Study and exploitation of diverse soil environments for novel natural product discovery using metagenomic approaches

Natural products with antimicrobial activity have played an important role in the treatment of infection since their discovery. The increasing emergence of pathogens resistant to multiple antibiotics has raised awareness of the urgent need for novel antibiotics. Soil microorganisms are the major source of antibiotics and Actinobacteria in particular have an impressive capacity for production of diverse bioactive secondary metabolites. However, culture-independent studies have shown a greater microbial diversity present in soil with potential for novel chemical structures and these can be explored further using metagenomic approaches capturing genes without the need to cultivate the host. Different metagenomic tools were used to study and explore microbial secondary metabolite diversity in soil. In particular, amplicon sequencing of 16S rRNA gene, NRPS and PKS biosynthetic genes allowed the identification of novel potential phylogenetic drivers of secondary metabolite diversity in the less characterized phyla Verrucomicrobia and Bacteroidetes and potential geographic hotspots harbouring unique biosynthetic diversity such as Antarctica and Cuba. The exploitation of these hotspots presented some bottlenecks in the form of DNA extraction efficiency, library creation, screening and heterologous expression. These were overcome by comparative analysis of different eDNA extraction methods to optimise fragment size and purity combined with development of new cloning tools for both DNA capture and expression. Modification of the microbial community through the amendment of the soil with chitin, highlighted the beneficial effect of microbial enrichment allowing a higher recovery of eDNA and higher detection of the biosynthetic gene of interest related to secondary metabolite production. Further additions were made to the metagenomic molecular toolbox in the form of BAC vectors (pBCaBAC and pBCkBAC) which were tested with suitable heterologous host systems (Streptomyces sp. and the engineered Pseudomonas putida species) potentially facilitating heterologous expression. In conclusion this is the first study to identify the drivers of microbial secondary metabolite diversity in situ and provided a comparative analysis of a range of diverse soil types. This approach paired with new developments in metagenomic technologies will make a substantial contribution to improving the likelihood for discovery and exploitation of new drugs for treating multi-resistant pathogenic bacteria

Metastasis to parotid gland from non Head and Neck tumors

Most primary tumors spreading metastasis to the parotid gland are usually located in the head and neck region, nonetheless, rarely, parotid gland can also be the target of metastatic localization site of distant primary tumors. The purpose of this study was to describe a clinical series of metastasis to the parotid gland from distant primary tumors (non Head & Neck)

Microbial community drivers of PK/NRP gene diversity in selected global soils

Background The emergence of antibiotic-resistant pathogens has created an urgent need for novel antimicrobial treatments. Advances in next-generation sequencing have opened new frontiers for discovery programmes for natural products allowing the exploitation of a larger fraction of the microbial community. Polyketide (PK) and non-ribosomal pepetide (NRP) natural products have been reported to be related to compounds with antimicrobial and anticancer activities. We report here a new culture-independent approach to explore bacterial biosynthetic diversity and determine bacterial phyla in the microbial community associated with PK and NRP diversity in selected soils. Results Through amplicon sequencing, we explored the microbial diversity (16S rRNA gene) of 13 soils from Antarctica, Africa, Europe and a Caribbean island and correlated this with the amplicon diversity of the adenylation (A) and ketosynthase (KS) domains within functional genes coding for non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), which are involved in the production of NRP and PK, respectively. Mantel and Procrustes correlation analyses with microbial taxonomic data identified not only the well-studied phyla Actinobacteria and Proteobacteria, but also, interestingly, the less biotechnologically exploited phyla Verrucomicrobia and Bacteroidetes, as potential sources harbouring diverse A and KS domains. Some soils, notably that from Antarctica, provided evidence of endemic diversity, whilst others, such as those from Europe, clustered together. In particular, the majority of the domain reads from Antarctica remained unmatched to known sequences suggesting they could encode enzymes for potentially novel PK and NRP. Conclusions The approach presented here highlights potential sources of metabolic novelty in the environment which will be a useful precursor to metagenomic biosynthetic gene cluster mining for PKs and NRPs which could provide leads for new antimicrobial metabolites

EMI and Beyond

This collection presents the state of the art on English-medium instruction (EMI) / Integrating content and language (ICL) in Italian higher education, drawing attention to different critical aspects of the teaching/learning experience and highlighting the perspectives of various educational stakeholders regarding the effectiveness of tertiary study in a foreign language. The chapters draw on a range of methodologies, from multimodal participant observation, to action research, to video-stimulated recall (VSR), to questionnaires and interviews, in examining language policies and practices across various educational settings. Overall, the volume suggests that internationalisation succeeds best when the form of lessons (language) and the content of lessons (disciplinary concepts) are constructively aligned in curriculum planning and delivery. This integration process requires the strategic support of educators to guarantee the quality of learning in multilingual education.

