Host and environmental factors shape upper airway microbiota and respiratory health across the human lifespan.

Our understanding of the normal variation in the upper respiratory tract (URT) microbiota across the human lifespan and how these relate to host, environment, and health is limited. We studied the microbiota of 3,104 saliva (<10 year-olds)/oropharynx (≥10 year-olds) and 2,485 nasopharynx samples of 3,160 Dutch individuals 0-87 years of age, participating in a cross-sectional population-wide study (PIENTER-3) using 16S-rRNA sequencing. The microbiota composition was strongly related to age, especially in the nasopharynx, with maturation occurring throughout childhood and adolescence. Clear niche- and age-specific associations were found between the microbiota composition and host/environmental factors and health outcomes. Among others, social interaction, sex, and season were associated with the nasopharyngeal microbial community. By contrast, the oral microbiota was more related to antibiotics, tobacco, and alcohol use. We present an atlas of the URT microbiota across the lifespan in association with environment and health, establishing a baseline for future research.

Human Monocytes Exposed to SARS-CoV-2 Display Features of Innate Immune Memory Producing High Levels of CXCL10 upon Restimulation.

INTRODUCTION: A role for innate immune memory in protection during COVID-19 infection or vaccination has been recently reported. However, no study so far has shown whether the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can train innate immune cells. The aim of this study was to investigate whether this virus can induce trained immunity in human monocytes. METHODS: Monocytes were exposed to inactivated SARS-CoV-2 (iSARS-CoV-2) for 24 h, followed by a resting period in the medium only and a secondary stimulation on day 6 after which the cytokine/chemokine and transcriptomic profiles were determined. RESULTS: Compared to untrained cells, the iSARS-CoV-2-trained monocytes secreted significantly higher levels of IL-6, TNF-α, CXCL10, CXCL9, and CXCL11 upon restimulation. Transcriptome analysis of iSARS-CoV-2-trained monocytes revealed increased expression of several inflammatory genes. As epigenetic and metabolic modifications are hallmarks of trained immunity, we analyzed the expression of genes related to these processes. Findings indicate that indeed SARS-CoV-2-trained monocytes show changes in the expression of genes involved in metabolic pathways including the tricarboxylic acid cycle, amino acid metabolism, and the expression of several epigenetic regulator genes. Using epigenetic inhibitors that block histone methyl and acetyltransferases, we observed that the capacity of monocytes to be trained by iSARS-CoV-2 was abolished. CONCLUSION: Overall, our findings indicate that iSARS-CoV-2 can induce properties associated with trained immunity in human monocytes. These results contribute to the knowledge required for improving vaccination strategies to prevent infectious diseases.

Higher off-target amplicon detection rate in MiSeq v3 compared to v2 reagent kits in the context of 16S-rRNA-sequencing.

One of the most widely used techniques in microbiota research is 16S-rRNA-sequencing. Several laboratory processes have been shown to impact sequencing results, especially in low biomass samples. Low biomass samples are prone to off-target amplification, where instead of bacterial DNA, host DNA is erroneously amplified. Knowledge on the laboratory processes influencing off-target amplification and detection is however scarce. We here expand on previous findings by demonstrating that off-target amplification is not limited to invasive biopsy samples, but is also an issue in low bacterial biomass respiratory (mucosal) samples, especially when below 0.3 pg/µL. We show that off-target amplification can partly be mitigated by using gel-based library purification methods. Importantly, we report a higher off-target amplicon detection rate when using MiSeq reagent kit v3 compared to v2 (mean 13.3% vs 0.1% off-target reads/sample, respectively), possibly as a result of differences in reagents or sequencing recipes. However, since after bioinformatic removal of off-target reads, MiSeq reagent kit v3 still results in a twofold higher number of reads when compared to v2, v3 is still preferred over v2. Together, these results add to the growing knowledge base on off-target amplification and detection, allowing researchers to anticipate this problem in 16S-rRNA-based microbiome studies involving low biomass samples.

Associations and recovery dynamics of the nasopharyngeal microbiota during influenza-like illness in the aging population.

