Abbreviations: AAI, pairwise amino acid identity; ANI, average nucleotide identity; cAAI, pairwise amino acid identity of conserved genes. †These authors also contributed equally to this work †These authors share senior authorship. Six supplementary tables and seven supplementary figures are available with the online version of this article.

To improve our understanding of upper respiratory tract (URT) diseases and the underlying microbial pathogenesis, a better characterization of the healthy URT microbiome is crucial. In this first large-scale study, we obtained more insight in the URT microbiome of healthy adults. Hereto, we collected paired nasal and nasopharyngeal swabs from 100 healthy participants in a citizen-science project. High-throughput 16S rRNA gene V4 amplicon sequencing was performed and samples were processed using the Divisive Amplicon Denoising Algorithm 2 (DADA2) algorithm. This allowed us to identify the bacterial richness and diversity of the samples in terms of amplicon sequence variants (ASVs), with special attention to intragenus variation. We found both niches to have a low overall species richness and uneven distribution. Moreover, based on hierarchical clustering, nasopharyngeal samples could be grouped into some bacterial community types at genus level, of which four were supported to some extent by prediction strength evaluation: one intermixed type with a higher bacterial diversity where Staphylococcus, Corynebacterium, and Dolosigranulum appeared main bacterial members in different relative abundances, and three types dominated by either Moraxella, Streptococcus, or Fusobacterium. Some of these bacterial community types such as Streptococcus and Fusobacterium were nasopharynx-specific and never occurred in the nose. No clear association between the nasopharyngeal bacterial profiles at genus level and the variables age, gender, blood type, season of sampling, or common respiratory allergies was found in this study population, except for smoking showing a positive association with Corynebacterium and Staphylococcus. Based on the fine-scale resolution of the ASVs, both known commensal and potential pathogenic bacteria were found within several genera – particularly in Streptococcus and Moraxella – in our healthy study population. Of interest, the nasopharynx hosted more potential pathogenic species than the nose. To our knowledge, this is the first large-scale study using the DADA2 algorithm to investigate the microbiota in the “healthy” adult nose and nasopharynx. These results contribute to a better understanding of the composition and diversity of the healthy microbiome in the URT and the differences between these important URT niches.Trial Registration: Ethical Committee of Antwerp University Hospital, B300201524257, registered 23 March 2015, ClinicalTrials.gov Identifier: NCT02 933983.

The closely related species of the Lactobacillus casei group are extensively studied because of their applications in food fermentations and as probiotics. Our results show that many strains in this group are incorrectly classified and that reclassifying them to their most closely related species type strain improves the functional predictive power of their taxonomy. In addition, our findings may spark increased interest in the L. casei species. We find that after reclassification, only 10 genomes remain classified as L. casei. These strains show some interesting properties. First, they all appear to be catalase positive. This suggests that they have increased oxidative stress resistance. Second, we isolated an L. casei strain from the human upper respiratory tract and discovered that it and multiple other L. casei strains harbor one or even two large, glycosylated putative surface adhesins. This might inspire further exploration of this species as a potential probiotic organism.

Highlights d Lactic acid bacteria are enriched in the healthy human nose and nasopharynx d Lactobacillus casei AMBR2 is functionally adapted to the upper respiratory tract d L. casei AMBR2 has antimicrobial and immunomodulatory properties d Live L. casei AMBR2 is safe for intranasal application in healthy humans

There is a clear need to better understand the pathology and specific microbiome features in chronic rhinosinusitis patients, but little is known about the bacterial topography and continuity between the different niches of the upper respiratory tract. Our work showed that the anterior nares could be an important reservoir for potential sinus pathobionts. This has implications for the diagnosis, prevention, and treatment of CRS. In addition, we found a potential pathogenic role for the Corynebacterium tuberculostearicum, Haemophilus influenzae/H. aegyptius, and Staphylococcus taxa and a potential beneficial role for Dolosigranulum. Finally, a decreased microbiome diversity was observed in patients with chronic rhinosinusitis without nasal polyps compared to that in healthy controls but not in chronic rhinosinusitis patients with nasal polyps. This suggests a potential role for the microbiome in disease development or progression of mainly this phenotype.

