Evolutionary dynamics of Erinaceus coronavirus in Italy: spike protein adaptation and variable CD200 ortholog insertion Luca De Sabato, Ilaria Di Bartolo, Irene Berselli, Giovanni Ianiro, Marina Monini, Monica Giammarioli, Marco Gobbi, Riccardo Orusa, Serena Robetto, Maria Lucia Mandola, Chiara Nogarol, Ana Moreno, Davide Lelli, Claudia Cotti, Mauro Delogu, Maria Alessandra De Marco, Stefania Leopardi, Francesca Festa, Manuela Marra, Valentina Libri, Stefania Pulcini, Francesca Manzia, Roberto Nardini, Maria Teresa Scicluna, Antonella Cersini, Claudia Eleni, Giulia Pacchiarotti, Gianluca Nocco, Maria Teresa Capucchio, Gabriele Vaccari Frontiers in Virology, 2026 Introduction In 2013, a novel coronavirus (CoV) species was identified in European hedgehogs ( Erinaceus europaeus ) in Germany. Since then, only a limited number of studies reported the circulation of this virus, recognized as Erinaceus coronavirus (EriCoV), and demonstrated its evolutionary relationship with Middle East respiratory syndrome coronavirus (MERS-CoV). Given that MERS-CoV is one of the three zoonotic coronaviruses known to cause severe disease in humans, these findings raise concerns about the zoonotic potential of EriCoV strains. In a previous study conducted in Italy, eight complete EriCoV genomes were obtained from hedgehogs sampled in Northern Italy, revealing substantial genetic heterogeneity among circulating strains. Genome analyses also identified the insertion of a gene encoding the CD200 ortholog, containing a short region potentially involved in host immune modulation. The present study aimed to investigate the circulation and genetic diversity of coronaviruses in hedgehogs from different areas of Italy and to assess the occurrence and distribution of the CD200 ortholog insertion. Methods Complete EriCoV genomes were obtained from 35 hedgehogs using Next Generation Sequencing (NGS) by Ion Torrent machine. For an additional 20 animals, full genome sequences could not be generated; therefore, sequences were obtained by nested RT-PCR amplifying partial sequences of the RNA-dependent RNA polymerase (RdRp) gene and, when present, on the CD200 insertion. Phylogenetic analysis of sequences obtained were performed. Results Phylogenetic analysis revealed no clear geographic clustering, with sequences from Northern Italy interspersed with those from Central and Southern Italy. Analysis of the spike (S) protein identified 25 sites evolving under positive selection, three of which were located within the receptor-binding domain (RBD). The CD200 ortholog was confirmed in 15 complete genomes and in 5 additional samples by RT-PCR, collected from both Northern and Southern Italy. Discussion Overall, the study confirms that EriCoV related strains are found across the country. A CD200 insertion appears in both Northern and Southern regions, but it is not consistently present, indicating that this insertion is not fixed within the viral population. Sites found under positive selection in spike protein, including three in the receptor-binding domain, that may be relevant for host–virus interactions and merit further investigation in the context of cross-species transmission risks.
Investigation of dipyridamole-elicited signaling in the brain of Niemann Pick type C mice: A multi-omic study Sabrina Tait, Federica Fratini, Zaira Boussadia, Lucia Gaddini, Manuela Marra, Loredana Le Pera, Gloria Venturini, Antonella Ferrante Brain Research Bulletin, 2026 mice administered with DIP were those related to cGMP-PKG activation and to mitochondrial function. Our results paved the way to test DIP in experimental models of other neurodegenerative disorders, such as Alzheimer's disease that is similarly marked by hippocampal and mitochondrial dysfunctions.