Biosynthetic potential of uncultured Antarctic soil bacteria revealed through long-read metagenomic sequencing

The growing problem of antibiotic resistance has led to the exploration of uncultured bacteria as potential sources of new antimicrobials. PCR amplicon analyses and short-read sequencing studies of samples from different environments have reported evidence of high biosynthetic gene cluster (BGC) diversity in metagenomes, indicating their potential for producing novel and useful compounds. However, recovering full-length BGC sequences from uncultivated bacteria remains a challenge due to the technological restraints of short-read sequencing, thus making assessment of BGC diversity difficult. Here, long-read sequencing and genome mining were used to recover >1400 mostly full-length BGCs that demonstrate the rich diversity of BGCs from uncultivated lineages present in soil from Mars Oasis, Antarctica. A large number of highly divergent BGCs were not only found in the phyla Acidobacteriota, Verrucomicrobiota and Gemmatimonadota but also in the actinobacterial classes Acidimicrobiia and Thermoleophilia and the gammaproteobacterial order UBA7966. The latter furthermore contained a potential novel family of RiPPs. Our findings underline the biosynthetic potential of underexplored phyla as well as unexplored lineages within seemingly well-studied producer phyla. They also showcase long-read metagenomic sequencing as a promising way to access the untapped genetic reservoir of specialised metabolite gene clusters of the uncultured majority of microbes

Designing and implementing an assay for the detection of rare and divergent NRPS and PKS clones in European, Antarctic and Cuban soils

The ever increasing microbial resistome means there is an urgent need for new antibiotics. Metagenomics is an underexploited tool in the field of drug discovery. In this study we aimed to produce a new updated assay for the discovery of biosynthetic gene clusters encoding bioactive secondary metabolites. PCR assays targeting the polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) were developed. A range of European soils were tested for their biosynthetic potential using clone libraries developed from metagenomic DNA. Results revealed a surprising number of NRPS and PKS clones with similarity to rare Actinomycetes. Many of the clones tested were phylogenetically divergent suggesting they were fragments from novel NRPS and PKS gene clusters. Soils did not appear to cluster by location but did represent NRPS and PKS clones of diverse taxonomic origin. Fosmid libraries were constructed from Cuban and Antarctic soil samples; 17 fosmids were positive for NRPS domains suggesting a hit rate of less than 1 in 10 genomes. NRPS hits had low similarities to both rare Actinobacteria and Proteobacteria; they also clustered with known antibiotic producers suggesting they may encode for pathways producing novel bioactive compounds. In conclusion we designed an assay capable of detecting divergent NRPS and PKS gene clusters from the rare biosphere; when tested on soil samples results suggest the majority of NRPS and PKS pathways and hence bioactive metabolites are yet to be discovere

EMI and Beyond

This collection presents the state of the art on English-medium instruction (EMI) / Integrating content and language (ICL) in Italian higher education, drawing attention to different critical aspects of the teaching/learning experience and highlighting the perspectives of various educational stakeholders regarding the effectiveness of tertiary study in a foreign language. The chapters draw on a range of methodologies, from multimodal participant observation, to action research, to video-stimulated recall (VSR), to questionnaires and interviews, in examining language policies and practices across various educational settings. Overall, the volume suggests that internationalisation succeeds best when the form of lessons (language) and the content of lessons (disciplinary concepts) are constructively aligned in curriculum planning and delivery. This integration process requires the strategic support of educators to guarantee the quality of learning in multilingual education.

Recovery from olfactory and gustatory dysfunction following COVID-19 acquired during Omicron BA.1 wave in Italy

Background: Despite alterations in the sense of smell and taste have dominated the symptoms of SARS-CoV-2 infection, the prevalence and the severity of self-reporting COVID-19 associated olfactory and gustatory dysfunction has dropped significantly with the advent of the Omicron BA.1 subvariant. However, data on the evolution of Omicron-related chemosensory impairment are still lacking. Objective: The aim of the present study was to estimate the prevalence and the recovery rate of self-reported chemosensory dysfunction 6-month after SARS-CoV-2 infection acquired during the predominance of the Omicron BA.1 subvariant in Italy. Methods: Prospective observational study based on the sino-nasal outcome tool 22 (SNOT-22), item "sense of smell or taste" and additional outcomes conducted in University hospitals and tertiary referral centers in Italy. Results: Of 338 patients with mild-to-moderate COVID-19 completing the baseline survey, 294 (87.0 %) responded to the 6-month follow-up interview. Among them, 101 (34.4 %) and 4 (1.4 %) reported an altered sense of smell or taste at baseline and at 6 months, respectively. Among the 101 patients with COVID-19-associated smell or taste dysfunction during the acute phase of the disease, 97 (96.0 %) reported complete resolution at 6 months. The duration of smell or taste impairment was significantly shorter in vaccinated patients (p = 0.007). Conclusions: Compared with that observed in subjects infected during the first wave of the pandemic, the recovery rate from chemosensory dysfunctions reported in the present series of patients infected during the predominance of the Omicron BA.1 subvariant was more favorable with a shorter duration being positively influenced by vaccination

