Nanopore sequencing is a promising technology used for 16S rRNA gene amplicon sequencing as it can provide full-length 16S reads and has a low up-front cost that allows research groups to set up their own sequencing workflows. To assess whether Nanopore with the improved error rate of the Kit 12 chemistry should be adopted as the preferred sequencing technology instead of Illumina for 16S amplicon sequencing of the gut microbiota, we used a mock community and human faecal samples to compare diversity, richness, and species-level community structure, as well as the replicability of the results. Nanopore had less noise, better accuracy with the mock community, a higher proportion of reads from the faecal samples classified to species, and better replicability. The difference between the Nanopore and Illumina results of the faecal bacterial community structure was significant but small compared to the variation between samples. The results show that Nanopore is a better choice for 16S rRNA gene amplicon sequencing when the focus is on species-level taxonomic resolution, the investigation of rare taxa, or an accurate estimation of richness. Illumina 16S sequencing should be reserved for communities with many unknown species, and for studies that require the resolution of amplicon sequence variants.

Aim: The aim of the present study was to investigate and correlate the prevalence of Enterococcus faecalis in saliva and in root canals with different pulpal and periapical conditions.Methodology: Sixty-seven patients were divided into five groups based on pulpal and periapical tissue status: healthy vital teeth (HVT, n=7), healthy treated teeth without lesion (HTT, n=9), irreversible pulpitis (IP, n=13), necrosis (N, n=18), and post-treatment apical periodontitis (PTAP, n=20). Saliva, rubber dam, sterility control and pre-treatment root canal samples were collected and microbiologically processed by culture method. The phylogenetic relationship of E. faecalis isolates collected from root canals and saliva were investigated by whole genome sequencing. Fisher’s exact test was used to correlate the presence of E. faecalis in root canals or saliva with clinical and/or radiographic findings. Linear/logistic regression analyses were performed to establish the relationship between the presence of E. faecalis in root canals, saliva, and the status of periapical tissues.Results: E. faecalis was found in 18 root canal and saliva samples. E. faecalis root canal isolates were recovered with the highest frequency from post-treatment apical periodontitis. The occurrence of E. faecalis in saliva was strongly associated with its detection in the root canals (P < 0.001). The pretreatment presence of E. faecalis in root canals was associated with significantly higher odds of having periapical lesions (OR=11.03; 95% CI, 1.27-95.70; p < 0.05). Saliva and root canal isolates from the same patient were highly correlated at the phylogenetic level (Jaccard index >0.95).Conclusion: This pilot study confirms the role of E. faecalis in developing peri-radicular lesions in secondary endodontic infections and suggests that saliva could be the main source of infection. Further studies are needed to investigate the exact origin of this bacteria and its true role in the pathogenesis of secondary/persistent endodontic infections.

Streptococcus mitis is a Gram-positive bacterium, member of the oral commensal microbiota, which can occasionally be the etiologic agent of diseases such as infective endocarditis, bacteraemia and septicaemia. The highly recombinogenic and repetitive nature of the S. mitis genome impairs the assembly of a complete genome relying only on short sequencing reads. Oxford Nanopore sequencing can overcome this limitation by generating long reads, enabling the resolution of genomic repeated regions and the assembly of a complete genome sequence. Since the output of a Nanopore sequencing run is strongly influenced by genomic DNA quality and molecular weight, the DNA isolation is the crucial step for an optimal sequencing run. In the present work, we have set up and compared three DNA isolation methods on two S. mitis strains, evaluating their capability of preserving genomic DNA integrity and purity. Sequencing of DNA isolated with a mechanical lysis-based method, despite being cheaper and quicker, did not generate ultra-long reads (maximum read length of 59516 bases) and did not allow the assembly of a circular complete genome. Two methods based on enzymatic lysis of the bacterial cell wall, followed by either (i) a modified CTAB DNA isolation procedure, or (ii) a DNA purification after osmotic lysis of the protoplasts allowed the sequencing of ultra-long reads up to 107294 and 181199 bases in length, respectively. The reconstruction of a circular complete genome was possible sequencing DNAs isolated using the enzymatic lysis-based methods.