Aeromonas spp. as possible bacterial indicator for monitoring antibiotic resistance in seafood Elisa Albini, Biagio Caponi, Silvia Pieralisi, Serenella Orsini, Francesca Blasi, Luisa Massaccesi, Carmen Maresca, Eleonora Scoccia, Alessandro Fiorucci, Valeria Michelacci, Paola Chiani, Manuela Marra, Maria Carollo, Francesca Romana Massacci, Giovanni Pezzotti, Francesca Leoni, Chiara Francesca Magistrali Frontiers in Microbiology, 2026 Fishery and aquaculture products increasingly represent an important food source for the world population. The intensification of aquaculture guided an increase in the use of antimicrobials in this sector as well. The aquatic environment represents a link among humans, animals and environmental antibiotic resistance. The rising problem of antibiotic resistance leads to the need for the identification of indicator bacteria and the development of monitoring methods, which are poorly standardized for the aquatic environment. Aeromonas spp., a ubiquitous bacterium in aquatic environments, is among the possible candidates as bacterial indicator. The goals of this study were to: (i) evaluate the antibiotic-resistance profile of different antimicrobial classes in Aeromonas spp. collected from seafood sold in superstores of central Italy; (ii) propose possible epidemiological cut-offs for the genus Aeromonas and evaluate its effectiveness as a possible bacterial indicator for monitoring antibiotic resistance in seafood. The results obtained revealed a high presence of Aeromonas spp. in seafood categories, highlighting its candidature as a bacterial indicator. The presence of isolates resistant to some of the tested antibiotics has been described, even if in rare occasion. This data raises concerns about the food safety of seafood for consumption, which may represent a risk for public health and consumers. In this study, we described the epidemiological cut-offs which agree with the literature, representing a contribution to the definition of reliable epidemiological cut-offs for the genus Aeromonas spp.
Genomic insights into multidrug-resistant Enterobacter hormaechei isolates from seafood at retail carrying the mcr-9 gene and other resistance determinants on IncHI2 plasmids Valeria Michelacci, Silvia Pieralisi, Elisa Albini, Francesca Romana Massacci, Gabriele Angelico, Paola Chiani, Manuela Marra, Maria Carollo, Chiara Francesca Magistrali, Francesca Leoni Journal of Global Antimicrobial Resistance, 2025 OBJECTIVES: We aimed to characterise the genomic features of two multidrug-resistant (MDR) isolates from retail seafood samples. METHODS: Antimicrobial susceptibility was determined by minimum inhibitory concentration (MIC) tests. Genome sequences were obtained by hybrid sequences of short and long. RESULTS: The two MDR (ampicillin, cefotaxime, ciprofloxacin, ceftazidime, nalidixic acid, gentamicin, tetracycline, sulfamethoxazole, cefoxitin, and trimethoprim) Enterobacter hormaechei (E. hormaechei) isolates belonged to ST114. Nineteen antibiotic resistance genes (ARGs), including mcr-9, were identified in the genomes. The mcr-9 and other resistance determinants were on an IncHI2 plasmid. Sequence analysis showed mutations in genes implicated in colistin resistance, of which a non-conservative one was present in the qseB. Virulence genes of T6SS or involved in motility were identified in both genomes. Comparison of mcr-9 plasmids allowed us to identify similar plasmids in the isolates from Italy and in the five best hits of the PLSDB database, with some regions of difference in the MDR region. A genome cluster analysis grouped our isolates into the same clade of ST114 E. hormaechei isolated from humans in Guadeloupe in 2018 and Switzerland in 2019, most of which harboured an IncHI2 replicon but not the mcr-9 gene. CONCLUSIONS: The finding of MDR E. hormaechei in seafood, carrying the mcr-9 and other resistance determinants on IncHI2 plasmids, enhances our understanding of antimicrobial resistance (AMR) and highlights the importance of gaining more data on the occurrence and circulation of AMR and ARGs in aquatic environments, in order to better understand sources, transmission routes, and evolution of AMR mechanisms.
