Biology subjects

Porcellato, D.

Publications and source records attributed to Porcellato, D..

3 recordsLinked to original sources

bioRxiv2025

Longitudinal study of the udder microbiome of Norwegian red dairy cows using metataxonomic and shotgun metagenomic approaches: Insights into pathogen-driven microbial adaptation and succession

Bovine mastitis remains the most significant disease affecting dairy herds globally, driven by its multi-etiological nature and the complex dynamics of udder immunity and infection. While research addressing the microbial and immunological aspects of the bovine udder is limited, optimizing the udder microbiome has emerged as a promising strategy for preventing mastitis. This longitudinal study aimed to investigate the udder microbiome throughout lactation using both metataxonomic and shotgun metagenomic approaches, including analysis at the metagenome-assembled genome (MAG) level. The use of such methodologies provides a deeper understanding of the microbial composition and dynamics within the udder, bridging critical gaps in knowledge and revealing potential interactions between the microbiota and host. Milk samples were collected from 342 individual quarters of 24 Norwegian Red dairy cows. Significant variations in somatic cell count and microbiota composition were observed across lactation stages. Quarters with low somatic cell count were notably higher during early lactation (80%) and mid-lactation (78.9%) compared to dry-off (53.1%) and late lactation (53%), with high somatic cell countobserved in 20-47% of samples. Diversity indices based on Shannon and Simpson metrics indicated significant effects of lactation stage, somatic cell count, and individual animal variability on microbial diversity. PERMANOVA analyses confirmed that individual animal variability (15.73%) and lactation period (5.52%) were the strongest factors influencing microbiota structure, with dysbiosis linked to mastitis-causing pathogens contributing 7.17% of the variance. Key pathogens identified included Enterococcus faecalis, Staphylococcus aureus, Streptococcus uberis, and Staphylococcus chromogenes, with persistent infections observed for S. aureus and S. chromogenes, but not S. uberis. Samples with low somatic cell count were enriched in beneficial genera such as Corynebacterium, Bradyrhizobium, and Lactococcus, while Staphylococcus predominated in milk samples with high somatic cell count. Dimensionality reduction integration with culturomics enhanced milk microbiota classification, providing novel insights into udder microbiota dynamics and their role in bovine mastitis. Metagenomic shotgun sequencing revealed pathogen-specific metabolic signatures in the bovine mammary gland, identifying 289 MetaCyc pathways. Genome-centric analysis reconstructed 142 metagenome-assembled genomes, including 26 from co-assembly and 116 from individual assembly. Multi-locus sequence typing, virulence factors, and antimicrobial resistance gene profiling provided insights into pathogen adaptation and persistence in the bovine mammary gland. Notably, 27 bacteriocin gene clusters and 322 biosynthetic gene clusters were predicted using genome mining tools. Our findings contribute to the establishment of pathogen-based therapies and enhance our understanding of the milk microbiome, which remains far from fully characterized. Such insights are vital for developing effective strategies to combat mastitis and improve dairy cattle health.

microbiology↗

bioRxiv2024

An antibiotic-free antimicrobial combination of bacteriocins and a peptidoglycan hydrolase: in vitro and in vivo assessment of its efficacy

Mastitis is an inflammatory disease of the mammary gland commonly brought about by bac-terial pathogens that gain physical access to the glandular epithelium through the teat canal. In bovines, common mastitis-causing agents are environmental or pathogenic bacterial spe-cies, including staphylococci, streptococci, enterococci, and Gram-negative bacteria such as Escherichia coli. Current therapeutic strategies for bovine mastitis typically involve the ad-ministration of antibiotic formulations within the infected udder, possibly resulting in in-creased selection of antibiotic resistance and the accumulation of antibiotic residues within the milk. In this study, we sought to design an antibiotic-free antimicrobial formulation to treat bovine mastitis based on bacterial antimicrobial peptides (bacteriocins) and proteins (pepti-doglycan hydrolases). Using a combination of in vitro assays with a range of bacteriocins, we show that the combination of the thiopeptide micrococcin P1 (MP1) and the lantibiotic nisin A (NisA) is a robust antimicrobial formulation that effectively inhibits the growth of bo-vine mastitis-derived bacteria, both in planktonic and biofilm-associated growth modes. The addition of AuresinePlus (Aur, a staphylococcus-specific PGH) further increased the antimi-crobial potency against S. aureus. Furthermore, using two mouse models, a skin infection model and a mastitis model, we show that the combination MP1-NisA-Aur effectively inhibits methicillin-resistant S. aureus (MRSA) in vivo. We discuss the potential and challenges of using antibiotic-free antimicrobial combinations in the treatment of bacterial infections.

microbiology↗

bioRxiv2023

Genome-wide CRISPRi screens reveal the essentialome and determinants for susceptibility to dalbavancin in Staphylococcus aureus

Antibiotic resistance and tolerance remain a major problem for treatment of staphylococcal infections. Knowing genes that influence antibiotic susceptibility could open the door to novel antimicrobial strategies, including targets for new synergistic drug combinations. Here, we developed a genome-wide CRISPR interference library for Staphylococcus aureus, demonstrated its use by quantifying the essentialome in different strains through CRISPRi-seq, and used it to identify genes that modulate susceptibility to the lipoglycopeptide dalbavancin. By exposing the library to sublethal concentrations of dalbavancin using both CRISPRi-seq and direct selection methods, we found genes previously reported to be involved in antibiotic susceptibility, but also identified genes thus far unknown to affect antibiotic tolerance. Importantly, some of these genes could not have been detected by more conventional knock-out approaches because they are essential for growth, stressing the complementary value of CRISPRi-based methods. Notably, knockdown of a gene encoding the uncharacterized protein KapB specifically sensitizes the cells to dalbavancin, but not to other antibiotics of the same class, while knockdown of the Shikimate pathway surprisingly has the opposite effect. The results presented here demonstrate the potential of CRISPRi-seq screens to identify genes and pathways involved in antibiotic susceptibility and pave the way to explore alternative antimicrobial treatments through these insights.

microbiology↗