Background: Timely and accurate identification of bloodstream pathogens is critical for targeted antimicrobial therapy in sepsis. Conventional blood cultures remain the Standard-of-Care (SoC) for pathogen identification but are limited by low sensitivity and prolonged turnaround times, hampering timely and targeted antimicrobial stewardship. Advances in next-generation sequencing (NGS) offer potential for culture-independent, rapid, and comprehensive detection of pathogens and prediction of antimicrobial resistance. This study evaluated the diagnostic performance of PISTE™ technology, an NGS-based diagnostic workflow combining full-length 16S rRNA gene sequencing and metagenomic analysis for the diagnosis of circulating bacteria in sepsis.Methods: In this prospective, multicenter, phase IIa proof-of-concept study, adult patients with suspected sepsis were enrolled from four hospitals in Athens, Greece. Blood samples were collected prior to antibiotic initiation and processed using SoC cultures and PISTE platform. PISTE integrates automated DNA purification (KingFisher, Thermo Fisher Scientific), full-length 16S rRNA gene sequencing, metagenomics analysis (SQK-PRB114.24, Oxford Nanopore Technologies), and real-time sequencing using Oxford Nanopore GridION Mk1b device. A dedicated analysis pipeline was developed for accurate pathogen detection and prediction of antimicrobial resistance profiles. The primary endpoint was the diagnostic concordance between PISTE and SoC cultures.Results: A total of 100 patients (median age 79 years, median Charlson’s Comorbidity Index 5) were enrolled. Of these, 71 patients met Sepsis-3 criteria. In this subgroup, PISTE showed an overall accuracy of 95.7%, with a sensitivity of 91.7%, specificity of 96.5%, positive predictive value of 84.6%, and negative predictive value of 98.2% compared to SoC. The median time to pathogen identification and Antimicrobial Susceptibility Testing (AST) with PISTE was 12.0 hours, significantly faster than in SoC cultures (30.4 hours, p < 0.0001). Resistance gene profiling showed strong agreement with SoC AST results, particularly for β-lactam and carbapenem resistance.Conclusions: PISTE technology exhibited high diagnostic accuracy and significantly reduced turnaround time compared to conventional cultures, supporting its potential as a short turnaround time and reliable diagnostic tool for bloodstream infections. Further optimization and validation in larger cohorts are warranted to enhance clinical implementation and improve antimicrobial stewardship in sepsis management.

The human respiratory tract is colonized by a complex microbial community that helps maintain respiratory health and plays a crucial role in defending the host from infections. Respiratory viruses have been demonstrated to alter microbiota composition, resulting in opportunistic species expansion, and increasing the disease severity and host susceptibility to bacterial co-infections. This study aims to examine the compositional differences in the nasal microbiota between SARS-CoV-2-infected and non-infected patients. We conducted Oxford Nanopore full-length 16S rRNA sequencing on nasal swabs from 94 COVID-19 negative and 85 COVID-19 positive patients collected during the SARS-CoV-2 pandemic in Malta. Our analysis identified significant alpha and beta diversity differences in the nasal microbiota composition among our study groups. We observed a trend toward decreased microbial richness and evenness in the COVID-Positive cohort with and increased abundance of common nasal opportunistic species including Citrobacter koseri, Dolosigranulum pigrum, Haemophilus influenzae, Klebsiella pneumoniae, and Moraxella catarrhalis. The findings from this study are in line with previously published papers identifying key alterations in the nasal microbiota composition associated with SARS-CoV-2 infection. Understanding these microbiome-driven mechanisms could present novel prognostic markers or offer new approaches for disease prevention and treatment.

Acne vulgaris is a common dermatological condition strongly associated with disruptions in the skin microbiota, specifically involving key species such as Cutibacterium acnes and Staphylococcus epidermidis. This study investigates the efficacy of SkinDuo^TM, a topical probiotic containing Lactiplantibacillus plantarum, in modulating the skin microbiota and improving clinical outcomes in patients with acne vulgaris. Over a 4-week to 8-week observational study period, microbial composition and diversity shifts were analyzed using full-length 16S rRNA sequencing. Patient responses were categorized into “good” responders (showing significant clinical improvement) and “no_change” responders (with minimal or no improvement). SkinDuo^TM treatment resulted in lower post-treatment Cutibacterium acnes abundance in the “good” group compared to the “no_change” group. The “good” group maintained a stable level of alpha diversity following treatment. In contrast, the “no_change” group exhibited a marked reduction in microbial diversity. Beta diversity analysis revealed distinct clustering patterns associated with improved clinical outcomes. These findings suggest that the preservation of microbial richness and evenness may serve as a potential biomarker for positive response to probiotic therapy. This study highlights the potential of SkinDuo^TM to restore microbial balance and alleviate acne symptoms, contributing to the growing body of evidence supporting microbiome-based therapeutic strategies in dermatology.