Influenza-like illness (ILI), a disease caused by respiratory pathogens including influenza virus, is a major health concern in older adults. There is little information on changes and recovery dynamics of the nasopharyngeal (NP) microbiota of older adults associated with an ILI. Here, we compared the NP microbiota in older adults reporting (n = 240) or not (n = 157) ILI during the 2014-2015 influenza season at different times of the ILI event. A small but significant effect of the ILI was observed on the microbiota community composition and structure when compared to controls and samples collected at recovery. Corynebacterium was negatively associated with ILI and its abundance increased after recovery. Potential pathobionts such as Haemophilus, Porphyromonas and Gemella had higher abundances during acute-ILI. Stability and changes in the NP microbial community showed individual dynamics. Key core genera, Corynebacterium, Moraxella and Dolosigranulum exhibited higher inter-individual variability in acute-ILI, but showed comparable variability to controls after recovery. Participants in the ILI group with higher core microbiota abundances at the acute phase showed higher microbiota stability after recovery. Our findings demonstrate that acute-ILI is associated with alterations in the phylogenetic structure of the NP microbiota in older adults. The variation in the core microbiota suggests imbalances in the ecosystem, which could potentially play a role in the susceptibility and recovery of the NP microbiota after an ILI event.

Benchmarking laboratory processes to characterise low-biomass respiratory microbiota.

The low biomass of respiratory samples makes it difficult to accurately characterise the microbial community composition. PCR conditions and contaminating microbial DNA can alter the biological profile. The objective of this study was to benchmark the currently available laboratory protocols to accurately analyse the microbial community of low biomass samples. To study the effect of PCR conditions on the microbial community profile, we amplified the 16S rRNA gene of respiratory samples using various bacterial loads and different number of PCR cycles. Libraries were purified by gel electrophoresis or AMPure XP and sequenced by V2 or V3 MiSeq reagent kits by Illumina sequencing. The positive control was diluted in different solvents. PCR conditions had no significant influence on the microbial community profile of low biomass samples. Purification methods and MiSeq reagent kits provided nearly similar microbiota profiles (paired Bray-Curtis dissimilarity median: 0.03 and 0.05, respectively). While profiles of positive controls were significantly influenced by the type of dilution solvent, the theoretical profile of the Zymo mock was most accurately analysed when the Zymo mock was diluted in elution buffer (difference compared to the theoretical Zymo mock: 21.6% for elution buffer, 29.2% for Milli-Q, and 79.6% for DNA/RNA shield). Microbiota profiles of DNA blanks formed a distinct cluster compared to low biomass samples, demonstrating that low biomass samples can accurately be distinguished from DNA blanks. In summary, to accurately characterise the microbial community composition we recommend 1. amplification of the obtained microbial DNA with 30 PCR cycles, 2. purifying amplicon pools by two consecutive AMPure XP steps and 3. sequence the pooled amplicons by V3 MiSeq reagent kit. The benchmarked standardized laboratory workflow presented here ensures comparability of results within and between low biomass microbiome studies.

Behçet's Disease Under Microbiotic Surveillance? A Combined Analysis of Two Cohorts of Behçet's Disease Patients.

