analysis remains perfectly consistent performing the analysis using the MeltingPlot tool separately on the data from the three instruments. ConclusionsThe HRM protocol under study was highly repeatable and thus reliable for large studies, even when several operators are involved. Significance and Impact of the StudyOur results show that the wzi-HRM protocol for K. pneumoniae typing is suitable for multicenter studies, even if different instruments are used.

microbiology↗

bioRxiv2020

Identification of variable sites in Sars-CoV-2 and their abundance profiles in time

The SARS-CoV-2 pandemic that we are currently experiencing is exerting a massive toll both in human lives and economic impact. One of the challenges we must face is to try to understand if and how different variants of the virus emerge and change their frequency in time. Such information can be extremely valuable as it may indicate shifts in aggressiveness, and it could provide useful information to trace the spread of the virus in the population. In this work we identified and traced over time 7 amino acid variants that are present with high frequency in Italy and Europe, but that were absent or present at very low frequencies during the first stages of the epidemic in China and the initial reports in Europe. The analysis of these variants helps defining 6 phylogenetic clades that are currently spreading throughout the world with changes in frequency that are sometimes very fast and dramatic. In the absence of conclusive data at the time of writing, we discuss whether the spread of the variants may be due to a prominent founder effect or if it indicates an adaptive advantage.

bioinformatics↗

bioRxiv2019

Fast Klebsiella pneumoniae typing for outbreak reconstruction: an highly discriminatory HRM protocol on wzi capsular gene developed using EasyPrimer tool

In this work we present EasyPrimer, a user-friendly online tool developed to assist pan-PCR and High Resolution Melting (HRM) primer design. The tool finds the most suitable regions for primer design in a gene alignment and returns a clear graphical representation of their positions on the gene. EasyPrimer is particularly useful in difficult contexts, e.g. on gene alignments of hundreds of sequences and/or on highly variable genes. HRM analysis is an emerging method for fast and cost saving bacterial typing and an HRM scheme of six primer sets on five Multi-Locus Sequence Type (MLST) genes is already available for Klebsiella pneumoniae. We validated the tool designing a scheme of two HRM primer sets on the hypervariable gene wzi of Klebsiella pneumoniae and compared the two schemes. The wzi scheme resulted to have a discriminatory power comparable to the HRM MLST scheme, using only one third of primer sets. Then we successfully used the wzi HRM primer scheme to reconstruct a Klebsiella pneumoniae nosocomial outbreak in few hours. The use of hypervariable genes reduces the number of HRM primer sets required for bacterial typing allowing to perform cost saving, large-scale surveillance programs.

epidemiology↗

bioRxiv2018

Gene composition as a potential barrier to large recombinations in the bacterial pathogen Klebsiella pneumoniae

Klebsiella pneumoniae (Kp) is one of the most important nosocomial pathogens world-wide, being responsible for frequent hospital outbreaks and causing sepsis and multi-organ infections with a high mortality rate and frequent hospital outbreaks. The most prevalent and widely disseminated lineage of K. pneumoniae is clonal group 258 (CG258), which includes the highly resistant \"high-risk\" genotypes ST258 and ST11. Recent studies revealed that very large recombination events have occurred during the recent emergence of Kp lineages. A striking example is provided by ST258, which has undergone a recombination event that replaced over 1 Mb of the genome with DNA from an unrelated Kp donor. Although several examples of this phenomenon have been documented in Kp and other bacterial species, the significance of these very large recombination events for the emergence of either hyper-virulent or resistant clones remains unclear. Here we present an analysis of 834 Kp genomes that provides data on the frequency of these very large recombination events (defined as those involving >100Kb), their distribution within the genome, and the dynamics of gene flow within the Kp population. We note that very large recombination events occur frequently, and in multiple lineages, and that the majority of recombinational exchanges are clustered within two overlapping genomic regions, which result to be involved by recombination events with different frequencies. Our results also indicate that certain non-CG258 lineages are more likely to act as donors to CG258 recipients than others. Furthermore, comparison of gene content in CG258 and non-CG258 strains agrees with this pattern, suggesting that the success of a large recombination depends on gene composition in the exchanged genomic portion.\n\nAuthor SummaryKlebsiella pneumoniae (Kp) is an opportunistic bacterial pathogen, a major cause of deadly infections and outbreaks in hospitals worldwide. This bacterium is able to exchange large genomic portions (up to a fourth of the entire genome) within a single recombination event. Indeed, the most epidemiologically important Kp clone, is actually a hybrid which emerged after a > 1Mb recombination event. In this work, we investigated how recombinations affected the evolution of the most studied Kp Clonal Group, CG258. We found that large recombinations occurred frequently during Kp evolution, and occurred preferentially in a well-delimited genomic region. Furthermore, we found that four epidemiologically important clones emerged after large recombinations. We identified the donors of several large recombinations: despite many Kp lineages acted as donors during CG258 evolution, two of them have been involved more frequently. We hypothesize that the observed pattern of donors-recipients in recombinations, and the presence of a large recombinogenic region in Kp genome, could be related to gene composition. Indeed, genomic analyses showed a pattern compatible with this hypothesis, suggesting that gene content can represent a main factor in the success of a large recombination.

evolutionary biology↗