QIIME2 enhances multi-amplicon sequencing data analysis: a standardized and validated open-source pipeline for comprehensive 16S rRNA gene profiling Armando G. Licata, Marica Zoppi, Chiara Dossena, Federico Rossignoli, Davide Rizzo, Manuela Marra, Giorgio Gargari, Giacomo Mantegazza, Simone Guglielmetti, Luca Bergamaschi, Olga Nigro, Stefano Chiaravalli, Maura Massimino, Loris De Cecco Microbiology Spectrum, 2025 Multi-amplicon sequencing is a cost-effective method for profiling multiple regions of the 16S rRNA gene, offering a more comprehensive view of microbial diversity. However, implementing such pipelines on open-source platforms (e.g., QIIME2) is often hindered by limited documentation and lack of validation against established tools. This lack of standardization poses challenges for researchers, particularly in clinical and experimental settings. This study aims to: (i) develop and benchmark a standardized, open-source QIIME2- and R-based pipeline for 16S rRNA gene profiling using semiconductor-based sequencing, comparing it with a commercial, closed-source software; and (ii) validate its effectiveness in a pediatric cancer cohort to examine parental influence on the microbiome and child-caregiver microbial relationships. We generated 16S rRNA profiles from 5 mock communities and 12 child-caregiver fecal sample pairs. Benchmarking against commercial software showed that the multi-region (V2–9) approach produced microbial profiles nearly identical to proprietary outputs, with higher sequencing depth and improved taxonomic resolution compared to single-region analyses. Both approaches demonstrated similar microbial richness, accurate mock community reconstruction, and high reproducibility ( R = 0.99, P < 0.0001). These findings were further validated using fecal samples. Application of the pipeline to pediatric samples revealed distinct, differentially abundant Bifidobacterium bifidum and Bifidobacterium adolescentis variants in children whose microbiota closely resembled that of their caregivers. Overall, this study presents a validated, open-source QIIME2 and R pipeline for multi-amplicon sequencing, providing a standardized and reproducible framework for 16S rRNA gene profiling in clinical and research contexts. IMPORTANCE Multi-amplicon sequencing comprehensively characterizes microbial communities by targeting multiple regions of the 16S rRNA gene. However, analytical workflows and reference databases provided by commercial library preparation kits frequently rely on proprietary primers and closed-source pipelines, which can limit transparency, reproducibility, and adaptability. To address these limitations, we developed and validated an open-source bioinformatics pipeline utilizing QIIME2 and R. Our pipeline integrates data from all targeted 16S regions, generating microbial profiles comparable to those produced by proprietary software. Validation was performed using mock samples and fecal samples collected from pediatric cancer patients and their caregivers, confirming the pipeline’s reliability and broad applicability. Furthermore, our pipeline enables detailed analysis of microbial variants, surpassing traditional genus-level restrictions and fully leveraging the enhanced coverage offered by multi-amplicon sequencing. Our findings highlight the necessity of adopting open-source solutions to ensure scientific reproducibility and adaptability to emerging methodologies.
Genomic analysis of Neisseria meningitidis serogroup W causing invasive meningococcal disease in four travellers from Saudi Arabia, Italy, 2025 Arianna Neri, Elsa Taviani, Federica Ferraro, Francesco Maraglino, Manuela Marra, Laura Daprai, Caterina Vocale, Elisa Vian, Anna Teresa Palamara, Paola Stefanelli Eurosurveillance, 2025 Genomic analysis was performed on Neisseria meningitidis isolates from four cases of invasive meningococcal disease (IMD) identified in Italy after travel to Saudi Arabia in 2025. The cases were not epidemiologically related. The isolates from the cases were whole genome sequenced and belonged to serogroup W, sequence type (ST)-11, clonal complex (CC)11 and were susceptible to ceftriaxone, ciprofloxacin, penicillin G and rifampicin. Phylogenetic analysis grouped the four genomes with the Hajj strain sublineage.
Pathogenic Escherichia coli Virulome in Top Soil Improvers and Irrigation Waters Devoted to Leafy Vegetable Production: Hazard Assessment From a Case Study in Italy Giorgia Barbieri, Federica Gigliucci, Sofia Palma, Fabio Minelli, Paola Chiani, Manuela Marra, Maria Carollo, Arnold Knijn, Gianfranco Brambilla, Stefano Morabito Food Safety and Health, 2025 Top soil improvers (TSIs) and reused water from municipal wastewater treatment plants (WWTPs) represent useful and sustainable sources of organic carbon and nutrients for agriculture. However, from their regular use, emerging Escherichia coli enterophatotypes, particularly Shiga toxin‐producing E. coli (STEC), were responsible for severe foodborne outbreaks from contaminated ready‐to‐eat vegetables. We report the hazard characterization of 31 top soil improvers (TSIs) and 15 irrigation water samples of different origins, sampled at the farm level. The increased PCR signals of the marker virulence genes stx1, stx2, aggR, aaiC, ipaH, lt, stp, and sth for enteropathogenic, enteroaggregative, enteroinvasive, enterotoxigenic, and Shiga toxin‐producing E. coli from post‐enrichment samples were considered proof of the presence of viable bacteria. Despite positive signals from TSI‐enriched samples, we isolated one enteropathogenic and one enterotoxigenic strain from the same pig slurry sample and one STEC strain carrying the enterotoxigenic heat‐stable enterotoxin from bovine slurry. From irrigation water samples, the most detected genes were stx1, stx2, and eae, followed by aggR, aaiC, ipaH, lt, stp, and sth, leading to one STEC (stx1+) isolation. E. coli pathotype virulotyping could be proposed to assess if combined TSI and water inputs would potentially lead to hybrid strains with shuffled virulence features to support safe and sustainable ready‐to‐eat vegetable production.