Highlights d Lactic acid bacteria are enriched in the healthy human nose and nasopharynx d Lactobacillus casei AMBR2 is functionally adapted to the upper respiratory tract d L. casei AMBR2 has antimicrobial and immunomodulatory properties d Live L. casei AMBR2 is safe for intranasal application in healthy humans

Spontaneous vegetable fermentations, with their rich flavors and postulated health benefits, are regaining popularity. However, their microbiology is still poorly understood, therefore raising concerns about food safety. In addition, such spontaneous fermentations form interesting cases of man-made microbial ecosystems. Here, samples from 38 carrot juice fermentations were collected through a citizen science initiative, in addition to three laboratory fermentations. Culturing showed that Enterobacteriaceae were outcompeted by lactic acid bacteria (LAB) between 3 and 13 days of fermentation. Metabolite-target analysis showed that lactic acid and mannitol were highly produced, as well as the biogenic amine cadaverine. High-throughput 16S rRNA gene sequencing revealed that mainly species of Leuconostoc and Lactobacillus (as identified by 8 and 20 amplicon sequence variants [ASVs], respectively) mediated the fermentations in subsequent order. The analyses at the DNA level still detected a high number of Enterobacteriaceae, but their relative abundance was low when RNA-based sequencing was performed to detect presumptive metabolically active bacterial cells. In addition, this method greatly reduced host read contamination. Phylogenetic placement indicated a high LAB diversity, with ASVs from nine different phylogenetic groups of the Lactobacillus genus complex. However, fermentation experiments with isolates showed that only strains belonging to the most prevalent phylogenetic groups preserved the fermentation dynamics. The carrot juice fermentation thus forms a robust man-made microbial ecosystem suitable for studies on LAB diversity and niche specificity.IMPORTANCE The usage of fermented food products by professional chefs is steadily growing worldwide. Meanwhile, this interest has also increased at the household level. However, many of these artisanal food products remain understudied. Here, an extensive microbial analysis was performed of spontaneous fermented carrot juices which are used as nonalcoholic alternatives for wine in a Belgian Michelin star restaurant. Samples were collected through an active citizen science approach with 38 participants, in addition to three laboratory fermentations. Identification of the main microbial players revealed that mainly species of Leuconostoc and Lactobacillus mediated the fermentations in subsequent order. In addition, a high diversity of lactic acid bacteria was found; however, fermentation experiments with isolates showed that only strains belonging to the most prevalent lactic acid bacteria preserved the fermentation dynamics. Finally, this study showed that the usage of RNA-based 16S rRNA amplicon sequencing greatly reduces host read contamination.

Background: The popularity of Galleria mellonella as invertebrate model is increasing rapidly, because it forms an attractive alternative to study bacterial, fungal and viral infections, toxin biology, and to screen antimicrobial drugs. For a number of vertebrate and invertebrate animal and plant models, it has been established that the commensals present within the microbial communities on various host surfaces will influence the host's immune and growth development state and the colonization capacity of newly introduced microorganisms. The microbial communities of Galleria mellonella larvae have, however, not yet been well characterized. Results: In this study, we present the bacterial communities that were found by 16S rRNA amplicon sequencing on different body sites of G. mellonella larvae. These communities showed very little diversity and were mostly dominated by one Enterococcus taxon. In addition, we found that the production conditions (as 'bait' for fishing or under more controlled 'research grade' conditions-with or without hormones and antibiotics) appear to have little impact on the microbiota of the larvae. Conclusions: Establishment of the simplicity of the microbiota of G. mellonella larvae underlines the potential of the larvae as a model host system for microbiome-host interactions.