Background: In Behçet's disease (BD), an auto-inflammatory vasculitis, an unbalanced gut microbiota can contribute to pro-inflammatory reactions. In separate studies, distinct pro- and anti-inflammatory bacteria associated with BD have been identified. Methods: To establish disease-associated determinants, we performed gut microbiome profiling in BD patients from the Netherlands (n = 19) and Italy (n = 13), matched healthy controls (HC) from the Netherlands (n = 17) and Italy (n = 15) and oral microbiome profiling in Dutch BD patients (n = 18) and HC (n = 15) by 16S rRNA gene sequencing. In addition, we used fecal IgA-SEQ analysis to identify specific IgA coated bacterial taxa in Dutch BD patients (n = 13) and HC (n = 8). Results: In BD stool samples alpha-diversity was conserved, whereas beta-diversity analysis showed no clustering based on disease, but a significant segregation by country of origin. Yet, a significant decrease of unclassified Barnesiellaceae and Lachnospira genera was associated with BD patients compared to HC. Subdivided by country, the Italian cohort displays a significant decrease of unclassified Barnesiellaceae and Lachnospira genera, in the Dutch cohort this decrease is only a trend. Increased IgA-coating of Bifidobacterium spp., Dorea spp. and Ruminococcus bromii species was found in stool from BD patients. Moreover, oral Dutch BD microbiome displayed increased abundance of Spirochaetaceae and Dethiosulfovibrionaceae families. Conclusions: BD patients show decreased fecal abundance of Barnesiellaceae and Lachnospira and increased oral abundance of Spirochaetaceae and Dethiosulfovibrionaceae. In addition, increased fecal IgA coating of Bifidobacterium, Ruminococcus bromii and Dorea may reflect retention of anti-inflammatory species and neutralization of pathosymbionts in BD, respectively. Additional studies are warranted to relate intestinal microbes with the significance of ethnicity, diet, medication and response with distinct pro- and inflammatory pathways in BD patients.

Roux-Y Gastric Bypass and Sleeve Gastrectomy directly change gut microbiota composition independent of surgery type.

Bariatric surgery in morbid obesity, either through sleeve gastrectomy (SG) or Roux-Y gastric bypass (RYGB), leads to sustainable weight loss, improvement of metabolic disorders and changes in intestinal microbiota. Yet, the relationship between changes in gut microbiota, weight loss and surgical procedure remains incompletely understood. We determined temporal changes in microbiota composition in 45 obese patients undergoing crash diet followed by SG (n = 22) or RYGB (n = 23). Intestinal microbiota composition was determined before intervention (baseline, S1), 2 weeks after crash diet (S2), and 1 week (S3), 3 months (S4) and 6 months (S5) after surgery. Relative to S1, the microbial diversity index declined at S2 and S3 (p < 0.05), and gradually returned to baseline levels at S5. Rikenellaceae relative abundance increased and Ruminococcaceae and Streptococcaceae abundance decreased at S2 (p < 0.05). At S3, Bifidobacteriaceae abundance decreased, whereas those of Streptococcaceae and Enterobacteriaceae increased (p < 0.05). Increased weight loss between S3-S5 was not associated with major changes in microbiota composition. No significant differences appeared between both surgical procedures. In conclusion, undergoing a crash diet and bariatric surgery were associated with an immediate but temporary decline in microbial diversity, with immediate and permanent changes in microbiota composition, independent of surgery type.

Molecular surveillance of nasopharyngeal carriage of Streptococcus pneumoniae in children vaccinated with conjugated polysaccharide pneumococcal vaccines.

Following the introduction of pneumococcal conjugate vaccines (PCVs) for infants, surveillance studies on Streptococcus pneumoniae carriage have proven valuable for monitoring vaccine effects. Here, we compared molecular versus conventional diagnostic methods in prospective cross-sectional surveillances in vaccinated infants in the Netherlands. Nasopharyngeal samples (n = 1169) from 11- and 24-month-old children, collected during autumn/winter 2010/2011 and 2012/2013, were tested by conventional culture for S. pneumoniae. DNA extracted from all culture-plate growth was tested by qPCR for pneumococcal-specific genes (lytA/piaB) and selected serotypes (including PCV13-serotypes). qPCR significantly increased the number of carriers detected compared to culture (69% vs. 57%, p < 0.0001). qPCR assays targeting vaccine-serotypes 4 and 5 proved non-specific (results excluded). For serotypes reliably targeted by qPCR, the number of serotype-carriage events detected by qPCR (n = 709) was 1.68× higher compared to culture (n = 422). There was a strong correlation (rho = 0.980; p < 0.0001) between the number of serotypes detected using qPCR and by culture. This study demonstrates the high potential of molecular methods in pneumococcal surveillances, particularly for enhanced serotype detection. We found no evidence of a hidden circulation of vaccine-targeted serotypes, despite vaccine-serotypes still significantly contributing to invasive pneumococcal disease in unvaccinated individuals, supporting the presence of a substantial S. pneumoniae reservoir outside vaccinated children.