Characterization of the resistome and antibiotic-resistant bacteria in top soil improvers and irrigation waters devoted to food production: a case study from Italy Federica Gigliucci, Giorgia Barbieri, Marie Veyrunes, Paola Chiani, Manuela Marra, Maria Carollo, Arnold Knijn, Gianfranco Brambilla, Stefano Morabito Environmental Science and Pollution Research, 2025 Biosolids and reclaimed waters are valuable resources for reintroducing organic matter into agricultural soils and reducing the water footprint of intensive agricultural food system. While the circular economy is a sustainable practice, it may introduce vulnerabilities in the food chain, by exposing crops to zoonotic agents and antimicrobial resistance determinants. This option is far from being a speculation and evidence start to accumulate indicating that the risk is tangible. This study provides further evidence that the circular economy practices of reusing biomass and reclaimed waters in agricultural setting may be vectors for the spreading of antibiotic resistance genes (ARGs) targeting molecules used to treat human bacterial infections. We screened biosolid and water samples for ARGs presence using shotgun metagenomic sequencing. We demonstrated that the identified ARGs are present in live bacterial organisms, harbouring multidrug-resistant gene clusters, confirmed through phenotypic testing and whole-genome sequencing of isolated bacteria. Additionally, we observed that most of the antibiotic-resistant bacteria identified belonged to environmentally widespread species, which were not expected to be exposed to the antimicrobials, suggesting that inter-species transfer of resistance genes.
Distinct gut microbiota composition in pediatric patients with central nervous system (CNS) tumors: A comparative study Chiara Dossena, Luca Bergamaschi, Federico Rossignoli, Armando Giuseppe Licata, Patrizia Gasparini, Lara Veronica Venturini, Manuela Marra, Oriani Matilde, Veronica Biassoni, Elisabetta Schiavello, Olga Nigro, Stefano Chiaravalli, Maura Massimino, Loris De Cecco Neuro Oncology Advances, 2025 Background Central nervous system (CNS) tumors are the leading cause of cancer-related deaths in children aged 0–14 years. Despite significant efforts, targeted therapies based on identified pathways have not improved survival rates. Research has shown that the gut microbiota (GM) can influence brain tumor cell proliferation, suggesting that the microbiota–gut–brain axis plays a role in CNS cancer. Our study aims to assess whether the GM composition in pediatric CNS tumors exhibits specific characteristics. Methods The study included 18 pediatric patients, 9 diagnosed with CNS tumors (CNS tumors group) and 9 with other tumor types (extra-CNS tumors group). Microbial DNA was extracted from stool samples, and 16S DNA libraries were generated and sequenced. GM composition was analyzed using amplicon sequence variant (ASV) tables. Results Alpha-diversity analysis, represented by the number of observed features, was lower in the CNS tumors group (P = .0054), while Pielou’s evenness index was similar between groups. LEfSe analysis revealed a significantly reduced abundance of the Firmicutes phylum in CNS tumors group, along with other taxa within this phylum, such as the Clostridia class, Clostridiales order, and Lachnospiraceae family, compared to extra-CNS tumors group. Further analysis using sPLS-DA showed a distinct pattern in GM composition in the CNS tumors group, with lower levels of several taxa, particularly the Firmicutes phylum, Lachnospiraceae family, Clostridiales order, Clostridia class, Ruminococcaceae and Coriobacteriaceae families, and Blautia genus. Conclusions Pediatric patients with CNS tumors have a distinct GM composition. The reduction of specific beneficial microbial taxa may contribute to tumor growth through the microbiota–gut–brain axis.
Survival and genomic stability of Yersinia enterocolitica in environmental Acanthamoeba spp Paola Chiani, Valeria Michelacci, Elisabetta Delibato, Eleonora Ventola, Samra Mannan, Manuela Marra, Valentina Libri, Rubén L. Rodríguez-Expósito, Maria Reyes-Batlle, Isabel de Fuentes, Jacob Lorenzo-Morales, Stefano Morabito, Margherita Montalbano Di Filippo Frontiers in Cellular and Infection Microbiology, 2025
Voacamine, an alkaloid extracted from Peschiera fuchsiaefolia, inhibits P-glycoprotein action in multidrug-resistant tumor cells International Journal of Oncology, 2005