Microbial hitchhikers harbouring antimicrobial-resistance genes in the riverine plastisphere

Background: The widespread nature of plastic pollution has given rise to wide scientific and social concern regarding the capacity of these materials to serve as vectors for pathogenic bacteria and reservoirs for Antimicrobial Resistance Genes (ARG). In- and ex-situ incubations were used to characterise the riverine plastisphere taxonomically and functionally in order to determine whether antibiotics within the water influenced the ARG profiles in these microbiomes and how these compared to those on natural surfaces such as wood and their planktonic counterparts. Results: We show that plastics support a taxonomically distinct microbiome containing potential pathogens and ARGs. While the plastisphere was similar to those biofilms that grew on wood, they were distinct from the surrounding water microbiome. Hence, whilst potential opportunistic pathogens (i.e. Pseudomonas aeruginosa, Acinetobacter and Aeromonas) and ARG subtypes (i.e. those that confer resistance to macrolides/lincosamides, rifamycin, sulfonamides, disinfecting agents and glycopeptides) were predominant in all surface-related microbiomes, especially on weathered plastics, a completely different set of potential pathogens (i.e. Escherichia, Salmonella, Klebsiella and Streptococcus) and ARGs (i.e. aminoglycosides, tetracycline, aminocoumarin, fluoroquinolones, nitroimidazole, oxazolidinone and fosfomycin) dominated in the planktonic compartment. Our genome-centric analysis allowed the assembly of 215 Metagenome Assembled Genomes (MAGs), linking ARGs and other virulence-related genes to their host. Interestingly, a MAG belonging to Escherichia –that clearly predominated in water– harboured more ARGs and virulence factors than any other MAG, emphasising the potential virulent nature of these pathogenic-related groups. Finally, ex-situ incubations using environmentally-relevant concentrations of antibiotics increased the prevalence of their corresponding ARGs, but different riverine compartments –including plastispheres– were affected differently by each antibiotic. Conclusions: Our results provide insights into the capacity of the riverine plastisphere to harbour a distinct set of potentially pathogenic bacteria and function as a reservoir of ARGs. The environmental impact that plastics pose if they act as a reservoir for either pathogenic bacteria or ARGs is aggravated by the persistence of plastics in the environment due to their recalcitrance and buoyancy. Nevertheless, the high similarities with microbiomes growing on natural co-occurring materials and even more worrisome microbiome observed in the surrounding water highlights the urgent need to integrate the analysis of all environmental compartments when assessing risks and exposure to pathogens and ARGs in anthropogenically-impacted ecosystems. 1SQe33MjkWBo3cdx_C_SmDVideo Abstrac

Advanced lung cancer inflammation index and its prognostic value in HPV-negative head and neck squamous cell carcinoma: a multicentre study

Purpose: The aim of this study is to evaluate the prognostic value of pre-treatment advanced lung cancer inflammation index (ALI) in patients with HPV-negative HNSCC undergoing up-front surgical treatment. Methods: The present multi-centre, retrospective study was performed in a consecutive cohort of patients who underwent upfront surgery with or without adjuvant (chemo)-radiotherapy for head and neck squamous cell carcinoma (HNSCC). Patients were stratified by ALI, and survival outcomes were compared between groups. In addition, the prognostic value of ALI was compared with two other indices, the prognostic nutritional index (PNI) and systemic inflammatory index (SIM). Results: Two hundred twenty-three patients met the inclusion criteria (151 male and 72 female). Overall and progression-free survival were significantly predicted by ALI < 20.4 (HR 3.23, CI 1.51–6.90 for PFS and HR 3.41, CI 1.47–7.91 for OS). Similarly, PNI < 40.5 (HR = 2.43, 95% CI: 1.31–4.51 for PFS and HR = 2.40, 95% CI: 1.19–4.82 for OS) and SIM > 2.5 (HR = 2.51, 95% CI: 1.23–5.10 for PFS and HR = 2.60, 95% CI: 1.19–5.67 for OS) were found to be significant predictors. Among the three indices, ALI < 20.4 identified the patients with the worst 5-year outcomes. Moreover, patients with a combination of low PNI and low ALI resulted to be a better predictor of progression (HR = 5.26, 95% CI: 2.01–13.73) and death (HR = 5.68, 95% CI: 1.92–16.79) than low ALI and low PNI considered alone. Conclusions: Our results support the use of pre-treatment ALI, an easily measurable inflammatory/nutritional index, in daily clinical practice to improve prognostic stratification in surgically treated HPV-negative HNSCC